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Non-Diabetic Hypoglycemia

ABSTRACT

 

Objective: To review the diagnosis, evaluation, and management of non-diabetic hypoglycemia in adults. Methods: A literature review using PubMed and Google Scholar was performed. In absence of data, clinical expert opinion was provided. Results: Hypoglycemia in an individual without diabetes is uncommon mainly because of a tightly regulated counterregulatory physiological response. A detailed medical history, review of medications and physical exam findings are critical first steps in providing guidance for further investigation in a non-diabetic person with documented hypoglycemia based on Whipple’s triad (presence of symptoms when plasma glucose concentrations are low and absence of symptoms with normalized glycemia). In this review, we highlight strategies to diagnose and treat hypoglycemic disorders in non-diabetic individuals based on underlying mechanisms. Conclusion: Evaluation and management of non-diabetic hypoglycemia should be individualized based on clinical presentation and suspected diagnoses.

 

INTRODUCTION

 

In healthy humans, glucose concentrations are efficiently maintained within a narrow range by the physiological mechanisms that respond to intermittent exogenous nutrient ingestion by enhancing glucose utilization and respond to intervals of nutrient deprivation by enhancing glucose production. Deviation of glucose from the normal range in both hyper or hypoglycemia only occurs when physiological mechanisms involved in maintaining the balance between the glucose utilization and the glucose production fail. As such, hypoglycemia is a manifestation of a heterogeneous group of underlying disorders that increase glucose utilization or reduce glucose production or a combination of both.

 

While hypoglycemia in persons without diabetes is relatively rare (1), the clinical relevance of this condition regarding patient safety, cognitive function, and quality of life is undeniable. Depending on severity and duration, hypoglycemia also can be fatal (2). Furthermore, hypoglycemia blunts defense against subsequent hypoglycemia leading to a vicious cycle of recurrent hypoglycemia (3), which in turn not only exaggerates related morbidities but also makes the diagnosis more complex.

 

Here, we review the current recommendations regarding diagnosis, pathophysiology, and management of hypoglycemia in non-diabetic individuals. Hypoglycemia in the pediatric population, hypoglycemia caused by anti-diabetic medications, and a comprehensive review of insulinomas can be found in the Endotext chapters entitled “Hypoglycemia in Neonates, Infants, and Children” (4), “Hypoglycemia During Therapy of Diabetes” (5) and “Insulinoma” (6), respectively.

 

PHYSIOLOGY / PATHOPHYSIOLOGY

 

Glucose is the main fuel for the brain since it cannot store glycogen or synthesize glucose (7,8). To minimize any disturbance in glucose supplies to the brain and cerebral function, therefore, redundant but very efficient physiological counter-regulatory responses are in place to prevent or correct hypoglycemia (8-10).

 

Prandial

 

In the prandial condition, the extent of glycemic excursion (difference between glycemic peak and nadir concentrations) is determined by the pace of food transition from the stomach into the gut (gastric emptying) as well as the net hepatic and extrahepatic glucose uptake (11). In healthy subjects, during the first 30-60 min of an oral glucose or mixed tolerance meal test, plasma glucose and insulin concentrations rise, shifting the hepatic net glucose output during fasting condition to net glucose uptake during the prandial state (12). As a result of changes in glucose kinetics in the early absorptive phase of glucose/mixed meal ingestion, plasma glucose concentrations start to decline in the latter absorptive phase falling below premeal levels in parallel with reduction in ingested glucose delivery to the gut. The regulatory mechanisms that are responsible for preventing hypoglycemia and restoring euglycemia during the transition of glucose flux from the ingested glucose delivery to the hepatic glucose production are not fully characterized, but both hormonal and non-hormonal factors play a role (13).

 

Fasting

 

In the fasting or postabsorptive state (4-6 hours after nutrient ingestion), plasma glucose  concentrations range from 80-90 mg/dl and rates of glucose utilization and production are equal (2mg/kg/min) (14). Glucose homeostasis during fasting is tightly regulated by a reciprocal bihormonal response, in which reduction in glucose concentrations below baseline reduces β-cell insulin secretion and stimulates α-cell glucagon release (15). While the full range of glucoregulatory effects of glucagon in the prandial state is unclear, the main function of glucagon in the fasting state is to counterbalance the action of insulin on hepatic glucose production (15-17). Glucose production is mainly (~80%) attributed to hepatic glycogenolysis with a smaller contribution (~20%) from hepatic gluconeogenesis primarily from amino acids and lactate (11). After an overnight fast, the liver contains ~ 50 g of glycogen storage, which can supply glucose for 24 hours after complete depletion. With prolonged fasting, hyperglucagonemia enhances gluconeogenesis and hypoinsulinemia promotes lipolysis.  Lipolysis releases glycerol, a gluconeogenic substrate, and free fatty acids that are converted to ketones, mainly beta hydroxybutyrate (BOHB) and acetoacetate, in the liver to be used as an alternate fuel by the brain.

 

Using a hyperinsulinemic hypoglycemic clamp in the fasting condition in normal humans, a decrement in plasma glucose concentration from the physiological range, in a hierarchical manner: (1) suppresses endogenous insulin secretion to lower glucose utilization, (2) increases glucagon response to increase glucose production, and (3) enhances epinephrine secretion (more relevant in absence of glucagon secretion) as well as cortisol and growth hormone release (more relevant during prolonged hypoglycemia) (8). Under physiological conditions during the fasting state, this counterregulatory response can reestablish euglycemia and prevent symptoms. A greater decline in plasma glucose concentration, though, would result in a symptomatic autonomic response to warn of low glucose and prompt the person to correct it by eating. However, if the individual did not (or could not) intervene, such as in the presence of overwhelming hyperinsulinemia) or blunted glucose counterregulatory responses, plasma glucose concentrations would drop further and neuroglycopenic symptoms and cognitive dysfunction would occur (Table 1) (18).

 

Table 1. Symptoms of Hypoglycemia

Autonomic (neurogenic)

Neuroglycopenic

Sweating

Anxiety

Tremor

Palpitation

Hunger

Tingling

Ill-defined symptoms

Warmth

Behavioral changes

Blurred vision

Confusion/difficulty speaking

Dizziness/lightheadedness

Lethargy and weakness

Seizure

Loss of consciousness/coma

 

It is unclear whether this hierarchy in hormonal responses or glycemic thresholds described during the fasting state would also apply to the prandial state.  Here, we classify the hypoglycemic disorders based on timing from meal ingestion (fasting versus prandial) given the differences in the regulatory factors involved in glucose metabolism between the two conditions.

 

DIAGNOSIS AND EVALUATION

 

Diagnosis of hypoglycemia should be made when symptoms accompany low plasma glucose concentrations (chemical hypoglycemia) but symptoms are absent when plasma glucose levels are normalized (Whipple’s triad (19)).

 

Chemical hypoglycemia has been defined based on a glucose threshold that can evoke a counterregulatory response. In the fasting state, a decline in glucose below 55 mg/dl causes neurogenic symptoms while insulin secretion is maximally suppressed (insulin < 3 uU/ml and C-peptide < 0.6 ng/ml) and glucagon response is maximized (9).

 

Awareness of hypoglycemia alerting individuals to correct hypoglycemia is mainly because of increased autonomic nervous system activity triggered by hypoglycemia. The autonomic (neurogenic) hypoglycemic symptoms can be adrenergic (such as palpitation, tremor, and anxiety) or cholinergic (such as sweating, hunger, and paresthesia) (18). As glucose concentrations drop below 48-50 mg/dl during the fasting state, the neuroglycopenic symptoms (caused by brain glucose deprivation) manifest; these symptoms range from behavioral changes, fatigue, and confusion to loss of consciousness or seizure (18,20-22).

 

Diagnosis of hypoglycemia in the prandial state is much more complex since the glycemic threshold to define hypoglycemia in the prandial state has not been well characterized. Using an oral glucose challenge in 650 healthy individuals in a previous study (23), 10% of subjects developed postprandial nadir glucose concentrations below 47 mg/dl without associated symptoms, suggesting that asymptomatic low glucose events are relatively common following an oral glucose load in normal humans. Hence, the mixed meal test (described below) is the preferred provocative test to diagnose prandial hypoglycemia. Furthermore, recurrent postprandial symptoms suggestive of hypoglycemia but not associated with low glucose concentrations have also been observed in normal individuals (24), indicating that other factors beyond hypoglycemia play a role in provoking autonomic symptoms.

 

Therefore, after obtaining a detailed medical history and physical exam, diagnosis of hypoglycemia should be confirmed by verification of low glucose concentration associated with symptoms or signs that are relieved by raising glucose values (Whipple’s triad).

 

A careful history of nutritional status, current medication use, and concurrent multisystem illnesses such as liver, heart, kidney failure, or sepsis, as well as a thorough physical exam and laboratory data, can point to existing primary conditions that predispose to hypoglycemia. This is especially crucial in patients who are often too ill to be subjected to extensive evaluation. For healthy subjects who lack any background predisposing illnesses, the details about timing (relationship to food ingestion, physical activities, day versus nocturnal time), severity (frequency, presence of neuroglycopenia, and requiring assistance to treat), and time of onset of hypoglycemic episodes are critical in differential diagnosis. To understand the pattern of hypoglycemic episodes, reviewing the records of symptoms, activity, food intake along with capillary blood or interstitial glucose levels measured by glucometer or continuous glucose monitoring (CGM), respectively, may be helpful. However, the accuracy of glucometer and CGM is low in the hypoglycemic range, and they should not be used for diagnostic purposes.  On the other hand, masked (blinded) monitoring by CGM can provide insights into patterns of hypoglycemic episodes and triggering factors during patients’ daily routine (25).

 

The flowchart in Figure 1 demonstrates the suggested approach for evaluation in healthy appearing patients after a careful medical history, physical exam, and laboratory data excludes an underlying illness that can predispose to hypoglycemia. Disorders that may cause hypoglycemia are listed in Table 2.

 

To confirm the diagnosis and explore etiology, it is necessary to collect blood samples during hypoglycemia, whether it occurs spontaneously or by provoked testing that can be selected based on clues from the medical history.

 

In asymptomatic patients with documented chemical hypoglycemia, artifactual hypoglycemia due to conditions such as reticulocytosis (polycythemia, sickle cell anemia), leukocytosis (leukemia), and thrombocytosis that increase in vitro glycolysis in the blood sample while awaiting laboratory analysis should be considered (26).  Also, nadir glucose levels in the prandial state can be low without any associated symptoms, particularly in persons with a history of upper gastrointestinal (GI) surgery (25). A potential diagnostic challenge in using clinical criteria remains in patients who are adapted to recurrent hypoglycemia by blunted autonomic response, so called hypoglycemic unawareness (27).  It has been well recognized that antecedent insulin-induced hypoglycemia impairs counterregulatory glucose responses and blunts hypoglycemia symptoms (mainly autonomic symptoms) in normal humans (28). Therefore, in patients with a high index of clinical suspicion, monitoring of symptoms and signs of neuroglycopenia, which is less likely to be affected by recurrent hypoglycemia, and reevaluation over time should be considered.

 

Figure 1. Evaluation of non-diabetic hypoglycemia in healthy appearing adults.

 

 

Table 2. Causes of Hypoglycemia

Artifactual Hypoglycemia (without symptoms)

Reticulocytosis (polycythemia, sickle cell anemia)

Leukocytosis (leukemia)

Thrombocytosis

Fasting Hypoglycemia (> 5 hour from the last meal)

High Insulin, Low beta-hydroxy butyrate, High glucagon response

Insulinoma

Auto immune syndrome (antibodies to insulin or the insulin receptor)

Factitial due to exogenous insulin 

Factitial due to insulin secretagogues

Induced by non-diabetic medications

Low Insulin, High beta-hydroxy butyrate, Low glucagon response

Ketotic hypoglycemia

Prolonged exercise

Alcohol induced

Glycogen storage diseases

Post Prandial Hypoglycemia (within 5 hours from the last meal)

Bariatric surgery

Nesidioblastosis

Hereditary fructose intolerance

Associated with Other Disorder

Critical illness (liver failure, congestive heart failure, sepsis, renal failure, etc.)

Malnutrition

Adrenal insufficiency

Non-islet cell tumors

 

Fasting Hypoglycemia

 

In patients with concern for fasting hypoglycemia, confirmation of Whipple’s triad and exploration of the cause is recommended during an episode of spontaneous hypoglycemia or with a supervised fast of up to 72 hours (9)(Figure.1).  During the fast, patients can consume non-caloric caffeine-free beverages with all non-essential medications discontinued.  Plasma glucose, insulin, c-peptide, and BOHB are collected every 6 hours until plasma glucose is < 60 mg/dL; at that time, frequency of blood collection should be increased to every 1-2 hours. The fast is terminated after collecting the last blood sample when the plasma glucose is < 45 mg/dL and the patient has signs and/or symptoms of hypoglycemia or if the patient has not exhibited symptoms after 72 hours have elapsed.  Alternatively, the fast can be terminated when plasma glucose  is < 55 mg/dL in men and < 35 mg/dl in women, given the sex differences in abnormal fasting glycemic concentrations (29), without signs/symptoms if Whipple’s triad was documented previously but blood samples were not collected (9). At the end of the fast, glucose response to 1 mg of glucagon IV bolus injection will be measured every 10 minutes for a 30-minute period and then the patient is fed. Insulin antibodies from baseline blood samples as well as hypoglycemic anti-diabetic medications (sulfonylureas and meglitinides) screening from baseline blood and urine samples are also collected. After confirmation of diagnosis, the results of the fasting test will help to differentiate hypoglycemia mediated by insulin- versus non-insulin factors.  

 

INSULIN-DEPENDENT HYPOGLYCEMIA (HIGH PLASMA INSULIN CONCENTRATION)

 

C-peptide is secreted from β-cells at an equimolar ratio to insulin (30). Approximately half of the insulin which is secreted into the portal vein is removed by the liver (31). Therefore, plasma insulin concentration reflects not only insulin secretion or exogenous insulin administration, but also hepatic insulin degradation. In contrast to insulin, c-peptide undergoes minimal extraction by the liver and other organs (32), therefore c-peptide concentration represents endogenous insulin secretion (32,33). It has been well documented that exogenous insulin administration during euglycemia or hypoglycemia inhibits endogenous insulin secretion (34,35). In fact, this physiological phenomenon has been used to support the diagnosis of insulinoma using hyperinsulinemic hypoglycemic or euglycemic clamp (36,37).

 

Therefore, low plasma concentration of c-peptide in presence of elevated insulin values during hypoglycemia indicates factitial hypoglycemia due to exogenous insulin administration, whereas elevated c-peptide and insulin represents inappropriately greater endogenous insulin secretion due to insulin secretagogues (sulfonylurea or meglitinides), autoimmune syndromes (insulin antibody syndrome or type B insulin resistance) or an insulin-producing tumor (insulinoma).

 

Further, hypoglycemia induced by hyperinsulinemia is associated with low BOHB and glycemic response >25 mg/dl to glucagon injection. Insulin increases fatty acid synthesis and esterification and decreases fatty acid oxidation and ketogenesis in the liver, leading to lower plasma concentrations of BOHB. Enhanced insulin signaling in the liver activated by hyperinsulinemia or any non-insulin ligands, such as insulin-like growth factor—2 (IGF-2), also results in higher glycogen storage, hence a larger glycemic response to glucagon injection.

 

Factitial Hypoglycemia

 

Factitial hypoglycemia due to exogenous insulin (high insulin and low c-peptide) or insulin secretagogues medications (high insulin and c-peptide) remains a diagnostic challenge and often leads to extensive and costly investigation to rule out other causes because of limitations in biochemical assays (38), as well as patients’ denial of medication misuse. Factitial hypoglycemia is observed more often in patients who work in the medical health care system, have relatives with diabetes living in the same household, and those with underlying mental illness such as major depression. Therefore, obtaining detailed information regarding the patient’s medication list including herbal preparations that can be contaminated with sulfonylurea as well as family history is essential (39).

 

Hypoglycemia due to exogenous insulin is characterized by elevated plasma insulin, suppressed c-peptide and low BOHB, as well as an increase in plasma glucose > 25mg/dL after glucagon challenge (9).  Insulin antibodies may also be positive (40). 

 

Insulin-induced hypoglycemia due to insulin secretagogues (sulfonylurea or meglitinide) has a similar biochemical profile except that the c-peptide is elevated.  Therefore, the only way to differentiate anti-diabetic factitial hypoglycemia from insulinoma is by detecting the drug in blood or urine.

 

In a single-center retrospective study, factitious hypoglycemia accounted for 11 of 70 (16%) of admissions for evaluation of hypoglycemia. (41).  Prognosis is poor based on a small-size study, in which only 30% of affected patients during several years of follow-up recovered (42). Treatment requires a multisystemic treatment team led by a psychiatrist (42).

 

Autoimmune Syndromes

 

Autoimmune syndromes are a rare cause of hypoglycemia characterized by high concentrations of insulin autoantibodies (insulin autoimmune syndrome [IAS]) or anti-insulin receptor antibodies (type B insulin resistance) (43,44). While IAS is the third leading cause of hypoglycemia in Japan, it is very uncommon in the non-Asian population; type B insulin resistance is even less common (43). 

 

Insulin autoantibodies (IAAs) are mainly immunoglobulins (Ig) directed against endogenously released insulin in response to nutrient ingestion with a high binding capacity but low affinity to insulin. As a result, patients may manifest hyperglycemia in the early absorptive phase of meal or oral glucose intake, when exogenous glucose appearance into circulation is maximal, followed by hypoglycemia in a few hours during the late prandial condition or postabsorptive state. Hypoglycemia, in IAS, is caused by the binding and release of insulin from the antigen-antibody complex independently of changes in glucose concentrations. Therefore, insulin and c-peptide are both elevated at the time of low plasma glucose concentrations.  IAAs are different from insulin antibodies produced against exogenous insulin that are generally low binding and high affinity, thus, unable to cause hypoglycemia.

 

In non-Asian patients, IAS is mainly reported in individuals with autoimmune (lupus, rheumatoid arthritis) /hematological diseases (multiple myeloma, benign monoclonal gammopathy), who are exposed to triggering factors, such as medications (captopril, propylthiouracil, penicillin G) and viral infections (measles, mumps, rubella, varicella zoster, coxsackie B, and hepatitis C) (43,44).  In Japanese patients, IAS is commonly associated with exposure to medications with a sulphydryl group (methimazole) (43). The prognosis is relatively good with self-remission reported in 82% of patients (44). Treatment is often dietary modification (small, frequent low-carbohydrate meals and uncooked cornstarch) and occasionally requires medications to decrease insulin secretion (somatostatin analogues, diazoxide) or immunosuppressants (high -dose corticosteroids, azathioprine, rituximab) (43,44).

 

Type B insulin resistance is caused by anti-insulin receptor antibodies. Affected patients tend to be middle-aged women of Black race with obesity, acanthosis nigricans, and hyperandrogenism.  Co-occurrence of systemic autoimmune disease (i.e., lupus) is common (43). Patients typically present with hyperglycemia; however, a subset of patients (8 of 34 patients in an NIH cohort) experience fasting or postprandial hypoglycemia after period of hyperglycemia or without a history of hyperglycemia (43). The autoantibodies are believed to be partial agonists for the insulin receptor. Hyperglycemia or hypoglycemia ensues depending on the antibody titer: high titers antagonize the receptor, resulting in hyperglycemia and high insulin and c-peptide levels to compensate for the resistance; low titers activate the receptor, leading to hypoglycemia. There are diagnostic challenges as immunoprecipitation, the gold standard method to detect insulin receptor autoantibodies, is generally not commercially available (45).  Unlike IAS, prognosis of type B insulin resistance is poor with high mortality especially in patients that transition from a hyperglycemic to hypoglycemic phase.  Deaths are related to hypoglycemia and other causes (lupus, renal failure, cancer, cardiovascular events).  Therapy can include immunosuppressants, but response is variable or poor (43).

 

Insulinoma

 

Neuroendocrine insulin-producing tumors (insulinoma) are relatively rare with an estimated incidence of ~ 1 – 4 new cases per million people/year (46). Less than 10% of insulinomas are malignant, 10% multiple, and 4% associated with multiple endocrine neoplasia type 1 (MEN-1) syndrome (47).  They primarily manifest in the 5th decade of life and are slightly more common in females (48).  Typically, patients experience episodes of hypoglycemia fasting or after exercise, but some individuals may experience fasting and prandial hypoglycemia (49).  Less frequently (6% of 237 patients in a Mayo Clinic cohort), patients present solely with prandial hypoglycemia (50).  Diagnosis relies on biochemical testing. The 72-hour fast is usually successful in capturing hypoglycemia in patients with insulinoma, as 65% of patients will experience hypoglycemia within 24 hours, 93% within 48 hours, and 99% within 72-hours (51).

 

Individuals with confirmed fasting (or postprandial) hyperinsulinemic hypoglycemia, negative screening for oral hypoglycemic medications, and negative insulin autoantibody testing should undergo diagnostic tests to locate the insulinoma prior to surgery (9).  Non-invasive imaging can include transabdominal and endoscopic ultrasonography, abdominal computed tomography (CT), and magnetic resonance imaging (MRI) (52). CT has been shown to detect ~70-80% of tumors and MRI 85% (47). Given that insulinomas tend to be small in size (< 1 cm in diameter in 40% of diagnosed cases) (47), negative imaging does not rule out the diagnosis (9). If non-invasive imaging cannot determine preoperative localization, selective arterial calcium stimulation with hepatic venous sampling can been utilized (53). Calcium is injected into arteries supplying the pancreas, which stimulates insulin secretion from insulinomas; a ≥ 2-fold increase in insulin concentrations from baseline localizes the site of an insulinoma with a > 90% sensitivity (54,55).

 

Treatment is surgical enucleation of the tumor, performed more commonly via open surgery than laparoscopic (48). If not localized prior to surgery, intraoperative palpation by an experienced surgeon coupled with intraoperative ultrasound detects > 80% of tumors (48). Pancreatectomy (distal or central depending on location of tumor) is preferred over enucleation if the insulinoma is large or there is concern for malignancy or metastases. Pancreatic fistula is the most common complication in both open and laparoscopic surgeries.  As noted earlier, the hyperinsulinemic hypoglycemic or euglycemic clamp has also be utilized to differentiate insulinoma from other causes when conventional evaluation (prolonged fasting, imaging) were equivocal (37) or when surgery failed to detect an insulinoma but clinical suspicion was high (56).

 

Benign insulinomas have a high 5yr survival rate of 95-100% post-resection with relapse frequency 6% at 10yr and 8% at 20 yrs (48). Malignant insulinomas carry a poor prognosis with median life expectancy of 2 years (48). Inoperable cases may benefit from medical management such as diazoxide, streptozocin, verapamil, and phenytoin by reducing insulin secretion and corticosteroids by diminishing insulin action (48,57-59). Non-surgical procedures such as CT guided radiofrequency ablation, US-guided ablation with ethanol, peptide receptor radionuclide therapy, and robotic radiosurgery have also been utilized (48).

 

Non-Diabetic Medications

 

Non-diabetic medications such as quinolones, non-steroidal anti-inflammatory drugs, antipsychotics, and α and β blockers have been implicated in inducing hypoglycemia by stimulating insulin secretion (60).  Other medications, such as pentamidine, damage the β cells, resulting in transient hypoglycemia induced by cytolytic insulin release (60).  Risk factors related to hypoglycemia include higher medication doses, concomitant renal failure, older age, and poor nutrition  (60).  Treatment may include discontinuation of the medication and supportive care (60).  

 

INSULIN-INDEPENDENT HYPOGLYCEMIA (LOW PLASMA INSULIN CONCENTRATION)

 

In this group, plasma BOHB is elevated and glucose response to glucagon is small. In patients who appear healthy the following conditions should be considered and ruled out: 

 

Ketotic Hypoglycemia

 

Ketotic hypoglycemia is a relatively rare condition that can occur during extended periods of carbohydrate deprivation, as during fasting or starvation. Prolonged adherence to a ketogenic diet, which severely restricts carbohydrate intake to 20-50g/day to promote weight loss and increase plasma ketone bodies (61), combined with other factors interfering with counterregulatory response, such as alcohol intake, also can result in ketotic hypoglycemia (62). Hypoglycemia in this condition is mainly caused by reduction in hepatic glucose production by hyperketonemia, which outweighs the diminished glucose utilization (63).  Management is largely supportive depending on the severity of the presentation.

 

Prolonged Exercise

 

Prolonged exercise can result in plasma glucose to decline to the hypoglycemic range in 30-40% of healthy subjects, but these events are not associated with symptoms (Whipple’s triad) nor incompatible with continued exercise (64). While the mechanisms for lack of symptoms is not completely understood, studies in non-diabetic dogs using a hypoglycemic clamp with and without exercise have shown that (a) counterregulatory hormonal responses to exercise and hypoglycemia combined are greater than the response to either conditions alone, and (b) larger insulin action during exercise are negated by the counterregulatory response during hypoglycemia (both enhanced endogenous glucose production and reduced glucose utilization) (65). Thus, disturbed counterregulatory response in prolonged exercise combined with inadequate carbohydrate ingestion may result in clinical hypoglycemia (66).  Management is supportive.

 

Alcohol-Induced Hypoglycemia

 

Hypoglycemia due to alcohol has been attributed to inhibition of gluconeogenesis and blunting of growth hormone response to hypoglycemia (67). Management is mainly supportive depending on the severity and length of hypoglycemia. 

 

Glycogen Storage Diseases

 

Glycogen storage diseases (GSD) are rare genetic disorders that impair the breakdown of glycogen.  Although these diseases are commonly diagnosed in infancy, GSDs Type I (deficiency in glucose-6-phosphatase), III (deficiency in amylo-1,6-glucosidase), and 0 (lack of glycogen synthase) can present in adulthood or continue to persist in adulthood (68).  Adults with Type 1 GSD (most common form, annual incidence 1/100,000) may present with hypoglycemia, lactic acidosis, hyperuricemia, hypertriglyceridemia, and hepatomegaly (68).  Patients with glycogen storage disease are generally managed by frequent feeding with complex carbohydrates and cornstarch to prevent hypoglycemia.  Patients need to be followed long-term by a metabolic specialist.

 

Fatty Acid Oxidation (FAO) Disorders

 

FAO disorders are a rare group of autosomal recessive conditions characterized by impaired breakdown of fatty acids, leading to hypoketotic hypoglycemia and myopathy. FAO disorders also typically manifest in childhood but can continue through adulthood. Prognosis depends on the specific condition and severity. Treatment typically includes avoidance of fasting and high carbohydrate/low fat diets (68). Patients are managed by metabolic specialists. 

 

ASSOCIATED WITH OTHER DISORDERS

 

In ill-appearing patients with hypoglycemia but low insulin concentrations the following conditions should be considered:

 

Critical Illness

 

Critical illness including organ failure such as acute liver failure and congestive heart failure with hepatic congestion have been associated with hypoglycemia, likely due to impaired gluconeogenesis and depletion of hepatic glycogen stores (69,70).  Sepsis-induced hypoglycemia has been appreciated in humans and animal models with depleted glycogen stores, impaired gluconeogenesis, and increased peripheral glucose utilization implicated as contributing factors (71,72).  Hypoglycemia in non-diabetic people with end stage renal disease is attributed to concomitant adrenal insufficiency, certain medications, malnutrition, and infection (73,74).  Management of Ill-appearing individuals due to sepsis or organ failure is mainly treatment of underlying disorders and treatment of severe hypoglycemia by intravenous glucose administration.

 

Addison’s Disease

 

Addison’s disease is a rare disease that results in primary adrenal insufficiency characterized by glucocorticoid deficiency with or without mineralocorticoid deficiency.  Most cases are caused by autoimmune damage to the adrenal cortex by 21-hydroxylase antibodies (75).  Hypoglycemia, although rare, is likely due to cortisol deficiency that interferes with counterregulatory response during times of stress (76).  Addison’s disease should be considered in a hypoglycemic individual with hyperpigmentation, hyponatremia, hyperkalemia and acidosis (75). Further testing, such as ACTH stimulation test, may be warranted to confirm diagnosis (77).  Treatment for patients with primary adrenal insufficiency in adrenal crisis should be initiated by volume replacement and immediate treatment with intravenous or intramuscular hydrocortisone 100 mg followed by 100 mg every 6-8 hours until clinically stable.  Etiology of the precipitating adrenal crisis should be identified and treated (i.e., infection, hemorrhage, etc.) (75).

 

Non-Islet Cell Tumors

 

Nonislet cell tumors are rare mesenchymal and epithelial tumors that can be benign or malignant, are often large (> 10 cm), and clinically apparent.  Hypoglycemia results when the tumor overproduces incompletely processed IGF-2 (9,78-80). Tumors can secrete IGF-2 or its posttranslational precursor “big IGF” (81).  IGF-2 structurally is similar to insulin; high levels of IGF-2 can bind to the insulin receptor and mimic the action of insulin, resulting in hypoglycemia (82). Insulin and c-peptide levels are appropriately suppressed in response to the hypoglycemia.  Management of non-islet cell tumors may include surgery, radiotherapy, chemotherapy and medical therapy with glucocorticoids, GH, or octreotide (9).

 

Prandial Hypoglycemia

 

The glycemic threshold to define hypoglycemia after meal ingestion is unknown. However, symptoms associated with plasma glucose less than 50-55 mg/dL during mixed meal test that is relieved by normalization of glucose has been used to confirm meal-induced hypoglycemia (11,25).  In the prandial state, provocative testing should use a mixed meal containing protein, carbohydrates, and fat and not oral glucose. This is mainly because the oral glucose challenge has low specificity for detecting clinical hypoglycemia by causing asymptomatic low glucose nadirs as well as hypoglycemia symptoms (mainly autonomic) that do not correlate with low glucose concentrations (23,24).

 

Currently meal tests are not standardized as both solid and liquid mixed meals as well as variable carbohydrate content from 45 to 105 grams have been used (83). Regardless of approach, meal studies can increase the risk of inducing hypoglycemia, thus, these tests need to be done under supervision by personnel trained in a safe environment.

 

HYPOGLYCEMIA AFTER BARIATRIC SURGERY

 

Meal-induced hypoglycemia after upper GI tract (gastrectomy and pyloroplasty) (84) or bariatric surgery (85-87) (Roux-en y gastric bypass surgery [RYGB] and sleeve gastrectomy [SG]) are well documented. One in 10 bariatric subjects develop a late-complication of hypoglycemia (88,89), and one in 150 suffer from severe hypoglycemia requiring an emergency room visit or hospitalization (88). Hypoglycemia in this population is postprandial, progressive, often associated with cognitive impairment and occasionally with loss of consciousness or seizures and is only partially responsive to diet modification or available therapeutic options (25,86,90-92). Despite sporadic case reports of postprandial hypoglycemia after SG (85), this condition, in our experience, is less prevalent, and likely to be of lesser severity than RYGB. Severe hypoglycemia after bariatric surgery is debilitating as it compromises patient safety, cognition, and quality of life (both professional and personal). The long-term health outcomes of this debilitating complication are largely unknown. A recent study (93) using a driver simulator has demonstrated that driving performance and cognitive function is impaired following RYGB during prandial hypoglycemia without any changes in perception of symptoms (94,95).

 

Differentiating true hypoglycemia from those with prandial asymptomatic low glucose concentration or prandial symptoms without low glucose levels is more challenging in patients after GI surgery than non-operated individuals because of higher frequency of both conditions after bariatric surgery. Using CGM for 5 days has demonstrated that 70% of non-diabetic subjects after RYGB (n=40) had at least one episode of low interstitial glucose concentration (<55 mg/dl) (94). However, 80% of these low glucose events have been shown to be asymptomatic. Furthermore, it is well documented that a large proportion of bariatric patients experience dumping symptoms (91), which are almost identical with autonomic symptoms of hypoglycemia, but not associated with low glucose concentrations. Therefore, it is critical to document Whipple’s triad (neuroglycopenic rather than autonomic symptoms associated with low glucose) during free-living conditions or using mixed meal test to confirm hypoglycemia in this population.

 

Additional testing should be considered in ill-appearing patients after bariatric surgery to exclude adrenal insufficiency, other critical illnesses, and malnutrition (25). Post bariatric patients who experience fasting hypoglycemia (beyond 5 hours from previous meal ingestion) or hypoglycemia within 6-12 months from surgery should be evaluated for other causes of hypoglycemia such as insulinoma (25).

 

Underlying mechanisms by which rerouted gut after GI surgeries cause hypoglycemia is not completely understood. However, it is well documented that following RYGB, and to a smaller extent after SG, meal ingestion enhances glucose excursion leading to higher glucose peaks and lower nadir glucose concentrations mainly due to faster nutrient emptying from the stomach pouch/stomach to the gut (96) (Figure 2). Increased glucose delivery from the stomach pouch/ tube-like stomach to the gut after bariatric surgery is associated with hyperinsulinemia, which is exaggerated in RYGB patients with hypoglycemia compared to asymptomatic RYGB subjects (92) (Figure 2). Enhanced meal-induced beta-cell secretion in patients with hypoglycemia after RYGB has been attributed to not only a greater beta-cell sensitivity to increasing glycemia in the first absorptive phase, but also a lower insulin suppression during glycemic decline from peak to glucose nadir (87).

 

Prandial hyperinsulinemia after RYGB, particularly in patients with hypoglycemia, has been shown to be associated with greater prandial plasma concentration of glucagon-like peptide 1 (GLP-1), an insulinotropic gut hormone (87,90,97)(Figure 2). These observations hinted towards a key role for GLP-1 signaling beyond glycemic stimuli in meal-stimulated hyperinsulinemia after RYGB. In fact, we and others have shown that blocking the GLP-1 receptor (GLP-1R) corrects post-RYGB hypoglycemia (87,98).

 

Figure 2. The prandial glycemic effects of RYGB are exaggerated in patients with late-complication of hypoglycemia. RYGB enhances prandial glycemic excursion and increases insulin secretion rate (ISR) along with plasma GLP-1 concentrations. Patients with documented hypoglycemia after RYGB have greater insulin and GLP-1 secretin compared to those without. Following RYGB, glucagon response to meal ingestion is enhanced but there is no further increase in response to hypoglycemia. Adapted with permission from Salehi, JCEM, 2014.

 

Despite a larger meal-induced glucagon response after RYGB compared to non-operated individuals, there is no further increase in plasma glucagon concentration during prandial hyperglycemia (Figure 2), suggestive of dysregulated pancreatic α-cell response. In fact, we have shown that patients with RYGB and SG, glucagon response to insulin-induced hypoglycemia is smaller than healthy individuals without GI surgery (99,100). This data is aligned with a report demonstrating that counterregulatory hormonal response (glucagon, cortisol, and catecholamines) to hypoglycemia is significantly reduced after RYGB compared with before surgery (101). In prandial state using tracer technique, we also have shown that despite a larger prandial plasma glucagon concentration, endogenous glucose production response to hypoglycemia is smaller after RYGB compared to non-operated controls (102), suggestive of diminished liver sensitivity to glucagon. However, we have demonstrated that blocking the GLP-1R increases the prandial hepatic glucose production response to insulin-induced hypoglycemia in RYGB subjects but not in non-operated controls, suggesting that enhanced GLP-1 signal due to rerouted gut can potentially contribute to the impaired counterregulatory response to hypoglycemia (103).

 

Based on current pathophysiologic understanding treatment strategies that selectively reduce the pace of nutrient delivery to the gut and prandial insulin secretion or improve counterregulatory response are the most effective options. Because of limited therapeutic options at this point, dietary modification remains the cornerstone of management. The goal of dietary modification is to lower prandial glucose spikes while increasing glucose nadirs by (a) lowering the amount of carbohydrates for every meal (<30g) or snacks (<15 g), (b) avoiding simple carbohydrates with high-glycemic index, (c) adding fats and proteins to every meal and snack, and (d) changing the composition of carbohydrate from glucose to fructose (25). Uncooked starch has also been used in this population based on effectiveness in reducing hypoglycemic episodes in patients with diabetes mellitus (104).

 

The current medical interventions rely on drugs that had previously been used for treatment of other hypoglycemic conditions.  Acarbose, an antidiabetic medication, has been utilized as the first drug started with dietary modification. The effect of this intestinal alpha-glucosidase blocker is to block carbohydrate absorption and reduce prandial glycemic excursion after RYGB {Valderas, 2012 #6617}. Adverse effects include flatulence and bloating, especially if the dose is not gradually titrated. Other medications such as somatostatin analogues, diazoxide, and GLP-1R agonists also are used based on sporadic case reports (25). Unblinded CGM in a small size study of patients with post-RYGB hypoglycemia has also been shown to reduce hypoglycemic episodes, likely due to better self-assessment of glycemic excursion and the need for treatments (105). The investigational drugs that are in various phases of development include exendin-(9-30) (Eiger Biopharmaceutical, Paol Alto, CA, USA), a potent GLP-1R antagonist (87,106), glucagon-based drugs (107,108), somatostatin analogues (109) and sodium-glucose cotransporter-1 inhibitor (110).

 

OTHER PRANDIAL HYPOGLYCEMIC CONDITIONS (RARE)

 

Nesidioblastosis

 

Non-insulinoma pancreatogenous hypoglycemia is a rare condition that typically causes hypoglycemia in the postprandial state due to diffuse nesidioblastosis (β cell hypertrophy, islet hyperplasia, increase in β cell mass) (111). The pathogenesis in adults is largely unknown, but likely differs from nesidioblastosis that occurs in congenital hyperinsulinism that is caused by a genetic mutation (112,113). This etiology should be considered in a hypoglycemia patient with a negative 72 hour fast, positive mixed meal test concerning for endogenous hyperinsulinism without a history of GI surgery, and negative imaging for insulinoma. Selective arterial calcium stimulation demonstrates diffuse insulin secretion. Histopathology reveals nesidioblastosis (114-116). Ideal management is difficult to determine as the condition is rare; a majority of the publications are from case reports or case-series, and there is a lack of long-term follow up (82,117).  Management can include dietary interventions (low-carbohydrate frequent meals) or medical interventions with diazoxide, acarbose, verapamil or octreotide (82).  When these interventions fail, partial pancreatectomy can be performed in patients with severe neuroglycopenic symptoms (118).

 

Hereditary Fructose Intolerance

 

Hereditary fructose intolerance is a rare autosomal recessive disorder (<1-9/100,000 annual incidence) caused by fructose-1-phoshate deficiency that results in postprandial hypoglycemia after ingestion of fructose (fruits) or sucrose (sweet foods) that is usually diagnosed in childhood. The diagnosis should be suspected in ill-appearing adults with hypoglycemia associated with GI symptoms (nausea, vomiting, diffuse abdominal pain) after eating fruits or sugar. Clinical symptom resolution within days of elimination suggests hereditary fructose intolerance and can be confirmed by molecular diagnosis on DNA obtained from peripheral leukocytes (68).

 

CONCLUSION

 

Non-diabetic hypoglycemia is a rare phenomenon since in healthy individuals counterregulatory mechanisms prevent and correct hypoglycemia by reducing glucose uptake and by enhancing hepatic glucose production. These mechanisms are less well characterized in the prandial state compared to the fasting state. Nonetheless, hypoglycemia only occurs when impaired physiological responses offset the balance between glucose utilization and production. Evaluation of hypoglycemia starts with a detailed history, comprehensive review of medications and clinical presentation, and a thorough physical exam that guides the diagnostic approach in patients with documented hypoglycemia based on Whipple’s triad. In absence of confirmation of hypoglycemia during free living condition provoked testing, prolonged fast or mixed meal test for hypoglycemic conditions reported during fasting or prandial state, respectively, are indicated. Diagnostic and management strategies for non-diabetic hypoglycemia is individualized depending on specific pathophysiology and can include interventions that are dietary, medical, or surgical.

 

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Triglyceride Lowering Drugs

ABSTRACT

 

The two major goals of the treatment of hypertriglyceridemia are the prevention of cardiovascular disease and pancreatitis. Here we discuss the drugs used for the treatment of hypertriglyceridemia: (niacin, fibrates, omega-3-fatty acids, volanesorsen (available in Europe) and lipoprotein lipase gene therapy (alipogene tiparvovec- no longer available). Niacin decreases total cholesterol, TGs (20-50% decrease), LDL-C, and Lp(a). Additionally, niacin decreases small dense LDL resulting in a shift to large, buoyant LDL particles. Moreover, niacin increases HDL-C. Skin flushing, insulin resistance, and other side effects have limited the use of niacin. The enthusiasm for niacin has greatly decreased with the failure of AIM-HIGH and HPS-2 Thrive to decrease cardiovascular events when niacin was added to statin therapy. The omega-3-fatty acids eicosapentaenoic acid (C20:5n-3) (EPA) and docosahexaenoic acid (C22:6n-3) (DHA) lower TGs by 10-50% but do not affect total cholesterol, HDL-C, or Lp(a). LDL-C may increase with EPA + DHA when the TG levels are markedly elevated (>500mg/dL). EPA alone does not increase LDL-C. Omega-3-fatty acids have few side effects, drug interactions, or contraindications. Numerous studies of low dose omega-3-fatty acids on cardiovascular outcomes have failed to demonstrate a benefit. However, in the JELIS trial and REDUCE-IT trial high doses of EPA alone reduced cardiovascular events while in the STRENGTH trial high dose EPA+DHA did not reduce cardiovascular events. Fibrates reduce TG levels by 25-50% and increase HDL-C by 5-20%. The effect on LDL-C is variable. If the TG levels are very high (>500mg/dL), fibrate therapy may result in an increase in LDL-C, whereas if TGs are not markedly elevated fibrates decrease LDL-C by 10-30%. Fibrates also reduce apolipoprotein B, LDL particle number, and non-HDL-C and there may be a shift from small dense LDL towards large LDL particles. Fibrates do not have any major effects on Lp(a). Monotherapy with fibrates appears to reduce cardiovascular events in patients with high TG and low HDL-C levels. Whether the addition of fibrates to statin therapy will reduce cardiovascular disease is uncertain. In patients with diabetes fibrates appear to slow the progression of microvascular disease. Volanesorsen is an antisense oligonucleotide that inhibits the production of apolipoprotein C-III. In patients with the familial chylomicronemia syndrome (FCS) volanesorsen decreases TG by 77% (mean decrease of 1712 mg/dL) with 77% of the patients having TG levels less than 750 mg/dL. In addition, volanesorsen treatment resulted in decreases in non–HDL-C by 46%, and VLDL-C by 58% and increases in HDL-C by 46%, LDL-C by 136%, (LDL-C increased from 28 to 61 mg/dL), and total apolipoprotein B by 20%. Studies have suggested that volanesorsen may reduce episodes of pancreatitis. Patients with FCS have also reported that volanesorsen improved symptoms and reduced interference of FCS with work/school responsibilities. Of concern has been decreases in platelet levels with 47% of patients treated with volanesorsen developing platelet counts below100 x 109/L. Thus, a number of drugs are available for the treatment of hypertriglyceridemia and may be employed when lifestyle changes are not sufficient. 

 

INTRODUCTION

 

The two primary goals of the treatment of hypertriglyceridemia are the prevention of cardiovascular disease and the prevention of pancreatitis. The evaluation and guidelines for the management of hypertriglyceridemia are discussed in detail in the Endotext chapter “Risk of Fasting and Non-Fasting Hypertriglyceridemia in Coronary Vascular Disease and Pancreatitis” (1) and the approach to evaluating a patient with hypertriglyceridemia is discussed in the Endotext chapter “Approach to the Patient with Dyslipidemia” (2). The treatment of hypertriglyceridemia by diet and weight loss are discussed in detail in the Endotext chapter “The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels” and “Obesity and Dyslipidemia” (3,4). Lifestyle changes are recommended as the first line for therapy of hypertriglyceridemia, but drug therapy is often required. In this chapter we will discuss the drugs used for the treatment of elevated plasma TG levels. Statins, ezetimibe, PCSK9 inhibitors, bempedoic acid, lomitapide, mipomersen, and evinacumab, which are primarily used to lower LDL-C, are discussed in the chapter “Cholesterol Lowering Drugs” (5).  

 

NIACIN

 

Introduction

 

Niacin was the first drug approved to treat dyslipidemia. In 1955, Altschul et al showed that pharmacologic doses of niacin decreased plasma cholesterol levels (6). Several forms of niacin are available for clinical use. Immediate release niacin has a short duration of action and is typically given two or three times per day with meals, whereas sustained release niacin and extended-release niacin are once a day drugs usually given at bedtime. The extended release form of niacin exhibits release rates that are intermediate between immediate release niacin and sustained release niacin (7). While the effects of the various forms of niacin on plasma lipid levels are similar, the side effect profiles are different. Because of an increased risk of serious liver toxicity with sustained release niacin this preparation is no longer widely used to treat dyslipidemia. Over-the- counter “No flush” niacin is also available but is generally ineffective as a lipid-modifying agent because most of these preparations do not contain active nicotinic acid.    

 

Effect of Niacin on Lipid and Lipoprotein Levels

 

Table 1. Effect of Niacin on Lipid and Lipoproteins

Decreases Total Cholesterol

Decreases LDL-C

Decreases TGs

Decreases Non-HDL-C

Decreases Lp(a)

Increases HDL-C

Decreases Apolipoprotein B

Shifts Small Dense LDL to Large Buoyant LDL

 

Niacin decreases all the pro-atherogenic lipid and lipoprotein particles including total cholesterol, TG, LDL-C, and Lp(a) levels (Table 1) (8,9). Additionally, niacin has been shown to decrease small dense LDL resulting in a shift to large, buoyant LDL particles (10). Moreover, niacin increases HDL-C levels (8,9).

 

In a meta-analysis of 30 trials with 4,749 subjects treatment with immediate release, sustained release, or extended release niacin decreased total cholesterol by 10%, decreased TGs by 20%, decreased LDL-C by 14%, and increased HDL-C by 16% (11). All three niacin preparations were effective in decreasing total cholesterol, TG, and LDL-C levels and increasing HDL-C levels (11). At a dose of 1.5 grams per day, immediate release niacin and extended release niacin produced similar decreases in total cholesterol, TGs, and LDL-C and a similar increase in HDL-C (12). A meta-analysis of 14 studies with 9,013 subjects reported a 23% decrease in Lp(a) with extended release niacin treatment (13).

 

A small meta-analysis of 5 trials in 432 subjects compared the response to extended release niacin in men and women (14). The effect of niacin on LDL-C was greater in women than men at all niacin doses (1,000mg 6.8% decrease in women vs 0.2% in men, p = 0.006; 1,500mg 11.3% decrease vs 5.6% decrease, p = 0.013; 2,000 mg 14.8% decrease vs 6.9% decrease, p = 0.010; 3,000mg 28.7% decrease vs 17.7% decrease, p = 0.006). The effect of niacin on plasma TG levels also tended to be greater in women but the difference only reached statistical significance at the 1,500mg dose (28.6% vs 20.4%, p = 0.040). The mechanism for the more robust decrease in LDL-C and TGs in women is unknown but might be due to a smaller body mass in women leading to increased circulating niacin levels and hence a greater response. However, the effect of niacin on HDL-C and Lp(a) levels were similar in males and females. Not unexpectedly the effect of niacin is dose dependent with higher doses having a greater effect on plasma lipid and lipoprotein levels (Table 2) (14).

 

Table 2. Effect of Niacin Dose on Lipid and Lipoprotein Response in Women (percent change)

Niacin Dose

LDL-C

TG

HDL-C

Lp(a)

500mg

-5.2

-9.5

7.7

-2.6

1000mg

-6.8

-14.5

17.6

-11.5

1500mg

-11.3

-28.6

21.1

-4.0

2000mg

-14.8

-37.3

25.2

-24.7

2500mg

-28.7

-45.6

34.5

-28.6

3000mg

-28.7

-51.0

28.7

-29.9

 

Numerous studies have examined the effect of the addition of niacin to statin therapy. Combination therapy typically results in further reductions in atherogenic lipoprotein particles and an increase in HDL-C levels. An example of such a study is shown in Table 3 (15).

 

Table 3. Effect of the Addition of Niacin to Statin Therapy on Lipid and Lipoprotein Levels

 

 Change in Lipids with Addition of Extended-Release Niacin 2000mg/day to Simvastatin 20mg/day

LDL-C

7.1% Decrease

HDL-C

18.2% Increase

TG

22.7% Decrease

Non-HDL-C

15.1% Decrease

Lp(a)

17.4% Decrease

 

While a literature search did not find any studies comparing the combination of ezetimibe + niacin vs. monotherapy there is a large trial that has examined the effect of adding 2 grams niacin to ezetimibe/simvastatin 10/20 (16). In this study the addition of niacin improved the lipid profile with a marked decrease in TGs and an increase in HDL-C levels (table 4).

 

Table 4. Effect of the Addition of Niacin to Ezetimibe/Statin Therapy on Lipid and Lipoprotein Levels

 

 Change in Lipids with Addition of Niacin 2000mg/day to Ezetimibe/Simvastatin 10/20mg/day

LDL-C

4.8% Decrease

HDL-C

21.5% Increase

TG

17.6% Decrease

Non-HDL-C

7.3% Decrease

 

In patients with marked hypertriglyceridemia combining niacin with other drugs that also lower plasma TGs can be considered. Sixty patients with the metabolic syndrome were randomized to 16 weeks of treatment with placebo, omega-3-fatty acids (Lovaza 4 g/day), extended release niacin (2 g/day), or both drugs in combination (17). In the niacin group TGs were decreased by 30%, in the omega-3-fatty acids group by 22%, and in the combination group by 42% compared to the placebo group. Of note the beneficial effects of niacin on decreasing LDL and non-HDL-C levels were blunted by omega-3-fatty acids, which are known to raise LDL-C levels in patients with marked hypertriglyceridemia (see below). These results show that the combination of niacin and fish oil will lower TG levels more than either drug individually but at the expense of diminishing the effect of niacin on LDL and non-HDL-C levels.   

 

Surprisingly there are few large randomized trials examining the effect of combination therapy with niacin + fibrate vs. monotherapy. One very small trial reported that while both niacin monotherapy and bezafibrate monotherapy were effective in lowering serum TGs there was no statistically significant added benefit of combination therapy in reducing serum TG levels (18). However, a larger trial in HIV+ patients reported that the combination of niacin and fenofibrate was better at lowering TGs and non-HDL-C and increasing HDL-C levels than monotherapy with either niacin or fenofibrate (19). It would be informative if additional trials of combination therapy were carried out in patients with marked hypertriglyceridemia that can often be difficult to control with lifestyle changes and monotherapy.

 

Mechanisms Accounting for the Niacin Induced Lipid Effects

 

TRIGLYCERIDES

 

Early studies demonstrated that niacin inhibited the release of free fatty acids from cultured adipocytes and decreased circulating free fatty acid levels (20-22). The ability of niacin to inhibit adipose tissue lipolysis is mediated by the activation of GPR109A (hydroxycarboxylic acid 2 receptor), a G protein-coupled receptor that is highly expressed in adipose tissue (22-24). It was initially thought that the decrease in plasma TGs induced by niacin therapy was due to niacin inhibiting lipolysis in adipose tissue resulting in a decrease in the transport of fatty acids to the liver leading to the decreased availability of fatty acids for hepatic TG synthesis. However, studies have shown that while niacin acutely decreases plasma free fatty acid levels this inhibition is not sustained (25). Additionally, studies in mice lacking GPR109A have shown that niacin does not inhibit lipolysis but still decreases plasma TG and LDL-C levels (26). Moreover, studies in humans using GPR109A agonists lowered plasma free fatty acid levels but did not cause the expected effects on plasma TGs and LDL-C (26). Thus, the effects of niacin on adipose tissue lipolysis are no longer thought to mediate the niacin induced decrease in plasma TG levels.

 

Niacin has been shown to inhibit diglycerol acyltransferase 2 (DGAT2) activity in the liver (22,27). DGAT2 is the key enzyme that catalyzes the final step in TG synthesis. Inhibition of DGAT2 will reduce hepatic TG synthesis and the availability of TG for VLDL assembly and secretion (22). A decrease in TG will result in an increase in apolipoprotein B degradation in the liver. Kinetic studies in humans have shown that treatment with niacin decreases VLDL TG production (28,29).

 

In addition, in animal models, niacin reduces the hepatic expression of apolipoprotein C-III, which could result in the accelerated clearance of TG rich lipoproteins (30). Whether this plays a significant role in mediating the decrease in plasma TG levels induced by niacin therapy remains to be determined.

 

LOW DENSITY LIPOPROTEIN

 

The decrease in plasma LDL-C with niacin therapy is thought to be secondary to a reduction in VLDL and LDL formation and secretion by the liver (22).

 

HIGH DENSITY LIPOPROTEIN

 

There are multiple potential mechanisms by which niacin may increase HDL-C levels. Some of these changes may be anti-atherogenic while others may be pro-atherogenic. One hypothesis for the increase in HDL induced by niacin therapy is a decrease in the surface expression of hepatocyte beta chain ATP synthase, a receptor that has been proposed to be involved in the uptake of HDL particles by the liver (31). Studies have further shown that niacin inhibits HDL protein degradation by cultured hepatocytes but does not inhibit the selective uptake of cholesterol esters carried in HDL (22,32).

 

Some kinetic studies have shown that niacin decreases HDL and apolipoprotein A1 fractional catabolic rate (33,34). In contrast, other kinetic studies have shown that niacin increase apolipoprotein AI production (35).

 

In addition, in monocytes, niacin also increased the expression of ABCA1 and CD36 resulting in an increase in cholesterol efflux to HDL, which would increase HDL-C levels and likely have anti-atherogenic effects (36). Similarly, in vitro studies suggest that niacin may increase the transport of cholesterol and phospholipids via ABCA1 from the liver to lipid poor apolipoprotein A1 particles thereby decreasing the clearance of apolipoprotein A1, which might not be anti-atherogenic (22,37).

 

Finally, decreasing plasma TG levels may result in a reduction in CETP mediated exchange of TGs on VLDL for cholesterol on HDL leading to an increase in HDL-C levels. Additionally, studies have shown that niacin decreases the expression of CETP (38).

   

LIPOPROTEIN(a)

 

Niacin decreases the synthetic rate of Lp(a) but does not increase Lp(a) catabolism (39,40). In cell culture and animal studies niacin has been shown to decrease the expression of apo (a) (41).

 

Pharmacokinetics

 

Oral niacin is well absorbed with immediate release niacin resulting in a rapid increase in plasma levels while extended release and sustained release niacin result in a delayed peak in plasma levels. Niacin undergoes metabolism in the liver by two primary pathways; conjugation or amidation (7,42). The conjugative pathway is low affinity and high capacity that metabolizes niacin to nicotinuric acid while the amidation pathway is high affinity and low capacity that converts niacin into several oxidative-reductive intermediates, which can induce hepatic toxicity (7,42) (Figure 1). The clinical importance is that immediate release niacin results in high levels of niacin and therefore is primarily metabolized by the conjugative pathway (low affinity, high capacity), which does not result in toxic intermediates that can cause liver damage. In contrast, sustained release niacin results in lower levels of niacin for a longer period and therefore metabolism via the amidation pathway (high affinity, low capacity) is dominant leading to an increase in the formation of toxic intermediates that can induce hepatic injury (7,42). Extended-release niacin would be metabolized midway between immediate release and sustained release niacin (42).

 

Figure 1. Pathways of Niacin Metabolism.

 

Effect of Niacin on Cardiovascular Outcomes

 

MONOTHERAPY

 

The Coronary Drug Project, conducted between 1966 and 1975, was the first large randomized, double-blind clinical trial to show that lowering lipids reduced cardiovascular disease (43). This trial determined the effect of clofibrate (1.8g/day), dextrothyroxine (6mg/day), two doses of oral estrogen (2.5 or 5mg per day), or immediate release niacin (3 grams/day) vs. placebo in 8,341 men 30 to 64 years of age with an electrocardiogram documented myocardial infarction. The mean baseline total cholesterol level was 251mg/dL and the TG level was 183mg/dL. The two estrogen regimens and dextrothyroxine treatment groups were discontinued early because of increased adverse effects. Clofibrate treatment did not demonstrate clinical benefit. In the niacin treated patients there was an average 10% decrease in serum cholesterol and 26% decrease in serum TGs despite modest compliance with the study medication. Moreover, niacin treatment (n=1,119) decreased recurrent myocardial infarctions by 26%, stroke by 24%, and revascularization by 67% compared to placebo (n=2,789) but did not decrease total mortality, which was the primary endpoint. Long term follow-up (6.2 years during the study and 8.8 years post study after niacin was discontinued in most participants) demonstrated an 11% decrease in mortality in the niacin group vs. the placebo group (52.0 versus 58.2%; p = 0.0004) (44). The majority of this difference in mortality was accounted for by a decrease in coronary heart disease mortality (36.5% vs. 41.3%; p=0.005). Further analysis revealed that niacin reduced the risk of 6-year recurrent myocardial infarction and coronary heart disease death and 15-year total mortality similarly in patients at all levels of baseline fasting plasma glucose, including those with glucose levels ≥126mg/dL (i.e. patients with diabetes) (45). Additionally, the beneficial effect of niacin on cardiovascular events and total mortality was not diminished, even among those with one hour plasma glucose levels > 220mg/dL (45). Moreover, the beneficial effects of niacin on recurrent myocardial infarction and total mortality were similar in patients with or without the metabolic syndrome at baseline (46). These results demonstrate that immediate release niacin monotherapy decreases recurrent atherosclerotic cardiovascular events in a broad spectrum of patients with pre-existing cardiovascular disease (secondary prevention).

 

COMBINATION WITH FIBRATES

 

In the Stockholm Ischemic Heart Disease Secondary Prevention Study survivors of a myocardial infarction below 70 years of age were randomized to a control group (n = 276) (no placebo) and a group treated with clofibrate (2 grams) and immediate release nicotinic acid (up to 3 grams) (n = 279) (47). Serum cholesterol and TG was lowered by 13% and 19%, respectively, in the treatment group compared to the control group. Recurrent myocardial infarction was reduced by 50% within one year (48). Total mortality was decreased by 26% in the group treated with clofibrate + niacin (p< 0.05) while ischemic heart disease mortality was decreased by 36% (p< 0.01). Notably, the benefit of clofibrate + niacin was only observed in patients with a baseline TG level > 143mg/dL. In the age of statins, the clinical implications of this early study are unclear. 

 

COMBINATION WITH STATINS

 

The AIM-HIGH trial was designed to determine if the addition of Niaspan, an extended-release form of niacin, to aggressive statin therapy would result in a further reduction in cardiovascular events in patients with pre-existing cardiovascular disease (49). In this trial 3,314 patients were randomized to extended-release Niaspan (1500-2000mg/day) vs. placebo that contained 100-150mg of immediate release niacin. On trial, LDL-C levels were in the 60-70mg/dL range in both groups. As expected, HDL-C levels were increased in the Niaspan treated group (approximately 44mg/dL vs. 38mg/dL), while TGs were decreased (approximately 121mg/dL vs. 155mg/dL). However, there were no differences in the primary endpoint between the control and Niaspan treated groups (Primary endpoint consisted of the first event of death from coronary heart disease, nonfatal myocardial infarction, ischemic stroke, hospitalization for an acute coronary syndrome, or symptom-driven coronary or cerebral revascularization). There were also no differences in secondary endpoints except for a possible increase in strokes in the Niaspan treated group. The addition of Niaspan to statin therapy did not result in a significant increase in either muscle or liver toxicity. Thus, this study does not provide support for the addition of niacin to statins. However, most of the patients included in this study did not have a lipid profile that one would typically consider treating with niacin therapy. In the subset of patients with TG > 198mg/dL and HDL-C < 33mg/dL Niaspan treatment showed a trend towards benefit (hazard ratio 0.74; p=0.073), suggesting that if the appropriate patient population was studied the results may have been different (50).

 

HPS 2 Thrive also studied the effect of niacin added to statin therapy (51). This trial utilized extended-release niacin (2000mg/day) combined with laropiprant, a prostaglandin D2 receptor antagonist, which reduces the flushing side effect of niacin treatment. HPS 2 Thrive was a very large trial with over 25,000 patients randomized to either niacin therapy or placebo. As in the AIM HIGH study, the baseline LDL-C levels were low at 63mg/dL, the HDL-C levels were 44mg/dL, and the TGs were 125mg/dL at baseline. As expected, niacin therapy resulted in a modest reduction in LDL-C (10mg/dL), a modest increase in HDL-C (6mg/dL), and a marked reduction in TGs (33mg/dL) compared to placebo. However, despite these lipid changes there were no significant differences in major cardiovascular events between the niacin and control group (risk ratio 0.96 CI 0.90- 1.03). It is unknown whether laropiprant, the prostaglandin D2 receptor antagonist, might have effects that worsen atherosclerosis and increase event rates. Mice deficient in the prostaglandin D2 receptor have been noted to have an increase in atherogenesis in response to angiotensin II (52). Similar to the AIM-HIGH study, the group of patients included in the HPS 2 Thrive trial may not have been the ideal patient population to study for the beneficial effects of niacin treatment added to statin therapy. Ideally, patients with high TGs and high non-HDL-C levels coupled with low HDL-C levels should be studied.

 

Thus, these two studies have failed to demonstrate that adding niacin to statin therapy results in a decrease in cardiovascular events. It should be recognized that both the AIM-HIGH study and the HPS-2 Thrive study had limitations. First, the patient populations that were included in these studies were not ideal as the TG and non-HDL-C levels were not elevated in a range that one would usually consider adding niacin therapy. Second, in both trials a significant percentage of patients stopped niacin therapy (AIM-HIGH 25.4% discontinued niacin; HPS-2 Thrive 25.4% discontinued niacin). Third, the duration of these studies was relatively short and it is possible that the beneficial effects of niacin take longer to occur (AIM-HIGH 3 years; HPS-2 Thrive 3.9 years). Fourth, in the HPS-2 Thrive it is possible, as noted earlier, that laropiprant had adverse effects that increased the risk of cardiovascular events. Fifth, in the AIM-HIGH study the placebo contained a low dose of niacin, which may have resulted in beneficial effects. Finally, both of these trials used extended-release niacin, whereas the Coronary Drug Project and the Stockholm Ischemic Heart Disease Secondary Prevention Study used immediate release niacin. It is possible that these different formulations of niacin have different effects on cardiovascular events. Additional studies are required to definitively determine the effect of niacin added to a statin therapy on cardiovascular events.

 

Effect of Niacin on Atherosclerosis

 

Many of the initial niacin therapy imaging studies combined niacin with other drugs and compared these combinations vs. placebo. These studies showed that niacin in combination with other drugs reduced the progression and/or increased the regression of atherosclerosis. However, because of the use of other drugs it is impossible to determine if niacin therapy per se was beneficial (Table 5).

 

Table 5. Niacin Angiography Imaging Studies

Combination Studies

Drugs

Cholesterol Lowering Atherosclerosis Study (CLAS) (53)

Niacin + colestipol vs. placebo

Familial Atherosclerosis Treatment Study (FATS) (54)

Niacin + colestipol or lovastatin + colestipol vs. placebo

UCSF-SCORE (55)

Niacin + colestipol +/- lovastatin vs. placebo +/- low dose colestipol

HDL Atherosclerosis Study (HATS) (56)

Niacin + simvastatin vs. placebo

Armed Forces Regression Study (57)

Niacin + gemfibrozil + cholestyramine vs. placebo

Harvard Atherosclerosis Reversibility Project (HARP)  (58)

Niacin + pravastatin + cholestyramine + gemfibrozil as needed vs. placebo

 

However, there are studies that compared niacin to placebo or other drugs added to standard statin therapy that do provide useful insights (Table 6).

 

Table 6. Effect of Niacin Added to Statin Therapy on Atherosclerosis

ARBITER 2/3

(59,60)

ER niacin vs. placebo

Decrease in CIMT vs. placebo

ARBITER 6 (61)

ER niacin vs. ezetimibe

Decrease in CIMT vs. ezetimibe

Thoenes (62)

ER niacin vs. placebo

Decrease in CIMT vs. placebo

Lee (63)

Modified release niacin vs. placebo

Decrease in carotid wall area on MRI vs. placebo

 

The ARBITER 2 Trial was a double-blind randomized study of extended-release niacin (1000mg) vs. placebo added to background statin therapy in 167 patients with coronary heart disease and low HDL-C levels (<45mg/dL) (60). At the initiation of the study mean LDL-C levels were < 100mg/dL. The primary end point was the change in common carotid intima-media thickness (CIMT). As expected, plasma TGs decreased and HDL-C levels increased with niacin therapy. LDL-C levels were unchanged. After 12 months, mean CIMT increased significantly in the placebo group (P<0.001) and was unchanged in the niacin group (P=0.23). The overall difference in CIMT progression between the niacin and placebo groups was almost statistically significant (P=0.08). Cardiovascular events occurred in 3 patients treated with niacin (3.8%) and 7 patients treated with placebo (9.6%; P=0.20). ARBITER 3 was a 12-month extension and in the 57 patients that continued on niacin therapy there was an additional regression of CIMT (p = 0.001 vs. placebo) (59).

 

In ARBITER 6, patients with coronary heart disease or a coronary heart disease risk equivalent on long-term statin therapy with LDL-C level < 100mg/dL and an HDL-C level < 50mg/dL for men or 55mg/dL for women were randomly assigned to receive either extended-release niacin (target dose, 2000mg per day) or ezetimibe (10mg per day) (61). The primary end point was the change from baseline in the mean CIMT. LDL-C levels decreased in the ezetimibe group by −18mg/dL (~ 20%) and by −10.0mgdl (~ 12%) in the niacin group (P=0.01) while HDL-C levels were slightly decreased in the ezetimibe group −2.8mg/dL and increased by 7.5mg/dL (~18%) in the niacin group (P<0.001). TG levels were not markedly altered in the ezetimibe group but decreased by ~ 15-20% in the niacin group.  Notably niacin therapy resulted in a significant reduction of both mean (P = 0.001) and maximal CIMT (P < 0.001) while ezetimibe therapy significantly increased CIMT (P < 0.001). The incidence of major cardiovascular events was lower in the niacin group than in the ezetimibe group (1% vs. 5%, P = 0.04).

 

In a trial by Thoenes and colleagues fifty patients with the metabolic syndrome not on statin therapy were randomized to either extended-release niacin (1000mg/day) or placebo (62). Treatment with niacin decreased LDL-C by 17% and TGs by 23% and increased HDL-C levels by 24% without significant changes in the placebo group. After 52 weeks of treatment, there was an increase in CIMT of +0.009 +/- 0.003 mm in the placebo group and a decrease in CIMT of -0.005 +/- 0.002 mm in the niacin group (p = 0.021 between groups).

 

Finally, Lee and colleagues performed a double-blind, randomized study of 2 g daily modified-release niacin or placebo added to statin therapy in 71 patients with low HDL-C (<40mg/dL) and either: 1) type 2 diabetes with coronary heart disease; or 2) carotid/peripheral atherosclerosis (63). The primary end point was the change in carotid artery wall area, quantified by magnetic resonance imaging, after 1 year. Treatment with niacin increased HDL-C by 23% and decreased LDL-C by 19% and TGs by 11%. At 12 months, niacin significantly reduced carotid wall area compared with placebo (Mean change in carotid wall area was -1.1 +/- 2.6 mm2 for niacin vs +1.2 +/- 3.0 mm2 for placebo).

 

While these imaging studies provide data suggesting that niacin therapy when added to statin therapy may reduce atherosclerotic cardiovascular disease, one must recognize that the studies described above were relatively small studies and that decreases or the lack of progression in CIMT or carotid wall area are surrogate markers, which may not necessarily indicate that cardiovascular events will be decreased.  

 

Side Effects

 

Treatment with niacin frequently results in side effects and these side effects are a major limitation of niacin therapy.

 

SKIN FLUSHING

 

This is a very common side effect and is characterized by redness and warmth due to vasodilation of the blood vessels in the skin (8,64). It is often most apparent in the head and neck region. Itching can occur and a tingling and burning sensation may also be noted. Niacin induced flushing is usually not accompanied by diaphoresis. The cutaneous flushing usually lasts for approximately one hour and in some patients is extremely annoying. In a review of 30 studies, it was noted that flushing occurred in 85% of participants treated with immediate release niacin, 66% of participants treated with extended release niacin, and 26% of participants treated with slow release niacin (11).  The occurrence of flushing is related to a rapid increase in plasma nicotinic acid levels, which differs depending upon the niacin preparation. Flushing was the primary reason that subjects discontinued niacin therapy during studies and with either immediate release or extended release niacin approximately 20% of study participants discontinue niacin, which is twice the rate of discontinuation observed in the placebo groups (11). Continuous administration of niacin for approximately one- week results in tachyphylaxis and the flushing decreases. Unfortunately, if a patient skips taking niacin for a few days this tachyphylaxis is lost and the flushing returns.

 

The mechanism for the niacin induced skin flushing has been partially elucidated (8,64). Niacin activates GPR109A in dermal Langerhan cells (macrophages in the skin), which leads to the increased production of prostaglandin D2.  Additionally, niacin activates GPR 109A in keratinocytes, which leads to the production of prostaglandin E2.  The prostaglandins then interact with prostaglandin receptors on blood vessels resulting in vasodilation and the flushing phenomena. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDS) taken prior to niacin administration can decrease flushing by inhibiting the synthesis of prostaglandins (8,65). Laropiprant decreases flushing by blocking the D prostanoid receptor (8). Since flushing is related to rapid increases in plasma nicotinamide levels taking immediate release niacin with food slows absorption and thereby reduces flushing. Extended-release niacin is typically taken at bedtime so that the flushing will occur when the patient is asleep. Conditions that predispose to cutaneous vasodilatation such as alcohol intake, hot liquids, spicy foods, or hot showers should be avoided. One should increase the dose of niacin slowly to reduce the severity of flushing reactions and allow tolerance to develop.

 

HEPATIC TOXICITY

 

Sustained release niacin has a much greater propensity to induce hepatic toxicity than other niacin preparations and therefore is no longer widely used (7,42,66). The explanation for this difference is due to the increased metabolism of sustained release niacin by the amidation pathway described in the pharmacokinetics section, which results in toxic compounds that injure the liver (7,42). Patients who have developed signs of liver toxicity on sustained release niacin can often be treated with immediate release niacin without developing liver problems (67). Extended-release niacin can induce liver dysfunction but the rate is much lower than sustained release niacin. Because of the potential for liver disease, serum transaminase levels (SGOT and SGPT) should be monitored before treatment begins, every 6 to 12 weeks for the first year, and periodically thereafter (e.g., at approximately 6-month intervals).

 

It should be noted that there is some evidence that niacin may be beneficial for non-alcoholic fatty liver disease (NAFLD) but further studies are required (68).

 

MUSCLE SYMPTOMS

 

Myalgias and myopathy have not been a significant adverse effect with niacin monotherapy (11). In combination with statins, an increased risk of muscle symptoms has been observed in some studies. In the HPS-2 Thrive study the combination of simvastatin and extended-release niacin increased the risk of myopathy four-fold (1.2% of patients on combined therapy) (51). Of note, this increase occurred predominantly in Chinese participants. In contrast, in the AIM-HIGH trial muscle related symptoms were not increased with the simvastatin + niacin combination (49,69).

 

HYPERGLYCEMIA

 

It has been recognized for many years that niacin induces insulin resistance (70). The mechanisms by which niacin induces insulin resistance are unknown but possible mechanisms include a rebound increase in free fatty acids with niacin therapy or the accumulation of diacylglycerol (29,71). A recent analysis of the AIM-HIGH trial demonstrated that in subjects with normal glucose metabolism, subjects with impaired fasting glucose, and subjects with diabetes, treatment with extended release niacin resulted in only small increases in fasting glucose levels but increased serum insulin levels due to an increase in insulin resistance (72). Additionally, there was an increased risk of progressing from normal to impaired fasting glucose in subjects treated with niacin in the AIM-HIGH trial (niacin 58.6% vs placebo 41.5%; P < .001) (72).

 

A meta-analysis examined the effect of niacin therapy on the development of new onset diabetes (73). In 11 trials with 26,340 non-diabetic participants, niacin therapy was associated with a 34% increased risk of developing diabetes (RR of 1.34; 95% CIs 1.21 to 1.49). This increased risk results in one additional case of diabetes per 43 initially non-diabetic individuals who are treated with niacin for 5 years (0.47% ten-year risk or 4.7 per 1000 patient years). Results were similar in patients who were receiving niacin therapy in combination with statin therapy.

 

Studies have shown that niacin is usually well tolerated in diabetic subjects who are in good glycemic control (74,75). In patients with poor glycemic control, niacin is more likely to adversely impact glucose levels. A meta-analysis of 7 studies with 838 patients with diabetes found that niacin therapy did not result in a significant increase in fasting glucose levels in short term studies but in long term studies there was a very small increase in fasting glucose levels (1.5mg/dL) that was not clinically significant (76). An important caveat is that in most of these trials adjustments in diabetes therapy was permitted, which could blunt worsening of glycemic control. In contrast to these findings, the HPS-2 Thrive Trial reported that in the 8,299 participants who had diabetes at the time of randomization, treatment with niacin–laropiprant was associated with a 55% increase in serious disturbances in diabetes control, most of which led to hospitalization (11.1% vs. 7.5%, P<0.001) (51). The extent to which the latter was due to laropiprant is unknown. Thus, care must be used in treating patients with diabetes with niacin. In patients in whom adjustments in diabetic therapy can easily be carried out the risk of adverse effects will likely be limited whereas in patients in whom adjustments in diabetic therapy will be difficult the risks of niacin therapy are likely to be increased. Careful patient selection and education are important steps to reduce the risks of niacin therapy in patients with diabetes.

 

Thus, while niacin therapy may adversely affect glucose homeostasis one needs to balance these adverse effects with the potential benefits of niacin therapy. One should note that in the Coronary Drug Project participants with abnormal glucose metabolism also demonstrated a decrease in cardiovascular events with niacin therapy (45).  

 

URIC ACID  

 

Niacin may increase uric acid levels by inhibiting the secretion of uric acid (8,77). In susceptible patients niacin therapy can precipitate gouty attacks (8).   

 

GASTROINTESTINAL SYMPTOMS  

 

Niacin therapy can induce heartburn, indigestion, nausea, diarrhea, and abdominal discomfort (8). High dose niacin is more likely to cause these gastrointestinal disturbances. The mechanism for these symptoms is not clear. 

 

MISCELLANEOUS  

 

Recent trials have reported an increased incidence of infections with niacin therapy (51,69). A trial of niacin in combination with laropiprant found increased bleeding (51). The increased bleeding could be due to the approximately 10% decrease in platelet levels that can occur with niacin (see Niaspan Package Insert). However, a very large observational study that compared rates of major gastrointestinal bleeding and intracranial hemorrhage in patients treated with niacin (>200,000 subjects) to propensity matched subjects on fenofibrate did not observe an increase in bleeding (78). Niacin has been reported to induce cystoid macular edema, which resolves when the drug is stopped (79).

 

Contraindications

 

There are a number of contraindications to niacin therapy (Table 7).

 

Table 7. Contraindications for Niacin Therapy

Active gastritis or peptic ulcer disease

Impaired liver function (elevated transaminases 2-3X the upper limit or cholestasis)

Uncontrolled gout

Pregnancy

Lactation

Poorly controlled diabetes

Active bleeding

 

Summary

 

The enthusiasm for the use of niacin has greatly decreased with the failure of AIM-HIGH and HPS-2 Thrive to show a decrease in cardiovascular events when niacin was added to statin therapy. In the absence of definitive data showing benefits from niacin therapy when added to a statin it is hard to justify the use of this drug given the frequent side effects. The availability of ezetimibe, bempedoic acid, and PCSK9 inhibitors has greatly reduced the need to use niacin to lower LDL-C levels. Additionally, in patients with markedly elevated TG levels (>500mg/dL), niacin can be employed in combination with other drugs to reduce the risk of pancreatitis but fibrates and omega-3-fatty acids are the initial choices.

 

OMEGA-3-FATTY ACIDS (FISH OIL)

 

Introduction

 

The lipid lowering effects of fish oil are mediated by two omega-3-fatty acids; eicosapentaenoic acid (C20:5n-3) (EPA) and docosahexaenoic acid (C22:6n-3) (DHA). There are four prescription products approved by the FDA which contain various amounts of EPA and DHA (Table 8). Lovaza and Omacor contain a mixture of EPA and DHA fatty acid esters (ethyl esters), Vascepa contains only EPA fatty acid esters (ethyl esters), and Epanova contains a mixture of EPA and DHA free fatty acids (Epanova is currently not available in the US).

 

Table 8. Prescription Omega-3-fatty acid products (data from package inserts)

Generic Name

Omega-3-ethyl esters

Icosapent ethyl

Omega-3-carboxylic acid

Brand Name

Lovaza or Omacor

Vascepa

Epanova

EPA/capsule

0.465g

1.0g

See below

DHA/capsule

0.375g

---

See below

Daily Dose

4 capsules/day

4 capsules/day

2-4 capsules/day

1-gram capsules of Epanova contain at least 850mg of fish oil derived fatty acids including multiple omega-3-fatty acids with EPA and DHA being the most abundant

 

Fish oil is also sold as a food supplement. It should be recognized that dietary fish oil supplements are not approved by the FDA and quality control will not meet the same rigorous standards as prescription or over the counter drugs. The amount of EPA and DHA can vary greatly in these supplements and one needs to read the labels carefully, as products can contain less than 100mg of EPA/DHA per 1 gram capsule (80). It is helpful to have the patient bring their fish oil supplements to the clinic for verification of the actual amount of EPA and DHA in the product. Moreover, the amount of EPA and DHA indicated on the label may not be accurate (81). One needs to take a sufficient number of capsules to provide 2-4 grams of EPA/DHA per day to effectively lower plasma TG levels. Depending upon the fish oil supplement, the patient may be required to take a large number of capsules to obtain 2-4 grams of EPA/DHA per day. Furthermore, these supplements may contain other compounds in addition to omega-3-fatty acids, such as cholesterol, oxidized lipids, and saturated fatty acids. The major advantage of fish oil supplements is that they are much less expensive than prescription omega-3-fatty acid drugs. If one elects to use fish oil supplements, one should have the patient use a single brand to try to ensure as much consistency as possible.

 

Some omega-3 supplements contain alpha linolenic acid (C18:3n-3) (ALA), a plant omega-3-fatty acid rather than EPA/DHA. ALA can be converted to EPA and DHA but the conversion is limited and hence it is ineffective in lowering plasma TG levels or altering other lipid or lipoprotein levels (82).

 

Effect of Omega-3-Fatty Acids on Lipid and Lipoprotein Levels

 

Table 9. Effect of Fish Oil Supplements on Lipids and Lipoproteins

Decreases TGs

No Change in Total Cholesterol

No Change in LDL-C; if TGs are very high may increase LDL-C

No Change in HDL-C

No Change in Lp(a); modest decrease in some studies

Shift from Small Dense LDL to Large Buoyant LDL

 

Several meta-analyses have examined the effect of fish oil supplements on lipid and lipoprotein levels. A meta-analysis by Eslick and colleagues of 47 studies with 16,511 participants found that fish oil supplements significantly decreased plasma TG levels by approximately 14% without resulting in clinically significant changes in total, LDL-C, or HDL-C levels (83). These authors also reported that the reduction in plasma TG levels was directly related to baseline plasma TG levels (i.e., the higher the baseline TG level the greater the reduction in TGs with fish oil). Additionally, the higher the dose of EPA/DHA, the greater the reduction in plasma TGs, with clinically significant reductions occurring with approximately 3.25 grams per day. A meta-analysis by Balk and colleagues of 21 studies also found minimal effects of fish oil supplements on total, LDL-C, and HDL-C levels (< 5% change) with significant decreases in plasma TG levels (most of the studies in this meta-analysis had at least a 15% decrease) (84). Similar to the meta-analysis by Eslick et al, the higher the baseline TG levels the greater the reduction in TG levels. 

 

Several meta-analyses have focused on specific patient populations. In a meta-analysis of patients with diabetes, twenty three trials with1075 participants were analyzed and similar to patients without diabetes the major effect of fish oil supplements was a reduction in plasma TG levels with no change in total cholesterol or HDL-C (85). A small increase in LDL-C was observed (4.3mg/dL). Of note, fish oil supplementation did not alter fasting glucose or glycated hemoglobin levels indicating that fish oil supplementation does not adversely affect glycemic control. In a meta-analysis that included patients with type 2 diabetes or impaired glucose metabolism a decrease in TGs was observed without significant changes in total cholesterol, LDL-C, or HDL-C levels (86). Again, no adverse effects on glycemic control were observed.

 

In patients with end stage renal disease several meta-analyses have consistently shown a decrease in plasma TGs with fish oil administration but the effect on total, LDL-C, and HDL-C has been variable (87-89). This variability was likely due to the small changes that were observed. In patients with nephrotic syndrome a study has shown a reduction in plasma TGs and an increase in LDL-C levels without a change in total cholesterol or HDL-C levels (90). In patients with non-alcoholic fatty liver disease, omega-3-fatty acids have also been shown to decrease plasma TG levels (91). Finally, In HIV infected subjects, fish oil supplementation was also effective in lowering plasma TG levels (92,93).

 

Thus, fish oil supplementation in a variety of different patient populations lowers plasma TG levels. In patients with elevated TG levels treated with 3-4 grams of EPA/DHA one can expect an approximate 25% decrease. Total plasma cholesterol levels are usually not altered by fish oil supplementation. The exceptions are patients with high chylomicron and/or VLDL levels where a substantial portion of the plasma cholesterol is carried on these TG rich lipoproteins. Fish oil supplementation will decrease the levels of these TG rich lipoproteins and thereby result in a decrease in total plasma cholesterol levels. LDL-C levels are not markedly affected by fish oil supplementation except in patients with very high TG levels (>500mg/dL) where increases in LDL-C levels have been observed (94-96). If there are sufficient reductions in plasma TG levels a shift from small dense LDL to large buoyant LDL may be observed (97,98). The effect of fish oil supplements on HDL-C levels is minimal except if the patient has very high TG levels where significant elevations (>10%) have been reported (94-96). Finally, some but not all studies have shown that the administration of fish oil modestly lowers Lp(a) levels (99-103)

 

During the development of pharmacological omega-3-fatty acid drugs for approval by the FDA, extensive clinical trials were carried out and will be reviewed below (Tables 10 and 11). It should be noted that these studies are not directly comparable as they studied different patient populations at different times.

 

EPA + DHA FATTY ACID ESTERS (LOVAZA)  

 

In patients with marked elevations in plasma TG levels (500-2000mg/dL) a 6 week trial of EPA + DHA esters resulted in a 31% decrease in plasma TGs, a 21% increase in LDL-C levels, and a 12% increase in HDL-C levels compared to the placebo group (96). In a 16 week trial TG concentrations were decreased by 45% and LDL-C and HDL-C were increased by 31% and 13%, respectively (94). Studies have also been carried out in patients with moderate hypertriglyceridemia (200-500mg/dL) who were on statin therapy (104). EPA + DHA esters resulted in a 23% decrease in plasma TGs and a 7% decrease in non-HDL-C levels, and a 4.6% increase in HDL-C levels (104).

 

EPA FATTY ACID ESTER ALONE (VASCEPA)  

 

In patients with marked elevations in plasma TGs (500-2000mg/dL), 4 grams of EPA ester alone significantly decreased TG levels by 33.1% and non-HDL-C levels by 17.7% (105). In contrast to EPA and DHA fatty acid esters, LDL-C and HDL-C levels were not significantly altered by EPA fatty acid esters alone (105). Studies have also been carried out in patients with moderate hypertriglyceridemia (200-500mg/dL) who were on statin therapy. EPA esters resulted in a 21.5% decrease in plasma TGs, 13.6% decrease in non-HDL-C, 6.2% decrease in LDL-C, and a 4.5% decrease in HDL-C levels (106)

 

EPA + DHA FATTY ACIDS (EPANOVA)  

 

In patients with marked elevations in plasma TGs (500-2000mg/dL), 4 grams of EPA + DHA fatty acids decreased plasma TGs by 31% and non-HDL-C by 9.6% and increased LDL-C by 19% and HDL-C by 5.8% (107). Studies have also been carried out in patients with moderate hypertriglyceridemia (200-500mg/dL) who were on statin therapy. EPA + DHA fatty acids resulted in a 20.6% decrease in plasma TGs, 6.9% decrease in non-HDL-C with no significant changes in LDL-C or HDL-C levels (95).

 

These studies demonstrate that in patients on statin therapy with moderate elevations in plasma TG levels the effects of these three pharmaceutical products on lipids and lipoprotein levels are similar (table 11). However, in patients with marked elevations in plasma TG levels EPA ethyl esters alone do not increase LDL-C levels whereas products containing EPA and DHA result in a substantial increase in LDL-C levels (table 10). It should also be noted that the ability of omega-3-fatty acids to reduce plasma TGs and increase HDL-C levels is enhanced if baseline TG levels are markedly elevated.

 

Table 10: Effect of Omega-3-Fatty Acids on Lipids and Lipoprotein in Patients with Marked Hypertriglyceridemia (500-2000mg/dL)

 

TGs

Non-HDL-C

LDL-C

HDL-C

EPA+DHA ethyl esters-

6 weeks

31% decrease

ND

21% increase

12% increase

EPA+DHA ethyl esters

12 weeks

45% decrease

ND

31% increase

13% increase

EPA ethyl esters

33% decrease

18% decrease

NS

NS

EPA+DHA fatty acids

31% decrease

9.6% decrease

19% increase

5.8% increase

ND- not determined; NS- no significant change

 

Table 11: Effect of Omega-3-Fatty Acids on Lipids and Lipoprotein in Patients with Moderate Hypertriglyceridemia (200-500mg/dL) on Statin Therapy

 

TGs

Non-HDL-C

LDL-C

HDL-C

EPA+DHA ethyl esters

23% decrease

7% decrease

__

4.6% increase

EPA ethyl esters

22% decrease

14% decrease

6.2% increase

4.5% decrease

EPA+DHA fatty acids

21% decrease

6.9% decrease

NS

NS

NS- no significant change

 

HEAD-TO-HEAD COMPARISONS  

 

A meta-analysis of six studies has compared the effect of EPA alone vs. DHA alone on plasma lipids and lipoproteins (108). Administration of DHA increased LDL-C by 4.6mg/dL compared to EPA (95% CI 2.2- 7.1). In contrast, DHA reduced plasma TG levels to a greater extent than EPA (6.1mg/dL; 95% CI 2.5- 9.8). Finally, DHA increased HDL-C levels more than EPA (3.7mg/dL; 95% CI: 2.4- 5.1). Whether these very modest differences are clinically significant is unknown.

 

Tatsuno et al compared the effect of DHA + EPA ethyl esters vs. EPA ethyl esters alone on lipid and lipoprotein levels in patients with mean baseline plasma TG of 250-270mg/dL and mean LDL-C levels of 125-135mg/dL (109,110). These authors found that at equivalent doses there were no differences in effect on plasma TG, LDL-C, or HDL-C levels between DHA + EPA ethyl ester or EPA ethyl ester treatment.

 

These head-to-head studies indicate that in subjects with moderate hypertriglyceridemia the effects of EPA and DHA on lipid and lipoprotein levels are similar. Perhaps if the baseline TGs were markedly elevated differences in response might have been observed.

 

IN COMBINATION WITH FENOFIBRATE  

 

In patients with marked hypertriglyceridemia a single drug is often not sufficient to lower TGs into the desired range. In patients with TG levels > 500mg/dL the combination of fenofibrate 130mg per day and 4 grams of Lovaza reduced TG levels to a greater extent than monotherapy with fenofibrate alone (7% decrease; P = 0.059) (111). Not unexpectedly, LDL-C levels were increased to a greater extent with combination therapy (9% increase; p= 0.03). When subjects who had received 8 weeks of fenofibrate monotherapy were treated with Lovaza during the 8-week, open-label extension study, TG levels were reduced by an additional 17.5% (P = 0.003). These results indicate that the addition of omega-3-fatty acids to fenofibrate will further decrease TG levels.

 

IN COMBINATION WITH NIACIN

 

Sixty patients with the metabolic syndrome were randomized to 16 weeks of treatment with placebo, Lovaza (4 g/day), extended release niacin (2 g/day), or both drugs in combination (17). In the niacin group TGs were decreased by 30%, in the omega-3-fatty acids group by 22%, and in the combination group by 42% compared to the placebo group. Of note, the beneficial effects of niacin on decreasing LDL and non-HDL-C were blunted by omega-3-fatty acids. These results show that the combination of niacin and fish oil will lower TG levels more than either drug individually but at the expense of diminishing the effect of niacin on LDL and non-HDL-C levels.

 

Mechanism Accounting for the Omega-3-Fatty Acid Induced Lipid Effects

 

As noted above, the major effect of fish oil is to lower plasma TG levels. The predominant cause of the reduction in plasma TG levels is a decrease in the hepatic production and secretion of TG rich lipoproteins (112-115). In cultured hepatocytes, omega-3-fatty acids inhibit the assembly and secretion of VLDL and apolipoprotein B 100 (113,115-117).  The incorporation of TGs into VLDL is a key regulatory step in determining the rate of formation and secretion of VLDL and there are a number of mechanisms by which omega-3 fatty acids reduce the level of hepatic TGs available for VLDL formation (112,113,115). Studies in animal models have demonstrated that omega-3-fatty acids inhibit fatty acid synthesis and stimulate fatty acid oxidation in the liver, which would reduce the availability of fatty acids for TG synthesis (112-115). The increase in fatty acid oxidation is due to omega-3-fatty acids activating PPAR alpha, which stimulates fatty acid oxidation in the liver and other tissues (112,114,115,118). The decrease in fatty acid synthesis is due to omega-3-fatty acids inhibiting the expression of SREBP-1c, a key transcription factor that regulates fatty acid synthesis (114,115,118). In addition, omega-3-fatty acids decrease TG synthesis, which may be due to the decreased availability of fatty acids and an inhibition of the activity of DGAT, a key enzyme required for TG synthesis (112,114,115). Finally, omega-3-fatty acids also decrease the flux of free fatty acids from adipose tissue to the liver, which will lead to a decreased quantity of fatty acids available for TG synthesis in the liver (112). This decrease in flux of free fatty acids is due to omega-3-fatty acids reducing hormone sensitive lipase mediated intracellular lipolysis in adipose tissue (112). It is likely that these and perhaps other factors lead to the decreased availability of TGs resulting in a reduction in VLDL formation and secretion. In addition, the peroxidation of omega-3-fatty acids may stimulate the degradation of apolipoprotein B-100, which would provide another pathway that could contribute to a decrease in VLDL formation and secretion (115).

 

While not the primary mechanism for the decrease in plasma TGs, studies have shown that omega-3-fatty acids may increase the clearance of TG rich lipoproteins (112,119). Post heparin lipoprotein lipase activity is not increased by omega-3-fatty acid administration but the lipolytic activity of non-stimulated plasma is enhanced (112,119).  Additionally, apolipoprotein C-III levels are decreased with omega-3-fatty acid administration which could also contribute to an increase in the clearance of TG rich lipoproteins (120-123).

 

The increase in LDL-C levels that occurs in patients with marked hypertriglyceridemia treated with omega-3-fatty acids is thought to be due to the enhanced conversion of VLDL to LDL (114). The increase in HDL-C observed in studies in patients with very high TG levels may be due to the increased clearance of TG rich lipoproteins.    

 

Pharmacokinetics and Drug Interactions

 

Omega-3 ethyl esters and fatty acids are absorbed by the GI tract similar to other dietary lipids. It is worth noting that omega-3-free fatty acids (Epanova) are directly absorbed by the small intestine and are not dependent on pancreatic lipases for absorption. Thus, absorption of omega-3-fatty acids is not decreased in patients with pancreatic insufficiency and therefore may be preferred in patients with pancreatic disease. Additionally, the bioavailability of omega-3-fatty acids with a low fat diet was greater than omega-3-ethyl esters while there was little difference between these different formulations with a high fat diet (124,125).

 

Drug interactions have not been seen with omega-3-fatty acids (Package Inserts for Lovaza, Vascepa, and Epanova).

 

Effect of Low Dose Omega-3-Fatty Acids on Clinical Outcomes

 

Initial studies of the effect of low dose fish oil administration on cardiovascular outcomes were favorable, demonstrating a reduction in events including all-cause mortality. However, more recent studies have failed to confirm these favorable results. In these more recent studies the use of other drugs, such as statins, that reduce cardiovascular disease were more intensively utilized. The outcomes studies that will be described below were carried out with doses of EPA and DHA that are lower than the doses used to lower plasma TGs. We will limit our discussion to the administration of fish oil as a drug and not discuss diet studies, such as DART, which had patients increase fatty fish intake (126,127).

 

  • GISSI-Prevenzione trial was a randomized trial of 850-882mg of EPA and DHA ethyl esters per day in 11,323 participants with a recent myocardial infarction (< 3 months) for 3.5 years (128). The primary endpoint was death, non-fatal myocardial infarction, and stroke. No change in total cholesterol, LDL-C, or HDL-C was observed but plasma TG levels were decreased by 5%. Patients treated with EPA/DHA had a significant decreased risk of major cardiovascular events (RR 0.90), cardiac death (RR 0.78), and sudden death (RR 0.74). The decrease in sudden death occurred very quickly and was noted as early as 4 months after initiation of therapy. Interestingly, non-fatal cardiovascular events were not affected by EPA/DHA treatment (RR 0.98). The decrease in total mortality was driven by a reduction in sudden death suggesting an anti-arrhythmic effect of EPA/DHA.

 

  • GISSI-Heart Failure (GISSI-HF) trial was a randomized, double-blind, placebo-controlled trial in patients with chronic heart failure who were randomly assigned to 850-882mg of EPA and DHA ethyl esters per day (n=3,494) or placebo (n=3,481) (129). Patients were followed for a median of 3.9 years. Primary endpoints were time to death, and time to death or admission to the hospital for cardiovascular reasons. Omega-3-fatty acid treatment at these low doses resulted in a slight decrease in plasma TG levels with no change in total, LDL-C or HDL-C levels. In the omega-3-fatty acid group 27% patients died from any cause vs. 29% in the placebo group (HR 0.91; p=0.041). In the omega-3-fatty acid group 57% of patients died or were admitted to hospital for cardiovascular reasons vs. 59% in the placebo group (HR 0.92; p=0.009). No significant differences were observed in fatal or non-fatal myocardial infarctions or strokes. In this trial, similar to the GISSI-Prevenzione trial, the benefit was primarily due to a reduction in arrhythmic events and little benefit on atherothrombotic events was noted.

 

  • OMEGA was a randomized, placebo-controlled, double-blind, trial in 3,851 survivors of an acute myocardial infarction (130). Patients were randomized 3 to 14 days after an acute myocardial infarction to omega-3-acid ethyl esters, 1 gram/day (460mg EPA and 380mg DHA) or placebo capsules containing 1 gram of olive oil and followed for one year. The primary endpoint was rate of sudden death and secondary end points were total mortality and nonfatal clinical events. No significant differences were seen in the primary or secondary endpoints.

 

  • Alpha Omega was a double-blind, placebo-controlled trial in 4,837 patients between 60 and 80 years of age (78% men) who had had a myocardial infarction (131). Patients were randomized to receive for 40 months one of four trial margarines: a margarine supplemented with a combination of EPA and DHA (with a targeted additional daily intake of 400mg of EPA-DHA; actual intake 226mg EPA and 150mg DHA), a margarine supplemented with alpha-linolenic acid (ALA) (with a targeted additional daily intake of 2g of ALA), a margarine supplemented with EPA-DHA and ALA, or a placebo margarine. The primary end point was the rate of major cardiovascular events, which comprised fatal and nonfatal cardiovascular events and cardiac interventions. Neither low dose EPA-DHA, ALA, nor the combination of EPA/DHA and ALA significantly reduced the rate of major cardiovascular events or cardiac interventions.

 

  • FOL.OM3 Study was a double blind, randomized, placebo-controlled trial in 2,501 patients with a history of a myocardial infarction, unstable angina, or ischemic stroke in the past 12 months (132). Patients were randomized to a daily dietary supplement containing 5-methyltetrahydrofolate (560μg), vitamin B-6 (3mg), and vitamin B-12 (20μg) or placebo; and a dietary supplement containing omega 3 fatty acids (600mg of EPA and DHA) or placebo. Median duration of treatment was 4.7 years. The primary outcome was a composite of non-fatal myocardial infarction, stroke, or death from cardiovascular disease. Treatment with B vitamins or omega 3 fatty acids had no significant effect on major vascular events.

 

  • Origin was a double-blind study in 12,536 patients at high risk for cardiovascular disease who had impaired fasting glucose, impaired glucose tolerance, or diabetes (133). Patients were randomized to receive a 1-gram capsule containing at least 900mg of ethyl esters of omega-3 fatty acids (EPA 465mg and DHA 375mg) or placebo for approximately 6 years. The primary outcome was death from cardiovascular causes. TG levels were reduced by 14.5mg/dL in the group receiving omega-3-fatty acids compared to the placebo group (P<0.001), without a significant effect on other lipids. The incidence of the primary outcome was not significantly decreased among patients receiving omega-3-fatty acids as compared with those receiving placebo. The use of omega-3-fatty acids also had no significant effect on the rates of major vascular events, death from any cause, or death from arrhythmia.

 

  • Risk and Prevention Study was a double-blind, placebo-controlled trial in 12,513 men and women with multiple cardiovascular risk factors or atherosclerotic vascular disease but not myocardial infarctions (134). Patients were randomly assigned to 1-gram daily omega-3 fatty acids (EPA and DHA content not <85 %,) or placebo (olive oil) for 5 years. The initially specified primary end point was the rate of death, nonfatal myocardial infarction, and nonfatal stroke. At 1 year, after the event rate was found to be lower than anticipated, the primary end point was revised as time to death from cardiovascular causes or admission to the hospital for cardiovascular causes. Plasma TG levels decreased slightly more in the omega−3-fatty acid group than in those who received placebo (−28.2±1.3mg/dL vs. −20.1±1.3mg/dL; P<0.001). Total, LDL, and HDL-C levels were similar in the omega-3-fatty acid and placebo groups. No significant differences were observed between the omega-3-fatty acid group and placebo group for the primary endpoint or any of the secondary endpoints.

 

  • A Study of Cardiovascular Events in Diabetes (ASCEND) was a randomized, placebo controlled, double blind trial of 1-gram omega-3-fattys acids (400mg EPA and 300mg DHA ethyl esters) vs. olive oil placebo in 15,480 patients with diabetes without a history of cardiovascular disease (primary prevention trial) (135). The primary end point was serious vascular events (non-fatal myocardial infarction, non-fatal stroke, transient ischemic attack, or vascular death). Total cholesterol, HDL-C, and non-HDL-C levels were not significantly altered by omega-3-fatty acid treatment (changes in TG levels were not reported). After a mean follow-up of 7.4 years the composite outcome of a serious vascular event or revascularization occurred in 882 patients (11.4%) on omega-3-fatty acids and 887 patients (11.5%) on placebo (rate ratio, 1.00; 95% CI, 0.91 to 1.09). Serious adverse events were similar in placebo and omega-3-fatty acid treated groups.

 

  • The Vitamin D and Omega-3 Trial (Vital) was a randomized, double blind, placebo-controlled trial of 1-gram omega-3 fatty acids (465mg EPA and 375mg DHA ethyl esters) vs. placebo in 25,875 men (>50 years of age) and women (>55 years of age) that were not selected on the basis of an elevated risk (primary prevention) (136). Changes in lipid levels were not reported. The primary end point was major cardiovascular events, a composite of myocardial infarction, stroke, or death from cardiovascular causes. After a median follow-up of 5.3 years, major cardiovascular event occurred in 386 participants in the omega-3 fatty acid group and in 419 in the placebo group (hazard ratio, 0.92; 95% confidence interval (CI), 0.80 to 1.06; P=0.24). Serious adverse events were similar in placebo and omega-3-fatty acid treated groups.

 

  • Summary: The above results indicate that low dose fish oil (doses that do not greatly affect lipid levels) do not consistently reduce the risk of cardiovascular disease.

 

Effect of High Dose Omega-3-Fatty Acids on Clinical Outcomes

 

  • Japan EPA Lipid Intervention Study (JELIS) was an open label study without a placebo in patients with total cholesterol levels > 254mg/dL with (n= 3,664) or without cardiovascular disease (n=14,981) who were randomly assigned to be treated with 1800 mg of EPA (Vascepa) + statin (n=9,326) or statin alone (n= 9,319) with a 5-year follow-up (130). The primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal myocardial infarction, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. Total, LDL-C, and HDL-C levels were similar in the two groups but plasma TGs were modestly decreased in the EPA treated group (5% decrease in EPA group compared to controls; p = 0.0001). In the EPA group the primary endpoint occurred in 2.8% of the patients vs. 3.5% of the patients in the statin alone group (19% decrease; p = 0.011). Unstable angina and non-fatal coronary events were also significantly reduced in the EPA group but in this study sudden cardiac death and coronary death did not differ between groups. Unstable angina was the main component contributing to the primary endpoint and this is a more subjective endpoint than other endpoints such as a myocardial infarction, stroke, or cardiovascular death. In patients with high TG levels (>150 mg/dL) and low HDL-C levels (<40 mg/dL EPA treatment decreased the risk of CAD by 53% (HR: 0.47; P=0.043) (137). A subjective endpoint has the potential to be an unreliable endpoint in an open label study and is a limitation of the JELIS Study.

 

  • The Reduction of Cardiovascular Events with EPA – Intervention Trial (REDUCE-IT) was a randomized, double blind trial of 2 grams twice per day of EPA ethyl ester (icosapent ethyl) (Vascepa) vs. mineral oil placebo in 8,179 patients with hypertriglyceridemia (135mg/dL to 499mg/dL) and established cardiovascular disease or high cardiovascular disease risk (diabetes plus one risk factor) who were on stable statin therapy (138). The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina. The key secondary end point was a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. At baseline, the median LDL-C level was 75.0 mg/dL, HDL-C level was 40.0 mg/dL, and TG level was 216.0 mg/dL. The median change in TG level from baseline to 1 year was a decrease of 18.3% (−39.0 mg/dL) in the EPA group and an increase of 2.2% (4.5 mg/dL) in the placebo group. After a median of 4.9 years the primary end-point occurred in 17.2% of the patients in the EPA group vs. 22.0% of the patients in the placebo group (hazard ratio, 0.75; P<0.001), indicating a 25% decrease in events. The number needed to treat to avoid one primary end-point event was 21. The reduction in cardiovascular events was noted after approximately 2 years of EPA treatment. Additionally, the rate of cardiovascular death was decreased by 20% in the EPA group (4.3% vs. 5.2%; hazard ratio, 0.80; P=0.03). The cardiovascular benefits of EPA were similar across baseline levels of TGs (<150, ≥150 to <200, and ≥200 mg per deciliter). Moreover, the cardiovascular benefits of EPA appeared to occur irrespective of the attained TG level at 1 year (≥150 or <150 mg/dL), suggesting that the cardiovascular risk reduction was not associated with attainment of a normal TG level. An increase in hospitalization for atrial fibrillation or flutter (3.1% vs. 2.1%, P=0.004) occurred in the EPA group. In addition, serious bleeding events occurred in 2.7% of the patients in the EPA group and in 2.1% in the placebo group (P=0.06). There were no fatal bleeding events in either group and the rates of hemorrhagic stroke, serious central nervous system bleeding, and serious gastrointestinal bleeding were not significantly higher in the EPA group than in the placebo group.

 

It should be noted that in this trial mineral oil was used as the placebo. In the placebo group the LDL-C, non-HDL-C, and CRP levels were increased compared to the EPA group during the trial (LDL-C 96mg/dL vs 85mg/dL; non-HDL-C 130mg/dL vs. 113mg/dL; hsCRP 2.8mg/L vs. 1.8mg/L). The impact of these adverse changes on clinical outcomes is uncertain and whether they contributed to the apparent beneficial effects observed in the individuals treated with EPA is unknown.

 

  • The STRENGTH Trial was a double-blind, randomized, trial comparing 4 grams per day of a carboxylic acid formulation of omega-3 fatty acids (EPA and DHA; Epanova) (n = 6,539)) vs. corn oil placebo (n = 6539) in statin-treated participants with high cardiovascular risk, hypertriglyceridemia, and low levels of HDL-C (139). Approximately 55% of patients had established cardiovascular disease and approximately 70% had diabetes. Median LDL-C level was 75.0 mg/dL, median TG level was 240 mg/dL and median HDL-C level was 36 mg/dL. There were minimal differences in the change in LDL-C and HDL-C levels between the treated and placebo groups after treatment for 12 months but as expected there was a greater reduction in TG levels in the group treated with omega-3-fatty acids (−19.0% vs −0.9%). The primary endpoint was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina requiring hospitalization which occurred in 12.0% of individuals treated with omega-3 CA vs. 12.2% treated with corn oil (hazard ratio, 0.99; P = .84). There were no significant differences between the treatment groups with regard to the risk of the individual components of the primary end point over the 3-4 years of the study. Similar to the REDUCE-IT trial atrial fibrillation was increased with EPA + DHA treatment (HR 1.69 CI 1.29- 2.21). Thus, in contrast the JELIS and REDUCE-IT trials the STRENGTH trial did not demonstrate a benefit of treatment with a mixture of omega-3-fatty acids (EPA + DHA).

 

  • The OMEMI trial was a randomized trial of 1.8 grams per day of omega-3-fatty acids (930 mg EPA and 660 mg DHA) (n= 505) vs. corn oil placebo (509) in patients aged 70 to 82 years with a recent myocardial infarction (2-8 weeks) (140). Baseline LDL-C was approximately 76mg/dL, HDL-C was 49mg/dL, and TGs 110mg/dL. The primary endpoint was a composite of nonfatal myocardial infarction, unscheduled revascularization, stroke, all-cause death, and heart failure hospitalization after 2 years of follow-up. The primary endpoint occurred in 21.4% of patients on omega-3-fatty acids vs. 20.0% on placebo (hazard ratio, 1.08; P=0.60). TGs levels decreased 8.1% in the omega-3-fatty acid group and increased 5.1% in the placebo group (between group difference 13.2%; P<0.001) while changes in LDL-C were minimal in both groups. Thus, similar to the STRENGTH trial no benefits on cardiovascular disease were observed with EPA + DHA treatment.

 

Summary of Omega-3-Fatty Acid Clinical Outcome Trials

 

  • Low dose omega-3-fatty acids are not effective at decreasing cardiovascular outcomes.
  • High dose EPA (JELIS and REDUCE-IT) reduced cardiovascular outcomes while high dose EPA+DHA (STENGTH and OMEMI) did not decrease cardiovascular outcomes.
  • The decrease in TG levels is not a major contributor to the beneficial effect of high dose EPA as the combination of high EPA+DHA lowers TG levels to the same degree as EPA alone without benefit. Additionally, the JELIS trial only lowered TG levels by 5% but nevertheless reduced cardiovascular events. It is likely that the beneficial effects of EPA seen in the JELIS and REDUCE-IT trials are multifactorial with TG lowering making only a small contribution to the decrease in cardiovascular disease. Other actions of EPA, such as decreasing platelet function, anti-inflammation, decreasing lipid oxidation, stabilizing membranes, etc. could account for or contribute to the reduction in cardiovascular events (141). A large meta-analysis, excluding the REDUCE-IT trial, demonstrated that a 40mg/dL decrease in triglyceride levels resulted in a relative risk reduction of only 0.96 (4% decrease) indicating that one needs to markedly lower triglyceride levels to reduce cardiovascular events (142).
  • Whether EPA has special properties that resulted in the reduction in cardiovascular events in the REDUCE-IT trial or there were flaws in the trial design (the use of mineral oil as the placebo) is uncertain and debated. It should be noted that in the REDUCE-IT trial LDL-C and non-HDL-C levels were increased by approximately 10% in the mineral oil placebo group (138). Additionally, Apo B levels were increased by 7% (6mg/dL) by mineral oil (138). Finally, an increase in hsCRP (20-30%) and other biomarkers of atherosclerosis (oxidized LDL-C, IL-6, IL-1 beta, and lipoprotein-associated phospholipase A2) were noted in the mineral oil group (138,143). In the STRENGTH trial there were no differences in LDL-C, Non-HDL-C, HDL-C, Apo B, or hsCRP levels between the treated vs. placebo groups (139). Whether EPA has special properties compared to DHA leading to a reduction in cardiovascular events or the mineral oil placebo resulted in adverse changes increasing ASCVD in the placebo resulting in an artifactual decrease in the EPA group is debated (144,145). Ideally, another large randomized cardiovascular trial with EPA ethyl ester (icosapent ethyl) (Vascepa) using a placebo other than mineral oil would help resolve this controversy. In the meantime, clinicians will need to use their clinical judgement on whether to treat patients with modest elevations in TG levels with EPA (icosapent ethyl; Vascepa) balancing the potential benefits of treatment vs. the potential side effects.

 

Side Effects

 

Gastrointestinal side effects such as diarrhea, nausea, dyspepsia, abdominal discomfort, and eructation have been observed with fish oil therapy (Package Inserts for Lovaza, Vascepa, and Epanova).

 

At very high doses, omega-3-fatty acids can inhibit platelets and prolong bleeding time. However, at the recommended doses this has not been a major clinical problem but nevertheless when patients are on anti-platelet drugs one should be alert for the possibility of bleeding problems (Package Inserts for Lovaza, Vascepa, and Epanova). Increased bleeding was noted in the REDUCE-IT trial in the patients treated with icosapent ethyl 4 grams/day (EPA) (see above discussion of this trial). A recent review found no evidence for discontinuing the use of omega-3 fatty acid treatment before invasive procedures or when given in combination with other agents that affect bleeding (146).

 

As noted above an increase in atrial fibrillation was observed in the REDUCE-IT trial in the patients treated with icosapent ethyl 4 grams/day (EPA) and in the STRENGTH trial in the patients treated with EPA + DHA.

 

Contraindications

 

There are no contraindications to the use of omega-3-fatty acids. Lovaza, Omacor, and Vascepa are pregnancy category C drugs and they should only be used if the benefits to the mother outweigh the potential risks to the fetus.

 

Conclusions

 

Omega-3-fatty acids are effective drugs in reducing TG levels with few significant side effects, drug interactions, or contraindications.  High dose EPA (4 grams/day) reduced cardiovascular disease events in the REDUCE-IT trial and a moderate dose of EPA (1.8 grams/day) reduced cardiovascular events in the JELIS trial but trials of EPA and DHA have not produced cardiovascular benefits. The basis for these differences is debated and discussed in the “Summary of Omega-3-Fatty Acid Clinical Outcome Trials” section above. Finally, omega-3-fatty acids are effective in lowering TGs in patients with marked hypertriglyceridemia and while not proven will likely reduce the risk of development of pancreatitis.

 

FIBRATES

 

Introduction

 

The fibrate drug class includes clofibrate, gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate. Clofibrate was developed in the 1960s and was the first member of this class. Clofibrate is no longer available because of an increased risk of adverse effects. Gemfibrozil and fenofibrate are available in the United States while gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate are available in Europe. All of the fibrates work via activation of the nuclear hormone receptor PPAR alpha.

 

Effect of Fibrates on Lipid and Lipoprotein Levels

 

Table 12. Effect of Fibrates on Lipids and Lipoproteins

Decreases TG

Increases HDL-C

Decreases LDL-C; if TGs Very High can Increase LDL-C

Decreases Non-HDL-C

Decreases Apolipoprotein B

Decreases LDL Particle Number

Shift Small Dense LDL to Large Buoyant LDL

No Effect on Lp(a)

 

Fibrates reduce fasting TG levels by 25-50% (147-149). The magnitude of the reduction in TGs is dependent on the baseline TG levels. Patients with marked elevations in TGs have a greater reduction in TG levels (147,149,150). In addition, fibrates increase HDL-C levels by 5-20% (148,149). The increase in HDL-C levels is more robust if the TG levels are elevated and/or if the HDL-C levels are low (150). The effect on LDL-C is more variable (149). If the TG levels are very high (>400-500mg/dL), fibrate therapy may result in an increase in LDL-C levels whereas if TGs are not elevated fibrates decrease LDL-C by 10-30% (147). Given the decrease in plasma TGs and LDL-C levels, fibrates also reduce apolipoprotein B, LDL particle number, and non-HDL-C levels (149). Depending upon the TG level there may be a shift from small dense LDL towards large LDL particles (149). Fibrates do not have any major or consistent effects on Lp(a) levels (151). Table 13 below shows the effect of fenofibrate on lipid and lipoprotein levels in patients with different lipid profiles and illustrates some of the principles outlined above.

 

Table 13. Effect of Fenofibrate on Lipid and Lipoprotein Levels

 

TGs

LDL-C

HDL-C

Elevated TG Levels

 

 

 

Baseline Levels

~404mg/dL

~125mg/dL

~35mg/dL

Change with Fenofibrate

45% Decrease

2.5% Increase

16% Increase

Elevated LDL-C and TG Levels

 

 

 

Baseline Levels

232mg/dL

220mg/dL

46.7mg/dL

Change with Fenofibrate

37% Decrease

13% Decrease

12% Increase

Elevated LDL-C and Normal TG Levels

 

 

 

Baseline Levels

102mg/dL

228mg/dL

58.1mg/dL

Change with Fenofibrate

35% Decrease

29% Decrease

7% Increase

The values are adjusted for changes in the placebo group. Data modified from Tricor Package Insert.

 

In large, randomized, fibrate outcome trials similar changes in lipid and lipoprotein levels were noted (Table 14). These trials are discussed in detail in the effect of fibrates on cardiovascular outcomes section presented below.

 

Table 14. Effect of Fibrates on Lipid and Lipoprotein Levels in Large Outcome Studies*

 

TGs

LDL-C

HDL-C

Helsinki Heart Study- Gemfibrozil (152)

35% Decrease

11% Decrease

10% Increase

VA-HIT Study

Gemfibrozil (153)

31% Decrease

No Change

6% Increase

BIP Study

Bezafibrate (154)

21% Decrease

7% Decrease

18% Increase

Leader Study

Bezafibrate (155)

23% Decrease

8% Decrease

8% Increase

Field Study

Fenofibrate (156)

29% Decrease

12% Decrease

5% Increase

*The values are adjusted for changes in the placebo group.

 

The different fibrates in general cause similar changes in lipid and lipoprotein levels. There are only a few comparative trials of fibrates comparing their effects on lipid and lipoprotein levels and these trials have been very small. Comparisons of ciprofibrate and gemfibrozil have not shown any major differences between these two fibrates (157,158). In contrast, two very small trials have compared gemfibrozil vs. fenofibrate and reported that fenofibrate was more efficacious in lowering LDL levels than gemfibrozil (159,160).

 

In very rare instances fibrates can cause a paradoxical marked decrease in HDL-C levels (161-164). This rare paradoxical decrease in HDL-C typically occurs when fibrates are used in combination with a thiazolidinedione (rosiglitazone and pioglitazone) but can occur when fibrates are used alone or with ezetimibe (161-165). The decrease in HDL-C can be extreme with decreases of 50% to 88% reported and recovery to normal can take weeks after the fibrate is discontinued (162). The mechanism for this paradoxical effect is unknown.

 

Effect of Fibrates in Combination with Other Lipid Lowering Drugs on Lipid and Lipoprotein Levels

 

STATINS

 

Statins are the primary drugs used to treat most patients with dyslipidemia. Statins are very effective in lowering LDL-C levels but have only modest effects on TG and HDL-C levels. Therefore, it is appealing to add a fibrate to patients who on statin therapy have LDL-C levels at goal but still have elevated non-HDL-C and TG levels and decreased HDL-C levels. Therefore, there have been numerous studies examining the effect of the combination of statins and fibrates on lipid and lipoprotein levels. An example is the Safari Trial which compared the effect of simvastatin only (n=207) vs. simvastatin + fenofibrate (n=411) in patients with combined hyperlipidemia (166). The results of this trial are shown in table 15. As anticipated, adding a fibrate results in a further lowering of LDL-C, non-HDL-C, and TG levels with a further increase in HDL-C.

 

Table 15. Effect of Simvastatin Alone vs. Simvastatin + Fenofibrate on Lipid and Lipoprotein Levels

 

LDL

TG

Non-HDLC

HDL

Simvastatin

-26%

-20%

-26%

+10%

Simvastatin + Fenofibrate

-31%

-43%

-35%

+19%

 

A meta-analysis of 9 studies with over 1,200 participants compared the effect of statin alone vs. statin + fibrate on lipid and lipoprotein levels (167). The combination of statins and fibrates provided significantly greater reductions in total cholesterol, LDL-C, and TGs, and a significantly greater increase in HDL-C than treatment with statins alone. A larger meta-analysis of 13 randomized controlled trials, involving 7,712 patients, similarly demonstrated significant decreases in LDL-C (8.8mg/dL), TGs (58mg/dL), and total cholesterol (11.2mg/dL), and increases in HDL-C (4.65mg/dL) in patients receiving the combination of statins + fibrates compared with statin therapy alone (168). The combination of statins + fibrates also result in a shift of LDL particles from small dense particles to large buoyant particles whereas no change in LDL particle size was observed with statin monotherapy (169).  

 

A recent meta-analysis of 6 studies with over 400 participants compared the effect of adding a statin to fibrate therapy (fibrate alone vs. fibrate + statin) and showed similar changes (170).  The fibrate-statin combination produced significantly greater reductions in the levels of total cholesterol, LDL-C, and TGs compared to fibrate alone. In contrast there was no significant difference in HDL-C levels in the fibrate vs. fibrate + statins group.

 

EZETIMIBE

 

In patients unable to tolerate statin therapy one needs to use other drugs to treat dyslipidemia. In a study comparing the effect of ezetimibe 10mg alone, fenofibrate 145mg alone, or ezetimibe + fenofibrate the combination had a better effect on the lipid profile resulting in a greater decrease in LDL-C levels and increase in HDL-C levels than either drug alone (Table 16) (171).

 

Table 16. Effect of the Combination of Ezetimibe and Fenofibrate on Lipid and Lipoprotein Levels

 

LDL-C

HDL-C

TG

Ezetimibe

23% Decrease

2.2% Increase

10% Decrease

Fenofibrate

22% Decrease

7.5% Increase

38% Decrease

Ezetimibe + Fenofibrate

34% Decrease

11.5% Increase

38% Decrease

 

Similar results were observed in another randomized trial of ezetimibe 10mg and fenofibrate 160mg (172). Moreover, both fibrate therapy and the combination of ezetimibe and fenofibrate results in a shift of LDL particles from small dense LDL particles to large buoyant particles (172).

 

EZETIMIBE + STATIN

 

A large randomized trial has compared the effect of ezetimibe /simvastatin 10mg/20mg, fenofibrate 160mg, or ezetimibe/simvastatin + fenofibrate on lipid and lipoprotein levels. As one would expect triple drug therapy had a better effect on the lipid profile (Table 17) (173). While ezetimibe/simvastatin was very effective in lowering LDL-C levels and fenofibrate was very effective in lowering TGs and raising HDL-C levels the combination resulted in more favorable changes in TGs. In a similar study the addition of fenofibrate 135mg to atorvastatin 40 mg + ezetimibe 10 mg resulted in a greater reduction in TGs (-57% vs. -40%; p<0.001) and a greater increase in HDL (13% vs. 4.2%; p<0.001) than placebo (174).  Fibrate therapy and ezetimibe/simvastatin + fenofibrate also resulted in a shift of LDL particles from small dense LDL particles to large buoyant particles (173).

 

Table 17. Effect of the Combination of Ezetimibe/Simvastatin and Fenofibrate on Lipid and Lipoprotein Levels

 

LDL-C

HDL-C

TG

Placebo

-3.5%

+1.1

-3.1%

Ezetimibe/Simvastatin

-47%

+9.3%

-29%

Fenofibrate

-16%

+18.2

-41

Eze/Simva + Fenofibrate

-46%

+18.7

-50%

 

BILE ACID SEQUESTRANT  

 

Studies have also examined the effect of fibrates in combination with bile acid sequestrants. Participants receiving fenofibrate 160 mg/day were randomized to receive either colesevelam HCl 3.75 g/day or placebo (175). No significant differences in TG or HDL-C levels were observed between the two groups. However, LDL-C levels were decreased in the fenofibrate + colesevelam group compared to the fenofibrate + placebo group (12.4% greater decrease: p<0.001). A study of the combination of fenofibrate and colestipol also demonstrated a more marked decrease in LDL-C with that combination compared to either drug alone (colestipol -18%; fenofibrate -17%, colestipol + fenofibrate 37%) (176). The combination of both drugs did not blunt the effects of fenofibrate on VLDL and HDL. Other studies of the combination of a fibrate with a bile acid sequestrant have also demonstrated an enhanced effect in lowering LDL-C levels (177-179).

 

NIACIN

 

Surprisingly there are few large randomized trials examining the effect of combination therapy with niacin + fibrate vs. monotherapy. One very small trial did reported that while both niacin monotherapy and bezafibrate monotherapy were effective in lowering serum TGs there was no added benefit of combination therapy in reducing serum TG level although a large variance may have reduced the ability to detect statistically significant results (16). A larger trial in HIV+ patients reported that the combination of niacin and fenofibrate was better at lowering TGs and non-HDL-C and increasing HDL-C levels than monotherapy with either niacin or fenofibrate (17). It would be informative if additional trials of fibrate + niacin combination therapy were carried out in patients with marked hypertriglyceridemia that can often be difficult to control with lifestyle changes and monotherapy.

 

FISH OIL  

 

In patients with TG levels > 500mg/dL the combination of fenofibrate 130mg per day and 4 grams of Lovaza (DHA and EPA) reduced TG levels to a greater extent than monotherapy with fenofibrate alone (7% decrease; P = 0.059) (103). Not unexpectedly, LDL levels were increased to a greater extent with combination therapy (9% increase; p= 0.03). When subjects who had received 8 weeks of fenofibrate monotherapy were treated with Lovaza (DHA and EPA) during the 8-week, open-label extension study TG levels were reduced by an additional 17.5% (P = 0.003). These results indicate that the addition of omega-3-fatty acids to fenofibrate will further decrease TG levels.

 

Mechanisms Accounting for the Fibrate Induced Lipid Effects

 

Fibrates are ligands that bind and activate PPAR alpha, a member of the family of nuclear hormone receptors that are activated by lipids (180,181). PPAR alpha is highly expressed in the liver and other tissues important in fatty acid metabolism. PPAR alpha forms a heterodimer with RXR and together the PPAR alpha:RXR complex when activated binds to the PPAR response elements in a large number of genes and regulates the expression of these genes (180,181). The natural ligands of PPAR alpha are fatty acid derivatives formed during lipolysis, lipogenesis, or fatty acid catabolism (180,181).

 

TRIGLYCERIDES  

 

Fibrates lower plasma TG levels by decreasing VLDL production and by increasing the clearance of TG rich lipoproteins (182,183). The decrease in VLDL production is primarily due to PPAR alpha activation of the beta oxidation of fatty acids, which reduces the substrate available for the synthesis of TGs and the formation of VLDL (180,183). Additionally, a decrease in hepatic fatty acid synthesis may also contribute to the decrease in fatty acids (180,183). The increased clearance of TG rich lipoproteins is due to PPAR alpha stimulating the transcription of lipoprotein lipase, the key enzyme that catabolizes the TGs carried by VLDL and chylomicrons (180,183). In addition, activation of PPAR alpha also inhibits the transcription of APO C-III, which inhibits lipoprotein lipase activity (180,183). A decrease in Apo C-III enhances the clearance of TG rich lipoproteins by increasing lipoprotein lipase activity. Notably, a decrease in Apo C-III also decreases TG levels in patients deficient in lipoprotein lipase indicating that there are multiple mechanisms for its effects on TG metabolism (184). Recent studies suggest that Apo C-III inhibits the uptake of TG rich lipoproteins into the liver by the LDL receptors/ LDLR-related protein 1 axis (185). PPAR alpha activation also increases the transcription of Apo A-V, which would also facilitate the activity of lipoprotein lipase (180).

 

HIGH DENSITY LIPOPROTEINS

 

The increase in HDL induced by fibrates is due to PPAR alpha activation stimulating Apo A-I and A-II transcription (180,183). This leads to the increased production of HDL (182). In addition, a decrease in TG rich lipoproteins may result in a reduction in CETP mediated transfer of cholesterol from HDL to VLDL and of TG from VLDL to HDL (183). This would lead to less TG enrichment of HDL and a decrease in the opportunity of hepatic lipase to remove TG leading to small HDL particles that may be rapidly catabolized.

 

LOW DENSITY LIPOPROTEINS

 

As noted above the effect of fibrates on LDL-C levels is variable with increases in LDL seen in patients with high TG levels (>400mg/dL) and decreases in LDL-C levels in patients with lower TG levels. In patients with modest elevations in plasma TG levels the clearance of LDL is enhanced (182). The mechanism for this enhanced clearance could be due to a decrease in Apo C-III, as increased levels of this protein inhibits LDL receptor activity (185,186). Additionally, the shift from small dense LDL to large buoyant LDL would enhance the uptake of LDL by the LDL receptor (187). In patients with TG levels > 400mg/dL fibrate therapy decreases LDL clearance (182). Prior to treatment, patients with marked hypertriglyceridemia have hypercatabolism of LDL, which is likely due to increased uptake by the reticuloendothelial system (182). This increased clearance is LDL receptor independent. Treatment with fibrates lowers the plasma TGs leading to normalization of reticuloendothelial cell function and a decrease in LDL clearance resulting in an increase in LDL-C levels with fibrate therapy (182). In addition, the metabolism of VLDL to LDL may be enhanced by fibrates when the TG levels are markedly elevated.

 

Effect of Monotherapy with Fibrates on Cardiovascular Outcomes

 

There have been a number of studies that have examined the effect of monotherapy with a variety of different fibrates on cardiovascular disease. We will describe the major studies below.

 

  • Coronary Drug Project (CDP): CDP conducted between 1966 and 1975, was a randomized, double-blind clinical trial that determined the effect of clofibrate (1.8g/day), dextrothyroxine (6mg/day), two doses of oral estrogen (2.5 or 5mg per day), or immediate release niacin (3 grams/day) vs. placebo in 8,341 men aged 30 to 64 years of age with an electrocardiogram documented myocardial infarction on cardiovascular events and mortality (43). The mean baseline total cholesterol level was 251mg/dL and TG level was 183mg/dL. The two estrogen regimens and dextrothyroxine treatment groups were discontinued early because of increased adverse effects. Clofibrate treatment (n= 1,051) compared to placebo (n= 2,680) also did not demonstrate clinical benefit. The five-year mortality in subjects treated with clofibrate was 20.0% as compared with 20.9% in subjects on placebo therapy (P = 0.55). The results with niacin are discussed above in the section on niacin and cardiovascular outcomes.

 

  • WHO: WHO was a double-blind trial in middle-aged men, age 30-59 years of age, without evidence of heart or other major disease, who were treated with 1.6 grams/day clofibrate (n=5,000) or placebo (n=5,000) for an average of 5.3 years (188). Average serum cholesterol levels were approximately 248mg/dL and a mean reduction of approximately 9 per cent occurred in the clofibrate group. The incidence of ischemic heart disease was decreased by 20% in the clofibrate group compared to the control group (P <0.05). This decrease was confined to non-fatal myocardial infarcts which were reduced by 25% while the incidence of fatal heart attacks and angina was similar in the clofibrate and placebo groups. Importantly, the numbers of deaths and crude mortality rates from all causes were increased in the clofibrate-treated group compared to the control group (P < 0.05). The excess deaths were partially accounted for by increased deaths due to liver, biliary tract, and intestinal disease. There was also an increase in cholecystectomies in subjects treated with clofibrate. Because of increased toxicity clofibrate is no longer available.

 

  • Helsinki Heart Study (HHS): HSS was a randomized double-blind trial in middle aged men age 40-55 years of age without cardiovascular who had non-HDL-C levels greater than or equal to 200mg/dL (152). Subjects were randomized to receive 600mg gemfibrozil twice a day (n=2,051) or placebo (n=2,030) for five years. At initiation of the study total cholesterol was 289mg/dL, HDL-C 47mg/dL, non-HDL-C 242mg/dL, and TGs 176mg/dL. Gemfibrozil caused an increase in HDL-C (approximately 10%) and reductions in total (~10%), LDL-C (~11%), non-HDL-C (~14%), and TG levels (~35%). There were minimal changes in serum lipid levels in the placebo group. Fatal and non-fatal myocardial infarctions and cardiac death were the principal end points and the cumulative rate of these cardiac end points were reduced 34% in the gemfibrozil group (27.3 per 1,000 in the gemfibrozil group vs. 41.4 per 1,000 in the placebo group; P< 0.02). The decrease in cardiovascular disease in the gemfibrozil group became evident in the second year and continued throughout the remainder of the study. There was no difference in mortality between the gemfibrozil and placebo groups. The benefit of gemfibrozil therapy was greatest in participants with elevated TGs and decreased HDL-C levels (189,190). Risk reduction with gemfibrozil was 78% (P = .002) among those with BMI > 26 kg/m2 and dyslipidemia (TGs > ~200mg/dL and HDL-C < 42mg/dL) suggesting that certain types of patients are likely to derive greater benefit from fibrate treatment (191).

 

  • Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial (VA-HIT): VA-HIT was a double-blind trial in men with coronary heart disease who had an HDL-C level <40mg/dL and LDL-C level <140mg/dL (153). Subjects were randomized to gemfibrozil 1200mg per day (n=1,264) or placebo (n=1,267) for 5.1 years. Mean lipid levels at study initiation were HDL-C 32mg/dL, LDL-C 111mg/dL, total cholesterol 175mg/dL, and TGs 160mg/dL. At one year, the mean HDL-C level was 6 percent higher, the mean TG level was 31 percent lower, and the mean total cholesterol level was 4 percent lower in the gemfibrozil group than in the placebo group. LDL-C levels did not differ significantly between the groups. The primary study outcome was nonfatal myocardial infarction or death from coronary causes. The primary outcome occurred in 21.7% of patients in the placebo group and 17.3% of patients in the gemfibrozil group (22 percent decrease; P=0.006). A 24% reduction in the combined outcome of death from coronary heart disease, nonfatal myocardial infarction, and stroke was observed in the gemfibrozil group (P< 0.001). There were no significant differences in the rates of coronary revascularization, hospitalization for unstable angina, death from any cause, and cancer. Similar to HHS the beneficial effect of gemfibrozil did not become apparent until approximately two years after treatment. A low HDL-C (<33.5mg/dL) and high TGs (>180mg/dL) at baseline predicted a beneficial response to gemfibrozil therapy (192).

 

  • Bezafibrate Infarction Prevention Study (BIP): BIP was a double-blind study in male and female patients aged 45-74 with a previous myocardial infarction or stable angina (154). Patients were randomized to receive either 400 mg of bezafibrate per day (n=1,548) or a placebo (n=1,542) and were followed for 6.2 years. At the initiation of the study total cholesterol was 212mg/dL, LDL-C was 148mg/dL, HDL-C was 34.6mg/dL, and TGs were145mg/dL. Bezafibrate increased HDL-C by 18% and reduced TGs by 21%. There was a small 7% decrease in LDL-C. The primary end point was fatal or nonfatal myocardial infarction or sudden death. The primary end point occurred in 13. 6% of the bezafibrate group vs. 15.0% of the placebo (9.4% reduction; P=0.26). Total and non-cardiac mortality rates were similar. In a post hoc analysis in the subgroup with high baseline TGs (> or =200 mg/dL), the reduction in the primary end point in the bezafibrate group was 39.5% (P=0.02). Additionally, bezafibrate reduced cardiovascular events in patients with the metabolic syndrome (193). These results again suggest that patients with high TGs are likely to derive benefit from fibrate therapy.

 

  • Leader Trial: The Leader trial was a double blind placebo controlled randomized trial in men age 35 to 92 with lower extremity arterial disease (194,195). Subjects were randomized to bezafibrate 400mg per day (n=783) or placebo (n=785). At baseline total cholesterol levels were 218mg/dL, LDL-C levels 132mg/dL, HDL-C levels 44mg/dL, and TGs 187mg/dL. Bezafibrate therapy reduced total cholesterol levels by 7.6%, LDL-C by 8.1%, and TGs by 23% and increased HDL-C levels by 8%. The primary endpoint of coronary heart disease and strokes was not reduced by bezafibrate treatment. Neither major coronary events nor strokes were significantly reduced.

 .

  • Fenofibrate Intervention and Event Lowering in Diabetes Trial (FIELD): In the FIELD Trial patients with Type 2 diabetes between the ages of 50 and 75 with or without pre-existing cardiovascular disease not taking statin therapy were randomized to fenofibrate 200 mg daily (n=4,895) or placebo (n=4,900) and followed for approximately 5 years (156). At initiation of the study total cholesterol was 196mg/dL, LDL-C was 120mg/dL, HDL-C was 43mg/dL, and TGs were 152mg/dL. Fenofibrate therapy resulted in an 11% decrease in total cholesterol, a 12% decrease in LDL-C, a 29% decrease in TGs, and a 5% increase in HDL-C levels. The primary outcome was coronary events (coronary heart disease death and non-fatal MI), which were reduced by 11% in the fenofibrate group but this difference did not reach statistical significance (p= 0.16). However, there was a 24% decrease in non-fatal MI in the fenofibrate treated group (p=0.01) and a non-significant increase in coronary heart disease mortality. Total cardiovascular disease events (coronary events plus stroke and coronary or carotid revascularization) were reduced 11% (p=0.035). These beneficial effects of fenofibrate therapy on cardiovascular disease were observed in patients without a previous history of cardiovascular disease. In patients with a previous history of cardiovascular disease no benefits were observed. Additionally, the beneficial effect of fenofibrate therapy was seen only in those subjects less than 65 years of age. The beneficial effects of fenofibrate in this study may have been blunted by the increased use of statins in the placebo group, which reduced the differences in lipid levels between the placebo and fenofibrate groups. If one adjusted for the addition of lipid-lowering therapy, fenofibrate reduced the risk of coronary heart disease events by 19% (p=0.01) and of total cardiovascular disease events by 15% (p=0.004). Additionally, many patients in the Field trial did not have elevations in TGs and decreased HDL-C levels. In a post hoc analysis, patients with high TGs 200mg/dL) and low HDL levels (<40mg for men and <50mg/dL for women) derived greater benefit from fenofibrate therapy (196).

 

  • Summary: While the above monotherapy fibrate studies suggest that fibrates reduce cardiovascular event, particularly in patients with high TG and low HDL levels, the results are not as robust or consistent as the beneficial effects of statins on cardiovascular outcomes (5).

 

Effect of Combination Therapy of Fibrates and Statins on Cardiovascular Outcomes

 

Given the marked benefits of statin therapy it is essential to know if adding fibrates to statin therapy further reduces cardiovascular events. Two large trials described below have addressed this key question.

 

  • ACCORD LIPID Trial: The ACCORD-LIPID Trial was designed to determine if the addition of fenofibrate to aggressive statin therapy would result in a further reduction in cardiovascular disease in patients with Type 2 diabetes (197). In this trial, 5,518 patients on statin therapy were randomized to placebo or fenofibrate therapy. The patients had diabetes for approximately 10 years and either had pre-existing cardiovascular disease or were at high risk for developing cardiovascular disease. During the trial, LDL-C levels were approximately 80mg/dL. There was only a small difference in HDL-C with the fenofibrate groups having a mean HDL-C of 41.2mg/dL while the control group had an HDL-C of 40.5mg/dL. Differences in TG levels were somewhat more impressive with the fenofibrate group having a mean TG level of 122mg/dL while the control group had a TG level of 144mg/dL. First occurrence of nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular causes was the primary outcome and there was no statistical difference between the fenofibrate treated group and the placebo group. Additionally, there were also no statistically significant differences between the groups with regards to any of the secondary outcome measures of cardiovascular disease. Of note, the addition of fenofibrate to statin therapy did not result in an increase in either muscle or liver side effects. On further analysis there was a suggestion of benefit with fenofibrate therapy in the patients in whom the baseline TG levels were elevated (>204mg/dL) and HDL-C levels decreased (<34mg/dL). While this was a negative study, it must be recognized that most of the patients included in this study did not have the lipid profile that would typically lead to treatment with fibrates.

 

  • PROMINENT Trial: The PROMINENT trial studied the effect of pemafibrate, a new selective PPAR-alpha activator, in reducing cardiovascular outcomes in 10,497 patients (66.9% with previous ASCVD) with diabetes (198). This was a double-blind, randomized, controlled trial, in patients with Type 2 diabetes, with mild-to-moderate hypertriglyceridemia (TG level, 200 to 499 mg/dL), LDL-C < 100mg/dL, and HDL-C levels < 40 mg/dL who received either pemafibrate (0.2-mg tablets twice daily) or placebo in addition to statin therapy (96% on statins). The primary end point was a composite of nonfatal MI, ischemic stroke, coronary revascularization, or death from cardiovascular causes. Baseline fasting TG was 271 mg/dL, HDL-C 33 mg/dL, and LDL-C 78 mg/dL. Compared with placebo, pemafibrate decreased TG by 26.2%, while HDL-C increased 5.1% and LDL-C increased 12.3%. Notably non-HDL-C levels were unchanged and Apo B levels increased 4.8%. The primary endpoint was similar in the pemafibrate and placebo group (HR 1.03; 95% CI 0.91 to 1.15). The increase in LDL-C and Apo B levels may have accounted for the failure to reduce cardiovascular events.

 

  • Summary: The results of the ACCORD and PROMINENT trials were disappointing and have greatly reduced the enthusiasm for adding fibrates to statin therapy to cardiovascular events.

 

Effect of Fibrates on Non-Cardiovascular Outcomes

 

DIABETIC RETINOPATHY

 

Small studies in the 1960’s presented suggestive evidence that treatment with clofibrate improved diabetic retinopathy (199,200). Randomized trials have confirmed these observations.

 

The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study was a randomized trial in patients with Type 2 diabetes mellitus. Patients were randomly assigned to receive either fenofibrate 200 mg/day (n=4,895) or placebo (n=4,900). Laser treatment for retinopathy was significantly lower in the fenofibrate group than in the placebo group (3.4% patients on fenofibrate vs 4.9% on placebo; p=0.0002) (201). Fenofibrate therapy reduced the need for laser therapy to a similar extent for maculopathy (31% decrease) and for proliferative retinopathy (30% decrease). In the ophthalmology sub-study (n=1,012), the primary endpoint of 2-step progression of retinopathy grade did not differ significantly between the fenofibrate and control groups (9.6% patients on fenofibrate vs 12.3% on placebo; p=0.19). In patients without pre-existing retinopathy there was no difference in progression (11.4% vs 11.7%; p=0.87). However, in patients with pre-existing retinopathy, significantly fewer patients on fenofibrate had a 2-step progression than did those on placebo (3.1% patients vs 14.6%; p=0.004). A composite endpoint of 2-step progression of retinopathy grade, macular edema, or laser treatments was significantly reduced in the fenofibrate group (HR 0.66, 95% CI 0.47-0.94; p=0.022).

 

In the ACCORD Lipids Study a subgroup of participants were evaluated for the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale or the development of diabetic retinopathy necessitating laser photocoagulation or vitrectomy over a four-year period (202). At 4 years, the rates of progression of diabetic retinopathy were 6.5% with fenofibrate therapy (n=806) vs. 10.2% with placebo (n=787) (adjusted odds ratio, 0.60; 95% CI, 0.42 to 0.87; P = 0.006). Of note, this reduction in the progression of diabetic retinopathy was of a similar magnitude as intensive glycemic treatment vs. standard therapy.

 

A double-blind, randomized, placebo-controlled study in 296 patients with type 2 diabetes mellitus evaluated the effect of placebo or etofibrate on diabetic retinopathy (203). After 12 months an improvement in ocular pathology was more frequent in the etofibrate group vs the placebo group ((46% versus 32%; p< 0.001).

 

The MacuFen study was a small double-blind, randomized, placebo-controlled study in 110 subjects with diabetic macular edema who did not require immediate photocoagulation or intraocular treatment (204). Patients were randomized to fenofibric acid or placebo for 1 year. Patients treated with fenofibric acid had a modest improvement in total macular volume that was not statistically significant compared to the placebo group.

 

Taken together these results indicate that fibrates have beneficial effects on the progression of diabetic retinopathy (205). The mechanisms by which fibrates decrease diabetic retinopathy are unknown, and whether decreases in serum TG levels plays an important role is uncertain. Fibrates activate PPAR alpha, which is expressed in the retina (206). Diabetic PPARα KO mice developed more severe DR while overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation, suggesting that fibrates could have direct effects on the retina to reduce diabetic retinopathy (206).

 

DIABETIC KIDNEY DISEASE

 

The Diabetes Atherosclerosis Intervention Study (DAIS) evaluated the effect of fenofibrate therapy (n= 155) vs. placebo (n=159) on changes in urinary albumin excretion in patients with Type 2 diabetes (207). Fenofibrate significantly reduced the worsening of albumin excretion (fenofibrate 8% vs. placebo 18%; P < 0.05). This effect was primarily due to reduced progression from normal albumin excretion to microalbuminuria (fenofibrate 3% vs. 18% placebo; P < 0.001).

 

 In the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial, Type 2 diabetic patients aged 50 to 75 years were randomly assigned to fenofibrate (n = 4,895) or placebo (n = 4,900) for 5 years (208). Fenofibrate reduced urine albumin/creatinine ratio by 24% vs 11% in placebo group (p < 0.001), with 14% less progression and 18% more albuminuria regression (p < 0.001) in the fenofibrate group than in participants on placebo. As expected, fenofibrate therapy acutely increased plasma creatinine levels and decreased eGFR (209). However, over the long-term, the increase in plasma creatinine was lower in the fenofibrate group compared to the placebo group (14% decrease; p=0.01). Similarly, there was a slower annual decrease in eGFR in the fenofibrate group (1.19 vs 2.03 ml/min/1.73 m2annually, p < 0.001). End-stage renal disease, dialysis, renal transplant, and renal death were similar in the fenofibrate and placebo groups, likely due to the small number of events.

 

In the ACCORD-LIPID trial, 5,518 patients on statin therapy were randomized to placebo or fenofibrate therapy (197). The patients had diabetes for approximately 10 years and either had pre-existing cardiovascular disease or were at high risk for developing cardiovascular disease. The post-randomization incidence of microalbuminuria was 38.2% in the fenofibrate group and 41.6% in the placebo group (p=0.01) and post-randomization incidence of macroalbumuria was 10.5% in the fibrate group and 12.3% in the placebo group (p=0.04) indicating a modest reduction in the development of proteinuria in patients treated with fenofibrate (197). There was no significant difference in the incidence of end-stage renal disease or need for dialysis between the fenofibrate group and the placebo group, likely due to the small number of events.

 

A small randomized study in patients with Type 2 diabetes and hypertriglyceridemia compared the effect of fenofibrate (200mg/day) (n=28) vs. no treatment (n=28) on urinary albumin excretion (210). After 180 days urinary albumin/creatine ratio was decreased in the fenofibrate group vs. controls (control -8.15 vs fenofibrate -44.05 mg/g; P<0.05).

 

These studies suggest that fibrates may have a beneficial effect on diabetic kidney disease (211). One should recognize that reducing proteinuria is a surrogate marker and may not indicate a reduction in the development of end stage renal disease. The mechanisms accounting for the decrease in proteinuria are unknown.

 

AMPUTATIONS

 

In the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study, patients aged 50-75 years with Type 2 diabetes were randomly assigned to receive fenofibrate 200 mg per day (n=4,895) or matching placebo (n=4,900) for 5 years' duration (212). The risk of first amputation was decreased by 36% (p=0.02) and minor amputation events without known large-vessel disease by 47% (p=0.027) in the fenofibrate treated group (212). The reduction in amputations was independent of glucose control or dyslipidemia. No difference between the risks of major amputations was seen in the placebo and fenofibrate groups. The basis for this reduction in amputations is unknown.

 

GOUT

 

In the Field trial treatment, fenofibrate reduced uric acid levels by 20% and reduced episodes of gout by approximately 50% compared to placebo (HR 0·48, 95% CI 0·37-0·60; p<0·0001) (213). Interestingly, a meta-analysis of fibrate trials found that fenofibrate but not bezafibrate reduced serum uric acid levels suggesting that the reduction in uric acid levels is not a class effect (214).

 

SUMMARY

 

The above studies provide substantial evidence that fibrates have a favorable effect on diabetic microvascular disease (155). While fibrates are not approved specifically for the prevention or treatment of diabetic microvascular disease one should consider these potential beneficial effects when deciding on treatment choices. For example, in a patient with diabetes and microvascular disease and hypertriglyceridemia needing therapy one might elect to use fibrates to lower plasma TGs given their potential beneficial effects on slowing the progression of microvascular disease. 

 

Side Effects

 

RENAL

 

Fibrate therapy leads to an increase in serum creatinine and cystatin C levels (215-217). For example, in the Field Trial serum creatinine levels increased from 0.88mg/dL to 0.99mg/dL, a 12% increase (156). This increase in creatinine has been seen with all fibrates but appears to be less profound with gemfibrozil (215). The increase in cystatin C occurs with fenofibrate but not with other fibrates (216). It must be recognized that this increase in creatinine is reversible on stopping fibrate therapy and does not reflect kidney damage (215). In fact, careful measurements of renal function have not demonstrated a decrease in glomerular filtration rate despite the increase in serum creatinine (209,218,219). As discussed above, studies of renal function in patients with diabetes actually suggests that treatment with fibrates may be protective. The precise mechanism by which fibrates increase serum creatinine levels is unknown.

 

In patients with chronic renal disease fibrates should be used with caution and at lower doses (215). Fibrates are all excreted by the kidneys and thus the excretion of fibrates is decreased in patients with renal dysfunction (215). Therefore, one needs to adjust the fibrate dose depending upon renal function. The National Kidney Foundation recommends the dose adjustments shown in Table 18 (220).

 

Table 18. Fibrate Dose Adjustments in Renal Disease

 

No Kidney Disease

GFR 30-60

GFR < 30

Kidney Transplant

Bezafibrate

400-600mg

200mg

Avoid

Avoid

Ciprofibrate

1000-2000mg

?

Avoid

Avoid

Fenofibrate

150-200mg

40-60mg

Avoid

Avoid

Gemfibrozil

1200mg

1200mg

600mg

600mg

 

GALLBLADDER DISEASE

 

It is clear that clofibrate increases the risk of gallbladder disease. In both the WHO trial and the Coronary Drug Project, cholecystectomies occurred two to three times more often in the patients treated with clofibrate compared to placebo (43,188,221). Whether gemfibrozil, fenofibrate, or other fibrates increases the risk of gallbladder disease is uncertain. In the large randomized outcome studies presented earlier (Effect of fibrates on cardiovascular outcomes section) a statistically significant increase in either gallbladder disease or cholecystectomies were not observed. However, in a sub-study of 450 Helsinki Heart Study participants a trend toward a greater prevalence of gallstones during the study in the gemfibrozil group was observed (7.5% versus 4.9% for the placebo group, a 55% excess for the gemfibrozil group) (Lopid Package Insert). A trend toward a greater incidence of gallbladder surgery was also observed in the gemfibrozil group (17 versus 11 subjects, a 54% excess) (Lopid Package Insert). In a single epidemiological trial fibrate treatment independently correlated with the presence of gallstones with a relative risk of 1.7 (p=0.04) (222).

 

All fibrates alter the composition of bile resulting in an increase in the concentration of cholesterol, which will predispose to the formation of cholesterol gallstones (215). In a comparison of clofibrate and gemfibrozil it was observed that clofibrate resulted in changes in bile composition that would be more lithogenic than gemfibrozil (223).

 

The effect of combining fibrates with statins on the risk of gallbladder disease is unknown.  An increased risk of gallbladder disease or cholecystectomies was not reported in the ACCORD-LIPID trial where fenofibrate was added to statin therapy or the PROMINENT trial where pemafibrate was added to statin therapy (197,198).

 

While it is clear that clofibrate increases the risk of gallbladder disease the effect of other fibrates either as monotherapy or in combination with other drugs is less well defined.

 

PANCREATITIS  

 

In a meta-analysis of 7 fibrate trials involving 40,162 participants conducted over 5.3 years, 144 participants developed pancreatitis (84 assigned to fibrate therapy, 60 assigned to placebo) (RR, 1.39 (95% CI, 1.00-1.95; P = .053) (224). These observations raise the possibility that fibrates may increase the risk of pancreatitis.

 

CANCER

 

A large meta-analysis of 17 randomized controlled trials, involving 44,929 participants, with an average follow-up of 5.2 years has examined if fibrates lead to an increased risk of cancer. No increase in either cancer incidence (RR = 1.02, 95% CI 0.92-1.12) or cancer death (RR = 1.06, 95% CI: 0.92-1.22) was noted with fibrate treatment (225).

 

LIVER DISEASE

 

Fenofibrate has rarely been associated with idiosyncratic hepatotoxicity manifesting as hepatocellular to cholestatic disorders (226). The hepatitis may be acute self-limited or persistent chronic hepatitis. Liver abnormalities are very rare and in large trials such as the FIELD trial described above liver function test abnormalities were similar in the fenofibrate and placebo groups (156).   

 

GLYCEMIC PARAMETERS

 

A meta-analysis of 22 randomized placebo-controlled trials involving a total of 11,402 subjects demonstrated that fibrate therapy significantly decreased fasting plasma glucose, insulin levels, and insulin resistance measured by HOMA-IR, but did not effect HbA1c levels (227).

 

MUSCLE DISORDERS

 

Fibrate monotherapy has been reported to cause myopathy (215). In a large epidemiological study the incidence of hospitalization for rhabdomyolysis per 10,000 person-years for monotherapy with a fibrate was 2.82 (95% CI, 0.58-8.24) while in patients not exposed to lipid lowering drugs the incidence was 0 (95% CI, 0-0.48) (228). The risk of rhabdomyolysis was greater with gemfibrozil therapy than with fenofibrate. Interestingly the incidence of rhabdomyolysis was greater for patients treated with fibrate monotherapy than for patients treated with statin monotherapy (incidence for atorvastatin, pravastatin, or simvastatin was only 0.44 per 10,000 person-years). In an epidemiological study focusing on myopathy similar results were observed (229). The relative risks of myopathy in current users of fibrates and statins compared with nonusers were 42.4 (95% CI = 11.6-170.5) and 7.6 (95% CI = 1.4-41.3), respectively. It should be recognized though that in large randomized clinical trials the risk of muscle symptoms was low in patients treated with fibrates and not dissimilar to that seen in the patients treated with placebo (215). For example, in the Helsinki Heart Study over 2,000 patients were treated and in the VA-HIT over 1,000 patients were treated with gemfibrozil for five years and no cases of  myopathy were reported in either trial (152,153). In the Bezafibrate Infarction Prevention Study, seven patients in the placebo group and five patients in the bezafibrate group reported muscle pain, while CPK levels greater than 2x the upper range of normal was seen in four patients in the bezafibrate group and one patient in the placebo group (154). Finally, in the Field Trial, patients with diabetes were treated with fenofibrate (n=4,895) or placebo (n=4,900) (156). Myositis was observed in one patient treated with placebo and two patients treated with fenofibrate while rhabdomyolysis was observed in one patient treated with placebo and three patients treated with fenofibrate. Elevations in CPK levels values > 10x the upper range of normal were seen in three patients on placebo and 4 patients treated with fenofibrate. Thus, while fibrates can lead to significant muscle dysfunction this is a rare event and appears to occur only slightly more often in patients treated with a fibrate than in patients treated with a placebo. The risk of serious muscle disease appears to be increased in patients with renal failure, hypothyroidism, and in the elderly (215). The mechanism by which fibrates predispose to muscle disorders is unknown.

 

The effect of fibrates in combination with statins on muscle disorders will be discussed in detail in the section on drug interactions below.

 

Drug Interactions

 

STATINS

 

The combination a fibrate and a statin may increase the risk of developing muscle symptoms (215). The degree of risk is dependent on both the specific statin and the specific fibrate that is being used in combination (215). For example, the average incidence per 10,000 person-years for hospitalization for rhabdomyolysis with monotherapy with atorvastatin, pravastatin, or simvastatin was 0.44 (95 % CI, 0.20-0.84); with fibrate alone was 2.82 (95% CI, 0.58-8.24); and with combined therapy of atorvastatin, pravastatin, or simvastatin with a fibrate was 5.98 (95% CI, 0.72-216.0) (228). Of note, the average incidence per 10,000 person-years for hospitalization for rhabdomyolysis with the combination of cerivastatin with a fibrate was 1035 (95% CI, 389-2117), clearly demonstrating an increased risk of the cerivastatin-fibrate combination compared to other statin-fibrate combinations (228). A study by Alsheikh-Ali and colleagues looking at cases of rhabdomyolysis reported to the FDA relative to the total number of prescriptions reached the conclusion that the combination of cerivastatin with a fibrate markedly increased the risk of this complication (230). Additionally, it was noted that the risk of rhabdomyolysis was greater with gemfibrozil compared to fenofibrate and that the combination of cerivastatin and gemfibrozil was particularly toxic (230). Other studies have also noted a marked risk with the combination of cerivastatin and gemfibrozil (231). Cerivastatin is no longer available.

 

Studies comparing the risk of rhabdomyolysis with gemfibrozil-statin combination therapy compared to fenofibrate-statin combination therapy have shown an increased risk with gemfibrozil (215). For example, the number of cases of rhabdomyolysis reported with fenofibrate and statins other than cerivastatin was 0.58 per million prescriptions whereas with gemfibrozil and statins other than cerivastatin was 8.6 per million prescriptions (232). Reviews of the FDA’s adverse events reporting system database have estimated that the risk of myopathy for the combination of gemfibrozil with a statin was much greater than the risk with the combination of fenofibrate with a statin (230,232).  Additionally, studies that employed the combination of gemfibrozil and statins have reported a significant occurrence of muscle related symptoms whereas studies of fenofibrate in combination with statins have not shown an increase in muscle related symptoms (215). For example, the rate of myopathy in over 4,000 patients taking lovastatin was only 0.4% but in patients on the combination of lovastatin and gemfibrozil the frequency increased to 5% (233). In contrast, in the ACCORD-LIPID Trial over 5,000 patients on statin therapy were randomized to fenofibrate or placebo for 4.7 years and no increase in the incidence of muscle related symptoms was observed with fenofibrate therapy (197). Similarly, in the Field Trial approximately 1,000 patients were taking fenofibrate and a statin and with 5 years of follow-up no cases of rhabdomyolysis were reported (156). Finally, a meta-analysis by Geng and colleagues identified 13 randomized trials with 7,712 patients receiving combination fenofibrate-statin therapy compared with statin therapy alone (168). The incidence of elevated creatine kinase levels, muscle-associated adverse events, or withdrawals attributed to muscle dysfunction did not differ significantly between the fenofibrate + statin patients vs. the statin alone patients (168). The American College of Cardiology and American Heart Association Guidelines recommend against using the combination of a statin and gemfibrozil but recognize that the use of a statin and fenofibrate is appropriate under certain circumstances (234).

 

The increased risk of combining gemfibrozil with statins is due to alterations in statin metabolism leading to increases in the serum levels of statins and hence an increased risk of myopathy (215,235). In contrast, fenofibrate does not alter statin metabolism and therefore can be safely combined with statins (Table 19) (235).   

 

Table 19. Effect of Fibrates on Statin Pharmacokinetics (215,235,236)

Statin

Gemfibrozil

Fenofibrate

Atorvastatin

Increase in C-Max by 1.5-Fold

No Change

Simvastatin

Increase in C-Max by 2-Fold

No Change

Pravastatin

Increase in C-Max by 2-Fold

No Change

Rosuvastatin

Increase in C-Max by 2-Fold

No Change

Lovastatin

Increase in C-Max by 2.8-Fold

No Change

Pitavastatin

Increase in C-Max by 41%

Unknown

Fluvastatin

No Change

No Change

  

The explanation for the difference between gemfibrozil and fenofibrate is that gemfibrozil uses the same family of glucuronidation enzymes as the statins thereby inhibiting statin metabolism (215,237). In contrast, fenofibrate uses a different family of glucuronidation enzymes and does not inhibit statin metabolism (215).

 

COUMADIN ANTI-COAGULANTS

 

Gemfibrozil and fenofibrate can potentiate the effect of coumadin anti-coagulants leading to a prolongation of prothrombin time and an increased risk of bleeding. When starting a fibrate in patients on coumadin therapy the dose of coumadin should be decreased and prothrombin times should be closely monitored (Lopid and Tricor Package Inserts).    

 

REPAGLINIDE

 

Gemfibrozil in combination with rapaglinide increases blood levels of rapaglinide and therefore this combination should not be used because of the increased risk of hypoglycemia (Lopid Package Insert).

 

Contraindications

 

Fibrates are contraindicated in patients with severe hepatic dysfunction. Additionally, patients with pre-existing gallstones should not be treated with fibrates. Fenofibrate and gemfibrozil are pregnancy category C drugs and should only be used if the potential benefit justifies the potential risk to the fetus. The combination of gemfibrozil and a statin should be avoided.

 

Conclusions

 

Fibrates are effective drugs in reducing TG levels and modestly increase HDL-C levels. Additionally, they also reduce LDL-C and non-HDL-C levels. Fibrates have a number of side effects and one should avoid using gemfibrozil in combination with statins. In contrast, fenofibrate can be used in combination with statins. Studies have not consistently demonstrated that fibrate monotherapy therapy reduces cardiovascular events and the combination of fibrates and statins in two studies has not been shown to be beneficial. Therefore enthusiasm to use fibrates to reduce cardiovascular events has markedly diminished. In patients with diabetes fibrates appear to slow the progression of microvascular disease. Finally, fibrates are effective in lowering TGs in patients with marked hypertriglyceridemia and while not proven will likely reduce the risk of the development of pancreatitis.

 

VOLANESORSEN

 

Introduction 

 

Volanesorsen (Waylivra) is an antisense oligonucleotide inhibitor of apolipoprotein C-III (apo C-III) mRNA that is approved in Europe for the treatment of familial chylomicronemia syndrome (FCS). This drug has not been approved by the FDA for use in the United States. FCS is a rare metabolic disorder involving the impaired function of lipoprotein lipase (LPL) due to mutations in LPL, Apo C-II, Apo A-V, lipase maturation factor 1, and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) (238,239). For a detailed discussion of the diagnosis and treatment of FCS see the following references (238-240).

 

Effect of Volanesorsen on Lipid and Lipoprotein Levels

 

FAMILIAL CHYLOMICRONEMIA SYNDROME (FCS)

 

A double-blind, randomized 52-week trial (APPROACH study) evaluated the ability of volanesorsen (300 mg subcutaneously once weekly) vs. placebo to decrease TG levels in 66 patients with FCS (baseline TGs 2,209mg/dL) (241). The primary end point was the percentage change in fasting TG levels at 3 months. As expected, there was a marked reduction in Apo C-III levels (84% decrease) in the volanesorsen group and a small increase (6%) in the placebo group. Most importantly patients treated with volanesorsen had a 77% decrease at 3 months in TG levels (mean decrease of 1,712 mg/dL) whereas patients receiving placebo had an 18% increase in TG levels. The decrease in TGs in patients treated with volanesorsen persisted for 24 months (242). Significantly, 77% of the patients in the volanesorsen group vs. only 10% of patients in the placebo group had TG levels of less than 750 mg/dL, a level that would greatly reduce the risk of pancreatitis. In addition, patients who received volanesorsen had decreases in levels of chylomicron TG by 83%, apolipoprotein B-48 by 76%, non–HDL-C by 46%, and VLDL-C by 58% and increases in levels of HDL-C by 46%, apolipoprotein A1 by 14%, LDL-C by 136% (note LDL-C increased from 28 to 61 mg/dL), and total apolipoprotein B by 20%.

 

While the APPROACH study was not powered to examine the effect of volanesorsen on pancreatitis, during the study three patients in the placebo group had four episodes of acute pancreatitis, whereas one patient in the volanesorsen group had one episode. In patients with a history of recurrent pancreatitis events (≥ 2 events in the 5 years prior to study, n = 11), a reduction in pancreatitis attacks was seen in patients treated with volanesorsen compared with placebo (none of the 7 patients in the volanesorsen group and 3 of the 4 patients in the placebo group experienced a pancreatitis attack over the 52-week study period).

 

In a retrospective global web-based survey open to all patients with the FCS who received volanesorsen for ≥3 months, 22 patients responded and reported reductions in steatorrhea, pancreatic pain, and constant worry about an attack of pain/ acute pancreatitis (243). The patients also reported that volanesorsen improved overall management of symptoms and reduced interference of FCS with work/school responsibilities. Decreases in the negative impact of FCS on personal, social, and professional life were also reported.

 

HYPERTRIGLYCERIDEMIA

 

A randomized, double-blind, placebo-controlled, study evaluated volanesorsen in patients with hypertriglyceridemia (244). Patients who were not receiving TG-lowering therapy (n=57) were eligible if they had fasting TG level between 350 mg/dL and 2000 mg/dL and were assigned to volanesorsen 100, 200, or 300 mg or placebo. Patients who were receiving a fibrate (n=28) were eligible if they had a fasting TG level between 225 mg/dL and 2000 mg/dL and were randomly assigned to volanesorsen 200 or 300 mg or placebo. The study drug was administered as a single subcutaneous injection once a week for 13 weeks. Baseline TG levels were 581±291 mg/dL in patients not on fibrates and 376±188 mg/dL in patients on fibrates. In patients not on fibrates volanesorsen 300 mg decreased Apo C-III levels by 79.6% vs. an increase of 4.2% in the placebo group (P<0.001) and decreased TG levels by 70.9% compared with an increase of 20.1% in the placebo group (P<0.001). Additionally, HDL-C levels increased by 45.7% from baseline in the 300 mg group, as compared with an increase of 0.7% in the placebo group (P<0.001). LDL-C levels increased from 79.5±29.9 mg/dL to 127.8±44.9 mg/dL with 300 mg of volanesorsen and was associated with an increase in LDL particle size. However, non-HDL-C and total apo B levels remained relatively unchanged and similar to those in the placebo group. Similar changes in Apo C-III, TGs, HDL-C, non-HDL-C, VLDL-C, and total apo B levels were observed in the patients on fibrates treated with volanesorsen. Of note, LDL-C levels did not increase in the patients on fibrates treated with volanesorsen perhaps due to the lower baseline TG levels. 

 

The COMPASS study randomized 113 patients with fasting TGs ≥500 mg/dL (mean TG 1,261mg/dL) to receive either volanesorsen 300 mg or placebo subcutaneously once weekly for 26 weeks (245). Most of these patients had the multifactorial chylomicronemia syndrome but a small number had FCS. A 71% reduction in TGs from baseline after 3 months was observed in patients treated with volanesorsen vs. a 0.9% reduction in placebo-treated patients (P<0.0001). LDL-C levels increased 96% (64 to 111mg/dL), HDL-C increased 61% (25 to 39mg/dL) and non-HDL-C decreased 27% (232 to 158mg/dL) Notably pancreatitis episodes were reduced with 5 events in 3 patients occurring in the placebo group vs. none with volanesorsen treatment (P=0.036). 

 

DIABETES

 

A randomized, double-blind, placebo-controlled trial of volanesorsen 300 mg weekly or placebo was performed in 15 adult patients with type 2 diabetes (HbA1c >7.5%) and hypertriglyceridemia (TG >200 and <500 mg/dL) (246). Treatment with volanesorsen significantly reduced plasma apo C-III (-88%, P = 0.02) and TG (-69%, P = 0.02) levels and raised HDL-C (42%, P = 0.03) without altering LDL-C levels compared with placebo. These changes were accompanied by a 57% improvement in whole-body insulin sensitivity (P < 0.001) and decreases in HbA1c (-0.44%, P = 0.025) 3 months postdosing. The improvement in insulin sensitivity was strongly related to the decrease in plasma apo C-III and TGs.

 

FAMILIAL PARTIAL LIPODYSTROPY (FPL)

 

Patients with FPL were randomized to volanesorsen 300mg weekly (n=21) or placebo (n=19) (247). Median TG level was 781mg/dL in the placebo group and 749mg/dL in the volanesorsen group. Volanesorsen treatment at 3 months resulted in an 88% decrease in TG levels while in the placebo group TG levels decreased by 22% (net difference of −67%; P=0.0009). Non-HDL-HDL-C levels decreased while LDL-C and HDL-C levels increased.

 

Mechanisms Accounting for the Volanesorsen Induced Lipid Effects

 

Volanesorsen binds to apo C-III mRNA leading to increased degradation and thereby inhibits the hepatic synthesis of apo C-III protein resulting in a reduction in plasma apo C-III levels (248,249). Apo C-III has a number of important effects on the metabolism of TG rich lipoproteins (250). Apo C-III is an inhibitor of LPL and therefore decreasing apo C-III levels will enhance LPL activity. In patients with FCS this will not be important because patients with this disorder have defects in components of the LPL complex that result in the inability to increase LPL activity. However, in patients with increased TG levels not due FCS this would accelerate the clearance of TG rich lipoproteins. Studies have also shown that apo C-III stimulates the production and secretion of VLDL by the liver. This effect is also not likely to be of primary importance in patients with FCS as the very high TG levels are primarily due to chylomicrons and not VLDL. However, in other situations increased hepatic secretion of VLDL may be an important contributor to the hypertriglyceridemia. Whether apo C-III regulates chylomicron secretion by the intestine is unknown. Finally, Apo C-III inhibits the binding of TG rich lipoproteins to hepatic LDL receptors and LDL receptor–related protein 1 decreasing the clearance of TG rich lipoprotein particles. A decrease in apo C-III will accelerate the clearance of TG rich lipoproteins, which likely accounts for the ability of volanesorsen to decrease TG levels in patients with FCS.

 

Drug Administration and Pharmacokinetics

 

The recommended starting dose is 285 mg injected subcutaneously once weekly for 3 months after which the dose should be reduced to 285 mg every 2 weeks. If serum TGs decrease by less than 25% or are not below 2000 mg/dL (22.6 mmol/L) after 3 months on volanesorsen 285 mg weekly, treatment should be discontinued (package insert;https://www.ema.europa.eu/en/documents/product-information/waylivra-epar-product-information_en.pdf).

 

After 6 months of treatment one can consider increasing the dose frequency back to 285 mg weekly if the serum TG response has been inadequate and the platelet counts are in the normal range. Patients should return to 285 mg every 2 weeks if the higher 285 mg once weekly dose does not provide a significant additional TG reduction after 9 months (package insert).

 

Effect on Clinical Outcomes

 

As described above in the description of the effect of volanesorsen on lipid/lipoprotein levels in patients with FCS and marked hypertriglyceridemia there is suggestive evidence that lowering the very high TG levels with volanesorsen treatment will reduce the risk of pancreatitis and improve the quality of life.

 

Volanesorsen treatment reduced hepatic fat assessed by MRI in patients with FCS, severe hypertriglyceridemia, and familial partial lipodystrophy (251). The greater the hepatic fat the greater the decrease induced by volanesorsen.

 

The effect of volanesorsen on cardiovascular disease has not been determined. However, epidemiologic studies have demonstrated that increased Apo C-III levels are associated with an increased risk of cardiovascular events (252-254)and coronary artery calcification (255). Moreover, carriers of rare heterozygous loss-of-function mutations in Apo C-III have reduced TG levels and reduced cardiovascular disease risk (256-258). One can speculate that lowering Apo C-III and TG levels with volanesorsen will have beneficial effects on the development of cardiovascular disease.

 

Side Effects

 

Treatment with volanesorsen is very commonly associated with reductions in platelet count in patients with the FCS and may result in thrombocytopenia (package insert; https://www.ema.europa.eu/en/documents/product-information/waylivra-epar-product-information_en.pdf). Platelet counts below 140 x 109/L were observed in 75% of patients treated with volanesorsen vs. 24% of placebo patients. Reductions to below 100 x 109/L were observed in 47% of patients treated with volanesorsen compared with none of the patients in the placebo group. Bleeding secondary to low platelets may occur. Careful monitoring for thrombocytopenia is important during treatment and recommendations for adjustments to monitoring frequency and dosing are shown in table 20 (package insert). Platelet counts recover following drug discontinuation and administration of glucocorticoids where medically indicated.

 

Table 20.  Volanesorsen Monitoring and Treatment Recommendations

Platelet Count (x109/L)

Dose

Monitoring Frequency

Normal (≥140)

Starting dose: Weekly

After 3 months: Every 2 weeks

Every 2 weeks

100-139

Every 2 weeks

Weekly

75-99

Pause treatment for ≥4 weeks and resume treatment after platelet levels ≥ 100 x 109/L

Weekly

50-74

Pause treatment for ≥4 weeks and resume treatment after platelet levels ≥ 100 x 109/L

Every 2-3 days

Less than 50

Discontinue treatment

Glucocorticoids recommended

Daily

 

Renal toxicity has been observed after administration of volanesorsen. Monitoring for evidence of nephrotoxicity by routine urine dipstick is recommended on a quarterly basis. In the case of a positive assessment, one should measure serum creatinine and collect a 24-hour urine collection to quantify the proteinuria and assess creatinine clearance. Treatment should be discontinued if proteinuria ≥ 500 mg/24 hour is present, or an increase in serum creatinine ≥ 0.3 mg/dL that is >ULN occurs, or the creatinine clearance estimated by the CKD-EPI equation is ≤ 30 mL/min/1.73m2(package insert).

 

Elevations of liver enzymes have been observed after administration of volanesorsen. Serum liver enzymes and bilirubin should be monitored every 3 months. Treatment should be discontinued if there is a single increase in ALT or AST > 8 x ULN, or an increase > 5 x ULN, which persists for ≥ 2 weeks, or lesser increases in ALT or AST that are associated with total bilirubin > 2 x ULN or INR > 1.5 (package insert).

 

As expected, injection site reactions are frequently observed and were reported in 82% of patients (erythema, pain, pruritus, or local swelling) (package insert).

 

Contraindications

 

Treatment should not be initiated in patients with thrombocytopenia (platelet count <140 x 109/L). Safety and efficacy have not been established in patients with severe renal disease or patients with hepatic impairment (package insert). There are no data on the use of volanesorsen in pregnant women and it is preferable to avoid the use of volanesorsen during pregnancy (package insert).

 

Drug Interactions

 

Discontinuation of antiplatelet drugs/NSAIDs/anticoagulants should be considered for

platelet levels < 75 x 109/L. Treatment with these products must be discontinued at platelet levels < 50 x 109/L. No other drug interactions have been described (package insert)

 

Conclusions

 

Volanesorsen is a useful drug in patients with the FCS, particularly in patients who have repeated episodes of acute pancreatitis. Whether volanesorsen will be useful for the treatment of less severe hypertriglyceridemia remains to be determined, particularly given its potential side effects. Drugs similar to volanesorsen (Olezarsen) that do not adversely affect platelets are underdevelopment (259).  

 

ALIPOGENE TIPARVOVEC (GLYBERA)

 

Introduction

 

Alipogene tiparvovec is a gene therapy that was approved in Europe for adult patients with Familial Lipoprotein Lipase deficiency and a history of multiple or severe episodes of pancreatitis who have failed dietary therapy (260). The diagnosis of Familial Lipoprotein Lipase with loss of function mutations must be confirmed by genetic testing but patients need to have detectable levels of lipoprotein lipase protein (to avoid immunological reactions) (260). Alipogene tiparvovec is an adeno-associated virus gene therapy that results in the expression of the naturally occurring S447X variant of the human lipoprotein lipase gene that has increased lipoprotein lipase activity compared to “normal” lipoprotein lipase (260). Approximately 20% of Caucasians express this gene variant and these individuals have lower plasma TG levels and an increase in HDL-C levels (261,262). Because of the lack of long-term efficacy alipogene tiparvovec is no longer clinically available.

 

Effect of Alipogene Tiparvovec on Lipid and Lipoprotein Levels

 

In patients with plasma TG levels > 880mg/d, treatment with alipogene tiparvovec resulted in an approximately 40% decrease in fasting plasma TGs with half of the patients having > 40% decrease in fasting plasma TG levels at 3-12 weeks post treatment (263). By week 16-26, fasting TG levels returned to baseline values but chylomicron levels were reduced (263). While fasting TG levels returned to baseline, postprandial TG levels were reduced by approximately 60% suggesting that there are long term effects that are not reflected by fasting TG levels (264). In fact, in some patients treated with alipogene tiparvovec, lipoprotein lipase expression was demonstrated in muscle biopsies at 26 weeks (263).

 

Mechanisms Accounting for the Alipogene Tiparvovec Induced Lipid Effects

 

Gene therapy with alipogene tiparvovec results in the expression of lipoprotein lipase in muscle, which accelerates the clearance of chylomicrons (260,263). Studies have demonstrated a reduced peak level and a reduced area under the curve for postprandial chylomicrons (264).

 

Drug Administration and Pharmacokinetics

 

Alipogene tiparvovec is administered by multiple intramuscularly injections in the legs given at a single visit (260). The number of injections is > 40 and therefore the injections are given under spinal anesthesia (263). From 3 days before administration until 12 weeks after administration patients may be treated with cyclosporine (3mg/kg/day) and mycophenolate (2g/day) and on the day of administration methylprednisolone 1mg/kg) may be administered IV (260,263).

 

Effect on Clinical Outcomes

 

In patients with Familial Lipoprotein Lipase Deficiency the outcome of interest is pancreatitis. In a retrospective study of 19 patients treated with alipogene tiparvovec an approximate 50% decrease in pancreatitis was observed (265). In addition, patients treated with alipogene tiparvovec have reported benefits including discontinuing lipoprotein apheresis, increased energy, and the ability to liberalize their diet, which is difficult to comply with due to the marked limitation in dietary fat (263,266).

 

Conclusions

 

Alipogene tiparvovec may be a useful treatment for the rare patient with Familial Lipoprotein Lipase deficiency but the lack of long-term efficacy and the difficulty of giving the required injections led to this drug being removed from the market.  Because of the rarity of this disorder the information on patients treated with this drug is limited and randomized trials are impossible.

 

EVINACUMAB (EVKEEZA)

 

Introduction

 

Evinacumab is a human monoclonal antibody against angiopoietin-like protein 3 (ANGPTL3). It is approved for the treatment of Homozygous Familial Hypercholesterolemia. Evinacumab decreases LDL-C levels by mechanisms independent of LDL receptor activity. The recommended dose of evinacumab is 15 mg/kg administered by intravenous infusion over 60 minutes every 4 weeks. While it is not approved for TG lowering it is effective in lowering TG levels.

 

Effect on Evinacumab on TG Levels

 

For information on the effect of evinacumab on LDL-C levels see the Endotext chapter on “Cholesterol Lowering Drugs (5). Because of the difficulty in treating severe hypertriglyceridemia, I have focused on evinacumab in this group of patients. Phase 1 studies have shown that various doses of evinacumab lower TG levels in individuals with TG levels between 150-450mg/dL with maximal effects of approximately 80% reductions (267). As one would expect LDL-C and HDL-C levels also decreased in these individuals with modest hypertriglyceridemia.

 

A phase 2 study evaluated evinacumab in three groups of patients with severe hypertriglyceridemia; FCS patients with bi-allelic loss-of-function mutations in the lipoprotein lipase (LPL) pathway (n = 17), multifactorial chylomicronemia syndrome (MFCS) with heterozygous loss-of-function LPL pathway mutations (n = 15), and MFCS without LPL pathway mutations (n = 19) (268). Patients were randomized to evinacumab 15 mg/kg IV or placebo every 4 weeks over 12-weeks. The effect on TG and non-HDL-C levels are shown in table 21. Despite the very small number of patients the results suggest that evinacumab can lower TG levels in patients with MFCS but not in patients with FCS. This result Is not surprising based on the proposed mechanism of action of inhibiting ANGPTL3 (see below).

 

Table 21. Change in Lipid/Lipoprotein Parameters

 

FCS

MFCS/heterozygous LPL pathway mutations

MFCS/ without LPL pathway mutations

 

Placebo (n=5)

Evinacumab (n=12)

Placebo (n=8)

Evinacumab ((n=9)

Placebo (n=5)

Evinacumab (n=14)

Fasting TG

Baseline

3,918mg/dL

3,140mg/dL

1,351mg/dL

1,238mg/dL

1,030mg/dL

1,917mg/dL

% change

−22.9

−27.7

9.4

−64.8*

80.9

−81.7**

Non-HDL-C

Baseline

356mg/dL

345mg/dL

202mg/dL

220mg/dL

209mg/dL

296mg/dL

% change

−15.2

−34.2^

8.0

−31.0^^

48.4

−38.5^^^

*p= 0.0076, **p= 0.0418, ^p= 0.0074, ^^p= 0.0677, ^^^p= 0.1016.

FCS= familial chylomicronemia syndrome, MFCS= multifactorial chylomicronemia syndrome.

 

Mechanism Accounting for the Evinacumab Induced Decrease in TG

 

ANGPTL3 inhibits lipoprotein lipase (LPL) activity thereby slowing the clearance of VLDL and chylomicrons resulting in an increase in plasma triglyceride levels (269,270). Mice deficient in ANGPTL3 have lower plasma triglyceride levels while mice overexpressing ANGPTL3 have elevated plasma triglyceride levels (270). Evinacumab by inhibiting the ability of ANGPTL3 to decrease LPL activity results in an increases in LPL activity, which accelerates the clearance of TG rich lipoproteins decreasing plasma triglyceride levels (270). In patients with FCS who lack a functioning lipoprotein lipase clearance system evinacumab will not accelerate the clearance of TG rich lipoproteins. For information on the mechanism by which evinacumab lowers LDL-C and HDL-C see the Endotext chapter on “Cholesterol Lowering Drugs” (5).

 

Pharmacokinetics and Drug Interactions

 

There are no significant drug interactions.

 

Effect of Evinacumab on Clinical Outcomes

 

There are no cardiovascular outcome studies.

 

Homozygosity for loss-of-function mutations in ANGPTL3 is associated with significantly lower plasma levels of LDL-C, HDL-C, and triglycerides (familial combined hypolipidemia) (270,271). Heterozygous carriers of loss-of-function mutations in ANGPTL3, which occur at a frequency of about 1:300, have significantly lower total cholesterol, LDL-C, and triglyceride levels than noncarriers (270). Moreover, patients carrying loss-of-function variants in ANGPTL3 have a significantly lower risk of coronary artery disease (272,273). Additionally, in an animal model of atherosclerosis treatment with evinacumab decreased atherosclerotic lesion area and necrotic content (272). Taken together these observations suggest that inhibiting ANGPTL3 with evinacumab will reduce cardiovascular disease.

 

Side Effects

 

Serious hypersensitivity reactions have occurred with evinacumab. In clinical trials, 1 (1%) of evinacumab treated patients experienced anaphylaxis vs. 0% of patients who received placebo (package insert).

 

Contraindications

 

Based on animal studies, evinacumab may cause fetal harm when administered to pregnant patients (package insert). Patients should be advised of the potential risks to the fetus of pregnancy. Patients who may become pregnant should be advised to use effective contraception during treatment with evinacumab and for at least 5 months following the last dose.

 

Summary

 

Evinacumab lowers triglyceride levels in patients with severe hypertriglyceridemia due to multifactorial chylomicronemia syndrome and could be useful in selected patients with hypertriglyceridemia. Note it is not approved to treat severe hypertriglyceridemia and administration intravenously every 4 weeks will limit its use to special circumstances.

 

CLINICAL USE OF TRIGLYCERIDE LOWERING DRUGS

 

Marked Hypertriglyceridemia (>500mg/dL); Prevention of Pancreatitis

 

In patients with marked elevations in TG levels (>500-1000mg/dL) the major concern is an increased risk of pancreatitis (274,275). Because of this increased risk it is imperative to lower TG levels. The initial steps are to 1) treat any disease states that could be leading to an elevation in plasma TG levels, 2) if possible, discontinue any drugs that could be leading to an elevation in plasma TGs, and 3) initiate lifestyle changes (Table 22) (2,276).

 

Table 22. Causes of Secondary Hypertriglyceridemia

Lifestyle

Diseases

Medications

Excess calories

Poorly controlled diabetes

Corticosteroids

Excess dietary fat intake

Hypothyroidism

Oral estrogen

Excess simple sugars

Renal disease

Retinoic acid derivatives

Overweight/Obesity

HIV infection

Beta adrenergic blockers

Alcohol intake

Cushing’s syndrome

Thiazide diuretics

Pregnancy

Acromegaly

Protease inhibitors

 

Growth hormone deficiency

Bile acid sequestrants

 

Lipodystrophy

Anti-psychotic drugs

 

Paraproteinemia

Cyclosporine/tacrolimus

 

Nephrotic Syndrome

L-asparaginase

 

Inflammatory Disorders

Interferon alpha 2b

 

 

Cyclophosphamide

 

These initial steps are often sufficient to result in marked reductions in plasma TG levels eliminating the need for TG lowering medications. For example, in patients with diabetes in very poor glycemic control, treatment that results in good glycemic control can markedly lower TG levels (277). Similarly, the restoration of euthyroidism in a hypothyroid patient can also markedly lower lipid levels (278). If these initial steps do not result in a lowering of TGs into an acceptable range, then the use of drugs to lower plasma TG levels is indicated. There have been no randomized controlled trials demonstrating that treatment diminishes pancreatitis but most experienced clinicians believe that lowering TG levels to below 500-1000mg/dL reduces the risk of developing pancreatitis (274,275). The addition of either fibrates or fish oil to lifestyle changes are commonly used to lower markedly elevated TG levels. In some patients, combination therapy is required to lower plasma TGs to an acceptable range. In patients with Familial Chylomicronemia syndrome volanesorsen is a promising therapeutic tool.

 

Moderate Hypertriglyceridemia (150-500mg/dL); Prevention of Cardiovascular Disease

 

In the era of statin therapy, it is uncertain whether lowering TG levels in patients on statin therapy will further reduce cardiovascular events. As discussed in detail in the sections on individual drugs, the studies carried out so far have not shown that adding niacin or fibrates to statin therapy is beneficial with regards to cardiovascular disease. As also discussed, some of the available studies have major limitations because many of the patients in these outcome studies did not have substantial elevations in TGs. Nevertheless, at this time there is little enthusiasm for adding either fibrates or niacin to statins to lower the risk of cardiovascular event.

 

Notably, the REDUCE-IT trial, which tested the effect of high dose EPA (4 grams per day) in patients with elevated TG levels on statin therapy demonstrated a 25% reduction in cardiovascular events. However, the decrease in cardiovascular events was considerably greater than one would expect based on the reduction in TG levels suggesting that the decrease in cardiovascular events was not solely due to lowering TG levels and that other effects of EPA likely played a role. Additionally, as discussed in detail in the section discussing cardiovascular trials in the omega-3-fatty acid section there are concerns that the use of mineral oil as the placebo in the REDUCE-IT trial may have caused harmful effects leading to increased events. Thus, the role of EPA in reducing cardiovascular events is debated with some experts feeling that it is beneficial while others feeling that the evidence for benefit is very weak. Clearly additional studies are required to resolve this controversy. In the meantime, clinicians will need to use their clinical judgement on whether to treat patients with modest elevations in TG levels with EPA (icosapent ethyl; Vascepa) balancing the potential benefits of treatment vs. the potential side effects.

 

Some guidelines use non-HDL-C as a therapeutic goal and thus the use of omega-3-fatty acids and fibrates will often be required to lower TG levels to achieve these non-HDL-C goals. In contrast, other guidelines focus on LDL-C levels and the use of statins and thus de-emphasize the use of omega-3-fatty acids and fibrates. Given the absence of definitive data one needs to use clinical judgement. Consideration should also be given to the use of fenofibrate in hypertriglyceridemic patients with diabetes at high risk for microvascular disease given the studies that have shown that fibrates reduce the microvascular complications of diabetes. Because of the side effects of niacin, the use of niacin to lower TG levels has markedly diminished. In the past we used to use niacin to lower both LDL-C levels and TGs but with the availability of ezetimibe, bempedoic acid, and PCSK9 inhibitors the need to use niacin to lower LDL-C levels has markedly decreased.

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education.

 

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Introduction to Lipids and Lipoproteins

ABSTRACT

 

Cholesterol and triglycerides are insoluble in water and therefore these lipids must be transported in association with proteins. Lipoproteins are complex particles with a central core containing cholesterol esters and triglycerides surrounded by free cholesterol, phospholipids, and apolipoproteins, which facilitate lipoprotein formation and function. Plasma lipoproteins can be divided into seven classes based on size, lipid composition, and apolipoproteins (chylomicrons, chylomicron remnants, VLDL, VLDL remnants (IDL), LDL, HDL, and Lp (a)).  Chylomicron remnants, VLDL, IDL, LDL, and Lp (a) are all pro-atherogenic while HDL is anti-atherogenic. Apolipoproteins have four major functions including 1) serving a structural role, 2) acting as ligands for lipoprotein receptors, 3) guiding the formation of lipoproteins, and 4) serving as activators or inhibitors of enzymes involved in the metabolism of lipoproteins. The exogenous lipoprotein pathway starts with the incorporation of dietary lipids into chylomicrons in the intestine. In the circulation, the triglycerides carried in chylomicrons are metabolized in muscle and adipose tissue by lipoprotein lipase releasing free fatty acids, which are subsequently metabolized by muscle and adipose tissue, and chylomicron remnants are formed. Chylomicron remnants are then taken up by the liver. The endogenous lipoprotein pathway begins in the liver with the formation of VLDL. The triglycerides carried in VLDL are metabolized in muscle and adipose tissue by lipoprotein lipase releasing free fatty acids and IDL are formed. The IDL are further metabolized to LDL, which are taken up by the LDL receptor in numerous tissues including the liver, the predominant site of uptake. Reverse cholesterol transport begins with the formation of nascent HDL by the liver and intestine. These small HDL particles can then acquire cholesterol and phospholipids that are effluxed from cells, a process mediated by ABCA1 resulting in the formation of mature HDL.  Mature HDL can acquire addition cholesterol from cells via ABCG1, SR-B1, or passive diffusion. The HDL then transports the cholesterol to the liver either directly by interacting with hepatic SR-B1 or indirectly by transferring the cholesterol to VLDL or LDL, a process facilitated by CETP. Cholesterol efflux from macrophages to HDL plays an important role in protecting from the development of atherosclerosis.

 

INTRODUCTION

 

Because lipids, such as cholesterol and triglycerides, are insoluble in water these lipids must be transported in association with proteins (lipoproteins) in the circulation. Large quantities of fatty acids from meals must be transported as triglycerides to avoid toxicity. These lipoproteins play a key role in the absorption and transport of dietary lipids by the small intestine, in the transport of lipids from the liver to peripheral tissues, and the transport of lipids from peripheral tissues to the liver and intestine (reverse cholesterol transport). A secondary function is to transport toxic foreign hydrophobic and amphipathic compounds, such as bacterial toxins, from areas of invasion and infection (1). For example, lipoproteins bind endotoxin (LPS) from gram negative bacteria and lipoteichoic acid from gram positive bacteria thereby reducing their toxic effects (1). In addition, apolipoprotein L1, associated with HDL particles, has lytic activity against the parasite Trypanosoma brucei brucei and lipoproteins can neutralize viruses (2,3). Thus, while this article will focus on the transport properties of lipoproteins the reader should recognize that lipoprotein may have other important roles.

 

STRUCTURE OF LIPOPROTEINS (4)

 

Lipoproteins are complex particles that have a central hydrophobic core of non-polar lipids, primarily cholesterol esters and triglycerides. This hydrophobic core is surrounded by a hydrophilic membrane consisting of phospholipids, free cholesterol, and apolipoproteins (Figure 1). Plasma lipoproteins are divided into seven classes based on size, lipid composition, and apolipoproteins (Table 1 and Figure 2).

 

Figure 1. Lipoprotein Structure (figure modified from Biochemistry 39: 9763, 2000)

 

 

Table 1. Lipoprotein Classes

Lipoprotein

Density (g/ml)

Size (nm)

Major Lipids

Major Apoproteins

Chylomicrons

<0.930

75-1200

Triglycerides

Apo B-48, Apo C, Apo E, Apo A-I, A-II, A-IV

Chylomicron Remnants

0.930- 1.006

30-80

Triglycerides Cholesterol

Apo B-48, Apo E

VLDL

0.930- 1.006

30-80

Triglycerides

Apo B-100, Apo E, Apo C

IDL

1.006- 1.019

25-35

Triglycerides Cholesterol

Apo B-100, Apo E, Apo C

LDL

1.019- 1.063

18- 25

Cholesterol

Apo B-100

HDL

1.063- 1.210

5- 12

Cholesterol Phospholipids

Apo A-I, Apo A-II, Apo C, Apo E

Lp (a)

1.055- 1.085

~30

Cholesterol

Apo B-100, Apo (a)

 

Figure 2: Classes of Lipoproteins (figure modified from Advances Protein Chemistry 45:303, 1994)

 

Chylomicrons (5)

 

These are large triglyceride rich particles made by the intestine, which are involved in the transport of dietary triglycerides and cholesterol to peripheral tissues and liver. These particles contain apolipoproteins A-I, A-II, A-IV, A-V, B-48, C-II, C-III, and E. Apo B-48 is the core structural protein and each chylomicron particle contains one Apo B-48 molecule. The size of chylomicrons varies depending on the amount of fat ingested. A high fat meal leads to the formation of large chylomicron particles due to the increased amount of triglyceride being transported whereas in the fasting state the chylomicron particles are small carrying decreased quantities of triglyceride. The quantity of cholesterol carried by chylomicrons also can vary depending upon dietary intake.

 

Chylomicron Remnants (5-7)

 

The removal of triglyceride from chylomicrons by lipoprotein lipase in peripheral tissues results in smaller particles called chylomicron remnants. Compared to chylomicrons these particles are enriched in cholesterol and are pro-atherogenic.

 

Very Low-Density Lipoproteins (VLDL)

 

These particles are produced by the liver and are triglyceride rich. They contain apolipoprotein B-100, C-I, C-II, C-III, and E. Apo B-100 is the core structural protein and each VLDL particle contains one Apo B-100 molecule. Similar to chylomicrons the size of the VLDL particles can vary depending on the quantity of triglyceride carried in the particle. When triglyceride production in the liver is increased, the secreted VLDL particles are large. However, VLDL particles are smaller than chylomicrons.

 

Intermediate-Density Lipoproteins (IDL; VLDL Remnants) (6,7)

 

The removal of triglycerides from VLDL by muscle and adipose tissue results in the formation of IDL particles which are enriched in cholesterol. These particles contain apolipoprotein B-100 and E. These IDL particles are pro-atherogenic.

 

Low-Density Lipoproteins (LDL)

 

These particles are derived from VLDL and IDL particles and they are even further enriched in cholesterol. LDL carries the majority of the cholesterol that is in the circulation. The predominant apolipoprotein is B-100 and each LDL particle contains one Apo B-100 molecule. LDL consists of a spectrum of particles varying in size and density. An abundance of small dense LDL particles is seen in association with hypertriglyceridemia, low HDL levels, obesity, type 2 diabetes (i.e. patients with the metabolic syndrome) and infectious and inflammatory states. These small dense LDL particles are considered to be more pro-atherogenic than large LDL particles for a number of reasons (8). Small dense LDL particles have a decreased affinity for the LDL receptor resulting in a prolonged retention time in the circulation. Additionally, they more easily enter the arterial wall and bind more avidly to intra-arterial proteoglycans, which traps them in the arterial wall. Finally, small dense LDL particles are more susceptible to oxidation, which could result in an enhanced uptake by macrophages. 

 

High-Density Lipoproteins (HDL) (9-11)

 

These particles play an important role in reverse cholesterol transport from peripheral tissues to the liver, which is one potential mechanism by which HDL may be anti-atherogenic. In addition, HDL particles have anti-oxidant, anti-inflammatory, anti-thrombotic, and anti-apoptotic properties, which may also contribute to their ability to inhibit atherosclerosis. HDL particles are enriched in cholesterol and phospholipids. Apolipoproteins A-I, A-II, A-IV, C-I, C-II, C-III, and E are associated with these particles. Apo A-I is the core structural protein and each HDL particle may contain multiple Apo A-I molecules. In addition, using mass spectrometry proteins involved in proteinase inhibition, complement activation, and the acute-phase response have been found associated with HDL particles (12). HDL particles are very heterogeneous and can be classified based on density, size, charge, or apolipoprotein composition (Table 2).

 

Table 2. Classification of HDL

Method of classification

Types of HDL

Density gradient ultracentrifugation

HDL2, HDL3, very high-density HDL

Nuclear magnetic resonance

large, medium, and small

Gradient gel electrophoresis

HDL 2a, 2b, 3a, 3b, 3c

2-dimensional gel electrophoresis

pre-beta 1 and 2, alpha 1, 2, 3, 4

Apolipoprotein composition

A-I particles, A-I: A-II particles, A-I: E particles

 

Lipoprotein (a) (Lp (a)) (13-16)

 

Lp (a) is an LDL particle that has apolipoprotein (a) attached to Apo B-100 via a disulfide bond. Lp (a) contain Apo (a) and Apo B-100 in a 1:1 molar ratio. The size of Lp(a) particles can vary greatly based on the size of apolipoprotein (a). This particle is pro-atherogenic.

 

APOLIPOPROTEINS (17,18)

 

Apolipoproteins have four major functions including 1) serving a structural role, 2) acting as ligands for lipoprotein receptors, 3) guiding the formation of lipoproteins, and 4) serving as activators or inhibitors of enzymes involved in the metabolism of lipoproteins (Table 3). Apolipoproteins thus play a crucial role in lipoprotein metabolism.

 

Apolipoprotein A-I (19)

 

Apo A-I is synthesized in the liver and intestine and is the major structural protein of HDL accounting for approximately 70% of HDL protein. It also plays a role in the interaction of HDL with ATP-binding cassette protein A1 (ABCA1), ABCG1, and class B, type I scavenger receptor (SR-B1). Apo A-I is an activator of lecithin: cholesterol acyltransferase (LCAT), an enzyme that converts free cholesterol into cholesteryl ester. High levels of Apo A-I are associated with a decreased risk of atherosclerosis.

 

Apolipoprotein A-II (20)

 

Apo A-II is synthesized in the liver and is the second most abundant protein on HDL accounting for approximately 20% of HDL protein. The role of Apo A-II in lipid metabolism is unclear. Apo A-II is a strong predictor of risk for CVD.

 

Apolipoprotein A-IV (21)

 

Apo A-IV is synthesized in the intestine during fat absorption. Apo A-IV is associated with chylomicrons and high-density lipoproteins, but is also found in the lipoprotein-free fraction.  Its precise role in lipoprotein metabolism remains to be determined but studies have suggested a role for Apo A-IV in regulating food intake.

 

Apolipoprotein A-V (22,23)

 

Apo A-V is synthesized in the liver and associates with triglyceride rich lipoproteins. It is an activator of LPL mediated lipolysis and thereby plays an important role in the metabolism of triglyceride rich lipoproteins.

 

Apolipoprotein B-48 (24)

 

Apo B-48 is synthesized in the intestine and is the major structural protein of chylomicrons and chylomicron remnants. There is a single molecule of apo B-48 per chylomicron particle. There is a single apolipoprotein B gene that is expressed in both the liver and intestine. The intestine expresses a protein that is approximately ½ the size of the liver due to mRNA editing. The apobec-1 editing complex is expressed in the intestine and edits a specific cytidine to an uracil in the apo B mRNA in the intestine creating a stop codon that results in the cessation of protein translation and a shorter Apo B (Apo B-48). The portion of Apo-B that is recognized by the LDL receptor is not contained in Apo-B48 and therefore Apo B-48 is not recognized by the LDL receptor.

 

Apolipoprotein B-100

 

Apo B-100 is synthesized in the liver and is the major structural component of VLDL, IDL, and LDL. There is a single molecule of Apo B-100 per VLDL, IDL, LDL and Lp(a) particle. Apo B-100 is a ligand for the LDL receptor and therefore plays an important role in the clearance of lipoprotein particles. Certain mutations in Apo B-100 result in decreased binding to the LDL receptor and familial hypercholesterolemia (25). High levels of Apo B-100 are associated with an increased risk of atherosclerosis.

 

Apolipoprotein C (26-29)

 

The C apolipoproteins are synthesized primarily in the liver and freely exchange between lipoprotein particles and therefore are found in association with chylomicrons, VLDL, and HDL.

 

Apo C-II is a co-factor for lipoprotein lipase (LPL) and thus stimulates triglyceride hydrolysis and the clearance of triglyceride rich lipoproteins (26,29). Loss of function mutations in Apo C-II result in marked hypertriglyceridemia due to a failure to metabolize triglyceride rich lipoproteins (30).

 

Apo C-III is an inhibitor of LPL (31). Additionally, Apo C-III inhibits the interaction of triglyceride rich lipoproteins with their receptors (31). Recent studies have shown that loss of function mutations in Apo C-III lead to decreases in serum triglyceride levels and a reduced risk of cardiovascular disease. Interestingly, inhibition of Apo C-III expression results in a decrease in serum triglyceride levels even in patients deficient in lipoprotein lipase indicating that the ability of Apo C-III to modulate serum triglyceride levels is not dependent solely on regulating lipoprotein lipase activity (32).

 

Apolipoprotein E (33)

 

Apolipoprotein E is synthesized in many tissues but the liver and intestine are the primary source of circulating Apo E. Apo E exchanges between lipoprotein particles and is associated with chylomicrons, chylomicron remnants, VLDL, IDL, and a subgroup of HDL particles. There are three common genetic variants of Apo E (Apo E2, E3, and E4). ApoE2 differs from the most common isoform, Apo E3, by a single amino acid substitution where cysteine substitutes for arginine at residue 158. Apo E4 differs from Apo E3 at residue 112, where arginine substitutes for cysteine. Apo E3 and E4 are ligands for the LDL receptor while Apo E2 is poorly recognized by the LDL receptor. Patients who are homozygous for Apo E2 can develop familial dysbetalipoproteinemia (30). Apo E4 is associated with an increased risk of Alzheimer’s disease and an increased risk of atherosclerosis.

 

Apolipoprotein (a) (14,16)

 

Apo (a) is synthesized in the liver. This protein is a homolog of plasminogen and its molecular weight varies from 300,000 to 800,000. It is attached to Apo B-100 via a disulfide bond. High levels of Apo (a) are associated with an increased risk of atherosclerosis. Apo (a) is an inhibitor of fibrinolysis and can also enhance the uptake of lipoproteins by macrophages, both of which could increase the risk of atherosclerosis. The physiologic function of Apo (a) is unknown. Interestingly this apolipoprotein is found in primates but not in other species.

 

Table 3. Apolipoproteins

Apolipoprotein

MW

Primary Source

Lipoprotein Association

Function

Apo A-I

28,000

Liver, Intestine

HDL, chylomicrons

Structural protein for HDL, Activates LCAT

Apo A-II

17,000

Liver

HDL, chylomicrons

Structural protein for HDL, Activates hepatic lipase

Apo A-IV

45,000

Intestine

HDL, chylomicrons

Unknown

Apo A-V

39,000

Liver

VLDL, chylomicrons, HDL               

Promotes LPL mediated TG lipolysis

Apo B-48

241,000

Intestine

Chylomicrons

Structural protein for chylomicrons

Apo B-100

512,000

Liver

VLDL, IDL, LDL, Lp (a)

Structural protein, Ligand for LDL receptor

Apo C-I

6,600

Liver

Chylomicrons, VLDL, HDL

Activates LCAT

Apo C-II

8,800

Liver

Chylomicrons, VLDL, HDL

Co-factor for LPL

Apo C-III

8,800

Liver

Chylomicrons, VLDL, HDL

Inhibits LPL and uptake of lipoproteins

Apo E

34,000

Liver

Chylomicron remnants, IDL, HDL

Ligand for LDL receptor

Apo (a)

250,000- 800,00

Liver

Lp (a)

Inhibits plasminogen activation

                                

LIPOPROTEIN RECEPTORS AND LIPID TRANSPORTERS

 

There are several receptors and transporters that play a crucial role in lipoprotein metabolism.

 

LDL Receptor (34)

 

The LDL receptor is present in the liver and most other tissues. It recognizes Apo B-100 and Apo E and hence mediates the uptake of LDL, chylomicron remnants, and IDL, which occurs via endocytosis (Figure 3). After internalization, the lipoprotein particle is degraded in lysosomes and the cholesterol is released. The delivery of cholesterol to the cell decreases the activity of HMGCoA reductase and other enzymes required for the biosynthesis of cholesterol, and the expression of LDL receptors. LDL receptors in the liver play a major role in determining plasma LDL levels (a low number of receptors is associated with high plasma LDL levels while a high number of hepatic LDL receptors is associated with low plasma LDL levels). The number of LDL receptors is regulated by the cholesterol content of the cell (35). When cellular cholesterol levels are decreased the transcription factor SREBP is transported from the endoplasmic reticulum to the Golgi where proteases cleave and activate SREBP, which then migrates to the nucleus and stimulates the expression of LDL receptors (Figure 4). Conversely, when cellular cholesterol levels are high SREBP remains in the endoplasmic reticulum in an inactive form and the expression of LDL receptors is low. As discussed later PCSK9 regulates the rate of degradation of LDL receptors.

 

Figure 3. LDL Receptor Pathway (figure modified from Annual Review of Biochemistry 46: 897, 1977)

Figure 4. SREBP Pathway (figure modified from Journal of Lipid Research 50: Supp S15, 2009)

 

 

LRP-1 is a member of the LDL receptor family. It is expressed in multiple tissues including the liver. LRP-1 recognizes Apo E and mediates the uptake of chylomicron remnants and IDL (VLDL remnants).

 

VLDL Receptor (37)

 

The VLDL receptor is a member of the LDL receptor family. The VLDLR is expressed in the heart, skeletal muscle, adipose tissue, endothelium, brain, macrophages, and other tissues. Interestingly it is not usually expressed in the liver but hepatic expression can be induced by endoplasmic reticulum stress and PPAR alpha activation. Apo E but not Apo B bind to the VLDL receptor thereby allowing for the uptake of triglyceride rich lipoprotein particles (VLDL and chylomicrons).

 

Class B Scavenger Receptor B1 (SR-B1) (38)

 

SR-B1 is expressed in the liver, adrenal glands, ovaries, testes, macrophages, and other cells. In the liver and steroid producing cells, it mediates the selective uptake of cholesterol esters from HDL particles. In macrophages and other cells, it facilitates the efflux of cholesterol from the cell to HDL particles.

 

ATP-Binding Cassette Transporter A1 (ABCA1) (39)

 

ABCA1 is expressed in many cells including hepatocytes, enterocytes, and macrophages. It mediates the transport of cholesterol and phospholipids from the cell to lipid poor HDL particles (pre-beta-HDL).

 

ATP-Binding Cassette Transporter G1 (ABCG1) (40)

 

ABCG1 is expressed in many different cell types and mediates the efflux of cholesterol from the cell to HDL particles.

 

ATP-Binding Cassette Transporter G5 and G8 (ABCG5/ABCG8) (41,42)

 

ABCG5 and ABCG8 are expressed in the liver and intestine and form a heterodimer. In the intestine, these transporters mediate the movement of plant sterols and cholesterol from inside the enterocyte into the intestinal lumen thereby decreasing the absorption of cholesterol and limiting the uptake of dietary plant sterols. In the liver, these transporters play a role in the movement of cholesterol and plant sterols into the bile facilitating the excretion of sterols.

 

Niemann-Pick C1-Like 1 (NPC1L1) (41)

 

NPC1L1 is expressed in the intestine and mediates the uptake of cholesterol and plant sterols from the intestinal lumen into the enterocyte. NPC1L1 is also expressed in the liver where it mediates the movement of cholesterol from hepatocytes into the bile.

 

ENZYMES AND TRANSFER PROTEINS INVOLVED IN LIPOPROTEIN METABOLISM

 

There are several enzymes and transfer proteins that play a key role in lipoprotein metabolism.

 

Lipoprotein Lipase (LPL) (43)

 

LPL is synthesized in muscle, heart, and adipose tissue, then secreted and attached to the endothelium of the adjacent blood capillaries. This enzyme hydrolyzes the triglycerides carried in chylomicrons and VLDL to fatty acids, which can be taken up by cells. The catabolism of triglycerides results in the conversion of chylomicrons into chylomicron remnants and VLDL into IDL (VLDL remnants). This enzyme requires Apo C-II as a cofactor. Apo A-V also plays a key role in the activation of this enzyme. In contrast Apo C-III and Apo A-II inhibit the activity of LPL. Insulin stimulates LPL expression and LPL activity is reduced in patients with poorly controlled diabetes, which can impair the metabolism of triglyceride rich lipoproteins leading to hypertriglyceridemia (44).

 

Hepatic Lipase (45)

 

Hepatic lipase is localized to the sinusoidal surface of liver cells. It mediates the hydrolysis of triglycerides and phospholipids in IDL and LDL leading to smaller particles (IDL is converted to LDL; LDL is converted from large LDL to small LDL). It also mediates the hydrolysis of triglycerides and phospholipids in HDL resulting in smaller HDL particles.

 

Endothelial Lipase (46)

 

Endothelial lipase plays a major role in hydrolyzing the phospholipids in HDL.

 

Lecithin: Cholesterol Acyltransferase (LCAT) (47)

 

LCAT is made in the liver. In the plasma, it catalyzes the synthesis of cholesterol esters in HDL by facilitating the transfer of a fatty acid from position 2 of lecithin to cholesterol. This allows for the transfer of the cholesterol from the surface of the HDL particle (free cholesterol) to the core of the HDL particle (cholesterol ester), which facilitates the continued uptake of free cholesterol by HDL particles by reducing the concentration of cholesterol on the surface of HDL.

 

Cholesteryl Ester Transfer Protein (CETP) (48,49)

 

This protein is synthesized in the liver and in the plasma mediates the transfer of cholesterol esters from HDL to VLDL, chylomicrons, and LDL and the transfer of triglycerides from VLDL and chylomicrons to HDL. Inhibition of CETP activity leads to an increase in HDL cholesterol and a decrease in LDL cholesterol.

 

Microsomal Triglyceride Transfer Protein (MTTP) (50)

 

MTTP is expressed primarily in the liver and small intestine and plays a crucial role in the synthesis of lipoproteins in these tissues. MTTP mediates the transfer of triglycerides to apolipoprotein B-100 in the liver to form VLDL and to apolipoprotein B-48 in the intestine to form chylomicrons.

 

EXOGENOUS LIPOPROTEIN PATHWAY (CHYLOMICRONS)

 

Figure 5. Exogenous Lipoprotein Pathway

 

Fat Absorption (51-54)

 

The exogenous lipoprotein pathway starts in the intestine. Dietary triglycerides (approximately 100 grams per day) are hydrolyzed to free fatty acids and monoacylglycerol by intestinal lipases and emulsified with bile acids, cholesterol, plant sterols, and fat-soluble vitamins to form micelles. While the fatty acids in the intestine are overwhelmingly accounted for by dietary intake the cholesterol in the intestinal lumen is primarily derived from bile (approximately 800-1200mg of cholesterol from bile vs. 200-500mg from diet). Plant sterols account for approximately 25% of dietary sterol intake (approximately 100-150mg/day). The cholesterol, plant sterols, fatty acids, monoacylglycerol, and fat-soluble vitamins contained in the micelles are then transported into the intestinal cells. The uptake of cholesterol and plant sterols from the intestinal lumen into intestinal cells is facilitated by a sterol transporter, Niemann-Pick C1- like 1 protein (NPC1L1) (Figure 6). Ezetimibe, a drug which inhibits intestinal cholesterol and plant sterol uptake, binds to NPC1L1 and inhibits its activity. Once in the intestinal cell the cholesterol and plant sterols may be transported back into the intestinal lumen, a process mediated by ABCG5 and ABCG8, or converted to sterol esters by acyl-CoA cholesterol acyl transferase (ACAT), which attaches a fatty acid to the sterol. Compared to cholesterol, plant sterols are poor substrates for ACAT and therefore the formation of plant sterol esters does not occur as efficiently as the formation of cholesterol esters. In humans, <5% of dietary plant sterols are absorbed and the vast majority are transported out of the intestine cell, a process mediated by ABCG5 and ABCG8, which are very efficient at effluxing plant sterols from the intestinal cell into the intestinal lumen. Patients with sitosterolemia have mutations in either ABCG5 or ABCG8 and net absorption of dietary plant sterols is increased (20-30% absorbed vs. < 5% in normal subjects) (55). Thus, ABCG5 and ABCG8 along with ACAT serve as gate keepers and block the uptake of plant sterols and likely also play an important role in determining the efficiency of cholesterol absorption (humans typically absorb only approximately 50% of dietary cholesterol with a range of 25-75%).

 

Figure 6. Intestinal Cell and Sterol Metabolism. C= cholesterol, CE= cholesterol ester.

 

The pathway of absorption of free fatty acids is not well understood but it is likely that both passive diffusion and specific transporters play a role. The fatty acid transporter CD36 is strongly expressed in the proximal third of the intestine and is localized to the villi. While this transporter likely plays a role in fatty acid uptake by intestinal cells, this transporter is not essential as humans and mice deficient in this protein do not have fat malabsorption. However, in mice deficient in CD36 there is a shift in the absorption of lipid to the distal intestine, suggesting pathways that can compensate for the absence of CD36. Fatty acid transport protein 4 (FATP4) is also highly expressed in the intestine. However, mice deficient in FATP4 do not have abnormalities in fat absorption. It is likely that there are multiple pathways for the absorption of fatty acids into intestinal cells. The pathways by which monoacylglycerols are absorbed by intestinal cells remain to be defined.

 

Formation of Chylomicrons (51,54)

 

The absorbed fatty acids and monoacylglycerols are utilized to synthesize triglycerides. The key enzymes required for triglyceride synthesis are monoacylglycerol acyltransferase (MGAT) and diacylglycerol transferase (DGAT). MGAT catalyzes the addition of a fatty acid to monoacylglycerol while DGAT catalyzes the addition of a fatty acid to diacylglycerol resulting in triglyceride formation.  As noted above, the majority of the cholesterol absorbed by the intestine is esterified to cholesterol esters by ACAT. The triglycerides and cholesterol esters are packaged into chylomicrons in the endoplasmic reticulum. The size and composition of the chylomicrons formed in the intestine are dependent on the amount of fat ingested and absorbed by the intestine and the type of fat absorbed. Increased fat absorption results in larger chylomicrons. The formation of chylomicrons in the endoplasmic reticulum requires the synthesis of Apo B-48 by the intestinal cell (Figure 6).  Microsomal triglyceride transfer protein (MTTP) is required for the movement of lipid from the endoplasmic reticulum to the Apo B-48. The absence of MTTP results in the inability to form chylomicrons (Abetalipoproteinemia) (56). Lomitapide inhibits MTTP function and is used to treat patients with homozygous Familial Hypercholesterolemia (57).

 

Chylomicron Metabolism (26,31,43,58-62)

 

Chylomicrons are secreted into the lymph and delivered via the thoracic duct to the circulation. It should be noted that this results in the newly formed chylomicrons being delivered to the systemic circulation and not delivered directly to the liver via the portal circulation. This facilitates the delivery of the nutrients contained in the chylomicrons to muscle and adipose tissue. In muscle and adipose tissue lipoprotein lipase (LPL) is expressed at high levels. LPL is synthesized in muscle and adipocytes and transported to the luminal surface of capillaries. Lipase maturation factor 1 plays a key role in the stabilization and movement of LPL from muscle cells and adipocytes to the capillary endothelial cell surface. Glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) binds LPL and transports it to the capillary lumen and anchors LPL to the capillary endothelium. Activation of LPL by Apo C-II, carried on the chylomicrons, leads to the hydrolysis of the triglycerides that are carried in the chylomicrons resulting in the formation of free fatty acids, which can be taken up by the adjacent muscle cells and adipocytes for either energy production or storage.  Fatty acid transport proteins (FATPs) and CD36 facilitate the uptake of fatty acids into adipocytes and muscle cells. Some of the free fatty acids released from chylomicrons bind to albumin and can be transported to other tissues. Apo A-V also plays an important role in activating LPL activity. Loss of function mutations in LPL, Apo C-II, GPIHPB1, lipase maturation factor 1, and Apo A-V can result in marked hypertriglyceridemia (familial chylomicronemia syndrome) (30). In addition, there are proteins that inhibit LPL activity. Apo C-III inhibits LPL activity and loss of function mutations in this gene are associated with increases in LPL activity and decreases in plasma triglyceride levels. Similarly, angiopoietin like protein 3 and 4, which target LPL for inactivation, regulate LPL activity. Loss of function mutations in these proteins also are associated with decreases in plasma triglyceride levels. Finally, the expression of LPL by muscle cells and adipocytes is regulated by hormones (particularly insulin), nutritional status, and inflammation.

 

The metabolism of the triglycerides carried in the chylomicrons results in a marked decrease in the size of these particles leading to the formation of chylomicron remnants, which are enriched in cholesterol esters and acquire Apo E. As these particles decrease in size phospholipids and apolipoproteins (Apo A and C) on the surface of the chylomicrons are transferred to other lipoproteins, mainly HDL. The transfer of Apo C-II from chylomicrons to HDL decreases the ability of LPL to further breakdown triglycerides. These chylomicron remnants are cleared from the circulation by the liver. The Apo E on the chylomicron remnants binds to the LDL receptor and other hepatic receptors such as LRP and syndecan-4 and the entire particle is taken up by the hepatocytes. Apo E is crucial for this process and mutations in Apo E (for example homozygosity for the Apo E2 isoform) can result in decreased chylomicron clearance and elevations in plasma cholesterol and triglyceride levels (familial dysbetalipoproteinemia) (30).

 

The exogenous lipoprotein pathway results in the efficient transfer of dietary fatty acids to muscle and adipose tissue for energy utilization and storage. The cholesterol is delivered to the liver where it can be utilized for the formation of VLDL, bile acids, or secreted back to the intestine via secretion into the bile. In normal individuals, this pathway can handle large amounts of fat (100 grams or more per day) without resulting in marked increases in plasma triglyceride levels. In fact, in a normal individual, a meal containing 75 grams of fat results in only a very modest increase in postprandial triglyceride levels. 

 

ENDOGENOUS LIPOPROTEIN PATHWAY (VLDL AND LDL)

 

Figure 7. Endogenous Lipoprotein Pathway

 

Formation of VLDL (50,63,64) 

 

In the liver triglycerides and cholesterol esters are transferred in the endoplasmic reticulum to newly synthesized Apo B-100. Similar to the intestine this transfer is mediated by MTTP. The availability of triglycerides is the primary determinant of the rate of VLDL synthesis. If the supply of triglyceride is limited the newly synthesized Apo B is rapidly degraded. Thus, in contrast to many proteins the rate of synthesis of the Apo B-100 is not the major determinant of the rate of secretion. Rather the amount of lipid available determines whether Apo B-100 is degraded or secreted. MTTP is required for the early addition of lipid to Apo B-100 particles but additional lipid is added via pathways that do not require MTTP. Additionally, the size of the VLDL particles is determined by the availability of triglycerides. When triglycerides are abundant the VLDL particles are large.

 

The quantity of fatty acids available for the synthesis of triglycerides is the main determinant of   triglyceride synthesis in the liver. The major sources of fatty acids are a) de novo fatty acid synthesis, b) the hepatic uptake of triglyceride rich lipoproteins, and c) the flux of fatty acids from adipose tissue to the liver. Diabetes, obesity, and the metabolic syndrome are common causes of an increase in hepatic triglyceride levels and the increased secretion of VLDL (44,65).

 

Loss of function mutations in either Apo B-100 or MTTP result in the failure to produce VLDL and marked decreases in plasma triglyceride and cholesterol levels (familial hypobetalipoproteinemia or abetalipoproteinemia) (56). The precise pathway by which the newly synthesized VLDL particles are secreted from the hepatocyte into the circulation is not resolved.

 

VLDL Metabolism (6,58)

 

VLDL particles are transported to peripheral tissues where the triglycerides are hydrolyzed by LPL and fatty acids are released. This process is very similar to that described above for chylomicrons and there is competition between the metabolism of chylomicrons and VLDL. High levels of chylomicrons can inhibit the clearance of VLDL. The removal of triglycerides from VLDL results in the formation of VLDL remnants (Intermediate density lipoproteins (IDL)). These IDL particles are relatively enriched in cholesterol esters and acquire Apo E from HDL particles. In a pathway analogous to the removal of chylomicron remnants these IDL particles can be removed from the circulation by the liver via binding of Apo E to LDL and LRP receptors. However, while the vast majority of chylomicron remnants are rapidly cleared from the circulation by the liver, only a fraction of IDL particles are cleared (approximately 50% but varies). The remaining triglycerides in the IDL particles are hydrolyzed by hepatic lipase leading to a further decrease in triglyceride content and the exchangeable apolipoproteins are transferred from the IDL particles to other lipoproteins leading to the formation of LDL. These LDL particles predominantly contain cholesterol esters and Apo B-100.  Thus, LDL is a product of VLDL metabolism.

 

LDL Metabolism (34,66-69)

 

The levels of plasma LDL are determined by the rate of LDL production and the rate of LDL clearance, both of which are regulated by the number of LDL receptors in the liver. The production rate of LDL from VLDL is partially determined by hepatic LDL receptor activity with a high LDL receptor activity resulting in a decrease in LDL production due to an increase in IDL uptake. Conversely, low LDL receptor activity results in an increase in LDL production formation due to a decrease in IDL uptake. With regards to LDL clearance, approximately 70% of circulating LDL is cleared via hepatocyte LDL receptor mediated endocytosis with the remainder taken up by extrahepatic tissues. An increase in the number of hepatic LDL receptors therefore increases LDL clearance leading to a decrease in plasma LDL levels. Conversely, a decrease in hepatic LDL receptors slows LDL clearance leading to an increase in plasma LDL levels. Thus, the level of hepatic LDL receptors plays a key role in regulating plasma LDL levels. Many of the drugs used to lower plasma LDL levels, such as the statins, ezetimibe, PCSK9 inhibitors, bile acid sequestrants and bempedoic acid lower plasma LDL levels by increasing the number of hepatic LDL receptors (57).

 

The levels of LDL receptors in the liver are mainly regulated by the cholesterol content of the hepatocyte. As cholesterol levels in the cell decrease, inactive sterol regulatory element binding proteins (SREBPs), which are transcription factors that mediate the expression of LDL receptors and key genes involved in cholesterol and fatty acid metabolism, are transported from the endoplasmic reticulum to the Golgi where proteases cleave the SREBPs into active transcription factors (Figure 4). These active SREBPs move to the nucleus where they stimulate the transcription of the LDL receptor and enzymes required for cholesterol synthesis, including HMG-CoA reductase, the rate limiting enzyme in cholesterol synthesis. If cholesterol levels in the cell are high, then the SREBPs remain in the endoplasmic reticulum in an inactive form and do not stimulate LDL receptor synthesis. In addition, cholesterol in the cell is oxidized and oxidized sterols activate LXR, a nuclear hormone receptor that is a transcription factor, which stimulates the transcription of E3 ubiquitin ligase that mediates the ubiquitination and degradation of the low-density lipoprotein receptor (Inducible degrader of the low-density lipoprotein receptor (IDOL)). Thus, the cell can sense the availability of cholesterol and regulate LDL receptor activity. If the cholesterol content of the cell is decreased LDL receptor activity is increased to allow for the increased uptake of cholesterol. Conversely, if the cholesterol content of the cell is increased LDL receptor activity is decreased and the uptake of LDL by the cell is diminished. Statins, ezetimibe, bile acid sequestrants, and bempedoic acid decrease hepatic cholesterol levels thereby increasing LDL receptor levels and decreasing plasma LDL levels (57). Finally, the LDL receptor is targeted for degradation by PCSK9, a secreted protein that binds to the LDL receptor and enhances LDL receptor degradation in the lysosomes. Loss of function mutations in PCSK9 and drugs that inhibit PCSK9 result in increased LDL receptor activity and decreased LDL levels while gain of function mutations in PCSK9 lead to decreased LDL receptor activity and elevations in LDL levels.

 

Thus, the endogenous lipoprotein pathway facilitates the movement of triglycerides synthesized in the liver to muscle and adipose tissue. Additionally, it also provides a pathway for the transport of cholesterol from the liver to peripheral tissues.

 

HDL METABOLISM AND REVERSE CHOLESTEROL TRANSPORT (38-40,47,48,70,71)

 

Figure 8. HDL Metabolism

 

HDL Formation

 

Several steps are required to generate mature HDL particles. The first step involves the synthesis of the main structural protein contained in HDL, Apo A-I. Apo A-I is synthesized predominantly by the liver and intestine. After Apo A-I is secreted, it acquires cholesterol and phospholipids that are effluxed from hepatocytes and enterocytes. The efflux of cholesterol and phospholipids to the newly synthesized lipid poor Apo A-I (pre-beta HDL) is facilitated by ABCA1. Patients with loss of function mutations in ABCA1 (Tangiers disease) fail to lipidate the newly secreted Apo A-I leading to the rapid catabolism of Apo A-I and very low HDL levels (72). Using mice with targeted knock-out of ABCA1 it has been shown that HDL cholesterol levels are reduced by 80% in mice lacking ABCA1 in the liver and 30% in mice lacking ABCA1 in the intestine. While initially cholesterol and phospholipids are obtained from the liver and intestine, HDL also acquires lipid from other tissues and from other lipoproteins. Muscle cells, adipocytes, and other tissues express ABCA1 and ABCG1 and are able to transfer cholesterol and phospholipids to Apo A-I particles. Additionally, as noted above, newly formed HDL can also obtain cholesterol and phospholipids from chylomicrons and VLDL during their lipolysis by LPL. This accounts for the observation that patients with high plasma triglyceride levels due to decreased clearance frequently have low HDL cholesterol levels. Additionally, phospholipid transfer protein (PLTP) facilitates the movement of phospholipids between lipoproteins; mice lacking PLTP have a marked reduction in HDL cholesterol and Apo A-I levels. Finally, the lipolysis of triglyceride rich lipoproteins also results in the transfer of apolipoproteins from these particles to HDL.

 

HDL Cholesterol Esterification

 

As noted earlier the cholesterol in the core of HDL is esterified (cholesterol esters). The cholesterol that is effluxed from cells to HDL is free cholesterol and is localized on the surface of HDL particles. In order to form mature large spherical HDL particles with a core of cholesterol esters the free cholesterol transferred from cells to the surface of HDL particles must be esterified. LCAT, an HDL associated enzyme catalyzes the transfer of a fatty acid from phospholipids to free cholesterol resulting in the formation of cholesterol esters. The cholesterol ester formed is then able to move from the surface of the HDL particle to the core allowing additional free cholesterol to be transferred from cells to HDL particles. Apo A-I is an activator of LCAT and facilitates this esterification process. LCAT activity is required for the formation of large HDL particles. LCAT deficiency in humans results in decreased HDL cholesterol and Apo A-I levels and a higher percentage of small HDL particles (72).

 

HDL Metabolism

 

Lipases and transfer proteins play an important role in determining the size and composition of HDL particles. The cholesterol ester carried in the core of HDL particles may be transferred to Apo B containing particles in exchange for triglyceride. This transfer is mediated by CETP and results in HDL enriched in triglycerides which may then be metabolized by lipases. Humans deficient in CETP activity have very high HDL cholesterol levels and large HDL particles (72). CETP also impacts LDL cholesterol levels and the absence of CETP results in a decrease in LDL cholesterol. Mice do not have CETP and have relatively high HDL cholesterol levels and low LDL cholesterol levels. Hepatic lipase hydrolyzes both triglycerides and phospholipids in HDL. The triglycerides that are transferred to HDL by CETP activity are catabolized by hepatic lipase resulting in the formation of small HDL particles and Apo A-I more easily disassociates from small HDL resulting in the release of Apo A-I and increased Apo A-I degradation. Genetic deficiency of hepatic lipase results in a modest elevation in HDL cholesterol levels and larger HDL particles (72). Hepatic lipase activity is increased in insulin resistant states and this is associated with reduced HDL cholesterol levels. Endothelial cell lipase is a phospholipase that hydrolyzes the phospholipids carried in HDL particles. In mice increased endothelial lipase activity results in decreased HDL cholesterol levels while decreased endothelial lipase activity increases HDL cholesterol levels.

 

The cholesterol carried on HDL is primarily delivered to the liver. The uptake of HDL cholesterol by the liver is mediated by SR-BI, which promotes the selective uptake of HDL cholesterol. The HDL particle binds to SR-BI and the cholesterol in HDL is transported into the liver without internalization of the HDL particle. A smaller cholesterol depleted HDL particle is formed, which is then released back into the circulation. In SR-BI deficient mice there is a marked increase in HDL cholesterol levels. Interestingly the risk of atherosclerosis is increased in these SR-BI deficient mice despite an increase in HDL cholesterol levels. Notably, while HDL cholesterol levels are increased in SR-B1 deficient mice the reverse cholesterol transport pathway is actually reduced. While in mice the physiological importance of the hepatic SR-BI pathway is clear, the role in humans is uncertain. In mice, the movement of cholesterol from peripheral tissues to the liver is dependent solely on SR-BI while in humans CETP can facilitate the transport of cholesterol from HDL to Apo B containing lipoproteins, which serves as an alternative pathway for the transport cholesterol to the liver. 

 

Apo A-I is metabolized independently of HDL cholesterol. Most of the Apo A-I is catabolized by the kidneys with the remainder catabolized by the liver. Lipid free or lipid poor Apo A-I is filtered by the kidneys and then taken up by the renal tubules. The size of the Apo A-I particle determines whether it can be filtered by the kidneys and hence the degree of lipidation of Apo A-I determines the rate of catabolism. Conditions or disease states (for example Tangiers disease, which is due to a mutation in ABCA1, or LCAT deficiency) that result in lipid poor HDL led to the accelerated catabolism of Apo A-I by the kidney. Apo A-I binds to cubilin, which in conjunction with megalin, a member of the LDL receptor gene family, leads to the uptake and degradation of filtered Apo A-I by renal tubular cells. While the liver is also involved in the catabolism of Apo A-I, the mechanisms are poorly understood. HDL particles may contain Apo E and it is therefore possible that Apo E containing HDL particles are taken up via the LDL receptor and other Apo E receptors in the liver and degraded.

 

Reverse Cholesterol Transport (73-78)

 

Peripheral cells accumulate cholesterol through the uptake of circulating lipoproteins and de novo cholesterol synthesis. Most cells do not have a mechanism for catabolizing cholesterol. Cells that synthesize steroid hormones can convert cholesterol to glucocorticoids, estrogen, testosterone, etc. Intestinal cells, sebocytes, and keratinocytes can secrete cholesterol into the intestinal lumen or onto the skin surface thereby eliminating cholesterol. However, in order for most cells to decrease their cholesterol content reverse cholesterol transport is required. From a clinical point of view, the ability of macrophages in the arterial wall to efficiently efflux cholesterol into the reverse cholesterol transport pathway may play an important role in the prevention of atherosclerosis.

 

As noted earlier ABCA1 plays an important role in the efflux of cholesterol to lipid poor pre-beta Apo A-I particles (Figure 9). ABCG1 plays an important role in the efflux of cholesterol from cells to mature HDL particles. In some studies, SR-B1 also plays a role in the efflux of cholesterol to mature HDL particles. Additionally, passive diffusion of cholesterol from the plasma membrane to HDL may also contribute to cholesterol efflux. The levels of both ABCA1 and ABCG1 are increased by LXR activation. LXR is a nuclear hormone transcription factor that is activated by oxysterols. As the cholesterol levels in a cell increase the formation of oxysterols increases leading to the activation of LXR resulting in an increase in ABCA1 and ABCG1 expression, which will result in the enhanced efflux of cholesterol from the cell to HDL.  Additionally, ABCA1 and ABCG1 mRNAs are targeted for degradation by miR-33, a microRNA that is embedded within the SREBP2 gene. An increase in cellular cholesterol decreases the expression of SREBP2 leading to a decrease in miR-33 resulting in enhanced LXR expression. Thus, the decrease in SREBP2 transcription will lead to a decrease in LDL receptor activity and a reduction in cholesterol uptake, while simultaneously, a decrease in miR-33 will lead to an increase in LXR activity stimulating the expression of ABCA1 and ABCG1 resulting in increased cholesterol efflux. Conversely a decrease in cellular cholesterol levels will increase SREBP2 expression resulting in an increase in LDL receptor activity and an increase in miR-33, which will result in a decrease in LXR activity, decreased expression of ABCA1 and ABCG1, and a reduction in cholesterol efflux. Together changes in cholesterol uptake mediated by the LDL receptor and cholesterol efflux mediated by ABCA1 and ABCG1 will maintain cellular cholesterol homeostasis.

 

Figure 9. Cholesterol Efflux from Macrophages (modified from J. Clinical Investigation 116: 3090, 2006)

 

Once cholesterol is transferred from cells to HDL there are two pathways for the cholesterol to be transported and taken up by the liver. As discussed earlier, HDL can interact with hepatic SR-BI receptors resulting in the selective uptake of cholesterol from HDL particles. Alternatively, CETP can transfer cholesterol from HDL particles to Apo B containing particles with the subsequent uptake of Apo B containing lipoproteins by the liver. After the delivery of cholesterol to the liver there are several pathways by which the cholesterol can be eliminated. Cholesterol can be converted to bile acids and secreted in the bile. Alternatively, cholesterol can be directly secreted into the bile. ABCG5 and ABCG8 promote the transport of cholesterol into the bile and the expression of these genes is enhanced by LXR activation. Thus, an increase in hepatic cholesterol levels leading to increased oxysterol production will activate LXR resulting in the increased expression of ABCG5 and ABCG8 facilitating the secretion of cholesterol in the bile.

 

Evidence suggests that reverse cholesterol transport plays an important role in protecting from the development of atherosclerosis. It should be noted that HDL cholesterol levels may not be indicative of the rate of reverse cholesterol transport. As described above reverse cholesterol transport involves several steps and the level of HDL cholesterol may not accurately reflect these steps. For example, studies have shown that the ability of HDL to promote cholesterol efflux from macrophages can vary. Thus, the same level of HDL cholesterol may not have equivalent abilities to mediate the initial step of reverse cholesterol transport.  

 

LIPOPROTEIN (a) (14-16,79)

 

Figure 10. Lp (a)

 

Lp (a) consists of an LDL molecule and a unique apolipoprotein (a), which is attached to the Apo B-100 of the LDL via a single disulfide bound. Lp (a) contain Apo (a) and Apo B-100 in a 1:1 molar ratio. Like Apo B-100, apo (a) is also made by hepatocytes. Apo (a) contains multiple kringle motifs that are similar to the kringle repeats in plasminogen. The number of kringle repeats can vary and thus the molecular weight of apo (a) can range from 250,000 to 800,000.  The levels of Lp (a) in plasma can vary more than a 1000-fold ranging from undetectable to greater than 100mg/dl. Lp (a) levels largely reflect Lp (a) production rates, which are primarily genetically regulated and not greatly affected by environmental factors. Individuals with high molecular weight Apo (a) proteins tend to have lower levels of Lp (a) while individuals with low molecular weight Apo (a) tend to have higher levels. It is hypothesized that the liver is less efficient in secreting high molecular weight Apo (a). The mechanism of Lp (a) clearance is uncertain but does not appear to primarily involve LDL receptors. Therapies that accelerate LDL clearance and lower LDL levels do not lower Lp (a) levels (for example statin therapy). The kidney appears to play an important role in Lp (a) clearance as kidney disease is associated with delayed clearance and elevations in Lp (a) levels.

 

 Elevated plasma Lp(a) levels are associated with an increased risk of atherosclerosis. Apo (a) is an inhibitor of fibrinolysis and enhances the uptake of lipoproteins by macrophages, both of which could account for the increased the risk of atherosclerosis in individuals with elevated Apo (a) levels. Additionally, Lp (a) is the major lipoprotein carrier of oxidized phospholipids, which are inflammatory and could also increase the risk of atherosclerosis. The physiologic function of Apo (a) is unknown. Apo (a) is found in primates but not in other species.

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education.

 

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Monogenic Disorders Causing Hypobetalipoproteinemia

ABSTRACT

 

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia, abetalipoproteinemia, chylomicron retention disease, loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familiar Combined Hypolipidemia). This chapter describes the etiology, pathogenesis, clinical and laboratory findings, and the treatment of these rare monogenic disorders.

 

INTRODUCTION

 

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia (FHBL), abetalipoproteinemia (ABL), chylomicron retention disease (CMRD), loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familial Combined Hypolipidemia, FCH) (1). Increased understanding of the genetic and the molecular underpinnings of these disorders has allowed a focused prioritization of therapeutic targets for drug development. Table 1 summarizes genetic, lipid, and clinical features of the major hypobetalipoproteinemia syndromes and table 2 provides a new classification of these disorders. Of note the parental lipid profile is normal in abetalipoproteinemia and chylomicron retention disease.

 

It should be recognized that secondary, non-familial, forms of hypobetalipoproteinemia occur and include strict vegan diet, malnutrition, malabsorption, hyperthyroidism, malignancy, and chronic liver disease. In addition, hypobetalipoproteinemia can also be due to polymorphisms in multiple genes that together result in hypobetalipoproteinemia (polygenic etiology) (2-4). In a study of 111 patients with LDL-C levels below the fifth percentile 36% had monogenic hypobetalipoproteinemia, 34% had polygenic hypobetalipoproteinemia, and 30% had hypobetalipoproteinemia from an unknown cause (2). In a study of women with an LDL-C ≤1st percentile (≤50 mg/dL) 15.7% carried mutations causing monogenic hypocholesterolemia and 49.6% were genetically predisposed to a low LDL-C on the basis of an extremely low weighted polygenetic risk score (4). Of note individuals with monogenic hypobetalipoproteinemia are more likely to have liver steatosis than individuals without a monogenic disorder (2).

 

Table 1. Characteristics of the Hypobetalipoproteinemia Syndromes

 

Inheritance

Effected gene

Prevalence

Lipids

Clinical features

FHBL

ACD

Truncation mutations in Apo B

1:1000 – 1:3000

Apo B <5th percentile,

LDL-C 20- 50 mg/dL

Hepatic steatosis

Mild elevation of transaminases. Lower prevalence of ASCVD

ABL

 

FHBL

AR

 

AR

MTTP

 

Apo B

<1:1,000,000

Triglycerides < 30 mg/dL,

Cholesterol < 30 mg/dL),

LDL and Apo B undetectable

Hepatic steatosis

Malabsorption, steatorrhea, diarrhea, and failure to thrive.

Deficiency of fat-soluble vitamins.

PCSK9

ACD

Loss of function mutations in PCSK9

 

Heterozygous – mild to moderate reduction in LDL-C

Homozygous – LDL-C ~15 mg/dL

Normal health; significantly lower prevalence of ASCVD

FCH

ACD

Loss of function mutations in ANGPTL3

Very rare

Panhypolipidemia

Normal health; significantly lower prevalence of ASCVD

CMRD

AR

SAR1B

Very rare

LDL-C and HDL-C -decreased by 50%,

Triglycerides - normal

hypocholesterolemia associated with failure to thrive, diarrhea, steatorrhea, and abdominal distension

ACD- autosomal co-dominant; AR- autosomal recessive; FHBL- familial hypobetalipoproteinemia; ABL- abetalipoproteinemia; FCH- Familiar Combined Hypolipidemia; CMRD- chylomicron retention disease, MTTP- microsomal triglyceride transfer protein; ANGPTL3- angiopoietin-like protein 3; ASCVD- atherosclerotic cardiovascular disease.

 

Table 2. Classification of Disorders Causing Familial Hypocholesterolemia

New Name

Common Name

Gene Defect

Class I: Familial hypobetalipoproteinemia due to lipoprotein assembly and secretion defects

FHBL-SD1

Abetalipoproteinemia

Microsomal Triglyceride Transfer Protein

FHBL-SD2

Familial Hypobetalipoproteinemia

Apolipoprotein B

FHBL-SD3

Chylomicron retention disease

SAR1B

Class II: Familial hypobetalipoproteinemia due to enhanced lipoprotein catabolism

FHBL-EC1

Familial Combined Hypolipidemia

ANGPTL3

FHBL-EC2

 

PCSK9

Modified from (5).

 

FAMILIAL HYPOBETALIPOPROTEINEMIA  

 

Familial Hypobetalipoproteinemia (FHBL) is a relatively common autosomal semi-dominant disorder most commonly due to truncation mutations in the gene coding for Apo B (1,6-8). The prevalence of heterozygous FHBL is estimated to be 1 in 700 to 3000 (1). Variants that lead to truncated proteins that are 30% in length or shorter have more severe signs and symptoms than those with longer truncated proteins (6,7). The truncated forms of Apo B found in FHBL are generally non-functional (truncation decreases lipidation and secretion) and are catabolized quickly, resulting in markedly reduced levels in the plasma (Apo B <5th percentile and LDL-C typically between 20- 50 mg/dL) (7,8). Although there is one normal allele in heterozygous FHBL, plasma Apo B levels are approximately 25% of normal rather than the predicted 50% (8). These lower-than-expected levels result from a lower secretion rate of VLDL Apo B from the liver, decreased production of LDL Apo B, increased catabolism of VLDL, and extremely low secretion of the truncated Apo B (6-8). Given the reduced substrate (Apo B) for lipid (predominantly triglyceride) loading, fatty liver develops in these patients (6,9). Hepatic steatosis and mild elevation of liver enzymes are common in heterozygous FHBL (6,9). Interestingly, individuals with monogenic hypobetalipoproteinemia had a much greater prevalence of hepatic dysfunction than individuals with polygenic hypobetalipoproteinemia (2). In contrast to non-alcoholic fatty liver disease, FHBL is not associated with hepatic or peripheral insulin resistance (9). This observation, however, does not imply that hepatic steatosis associated with FHBL is benign. There are several reports of steatohepatitis, cirrhosis, and hepatocellular carcinoma in patients with FHBL and it is estimated that 5-10% of individuals with FHBL develop relatively more severe nonalcoholic steatohepatitis (6). Because of the risk of developing liver disease liver function tests should be checked every 1-2 years and a hepatic ultrasound in those with elevated liver transaminases (6). While hepatic fat accumulation is the rule, there is generally sufficient chylomicron production to handle dietary fat. However, oral fat intolerance and intestinal fat malabsorption have been reported (6). On the positive side the decrease in proatherogenic lipoproteins has been associated with a reduced risk of cardiovascular disease (10).

 

Given the association of FHBL and low LDL-C, Apo B has been an attractive target for drug development. Indeed, unraveling the genetic and molecular mechanisms of FHBL provided the motivation to pharmacologically antagonize Apo B synthesis for therapeutic gains. This culminated in the production of mipomersen, a synthetic single strand anti-sense oligonucleotide to Apo B (11,12). Essentially, anti-sense oligonucleotides contain approximately ~20 deoxyribonucleic acid (DNA) base pairs complementary to a unique messenger ribonucleic acid (mRNA) sequence. The hybridization of the anti-sense oligonucleotide to the mRNA of interest leads to its catabolism via RNase H1, with markedly reduced mRNA levels and ultimately reduced target protein levels. In this case, mipomersen binds to Apo B mRNA leading to reduced production of the protein, and mimicking (albeit to a lesser extent) FHBL. Mipomersen is the first anti-sense oligonucleotide approved by the United States Food and Drug Administration (FDA) and was commercialized in 2013 with a limited indication for adjunctive LDL-C lowering in patients with homozygous familial hypercholesterolemia (HoFH) (12). It is an injectable agent administered subcutaneously once a week. In the clinical trials, mipomersen was associated with a reduction of LDL-C by 21% in subjects with HoFH and 33% in subjects with heterozygous familial hypercholesterolemia (HeFH) (12). Interestingly, it was also found to lower Lp(a) by 21- 23% (12). While it is highly efficacious in LDL-C lowering, it has side effects, many of which can be predicted based on the experience with FHBL (e.g., hepatic steatosis, elevated liver enzymes) (12). It is also associated with injection site reactions in a considerable number of subjects (12). In May 2018 sales were discontinued due to safety concerns related to increased liver transaminases and fatty liver.

 

Homozygous hypobetalipoproteinemia (HHBL) is extremely rare (6). These patients are homozygous or compound heterozygous for mutations in the Apo B gene. The clinical manifestations mimic ABL (see below) (6).

 

ABETALIPOPROTEINEMIA  

 

Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder characterized by very low plasma concentrations of triglyceride and cholesterol (under 30 mg/dL) and undetectable levels of LDL and Apo B (1,7,13,14). The incidence of ABL is < 1 in 1,000,000. HDL-C levels are usually normal or modestly reduced. It is due to mutations in the gene that codes for microsomal triglyceride transfer protein (MTTP) (7,13-15). MTTP lipidates nascent Apo B in the endoplasmic reticulum to produce VLDL and chylomicrons in the liver and small intestine, respectively (15,16). Unlipidated Apo B is targeted for proteasomal degradation leading to the absence of Apo B containing lipoproteins in the plasma (and thus markedly reduced levels of LDL-C and triglycerides) (15,16). Similar to FHBL, VLDL production is inhibited (14). The reduced triglyceride export from the liver leads to hepatic steatosis, which rarely may progress to steatohepatitis, fibrosis, and cirrhosis (1,9,13). Additionally, lack of MTTP facilitated lipidation of chylomicrons in the small intestine results in lipid accumulation in enterocytes with associated malabsorption, steatorrhea, and diarrhea (1,7,13). The malabsorption and diarrhea lead to failure to thrive during infancy (1,7,13). A decrease in dietary fat can reduce the gastrointestinal symptoms. Acanthocytosis may encompass 50% of circulating red blood cells (red blood cells with spiked cell membranes, due to thorny projections) due to alterations in the lipid composition and fluidity of red cell membranes (1,13,14). An additional issue of importance related to ABL is deficiency of fat-soluble vitamins (1,13). Early diagnosis of ABL and homozygous hypobetalipoproteinemia is extremely important as vitamin E deficiency culminates in atypical retinitis pigmentosa, spinocerebellar degeneration with ataxia, vitamin K deficiency can lead to a significant bleeding diathesis, vitamin A deficiency can contribute to eye disorders, and vitamin D deficiency can lead to defects in bone formation (1,13). High dose supplementation with fat soluble vitamins early in life can prevent or delay these devastating complications (Table 3) (7,13). Additional treatment measures include a low-fat diet and supplementation with essential fatty acids (Table 3) (7,13).

 

 Table 3. Dietary Recommendations for Abetalipoproteinemia

Fat calories

Less than 10-15% (<15 g/day) of total daily caloric requirement. Increase as tolerated.

Essential fatty acids

Ensure 2-4% daily caloric intake of EFAs (alpha-linolenic acid/linoleic acid)

Medium chain triglycerides

May prevent or treat malnutrition

Vitamin E

100-300 IU/kg/day

Vitamin A

100-400 IU/kg/day

Vitamin D

800-1200 IU/day

Vitamin K

5-35 mg/week

Derived from (1)

 

Given the very low level of atherogenic lipoproteins and lipids associated with ABL, there was interest in inhibiting MTTP therapeutically. Lomitapide is an oral MTP inhibitor that has been developed over the course of many years (12,17). In early trials, it was tested at a relatively high dose and the side effect profile was prohibitive (nausea, flatulence, and diarrhea). The more recent clinical trial program tested lower doses with drug titration in subjects with Homozygote Familial Hypercholesterolemia (HoFH) (12,17). On an intention to treat basis, LDL-C was decreased by 40% and apolipoprotein B by 39% (12). In patients who were actually taking lomitapide, LDL-C levels were reduced by 50% (12). In addition to decreasing LDL-C levels, non-HDL-C levels were decreased by 50%, Lp(a) by 15%, and triglycerides by 45% (12). Lomitapide received the same limited indication as mipomersen for adjunctive treatment of patients with HoFH (12). Besides the gastrointestinal issues already alluded to, its side effect profile includes hepatic steatosis (12). Its long-term safety has not been established.

 

PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 (PCSK9)

 

Proprotein convertase subtilisin/ kexin type 9 (PCSK9) belongs to the proprotein convertase class of serine proteases (18-20). After synthesis, PCSK9 undergoes autocatalytic cleavage. This step is required for secretion, most likely because the prodomain functions as a chaperone and facilitates folding (18,19). PCSK9 is associated with LDL particles and the LDL-receptor (LDLR) (20). In 2003, Abifadel reported the seminal work that mapped PCSK9 as the third locus for autosomal dominant hypercholesterolemia (Familial Hypercholesterolemia- FH) (21). This finding revealed a previously unknown actor involved in cholesterol homeostasis and served to launch a series of investigations into PCSK9 biology. As it turns out, PCSK9 functions as a central regulator of plasma LDL-C concentration (18-20). It binds to the LDLR and targets it for destruction in the lysosome (18-20). Overactivity of PCSK9 results in a decrease in LDLR and an increase in LDL-C levels while decreased activity of PCSK9 results in an increase in LDLR and a decrease in LDL-C.

 

Since the discovery of gain-of-function mutations in PCSK9 as a cause of FH, investigators have also uncovered loss of function mutations of PCSK9. Loss-of-function mutations in PCSK9 are associated with low LDL-C levels and markedly reduced ASCVD (18,19). In African Americans 2.6 percent had nonsense mutations in PCSK9 that resulted in a 28 percent reduction in LDL-C and an 88 percent reduction in the risk of coronary heart disease (22). The hypolipidemia is not associated with liver abnormalities or other disorders. Interestingly, rare individuals homozygous or compound heterozygotes for loss of function mutations in PCSK9 have been reported with extremely low levels of LDL-C (~15 mg/dL), normal health and reproductive capacity, and no evidence of neurologic or cognitive dysfunction (20,23,24). Collectively, these observations served as further motivation to pursue antagonism of PCSK9 as a therapeutic target. Antagonizing PCSK9 would prolong the lifespan of LDLR, leading to significant reductions in plasma LDL-C levels. Two fully human monoclonal antibodies (alirocumab and evolocumab) targeting PCSK9 became commercially available in 2015 and inclisiran, a small interfering RNA that inhibits translation of PCSK9 is also available. Other approaches to inhibit PCSK9 are under investigation.  

 

FAMILIAL COMBINED HYPOLIPIDEMIA   

 

Familial combined hypolipidemia (FCH) is due to loss of function mutations in the gene encoding angiopoietin-like protein 3 (ANGPTL3) (25,26). ANGPTL3 inhibits various lipases, such as lipoprotein lipase and endothelial lipase (25,26). Therefore, loss of function mutations in ANGPTL3 relinquishes this inhibition increasing the activity of lipases resulting in more efficient metabolism of VLDL and HDL particles (25,26). In addition, to increasing VLDL clearance the secretion of VLDL is also decreased due to a decrease in free fatty acid flux to the liver (25). LDL clearance is increased but the mechanism remains to be fully elucidated (25). Studies have suggested that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production due to ANGPTL3 inhibition and increased endothelial lipase activity reducing VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation rather than being further metabolized to LDL (27).

 

Clinically, FCH manifests as panhypolipidemia (decreased triglycerides, LDL-C, HDL-C, apo B, and apo A-I) (25,26,28). Interestingly, heterozygotes for certain nonsense mutations in the first exon of ANGPTL3 have moderately reduced LDL-C and triglyceride levels while compound heterozygotes have significant reductions in HDL-C as well (25,26).  Homozygosity or compound heterozygosity for other loss-of-function mutations in exon 1 of ANGPTL3 have no detectable ANGPTL3 in plasma and striking reductions of atherogenic lipoproteins with HDL particles containing only apo A-I and preß-HDL. Individuals who are heterozygous for the loss of function mutations in ANGPTL3 have significantly reduced LDL-C and triglyceride levels and a reduced risk of atherosclerosis (25,26,28).

 

A pooled analysis of cases of familial combined hypolipidemia was published 2013 (29). One hundred fifteen individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were evaluated. Homozygotes and compound heterozygotes (two mutant alleles) had no measurable ANGPTL3 protein. In heterozygotes, ANGPTL3 was reduced by 34-88%, according to genotype. All cases (homozygotes and heterozygotes) demonstrated significantly lower concentrations of all plasma lipoproteins (except for Lp(a)) as compared to controls. Familial combined hypolipidemia is not associated with any comorbidity. In fact, the prevalence of fatty liver was the same as controls. However, ANGPTL3 deficiency is associated with a reduced risk of cardiovascular disease (25,30).

 

Recently, evinacumab, a human monoclonal antibody against ANGPTL3, was approved for the treatment of Homozygous Familial Hypercholesterolemia (12). Evinacumab decreases LDL-C levels by mechanisms independent of LDL receptor activity (12).

 

CHYLOMICRON RETENTION DISEASE

 

Chylomicron retention disease (CMRD), known also as Anderson’s disease for the individual who first described the condition in 1961, is a rare inherited lipid malabsorption syndrome (31,32). It is due to mutations in the SAR1B gene which codes for the protein SAR1b, a small GTPase, involved in intracellular protein trafficking (31). Mutations in SAR1b result in the failure of pre-chylomicrons to move from the endoplasmic reticulum to the golgi (31). This disorder usually presents in young infants with diarrhea, steatorrhea, abdominal distention, and failure to thrive, which can improve with a low-fat diet (1,31,32). Patients with CMRD demonstrate a specific autosomal recessive hypocholesterolemia that differs from other familial hypocholesterolemias. CMRD is associated with a 50% reduction in both plasma LDL-C and HDL-C with normal fasting triglyceride levels (31,32). Mutations in SAR1B do not affect VLDL secretion by the liver. The decrease in HDL-C is postulated to be due to a decrease in Apo A-I secretion and cholesterol efflux by the small intestine (31). The mechanism accounting for the decrease in LDL-C is not clear. The usual increase in triglycerides and chylomicron levels following a fat meal is blocked (31). The duodenal mucosa is white on endoscopy and intestinal biopsy reveals cytosolic lipid droplets and lipoprotein-sized particles in enterocytes (31). As one would expect the absorption of fat-soluble vitamins (A, D, K, and E) and essential fatty acids is impaired (31,32). Neurological and eye manifestations are milder and occur at an older age compared to abetalipoproteinemia (1). Red blood cell acanthosis is rare (1). Heterozygotes with mutations in SAR1B are unaffected.

 

Treatment for individuals with CMRD is similar to that described above for individuals with ABL (32).

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education.

 

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Hypertriglyceridemia: Pathophysiology, Role of Genetics, Consequences, and Treatment

ABSTRACT

 

Hypertriglyceridemia (HTG) can result from a variety of causes. Mild to moderate HTG tracks along with the metabolic syndrome, obesity and diabetes. HTG can be the result of multiple small gene variants or secondary to several diseases and drugs. Severe HTG with plasma triglyceride (TG) levels >1000-1500 mg/dL typically results from: (1) rare variants in the lipoprotein lipase (LPL) complex, where it is termed the familial chylomicronemia syndrome (FCS), and (2) the co-existence of genetic and secondary forms of HTG, termed the multifactorial chylomicronemia syndrome (MFCS), which is a much more common cause of severe HTG.  Mild to moderate HTG is associated with an increased risk of premature cardiovascular disease (CVD), while severe HTG can lead to pancreatitis as well as an increased risk of premature CVD. Appropriate management of the patient with HTG requires knowledge of the likely cause of the HTG, to prevent itscomplications.

 

PHYSIOLOGY

 

A detailed overview of lipoprotein physiology is provided in the Endotext chapter on Lipoprotein Metabolism (1).  Here we will briefly review some aspects the metabolism of the triglyceride (TG)-rich lipoproteins, very low-density lipoproteins (VLDL) and chylomicrons (CM) of particular relevance to this chapter.

 

Secretion of TG-rich Lipoproteins Into Plasma

 

TGs are transported through plasma as VLDL), which transport TGs primarily made in the liver, and as CM, which transport dietary (exogenous) fat.  VLDL secretion by the liver is regulated in several ways.  Each VLDL particle has one ApoB100 molecule, making ApoB100 availability a key determinant of the number of VLDL particles, and hence, TG secretion by the liver.  In addition to one molecule of ApoB-100, each VLDL particle contains multiple copies of other apolipoproteins, together with varied amounts of TGs, cholesteryl esters, and phospholipids.  The extent of TG synthesis is in part determined by the flux of free fatty acids (FFA) to the liver.  The addition of TG to the developing VLDL particle in the endoplasmic reticulum is mediated by the enzyme microsomal triglyceride transfer protein (MTTP).  The pool of ApoB100 in the liver is not typically regulated by its level of synthesis, which is relatively constant, but by its level of degradation, which can occur in several proteolytic pathways (2). Insulin also plays a role in the regulation of VLDL secretion -  it decreases hepatic VLDL production by limiting fatty acid influx into the liver and decreases the stability of, and promotes the posttranslational degradation of ApoB100 (3).  Recent studies have shown that ApoC-III, an Apolipoprotein thought to primarily play a role in inhibiting TG removal (see below), also is involved in the assembly and secretion of VLDL (4).  VLDL particles (containing ApoB100) also increase in plasma in the postprandial state as well as CM that contain ApoB48 (5).

 

Consumption of dietary fat results in the formation of CM by enterocytes.  Fatty acids and monoacylglycerols that result from digestion of dietary TGs by acid and pancreatic lipases are transported into enterocytes by mechanisms that are not completely understood.  In the enterocyte, monoacylglycerol and fatty acids are resynthesized into TGs by the action of the enzymes acyl-coenzyme A: monoacylglycerol acyltransferase and acyl-coenzyme A: diacylglycerol acyltransferase 1 and 2 (DGAT 1 and 2).  The resulting TGs are packaged with ApoB48 to form CM, a process also mediated by MTTP (6).   CM then pass into the thoracic duct from where they enter plasma and acquire additional apolipoproteins.  Of particular relevance to their clearance from plasma is the acquisition of ApoC-II and ApoC-III. 

 

Catabolism of the TG-rich Lipoproteins

 

TGs in both VLDL and CM are hydrolyzed by the lipoprotein lipase (LPL) complex.  LPL is synthesized by several tissues, including adipose tissue, skeletal muscle, and cardiac myocytes.  After secretion by adipocytes, the enzyme is transported by glycosylphosphatidylinositol-anchored high-density lipoprotein–binding protein 1 (GPIHBP1) to the luminal side of the capillary endothelium, where it becomes tethered to glycosaminoglycans (GAGs). This pool of LPL is referred to as “functional LPL”, since it is available to hydrolyze TGs in both VLDL and chylomicrons. LPL can be liberated from these GAG binding sites by heparin injection. Several other proteins, reviewed in (7), regulate LPL activity. These include ApoC-II, which activates LPL, and ApoC-III, which inhibits LPL in addition to its effect on VLDL secretion alluded to earlier. Both are produced by the liver and are present on TG-rich lipoproteins.  ApoC-III also inhibits the turnover of TG-rich lipoproteins through a hepatic clearance mechanism involving the LDL receptor/LDL receptor-related protein 1 (LDLR/LRP1) axis (8).  ApoE also is present on the TG-rich lipoproteins and plays an important role in the uptake and clearance of the remnants of the TG-rich lipoproteins that result from hydrolysis of TGs in these lipoproteins. Other activators of LPL include ApoA-IV (9), ApoA-V (10-12) and lipase maturation factor 1 (LMF1) (13, 14). In addition, several members of the angiopoietin-like (ANGPTL) protein family play a role in regulating LPL activity.  ANGPTL3 is produced by the liver and is an endocrine regulator by inhibiting LPL in peripheral tissues (7, 15, 16).  ANGPTL4 is produced in several tissues (7), where it inhibits LPL in a paracrine fashion (7, 17). Both ANPGTL3 and ANGPTL4 delay the clearance of the TG-rich lipoproteins (7).

 

The core TGs in VLDL and chylomicrons are hydrolyzed by ApoC-II activated LPL; FFA thus formed are taken up by adipocytes and re-incorporated into TGs for storage, or in skeletal and cardiac muscle, utilized for energy. Hydrolysis of chylomicron- and VLDL-TG results in TG-poor, cholesteryl ester and ApoE-enriched particles called chylomicron and VLDL remnants, respectively, which under physiological conditions are removed by the liver by binding to LDL receptors, LDL receptor related protein, and cell surface proteoglycans (12, 18). Hepatic TG lipase and ApoA-V also are involved in the remnant clearance process (10-12, 19, 20).

 

The clearance of TGs from plasma is saturable when plasma TGs exceed ~500-700 mg/dL (21).  When removal mechanisms are saturated, additional chylomicrons and VLDL entering plasma cannot readily be removed and hence accumulate in the plasma. As a result, plasma TGs can increase dramatically, resulting in very high levels and the accumulation of chylomicrons in plasma obtained after an overnight fast. 

 

NORMAL RANGE FOR PLASMA TRIGLYCERIDES AND DEFINITION OF HYPERTRIGLYCERIDEMIA

 

Plasma TG levels reflect the TG content of multiple lipoprotein particles, primarily chylomicrons and VLDL. Fasting TG levels less than 150 mg/dL has been generally accepted as “normal” (22, 23). A non-fasting TG of 175mg/dL represents ~75th percentile of the normal range, and levels of ~400mg/dL represent the 97th percentile (24). Plasma TGs are heavily skewed to the right in the general population with a tail towards highest levels and vary depending upon the population mix (25). The full range of TG extends from 30mg/dL to 10,000mg/dL (22). TG levels are different between sexes, being higher in males than in females, and increase with age and development of other coexisting conditions such as central adiposity, metabolic syndrome, and diabetes (24). TG levels also vary between geographic areas, among people of different ethnic backgrounds, with higher levels observed in certain populations such as Mexicans and South Asians. Lower TG levels have been observed in people of African descent and African-Americans but this may be changing due to adoption of urban lifestyles (26). Because of this skewed distribution, logarithmic transformation is required to establish statistical normal ranges of TG levels.  There is no current widely accepted definition of elevated non-fasting TG levels, but some groups have utilized 175mg/dL as a cut point (27, 28). Due to high variability of TG levels, precise definitions for non-fasting levels are difficult to establish. It is worthwhile noting that post-prandial TG levels rarely exceed 400mg/dL even after a high fat challenge.

 

Normal Range Based on Risk of Complications of Hypertriglyceridemia

 

The major complications of hypertriglyceridemia (HTG) are (1) acute pancreatitis and (2) increased risk of atherosclerotic cardiovascular disease (ASCVD). These two complications occur at different levels of TGs, the risk of pancreatitis occurring at much higher TG levels than the risk of premature ASCVD and are discussed in detail later in this chapter. 

 

Normal Range According to Guidelines

 

Despite concerns regarding establishment of an upper limit of normal for TGs, most guidelines define values for HTG, often without a strong biological rationale. Definitions for the diagnosis of HTG provided in several guidelines are shown in Table 1.   

 

Cut points for HTG were first defined by the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP). The terms mild, moderate and severe have been used based on degree of TG elevation (table 1). In general, mild to moderate HTG reflects TG levels under 500mg/dL. Severe hypertriglyceridemia (sHTG) has been arbitrarily defined by different national guidelines as either TG levels ≥500 mg/dL by the American Heart Association (AHA)/American College of Cardiology (ACC), Multispecialty Cholesterol and Canadian Cardiovascular Society Guidelines (29, 30) or TG levels ≥880 mg/dL according to the European Society of Cardiology guidelines (31). The Endocrine Society has used severe HTG for 1000 to 1999 mg/dL and very severe HTG for values >2000 mg/dL (23).  

 

Table 1. Definition of Hypertriglyceridemia According to Various Clinical Guidelines

Guideline

Classification

Triglyceride Levels

NCEP/ ATP III (32)

American Heart Association (33)

National Lipid Association (34)

Normal

Borderline-high TGs

High TGs

Very high TGs

<150 mg/dL (< 1.7 mmol/L)

150-199 mg/dL (1.7-2.3 mmol/L)

200-499 mg/dL (2.3-5.6 mmol/L)

≥500 mg/dL (≥5.6 mmol/L)

The Endocrine Society (35)

Normal

Mild HTG

Moderate HTG

Severe HTG

Very severe HTG

<150 mg/dL (< 1.7 mmol/L)

150-199 mg/dL (1.7-2.3 mmol/L)

200-999 mg/dL (2.3-11.2 mmol/L)

1000-1999 mg/dL (11.2-22.4 mmol/L)

≥2000 mg/dL (≥22.4 mmol/L)

European Society of Cardiology/European Atherosclerosis Society (36)

Normal

Mild-moderate HTG

Severe HTG

<1.7 mmol/L (<150mg/dL)

>1.7-< 10mmol/L (150-880 mg/dL)

> 10 mmol/L (> 880mg/dL)

Hegele (22)

Normal

Mild to moderate

Severe

<2.0 mmol/L (<175 mg/dL)

2.0-10 mmol/L (175- 885 mg/dL)

>10 mmol/dL (>885 mg/dL)

 

In summary, establishing a precise definition of what constitutes abnormal TG values is fraught with difficulty.  An acceptable level for the prevention of pancreatitis is likely to be quite different from that at which CVD risk might be increased. The impact of HTG on CVD risk needs to be evaluated in the context of the family history of premature CVD, associated abnormalities of lipids and lipoproteins, and other CVD risk factors, particularly those associated with the metabolic syndrome (see below).

 

CAUSES AND CLASSIFICATION OF HYPERTRIGLYCERIDEMIA

 

In general, HTG has been classified as primary, when a genetic or familial basis is suspected, or secondary, where other conditions that raise TG levels can be identified. However, this classification is likely overly simplistic. It has become clear in the past decade that the spectrum of plasma TG levels, ranging from mild elevation to very severe HTG, is modulated by a multitude of genes working in concert with non-genetic secondary and environmental contributors. Thus, in the vast majority of individuals, mutations in multiple genes with interaction from non-genetic factors result in altered TG-rich lipoprotein synthesis and catabolism and subsequent HTG.

 

Historical Perspective

 

Phenotypic heterogeneity among patients with HTG has been historically defined by qualitative and quantitative differences in plasma lipoproteins. In the pre-genomic era, the Fredrickson classification of hyperlipoproteinemia was based on electrophoretic patterns of lipoprotein fractions (37). The phenotypes are distinguished based on the specific class or classes of accumulated TG-rich lipoprotein particles, including chylomicrons, VLDL and VLDL-remnants. This classification included 6 phenotypes, five of which included HTG in their definition (except for Frederickson type 2 A hyperlipoproteinemia, which equates with genetic primary hypercholesterolemia). It has now become apparent that except for type 1 hyperlipoproteinemia (FCS), the HTG phenotypes, particularly Frederickson type 4 and type 5 hyperlipoproteinemia, are due to the accumulation of polygenic traits predisposing to HTG. However, this classification system is dated, has neither improved clinical or scientific insight, and therefore does not find wide use at this time (22).

 

In 1973, Goldstein and colleagues characterized a variable pattern of lipid abnormalities in families of survivors of myocardial infarction that they termed familial combined hyperlipidemia (FCHL) (38).  At the same time, this phenotype of mixed or combined hyperlipidemia was observed in another cohort, where it was called multiple-type familial hyperlipoproteinemia (39).  Affected family members can present with hypercholesterolemia alone, HTG alone, or with elevations in both TGs and LDL. This pattern was estimated to have a population prevalence of 1-2% (40), making it the most common inherited form of dyslipidemia.

 

In the aforementioned study, a pattern of isolated HTG, historically called familial HTG (FHTG) also was described (38). This condition was characterized by increased TG synthesis, with secretion of normal numbers of large TG-enriched VLDL particles (41), elevated VLDL levels, but normal levels of LDL and HDL cholesterol (42).  FHTG did not appear to be associated with an increased risk of premature CVD in an early study (43), but baseline TG levels predicted subsequent CVD mortality after 20 years of follow up among relatives in families classified as having FHTG (44, 45).  

 

FCHL and FHTG were initially believed to be monogenic disorders (38). However, more recent genetic characterization of individuals with familial forms of HTG indicates that these are not disorders associated with variation within a single gene, but rather polymorphisms in multiple genes associated with HTG, as detailed below. Therefore, classification of FCHL and FHTG is potentially misleading. Nevertheless, it is important to note that FCHL as originally described is associated with a very high prevalence of premature CVD (43, 44, 46).   

 

Genetic Forms of Hypertriglyceridemia

 

It is now evident that clinically relevant abnormalities of plasma TG levels appear to require a polygenic foundation of common or rare genetic variants (22).  Common small-effect gene variants confer a background predisposition that interact with rare large-effect heterozygous variants in genes that govern synthesis or catabolism of TG-rich lipoproteins, or nongenetic secondary factors, leading to the expression of a more severe TG phenotype (47). Recently, the most prevalent genetic feature underlying severe HTG was shown to be the polygenic accumulation of common (rather than rare) variants—more specifically, the accumulation of TG-raising alleles across multiple SNP loci (48).

 

Thus, mild to moderate hypertriglyceridemic states are complex, genetically heterogeneous disorders. Mild-to-moderate HTG is typically polygenic and results from the cumulative burden of common and rare variants in more than 30 genes, as quantified by genetic risk scores. All genetic forms can be exacerbated by non-genetic factors. Because they are a consequence of interaction between multiple susceptibility genes and lifestyle factors, individuals with moderate HTG should be considered as a single group without distinction, irrespective of concomitant lipoprotein disturbances (22). Because of the complexity of these disorders, routine genetic testing is not recommended.

 

Pathogenesis of Genetic Forms of Hypertriglyceridemia

           

Genetic forms of HTG without other lipoprotein disturbances (i.e., pure HTG) are characterized by increased TG synthesis, where normal numbers of large TG-enriched VLDL particles are secreted (41, 49-51). Reduced TG clearance also has been observed in some individuals (50-52).  Affected people have elevated VLDL levels, but normal levels of LDL, and are generally asymptomatic unless clinical CVD or severe HTG develops. 

 

A variety of metabolic defects that differ among families are associated with the combined hyperlipidemia phenotype. The characteristic lipoprotein abnormalities are increased ApoB levels and increased number of small dense LDL particles (42), a phenotype similar to that seen in the metabolic syndrome and type 2 diabetes (53). These primary defects occur due to 1) hepatic overproduction of VLDL particles (41) due to increased ApoB synthesis in the setting of disordered adipose metabolism (54, 55), insulin resistance (41, 56-58), and liver fat accumulation, and, 2) impaired clearance of ApoB containing particles (59, 60). Increased VLDL secretion results in an elevated plasma ApoB and HTG (56).  Long residence time of VLDL particles favors the formation of small dense LDL (59). An abundance of small dense LDL particles traditionally is associated with the presence of HTG; however, these LDL characteristics remain even after correction of the HTG by treatment with fibrates (61, 62).

 

In addition to Apo B abnormalities, other lipoprotein disturbances include abnormal expression of ApoA-II, ApoC-III, and PCSK9.  VLDL-TG levels in combined hyperlipidemia are modulated by ApoA-II and ApoC-III (63).  Plasma PCSK9 levels are higher in these patients, and levels correlate with TG and Apo B levels (64).

 

Visceral adiposity appears to be an important determinant of insulin resistance, which occurs commonly in subjects with both isolated HTG (65) and combined hyperlipidemia (65-69). Other abnormalities that have been reported in clinical FCHL include impaired lipolysis due to decreased cyclic AMP dependent signaling (54, 69), abnormal adipocyte TG turnover (70), fatty liver (71), increased arterial stiffness (72), and increased carotid intimal-medial thickness (73). 

 

In all of the phenotypes described above, severe HTG can occur when secondary causes of HTG such as untreated diabetes, marked weight gain, or use of TG-raising drugs are present concurrently, leading to the Multifactorial Chylomicronemia Syndrome (MFCS), described later (74).

 

Secondary Forms of Hypertriglyceridemia

 

These are described in greater detail in the chapters on Secondary Disorders of Lipid and Lipoprotein Metabolism (75-78).  However, in the section where we describe MFCS we will briefly touch on some aspects of secondary forms of HTG, since they assume importance in the pathogenesis of the severe HTG seen in the MFCS, where they often co-exist in individuals with genetic forms of HTG. In our experience, the commonest secondary forms of HTG that interact with genetic forms of HTG are type 2 diabetes (usually as part of the metabolic syndrome), obesity, recent weight gain, excessive alcohol consumption, the use of drugs that can raise TGs, and chronic kidney disease (CKD)(74, 79, 80). (Table 3)

           

Severe Hypertriglyceridemia and the Chylomicronemia Syndrome

 

In the late 1960s Fredrickson, Levy and Lees (37) classified HTG into types dependent on the pattern of lipoproteins on paper electrophoresis and the presence or absence of chylomicrons in fasting plasma. They recognized that acute pancreatitis and eruptive xanthomata occurred in the presence of chylomicronemia that accumulate in what they termed Type I and Type V hyperlipoproteinemia. Chylomicrons are present in the post-prandial state, and usually are present in fasting plasma when TG levels exceed 800 mg/dL, but absent in fasting plasma below that value (81). The term chylomicronemia syndrome was first used to describe a constellation of clinical findings such as abdominal pain, acute pancreatitis, eruptive xanthoma and lipemia retinalis that occurred in association with very high TG levels (82). Two groups of conditions can lead to severe HTG and clinical manifestations of the chylomicronemia syndrome; (1) familial chylomicronemia syndrome (FCS) due to variants in the LPL complex, and (2) multifactorial chylomicronemia syndrome (MFCS), in which genetic predisposition and secondary forms of HTG co-exist.

 

FAMILIAL CHYLOMICRONEMIA SYNDROME (FCS)

 

FCS is a monogenic disorder due to variants in one or more genes of the LPL complex that affect chylomicron catabolism. FCS incidence is very rare, with an estimated prevalence ranging from 1 in 20,000 to 300,000 (83).    

 

Genetics: Biallelic loss of function variants in five canonical genes lead to impaired hydrolysis of TG-rich lipoproteins, with subsequent increases in chylomicron particle numbers and markedly increased TG concentrations. The most common gene affected in FCS is LPL itself, in which patients are homozygous or compound heterozygous for two defective LPL alleles. Over 180 variants that result in LPL deficiency have been described with some clustered mutations due to founder effects (84-87). Loss of function variants account for over 90% of cases (83). Many are missense variants, some in catalytically important sites and some in regions that predispose to instability of the homodimeric structure of LPL required for enzyme activity (88). However, many common LPL gene variants have been described that have no clinical phenotype (89). Variants in the APOC2 gene, encoding ApoC-II, an activator of LPL, is another cause of FCS.  Variants have been described in several families (90, 91). In FCS thus far there is no known gene variant that affects synthesis or production of TG-rich lipoproteins.

 

FCS can also occur from biallelic loss of function variants in other components of the LPL complex, namely APOA5, LMF1, and GPIHBP1 genes (Table 2), each of which plays an important role in determining LPL function (92). The lipoprotein phenotype in these mimics that seen in classical LPL deficiency. Loss of function variants in GPIHBP1, which directs transendothelial LPL transport and helps anchor chylomicrons to the endothelial surface near LPL, thereby providing a platform for lipolysis, has been described in several families (83).  Autoantibodies to GPIHBP1 also can lead to chylomicronemia (93). A small number of individuals with homozygous variants in Apo A-V, which stabilizes  the lipoprotein–enzyme complex thereby enhancing lipolysis (10), have been described (94). Variants in LMF1, an endoplasmic reticulum chaperone protein required for post-translational activation of LPL, have also been identified in a few individuals (95).

 

Clinical presentation: FCS usually manifests in childhood or early adolescence with nausea, vomiting, failure to thrive and recurrent abdominal pain in infancy and childhood. Occasionally it can present in adulthood (87) but this is often due to delayed diagnosis with median age at diagnosis being due to unfamiliarity in most healthcare providers (96). Adults may report “brain fog” or transient confusion.

 

Classical clinical findings include eruptive xanthomas (often seen on buttocks, back, extensor surfaces of upper limbs), lipemia retinalis, and hepatosplenomegaly. Less common symptoms of FCS can include intestinal bleeding, anemia, and neurological features such as irritability and seizures. Patients present with TG levels ≥1000 mg/dL and often much higher, due to abnormal accumulation of chylomicrons, which can be detected by the appearance of lipemic/milky plasma.  Despite prolonged overnight fasting, plasma TG levels are >1000mg/dL due to the presence of chylomicrons in the circulation as a result of impaired clearance. The most serious concern, however, is the development of acute pancreatitis, which can lead to systemic inflammatory response syndrome, multi-organ failure, and death.

 

Table 2. Genetic Disorders Resulting in Familial Chylomicronemia Syndromes (FCS)

Disorder

Inheritance

Incidence

Lipid Phenotype

Underlying Defect

Clinical Features

LPL deficiency

Autosomal Recessive

1 in 1,000,000

Marked HTG/ chylomicronemia in infancy or childhood

Very low or absent LPL activity; circulating inhibitor of LPL

Hepato-splenomegaly; severe chylomicronemia

Apo C-II deficiency

Autosomal Recessive

Rare

Marked HTG/ chylomicronemia in infancy or childhood

Absent Apo C-II

Hepato-splenomegaly; severe chylomicronemia

Apo A-V mutation

Autosomal Recessive

Rare

Marked HTG/ chylomicronemia in adulthood

Defective or absent Apo A-V

Chylomicronemia

GPIHBP1 mutation

Autosomal Recessive

Rare

Marked HTG/ chylomicronemia in adulthood

Defective or absent GPIHBP1

Chylomicronemia

LFM1 mutation

Autosomal Recessive

Rare

Marked HTG/ chylomicronemia in adulthood

Defective or absent LFM1

Chylomicronemia

 Adapted from Ref (83)

 

MULTIFACTORIAL CHYLOMICRONEMIA SYNDROME (MFCS)

 

In contrast to FCS, MFCS is more common and complex. The prevalence of MFCS is much higher than FCS and estimated to be ~1:600-1000 (84).   

 

Genetics: MFCS has a genetic basis, but unlike FCS (where recessive or biallelic variants in the affected genes are causative), the genetic alteration does not always result in the phenotypic expression of the trait but only increases the possibility of the risk of developing the condition. Other factors, including non-genomic effects (epigenetics, methylation), gene-gene, or gene-environment interactions can also contribute. MFCS develops due to two main types of genetic factors that increase the odds that a patient will develop very high TG levels. First, heterozygous rare large-effect variants in one of the five canonical TG metabolism genes (LPL, Apo C-II, Apo A5, LMF-1 and GPIHBP-1) can contribute to TG elevations. These variants have variable penetrance, i.e.- clinical presentation can vary from normal to severe hypertriglyceridemia.

 

Secondly, the presence of a high burden of common small-effect TG-raising SNPs; cumulatively, these common SNP alleles increase susceptibility for developing hypertriglyceridemia. These SNPs may have an indirect impact of the metabolism of TG-rich

Lipoproteins. There is incomplete understanding of how an excess burden of SNPs contributes to TG levels, but their prevalence in patients with severe hypertriglyceridemia has been consistently demonstrated.

 

Several polygenic risk scores (PRS) for TG levels have been published (97). A recent study found that 32.0% of patients had a high polygenic score of TG-raising alleles across 16 loci compared to only 9.5% of normolipidemic controls (25). When the PRS is high, there is a significantly increased risk of developing HTG but this is not diagnostic or definitive.

 

Secondary Causes Contributing to Severe Hypertriglyceridemia in MFCS: The most common secondary cause in the past was undiagnosed or untreated diabetes (74), although earlier detection of diabetes may be making the association of marked hyperglycemia of untreated diabetes with very severe HTG less common. In addition, individuals with the metabolic syndrome and obesity have mild to moderate HTG which can become severe HTG; weight regain following successful weight loss can lead to marked HTG (23, 84). These patients almost always have relatives with genetic forms of HTG, whose TG levels are considerably lower than the index patient with severe HTG, in whom secondary forms of HTG also are present (74).  MFCS can result from the addition of specific drugs in patients with a genetic predisposition (23). These drugs include beta-adrenergic blocking agents (selective and non-selective) and/or diuretics (thiazides and loop-diuretics such as furosemide) used for hypertension, retinoid therapy for acne, oral estrogen therapy for menopause or birth control, selective estrogen receptor modulators (particularly raloxifene) for osteoporosis or breast cancer, protease inhibitors for HIV/AIDS, atypical anti-psychotic drugs, alcohol, and possibly sertraline (84).  Rarer causes of very severe HTG include autoimmune disease (sometimes with LPL- or GPIHBP1- specific antibodies), asparaginase therapy for acute lymphoblastic leukemia (98), (99) and bexarotene, a RXR agonist used in the treatment of cutaneous T cell lymphoma (100).  

 

Table 3. Secondary Causes That Can Contribute to Severe HTG

Conditions

Hypothyroidism

Suboptimally managed or new onset diabetes

Obesity

Sudden weight gain, weight regain after weight loss

Chronic kidney disease

Nephrotic syndrome

Pregnancy

Acute hepatitis

Sepsis

Inflammatory disorders

Cushing syndrome

Autoimmune chylomicronemia

            Systemic lupus erythematosis

            Anti-LPL antibodies

GPIHBP-1 antibodies

Rare Genetic Causes

Glycogen storage disorders

Lipodystrophies

            Congenital- generalized or partial

            Acquired- HIV, autoimmune

Drugs

Alcohol ingestion

Beta blockers

Thiazide diuretics

Oral estrogens

Selective estrogen reuptake modulators - tamoxifen, raloxifene, clomiphene

Androgens

Glucocorticoids

Atypical anti-psychotics

Sertraline

Bile acid resins

Sirolimus, tacrolimus

Cyclosporine

RXR agonists -bexarotene, isotretinoin, acetretin

HIV Protease inhibitors

L- asparaginase

Alpha-interferon

Propofol

Lipid emulsions

 

Following correction of treatable secondary forms of HTG in the MFCS, TG levels usually decrease to the moderately elevated levels seen in their affected relatives (101, 102).  

 

OTHER CONDITIONS RESULTING IN HYPERTRIGLYCERIDEMIA

 

Familial Dysbetalipoproteinemia (FDB or Remnant Removal Disease)

 

Familial dysbetalipoproteinemia, also referred to as remnant removal disease or type III hyperlipoproteinemia, is a rare autosomal recessive disorder that can present with elevated TG levels. This disorder is characterized by the accumulation of remnant lipoproteins.

 

PATHOGENESIS AND GENETICS

 

Remnant removal disease requires homozygosity for the ApoE2 genotype or a rare heterozygosity for a variant in the ApoE gene, which results in pathologic accumulation of remnant lipoproteins in the circulation due to impaired hepatic uptake of ApoE-containing lipoproteins (103). ApoE is a glycoprotein synthesized in the liver, brain and tissue macrophages and present on chylomicrons, VLDL and HDL. Apo E through interaction with the LDLR and heparan sulphate proteoglycans promotes the hepatic clearance of remnants of chylomicrons and VLDL (104); it also facilitates cholesterol efflux from macrophages to HDL (105). In humans, there are 3 common isoforms of ApoE , ApoE2, ApoE3, and ApoE4 (106).  Each differs in isoelectric point by one charge unit, ApoE4 being the most basic isoform and ApoE2 the most acidic.  ApoE3 (Cys112Arg158) is the commonest isoform.  ApoE2 (Arg158Cys) and ApoE4 (Cys112Arg) differ from ApoE3 by single amino acid substitutions at positions 158 and 112, respectively (107).  In the majority of cases (90%), remnant removal disease is associated with the E2/E2 genotype and results from impaired binding to the Apo E receptor. It is  an autosomal recessive disorder with the prevalence of ApoE2 homozygosity in Caucasian populations estimated to be about 1% (108).  Rarer ApoE variants such as ApoE3-Leiden (109) and ApoE2 (Lys1463Gln) that also can cause remnant accumulation are dominantly inherited (110) and account for 10% of cases (111, 112).  Rare APOE variants in the population, other than the APOE2 and APOE4 alleles, play an important role in the development of isolated hypercholesterolemia (113) and mixed hyperlipidemia, with and without familial dysbetalipoproteinemia (114). Thus it is becoming apparent that two different DBL phenotypes may exist- a genetic Apo E dysfunction and a multifactorial form (115). Modern prevalence of FDB is estimated at 1-2% (116).

 

In the absence of additional genetic, hormonal, or environmental factors, remnants do not accumulate to a degree sufficient to cause hyperlipidemia in ApoE2 homozygotes; in fact, lipid levels are commonly low. Remnant accumulation results when the E2/2 genotype is accompanied by a second genetic or acquired defect that causes overproduction of VLDL such as obesity or diabetes (117) (111, 118) , a decrease in remnant clearance, or a reduction in LDL receptor activity (e.g., hypothyroidism (119)). Thus, full phenotypic expression requires the presence of other environmental or genetic factors (120). In these circumstances, the reduced uptake of remnant lipoproteins by the liver results in reduced conversion of VLDL and intermediate density lipoproteins to LDL, with subsequent accumulation of remnant lipoproteins (121, 122), hence the term remnant removal disease.

 

DIAGNOSIS

 

Patients with remnant removal disease have roughly equivalent elevations in plasma cholesterol and TGs. The disease rarely manifests before adulthood, and in some individuals never manifests clinically. It is more common in men than in women, where expression seldom occurs before menopause, since estrogen has a protective effect in women who are ApoE2 homozygotes (108). Palmar xanthomas (Figure 1), orange lipid deposits in the palmar or plantar creases, are pathognomonic of remnant removal disease but are not always present (123). Tuberoeruptive xanthomas can be found at pressure sites on the elbows, knees and buttocks. The presence of remnant removal disease should be suspected when total cholesterol and TG levels range from 300 to 1000 mg/dL and are roughly equal in magnitude. Special diagnostic tests such as beta-quantification or lipoprotein electrophoresis are often required and are time consuming and not widely available. VLDL particles are cholesterol- enriched, which can be determined by isolation of VLDL by ultracentrifugation and by the demonstration of beta migrating VLDL on lipoprotein electrophoresis.  A VLDL-cholesterol/plasma TG ratio of <0.30 is usually observed (124).  A low ApoB/total cholesterol ratio of <0.33 also can be helpful in making the diagnosis (125). Simplified criteria for the diagnosis of DBL using a 3-step process has been proposed (126). The diagnosis of remnant removal disease should be confirmed by demonstrating the presence of the E2/E2 genotype. If the genotype result is not E2/E2, an autosomal dominant variant of APOE should be suspected. There is a high prevalence of premature coronary artery disease (127-129) and peripheral arterial disease (130-132).  Occasionally severe HTG and an increased risk of pancreatitis can develop in the presence of a concomitant secondary form of HTG or TG-raising drugs.

Figure 1. Palmar Xanthomas: Orange-yellow discoloration confined to the palmar creases.

 

Familial Partial Lipodystrophy (FPLD) Syndromes

 

A distinct entity that results in moderate and severe hypertriglyceridemia include partial lipodystrophy syndromes. Inherited lipodystrophies are a heterogeneous group of disorders considered to be rare, that manifest as complete or partial loss of white adipose tissue with accompanying severe metabolic dysregulation(133) and are reviewed elsewhere in the Endotext chapter on Lipodystrophies (134).  Loss of fat can be either localized to small discrete areas, in some cases partial with loss from extremities, or generalized with fat loss from nearly the entire body. Inherited lipodystrophies, while rare, can be autosomal dominant or recessive.  Some forms manifest at birth, while others become evident later in life.

 

Partial or generalized lipodystrophic disorders frequently are associated with significant metabolic derangements associated with severe insulin resistance, including HTG. The extent of fat loss sometimes determines the severity of metabolic complications (135).  HTG is a common accompaniment of many lipodystrophies, often in conjunction with low HDL-C levels.    The pathophysiology of hypertriglyceridemia in these subjects is possibly related to the reduced ability to deposit free fatty acids in adipose tissue due to its maldevelopment, and accelerated lipolysis with increased hepatic VLDL synthesis and delayed clearance (135).

 

Genetics: Several genes have been implicated in the manifestation of various forms including LMNA, PPARG, LIPE, CIDEC (136). In the Dunnigan variety, the most commonly identified genetic variant of FPLD, the commonest variants are in the LMNA gene and less frequently PPARG (133).  No specific genetic defect has been identified in Köbberling’s FPLD, although recent evidence suggests a heavy polygenic burden in these individuals (137, 138).

 

Diagnosis: Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder in which near total absence of subcutaneous adipose tissue is evident from birth.  HTG and hepatic steatosis are evident at a young age and are often difficult to control. Severe HTG, often associated with eruptive xanthoma and recurrent pancreatitis, can occur in patients with CGL. The prevalence of HTG in case series of CGL patients is over 70% (135, 139).  Plasma TGs are normal or slightly increased during early childhood, with severe HTG manifesting at puberty along with onset of diabetes mellitus. 

 

Familial partial lipodystrophies (FPLD) are complex metabolic disorders that are often not recognized clinically (140).  Partial lipodystrophies are characterized by partial loss of adipose tissue and significant metabolic derangements.  The Dunnigan variety of FPLD (FPLD type 2) is a rare autosomal dominant disorder in which fat loss mostly involves the extremities and the trunk.  Onset of fat loss in the buttocks and extremities occurs at puberty or late adolescence, with gain of fat to the face and neck. Acanthosis nigricans, calf muscle hypertrophy, and phlebomegaly (prominent veins) due to lack of subcutaneous fat, can be observed. Significant metabolic dysfunction including diabetes, which is often very insulin resistant, resistant hypertension, and HTG often severe and difficult to treat, can occur.  Myopathy, cardiomyopathy, and/or conduction system abnormalities can occur (141).  ASCVD risk also is increased (142, 143).

 

Some lipodystrophies, where fat loss appears to be proportionate to loss of total and lean body mass, do not result in dyslipidemia. Elevated TG levels have been reported in patients with atypical progeroid syndrome due to LMNA mutations (144, 145).  Of the acquired lipodystrophies, the HIV-associated form usually is characterized by more moderate HTG.  HIV-associated lipodystrophy occurs in patients receiving protease inhibitor containing highly active anti-retroviral therapy regimens (146).  Fat loss occurs in the face, buttocks, and extremities.

 

CONSEQUENCES OF HYPERTRIGLYCERIDEMIA

 

Atherosclerotic Cardiovascular Disease

 

EPIDEMIOLOGY

 

HTG has long been known to be a risk factor for ASCVD (33, 147-150), which has been confirmed in meta-analyses (45).  However, HTG also is frequently associated with low levels of HDL-cholesterol and an accumulation of remnants of the TG-rich lipoproteins, both known risk factors for ASCVD.  When adjusted for both HDL-C and non-HDL-C, which contains both remnants of the TG-rich lipoproteins and LDL, the association of TGs with ASCVD risk remained significant, although somewhat attenuated (151).  Postprandial TGs are elevated throughout the day in subjects with HTG, and postprandial TG-rich lipoproteins and their remnants also have been hypothesized to be important in the pathogenesis of atherosclerosis (150). It is therefore of interest that non-fasting TGs have been associated with ASCVD risk (150, 152, 153), despite non-fasting TGs being quite variable. However, unlike the situation with elevated LDL-C levels, the magnitude of the TG elevation does not appear to correlate with the extent of ASCVD risk. In particular, very severe HTG per se does not always appear to confer increased ASCVD risk, possibly because the chylomicrons that accumulate are too large to enter the arterial intima (154, 155). 

 

TRIGLYCERIDES IN THE PATHOGENESIS OF ASCVD

 

Although chylomicrons may be too large to enter the arterial intima, ApoE-and cholesterol-enriched remnants of the TG-rich lipoproteins can enter with ease (153) where they can bind to vascular proteoglycans, similar to LDL (156, 157).  Modification of these retained lipoproteins by either oxidative damage or enzyme digestion of some of the lipid components can liberate toxic by-products, which have been hypothesized to play a role in atherogenesis by facilitating local injury, generation of adhesion molecule, and cytokine expression and inflammation (157).  Remnants of the TG-rich lipoproteins also can be taken up by macrophages leading to the formation of foam cells, an important component of atherosclerotic plaques.  HTG also is associated with a preponderance of small, dense LDL, particles, reduced levels of HDL-C, and in the metabolic syndrome, with abnormalities of HDL composition (see earlier). Small, dense LDL can traverse the endothelial barrier more easily than large, buoyant LDL particles (158), are retained more avidly than large, buoyant LDL (159), and also are more readily oxidized (160, 161), all of which may facilitate atherogenesis. HDL particles in some hypertriglyceridemic states, e.g., in association with the metabolic syndrome, might be dysfunctional with respect to their cholesterol efflux, anti-inflammatory, and anti-oxidant properties. Moreover, a hypercoagulable state has been reported in association with both HTG and the metabolic syndrome (162). Thus, HTG might accelerate atherosclerosis by several mechanisms, all of which could increase CVD risk.

 

GENETIC EVIDENCE OF HYPERTRIGLYCERIDEMIA AND ATHEROSCLEROSIS

 

Recent human genetic studies have provided important insight into the contribution of TGs to ASCVD. Several genetic approaches, including candidate gene sequencing, GWAS of common DNA sequence variants, and genetic analysis of TG phenotypes have unraveled new proteins and gene variants involved in plasma TG regulation (163). Some genetic variants that influence TG levels appear to be associated with increased CVD risk even after adjusting for their effects on other lipid traits (164).  GWAS have identified common noncoding variants of the LPL gene locus associated with TG and CVD risk (165, 166).  A common gain-of-function mutation in the LPL gene, S447X (10% allele frequency), is associated with reduced TG levels and reduced risk of CVD (167) and an LPL variant associated with reduced TG and ApoB levels was associated with reduced CVD similar to LDL-C lowering variants, suggesting that the clinical benefit of lowering triglyceride and LDL-C levels may be proportional to the absolute change in ApoB (168).  Conversely, several loss-of-function LPL variants linked with elevated TG levels are associated with increased CVD risk (169). Variants in the TRIB1 locus have been associated with LDL, HDL-C and TG levels (166), hepatic steatosis (170) and coronary artery disease (171). Mutations that disrupt APOC3 gene function and reduce plasma ApoC-III concentration are associated with lower TG levels and decreased risk of clinical CVD (172, 173).  In contrast, carriers of rare mutations in APOA5, encoding ApoA-V, an activator of LPL, are associated with elevated TGs and with increased risk of myocardial infarction (174, 175).  Loss of function variants in ANGPTL4 that had lower TG levels also were associated with reduced CVD risk (176, 177).  Thus, exciting new human genetics findings have causally implicated TG and TG-rich lipoproteins in the development of CVD risk. In particular, the LPL pathway and its reciprocal regulators ApoC-III and ApoA-V appear to have an important influence on atherosclerotic CVD risk. However, despite this mountain of evidence demonstrating a causal relationship of TG with atherosclerosis, the possible involvement of a correlated trait, usually low HDL-C levels, or other unmeasured traits, cannot be ruled out.

 

CLINICAL TRIAL EVIDENCE OF TRIGLYCERIDE LOWERING AND ASCVD

 

In the pre-statin era, use of gemfibrozil monotherapy demonstrated cardiovascular benefit in men with coronary heart disease. However, since the advent of statins, drugs that specifically lower plasma triglyceride levels have not clearly been shown to have a benefit with cardiovascular risk reduction in clinical trials when added to background statin therapy. Reasons for this are unclear. In genetic studies to get a comparable reduction in Apo B and coronary heart disease (CHD) risk in clinical trials, a TG reduction of ~70mg/dL is required compared to a decrease in LDL-C of only 14mg/dL. Additionally, lipid alterations due to genetics are lifelong and result in much bigger reductions in CHD than a 5-year drug study. Based on genetic studies, the magnitude of TG reduction required to demonstrate cardiovascular benefit is quite large.

 

CARDIOVASCULAR DISEASE IN THE CHYLOMICRONEMIA SYNDROME

 

As described earlier, chylomicrons have been considered to be too large to penetrate the vascular endothelium and play a role in atherogenesis (152), although  remnants of the TG-rich lipoproteins may be atherogenic (152, 178-181). The incidence of CVD is low in individuals with FCS (182), although premature atherosclerosis has been documented in well characterized subjects with this disorder (183).  However, CVD risk clearly is increased in many patients with MFCS, although the exact frequency remains unclear. The frequency of CVD outcomes does not appear to relate to the magnitude of the TG elevations (184). It is not surprising that CVD is increased in MFCS considering the association between TGs and CVD that has been documented in many studies (reviewed in (150, 185, 186)).  Many subjects develop severe HTG due to the co-existence of polygenic mutations that result in mild to moderate HTG (22) with secondary causes of HTG.  Residual HTG due to these genetic disorders persists even after severely elevated TG levels have been reduced by treatment of the secondary forms of HTG and treatment of the HTG per se.  Moreover, many patients with the MFCS have other CVD risk factors such as diabetes, reduced levels of HDL-C, and hypertension, the latter resulting in use of diuretics and beta-blockers, which play a role in raising their TGs to levels at which chylomicrons accumulate due to saturation of clearance mechanisms. Therefore, strategies to prevent CVD need to be undertaken once the TGs have been lowered to a level where pancreatitis is unlikely to recur. 

 

Pancreatitis

 

Severe hypertriglyceridemia is the third most common cause of acute pancreatitis after alcohol and gallstones. The chylomicronemia syndrome describes a constellation of findings that occur with severe elevations of plasma TG levels. There is some lack of consensus as to what constitutes severe HTG, values >1000-1500 mg/dL are generally classified as severe, although some groups consider values in the 500-1000 mg/dL range also severe hypertriglyceridemia (187). 

 

Individuals with both FCS and MFCS often present with hypertriglyceridemia induced acute pancreatitis, which can be recurrent if triglyceride levels remain elevated persistently. Women with genetic HTG can develop severe HTG and pancreatitis during pregnancy particularly during the third trimester (188).

 

The pancreatitis that occurs with severe HTG can be recurrent.  In a prospective study of patients admitted with acute pancreatitis, the distribution of plasma TGs was bimodal when measured at the peak of the pain (101, 102).  TG levels <880 mg/dL were associated with gall bladder disease and chronic alcoholism, while those above 2000 mg/dL were associated with the simultaneous presence of familial and secondary forms of HTG.  It has been suggested that individuals become prone to the development of TG-induced pancreatitis at TG values between 1500-2000 mg/dL (189).  TG-induced pancreatitis has been reported with TG levels lower than 500 mg/dL(190, 191),  although in our experience this usually occurs when patients with severe HTG stopped eating some time prior to the blood draw. The frequency of severe HTG leading to acute pancreatitis varies widely from about 6-20% of subjects, possibly related to the type of patient presenting to different type of medical centers (192, 193).  Pancreatitis often is recurrent if HTG is not appreciated to be the cause and if TG levels are not adequately controlled (87). With long term multiple episodes of acute, recurrent pancreatitis, exocrine pancreatic insufficiency or insulin deficient secondary diabetes may occur. A meta-analysis of observational studies suggests that TG-induced pancreatitis has worse outcomes than pancreatitis from other causes, with an approximate doubling of renal and respiratory failure, a nearly 4-fold increase of shock and a near doubling of mortality (194). Pancreatitis due to very severe HTG also may occur during infusion of lipid emulsions for parenteral feeding (195) or with use of the anesthetic agent propofol, which is infused in a 10% fat emulsion (196). 

 

MECHANISM OF SEVERE HYPERTRIGLYCERIDEMIC PANCREATITIS

 

The mechanism by which very severe HTG leads to pancreatitis remains speculative. Suggested mechanisms include the local liberation of FFA from TGs and lysophosphatidylcholine from phosphatidycholine when pancreatic lipase encounters very high levels of TG-rich lipoproteins in the pancreatic capillaries (197). High local concentrations of FFA overwhelm the binding capacity of albumin with resultant aggregation into micellar structures with detergent properties.  Both FFA and lysophosphatidylcholine have been shown to cause chemical pancreatitis when infused into pancreatic arteries in animal models (198-200). This leads to local liberation of more lipases from the damaged pancreatic acini, resulting in a vicious cycle (198, 201).  It also has been hypothesized that increased plasma viscosity due to the presence of increased numbers of chylomicrons in the pancreatic microcirculation contributes to the development of pancreatitis (202). There also is recent evidence of gene associations in TG-induced pancreatitis; in a Chinese cohort with HTG, a CFTR variant and TNF alpha promoter polymorphism were found to be independent risk factors for developing pancreatitis (203), while another study found an increased frequency of ApoE4 (204).

 

DIAGNOSIS OF SEVERE HYPERTRIGLYCERIDEMIC INDUCED PANCREATITIS

 

The diagnosis of HTG-associated pancreatitis can be made by the presence of severely elevated TG levels in a patient with acute pancreatitis. Falsely low serum amylase levels can be encountered due to assay interference by the TG-rich lipoproteins (205). Pseudohyponatremia due to the presence of large numbers of TG-rich lipoproteins in plasma can be seen with very high TG levels. Interference with liver transaminase assays may also occur, giving spuriously high values making it difficult to exclude alcoholic liver disease (205).

 

With chronic chylomicronemia, patients may develop eruptive xanthomata (Figure 2). These xanthomas represent an inflammatory response to the deposition of chylomicron-associated lipids in tissues and are yellow-red papules that usually appear on the buttocks, back and extensor surfaces of the upper limbs. Histologically, these lesions contain lipid laden foamy macrophages (206).  

 

Figure 2. Eruptive Xanthomas. The commonest site is on the buttocks. The lesions are papular with an erythematous base. They often are itchy.

 

Lipemia retinalis, where the retinal vessels take on a whitish hue with pallor of the optic fundus and retina can be observed with very high TG levels (Figure 3).  There is no associated visual impairment.  

Figure 3. Lipemia retinalis. Note the pale color of the retinal vessels.

 

Acute recent memory loss and mental fogginess (82) can also occasionally be seen, but has not been extensively studied. Symptoms such as fatigue, blurred vision, dysesthesias, and transient ischemic attacks have been suggested to be related to hyperviscosity resulting from high TG levels (207, 208).  Hepatosplenomegaly is frequently present in FCS due to macrophage infiltration in response to the chylomicron accumulation. Fatty liver is a common finding on imaging in both FCS and MFCS.

 

MANAGEMENT OF SEVERE HYPERTRIGLYCERIDEMIA

 

Management of HTG by lifestyle and pharmacological means is discussed in detail in the Endotext chapters on The Effect of Diet on Cardiovascular Disease and Lipid and Lipoprotein Levels and Triglyceride Lowering Drugs (209, 210).  However, in this section we will make a few points specifically relevant to this chapter. 

 

Before initiating lifelong therapy for hypertriglyceridemia, evaluation for and treatment of reversible secondary disorders that can elevate plasma triglyceride levels is crucial. This includes appropriate management of diabetes and hypothyroidism and substituting drugs that can elevate triglyceride levels with lipid-neutral agents. Management of hypertension should include calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and alpha-adrenergic blockers rather than beta-adrenergic blocking agents and diuretics.

 

Cardiovascular Disease Prevention

 

ASCVD risk in HTG is modulated by the presence of several other factors, including other lipoprotein abnormalities, other CVD risk factors, and family history of CVD, with some families with HTG appearing to have a greater risk of CVD than others (44). The role of TG lowering by pharmacological means remains controversial, but there is consensus that the presence of HTG imparts residual risk after LDL has been adequately lowered with statins.

 

Statins: The best clinical trial data currently available for the prevention of ASCVD in patients with HTG demonstrate that statins are likely to confer the most benefit, even though their primary mode of action is not to reduce plasma TGs, nor are they very effective in so doing (211).  In patients with elevated TG levels statins will result in a significant decrease in TG levels. Based on the results of the IMPROVE-IT trial (212), the addition of ezetimibe may be of additional benefit.

 

Fibrates: Fibrates such as gemfibrozil and fenofibrate, are PPAR-α agonists, and very effective in lowering plasma TG levels (by up to 50%).  Several studies have failed to demonstrate a benefit of fibrates on ASCVD events, either alone or in combination with statins.  However, participants in these studies were not confined to individuals with HTG.  Nonetheless, post-hoc analysis showed that subgroups of subjects who had mild HTG >200mg/dL and LDL-C <34mg/dL had a significant reduction of ASCVD events (213-216).  In addition, the Action to Control Cardiovascular Risk in Diabetes (ACCORD)-LIPID trial, which was confined to subjects with diabetes, showed a similar outcome in the subgroups with HTG, although the trial was negative for all subjects (214).  Recently a novel selective peroxisome proliferator-activated receptor α modulator, pemafibrate, that possesses unique PPARα activity and selectivity (217), was evaluated in individuals with HTG and diabetes in the Pemafibrate to Reduce Cardiovascular OutcoMes by Reducing Triglycerides IN patiENts With diabetes (PROMINENT) trial. Patients with type 2 diabetes, triglyceride level 200 to 499 mg/dL and HDL-C of </=40 mg/dL were assigned to pemafibrate or placebo. Unfortunately, pemafibrate failed to demonstrate cardiovascular benefit in patients with type 2 diabetes and mild to moderate HTG despite significantly lowering triglyceride levels (218).  Thus, addition of a fibrate to a statin for cardiovascular risk reduction cannot be recommended at this time.

 

Omega-3 fish oil: Omega-3 (n-3) fatty acids are polyunsaturated fatty acids that lower TGs. The two main n-3 fatty acids are eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) which can lower VLDL secretion and are agonists of PPARa. However, their role in ASCVD prevention also has been controversial as several RCTs of various dosages of n-3 mixtures failed to demonstrate CV benefit in mild to moderate HTG (219). The Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial (REDUCE-IT), evaluated the addition of high dose icosapent ethyl (highly purified eicosapentanoic acid or EPA) compared to placebo in high-risk patients with mild to moderate HTG on statin therapy, demonstrated a surprising 25% lower CV risk in subjects. Notably this effect was independent of baseline TG levels and TG reduction.  (220).  Subsequently the STRENGTH (Statin Residual Risk Reduction With Epanova in High Cardiovascular Risk Patients with Hypertriglyceridemia) trial of n–3 fatty acid mixtures (EPA + DHA) failed to demonstrate cardiovascular benefit and was terminated early for futility (221). Similarly, use of an EPA/DHA mixture (1.8 g/d) in patients with a recent myocardial infarction in the OMega-3 fatty acids in Elderly with Myocardial Infarction (OMEMI) trial (222) also failed to meet its primary endpoint. Tissue EPA levels may be a contributor to the positive results in the REDUCE-IT study, as there is evidence that EPA inhibits inflammation, causes membrane stabilization, and decreases plaque volume. The REDUCE-IT trial has generated controversy due to use of mineral oil in the control group which resulted in an increase in both LDL-C and C-reactive protein (223, 224). Nonetheless, several current guidelines suggest addition of icosapent ethyl in addition to a statin for residual hypertriglyceridemia in high-risk individuals (those with known ASCVD or diabetes with additional risk factors).

.

Niacin: Niacin effectively lowers triglycerides and LDL-C while raising HDL-C. Niacin inhibits lipolysis in adipocytes, therefore decreasing the available fatty acids for VLDL synthesis. However, it has fallen out of favor for ASCVD risk reduction. Two RCTs, Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) and Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2-THRIVE) demonstrated no benefit to the addition of niacin to statins for decreasing cardiovascular risk. However, it is important to note that neither of these trials were confined to subjects with high TG levels. Therefore, due to the lack of efficacy and potential for increasing insulin resistance, niacin is not recommended for treatment of HTG.

 

Newer Therapies for HTG

 

Apo C-III Inhibitors: ApoC-III is an endogenous inhibitor of LPL, by displacing ApoC-II an activator of LPL. This leads to inhibition of lipolysis and elevations in TG levels. Apo C-III can also increase TGs by LPL independent mechanisms. Currently, ApoC-III inhibitors include volanesorsen (a second-generation antisense oligonucleotide (ASO)), olezarsen (a third generation ASO), and ARO-APOC3 (a small interfering RNA), all of which are directed at APOC3 gene (225). Volanesorsen has been studied in individuals with FCS and MFCS and has been found to decrease TG levels by up to 70% from baseline and potentially prevent pancreatitis. However, thrombocytopenia and injection site reactions are common. This drug is therefore not approved for use by the FDA due to concerns of bleeding but is approved for use by the European Medicines Agency.  

Olezarsen is a third generation ASO for which early studies have been completed.

 

ANGPTL-3 inhibitors: ANGPTL3 is a key regulator of lipoprotein metabolism and is able to repress LPL and endothelial lipase activity, with resultant increase in TGs and TG rich lipoprotein levels (226). Homozygous loss of function variants in ANGPTL3 result in combined hypolipidemia with low TG and LDL-C levels. ANGPTL3 inhibition results in decreased LDL-C, and TGs. ANGPTL3 inhibitors include evinacumab, a monoclonal antibody, vupanorsen, an antisense oligonucleotide (ASO), and ARO-ANG3, a small-interfering ribonucleic acid (siRNA). Evinacumab has been studied in patients with sHTG. A phase 2 study of evinacumab in patients with sHTG, demonstrated significant reductions in TGs only in patients with MFCS with and without LPL mutations, but not in patients with FCS, suggesting that ANGPTL3 inhibition is dependent on presence of some LPL activity (227). In the cohort with polygenic sHTG and MCS, evinacumab 15 mg/kg IV every 4 weeks resulted in an 81.7% reduction in TGs. It should be noted that evinacumab is currently only approved for homozygous familial hypercholesterolemia.

 

Management of Severe HTG-Induced Pancreatitis

 

Because of the low frequency of severe HTG in the general population, and because only some patients with severe HTG develop pancreatitis, large random controlled clinical trials are difficult to perform and unlikely to be undertaken in the foreseeable future. Therefore, therapeutic decisions need to be based on less stringent criteria than might otherwise be desirable.  However, keeping TG levels <500 mg/dL should prevent the onset of TG-induced pancreatitis (187, 228, 229). 

 

ACUTE MANAGEMENT

 

The clinical presentation of HTG-induced pancreatitis is similar to that from other causes of acute pancreatitis and can be preceded by episodic nausea, epigastric pain radiating through to the back, and increasing heart-burn. Individuals with recurrent acute pancreatitis may present without severe elevations in pancreatic enzymes (230). The immediate goal is to lower TGs in hospitalized patients.  Management is similar to the management of non-TG induced pancreatitis, which includes cessation of all oral intake for pancreatic rest, fluid resuscitation, pain management, and management of metabolic abnormalities. TGs fall rapidly with discontinuation of oral intake, often to under 1000mg/dL with cessation of oral intake. With clinical improvement, oral diet advancement should be done slowly and cautiously in the hospital as this can result in rebound TG elevations. Supportive care as needed should be instituted for organ failure. Lipid emulsions for parenteral feeding should be avoided since their use will further delay clearance and exacerbate the HTG. If long term nutrition is required for very ill individuals who cannot eat, total parental nutrition without lipids should be utilized.   

 

Heparin: Heparin will liberate LPL into plasma from its endothelial binding sites and hence rapidly lowers TGs (231). However, it also can cause rebound HTG due to rapid degradation of released LPL (232) and increases the risk of hemorrhagic pancreatitis. Therefore, the use of heparin is not recommended (233). 

 

Insulin: The rationale for the use of an IV insulin infusion of regular insulin (in conjunction with IV glucose administration as needed) is that it can activate LPL and enhance clearance of TG-rich lipoproteins (234). Intravenous insulin can be beneficial in individuals with diabetes needing glycemic control. Its use in TG--induced pancreatitis without diabetes has been reported in several case reports (235-239), and has become widespread but it is unclear whether similar changes would have occurred simply by restricting oral intake without the use of insulin.  Regular insulin at 0.1-0.2 units/kg/hour with a separate iv dextrose infusion to prevent hypoglycemia in individuals without diabetes is often used. IV insulin can be stopped when TG drops to below 1000mg/dL. However, TGs will increase when the individual consumes an oral diet; therefore, caution should be exercised with slow advancement of the diet. In a study of chylomicronemia with uncontrolled diabetes, insulin infusion lowered TGs more rapidly than plasmapheresis (240).

 

Plasmapheresis: The use of plasmapheresis to acutely lower TGs is controversial. Plasmapheresis is extracorporeal therapy where plasma is removed and replaced; plasma is separated from the blood and discarded removing chylomicrons. Substitute fluid is replaced to maintain blood volume. The procedure is highly effective in rapidly decreasing TG levels by 85% after 1 session. However, the effect is not persistent, without evidence of long-term efficacy or mortality and morbidity benefit demonstrated. Although recommended by some (241, 242), the current evidence for the benefit of use of plasmapheresis is limited to small uncontrolled anecdotal series (243) from which no firm conclusion can be made regarding its use in acute TG-induced pancreatitis (244). A recent retrospective analysis demonstrated no benefit on length of hospital stay or mortality when therapeutic plasma exchange was added to medical management of severely elevated plasma TGs (245). TG levels fall rapidly with cessation of oral intake and use of non-lipid-containing intravenous fluids.  Plasmapheresis requires a specialized center, needs central venous access, and transient anticoagulation; it only temporarily improves TG levels without addressing the underlying cause (83). Risks include line sepsis, deep vein thrombosis, and bleeding. Therefore, we do not recommend its routine use in this situation unless clinical circumstances necessitate plasmapheresis such as severe acute necrotizing pancreatitis (246), shock, or pregnancy (247). 

 

LONG-TERM MANAGEMENT TO PREVENT PANCREATITIS

 

After TG lowering in the setting of acute pancreatitis, it is essential to determine both the primary and secondary causes of the severe HTG that precipitated the acute pancreatitis.   Continued management of any secondary form of HTG, as well as lifestyle and drug therapy to maintain low TG levels is required to prevent recurrent pancreatitis. If fasting plasma TG levels remain above 1000 mg/dL after treating or removing the precipitating causes of the severe HTG, life-long therapy with fibrates or n-3 fatty acids, as described earlier, might be considered for these patients. Limited evidence suggests that orlistat, a gastrointestinal lipase inhibitor that decreases absorption of ingested fat, thereby reducing intestinal chylomicron synthesis, may be of benefit in reducing TG levels when used in conjunction with fibrate therapy (248, 249). TG and glucose control can be particularly challenging in individuals with familial partial lipodystrophy.

 

Management of Specific Syndromes that Accompany Severe HTG

 

FAMILIAL CHYLOMICRONEMIA SYNDROME (FCS)

 

Treatment of FCS includes management of an acute crisis (pancreatitis) and long-term management of HTG. Management of acute HTG-induced pancreatitis is described in the previous section. Long-term management of individuals with FCS involves patient education and maintaining a very low-fat diet. Consumption of even small amounts of fat can lead to severe HTG in FCS due to the absence of functional LPL. Infants with FCS presenting with abdominal pain or failure to thrive require discontinuation of breast feeding with replacement by very low-fat formula feeding to decrease TG levels and symptoms. In children and adults with FCS, dietary fat calories should be severely restricted to control the severe HTG and abdominal pain. This translates to about 5% to 10% of total daily calories from fat, which is a major burden for these patients (250).  Medium-chain TGs, which are taken up directly by the liver after absorption and do not enter plasma as chylomicrons via the thoracic duct, are a potential alternate fat source for these patients. n-3 fatty acids can aggravate the severe HTG of FCS and therefore are contraindicated in these individuals (251, 252).  Fibrates are not efficacious in FCS (253).  There are limited studies showing that orlistat might be beneficial in patients with FCS (254, 255).  Alcohol, oral contraceptives, and other TG-elevating drugs (see Table 3) can exacerbate severe HTG and precipitate acute pancreatitis in FCS.  Successful pregnancies in patients with FCS have become more common of late (256, 257).

 

Alipogene tiparvovec, an adeno-associated virus LPL gene therapy that was developed and resulted in significant improvement in postprandial chylomicron metabolism in patients with FCS has been abandoned and no longer available (258), Antisense oligonucleotide inhibitors of ApoC-III (volanesorsen is approved in Europe but no in the US) and of ANGPLT3 are in development for FCS (96).

 

MULTIFACTORIAL CHYLOMICRONEMIA SYNDROME (MFCS)

 

Management of acute HTG-induced pancreatitis is described in the previous section.

 

Long term management: To prevent acute HTG-induced pancreatitis in MFCS, the goal is to maintain TG levels below the threshold for pancreatitis, preferably <500 mg/dL. This requires instituting lifestyle adjustments, reversal of any secondary causes of HTG, such as treatment of suboptimally managed or undiagnosed diabetes, treating hypertension with lipid neutral agents such as ACE inhibitors, ARBs, calcium channel inhibitors, or alpha blockers rather than beta-adrenergic blockers and diuretics, and discontinuing other TG-raising drugs (table 3) where possible.  Alcohol intake should be limited or eliminated, since even small amounts of alcohol can substantially raise TG levels in individuals with baseline HTG.  Attention should be paid to avoid rebound weight gain that commonly occurs after successful weight loss. Oral estrogens should be substituted by transdermal or vaginal preparations, which raise plasma TGs to a lesser extent than oral estrogens (259, 260). Residual HTG should be treated with fibrates (229), which together with management of the secondary disorder or disorders, can reduce TG levels to below the threshold for developing pancreatitis. Other agents that can be used to lower TGs alone or in combination with fibrates, include n-3 fatty acids, and high-dose statins. We do not recommend using niacin due to risk of worsening insulin resistance and lack of clinical trial data for benefit. Lifestyle measures and weight loss are important, but patients should be educated on risks of rapid weight regain after successful weight loss can be associated with rebound severe HTG. Bariatric surgery also has been used to reduce severe HTG in refractory HTG (261). Inhibition of ApoC-III or ANGPTL3, which have been shown to lower TGs in patients with severe HTG (262), may have a role to play in the future the management of severe HTG in patients with MFCS. 

 

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Monogenic Disorders Altering HDL Levels

ABSTRACT

 

Very low HDL-C levels (<20mg/dL) may be due to severe elevations in triglycerides, very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, and certain medications such as anabolic steroids. Additionally, variants in multiple genes that each have a small effect but cumulatively lead to a decrease in HDL-C can result in very low HDL-C levels. Finally, rare monogenic disorders such as familial hypoalphalipoproteinemia, Tangier disease, and lecithin acyltransferase (LCAT) deficiency can lead to very low HDL-C levels. In this chapter we discuss the lipid abnormalities and clinical features of these monogenic disorders causing very low HDL-C levels. An elevated concentration of apo A-I and apo A-II is called hyperalphalipoproteinemia (HALP). HALP is classified as moderate (HDL-C levels between 80 and 100 mg/dL) or severe (HDL-C levels > 100 mg/dL). HALP is a heterogeneous condition caused by a variety of genetic and secondary conditions (for example ethanol abuse, primary biliary cirrhosis, multiple lipomatosis, emphysema, exercise, and certain drugs such as estrogens). In many individuals HALP has a polygenic origin. Monogenic HALP includes CETP deficiency, hepatic lipase deficiency, endothelial lipase deficiency, and loss of function mutations in SRB1. In this chapter we discuss the lipid abnormalities and clinical features of these monogenic disorders causing HALP.

 

LOW HDL CONDITIONS

 

The inverse relationship between HDL-C and ASCVD risk is well established but it should be recognized that while this association is consistently observed recent genetic and cardiovascular outcome studies suggest that this association is not causal (1). However, as discussed below major reductions in HDL-C induced by specific monogenic disorders may increase the risk of ASCVD.

 

Isolated low HDL-C levels can occur; however, it is more commonly found in association with hypertriglyceridemia and/or elevated apo B levels, typically as part of the obesity/metabolic syndrome (2). Patients with very low HDL-C (<20 mg/dL) in the absence of severe hypertriglyceridemia, very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, anabolic steroids, or a paradoxical response to PPAR agonists are very rare (<1% of the population) (3,4). These individuals may have a very rare monogenic disorder associated with marked HDL deficiency, including familial hypoalphalipoproteinemia, Tangier disease, and lecithin acyltransferase (LCAT) deficiency. Table 1 summarizes the genetic, lipid, and clinical features of these monogenic low HDL conditions. Inheritance is autosomal co-dominant with heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes (3). In some individuals the decrease in HDL-C can be polygenic i.e., variants in multiple genes that each have a small effect but cumulatively lead to a decrease in HDL-C (5).

 

Table 1. Characteristics of Monogenic Low HDL Syndromes

 

Effected genes

Lipids

Clinical features

Familial hypoalpha-lipoproteinemia

apo A-I/apo C-III/ apo A-IV

apo A-I/apo C-III

apo A-I

Apo AI undetectable, marked deficiency in HDL-C, low – normal triglycerides, normal LDL-C

Xanthomas Premature ASCVD Corneal manifestations

Tangier disease

ABCA1

HDL species exclusively preß-1, HDL-C <5 mg/dL

LDL-C low (half normal)

Hepatosplenomegaly

Enlarged tonsils

Neuropathy

ASCVD (6-7th decade)

LCAT deficiency

LCAT

HDL-C <10 mg/dL

apo A-I 20-30 mg/dL

<36% cholesteryl esters

Low LDL-C

Presence of Lp-X particles

FLD develop corneal opacities (“fish eye”), normochromic anemia and proteinuric end stage renal disease

 

FED only develop corneal opacities

Inheritance is autosomal co-dominant with heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes (3). FLD- Familial Lecithin: Cholesteryl Ester Acyltransferase Deficiency; FED- Fish Eye Disease

 

Familial Hypoalphalipoproteinemia  

 

Familial hypoalphalipoproteinemia is a heterogeneous group of apolipoprotein A-I (apo A-I) deficiency states. This disorder is the rarest cause of  monogenic severe HDL deficiency (6). These various conditions are characterized by the specific apolipoprotein genes that are affected on the apo A-I/C-III/A-IV gene cluster (3). The genes for these 3 apolipoproteins (apo A-I, apo C-III, and apo A-IV) are grouped together in a cluster on human chromosome 11. In patients with apo A-I/C-III/A-IV deficiency, apoA-1 is undetectable in the plasma and is associated with marked deficiency in HDL-C, low triglyceride levels (due to apo C-III deficiency), and normal LDL-C levels (3). Heterozygotes have plasma HDL-C, apo A-I, apo A-IV, and apo C-III levels that are about 50% of normal (3). This condition is associated with aggressive, premature ASCVD. Additionally, there is evidence of mild fat malabsorption due to deficiency of apo A-IV. Patients with apo A-I/C-III deficiency have undetectable apo A-I and a similar lipid profile as those with apo A-I/C-III/A-IV deficiency (3). This condition is also associated with premature ASCVD. It is distinguished from the former by presence of planar xanthomas and absence of fat malabsorption (since apo A-IV is present). Familial apo A-I deficiency is itself a heterogeneous group of disorders associated with numerous Apo A-I mutations (3). Common manifestations include undetectable plasma Apo A-I, marked HDL deficiency with normal LDL-C and triglyceride levels, xanthomas (planar, tendon, and/or tubero-eruptive depending on the specific gene mutation), and premature ASCVD. Some forms of the disease are also associated with corneal manifestations, including corneal arcus and corneal opacification. One of the interesting manifestations of familial apo A-I deficiency is that levels of apo A-IV and apo E containing HDL particles are only modestly reduced, with preserved electrophoretic mobility and particle size (7).

 

It is notable that familial hypoalphalipoproteinemia is associated with an increased risk of premature ASCVD presumably due to the marked deficiency in Apo A-I and HDL. Given the increased ASCVD risk associated with Apo A-I deficiency, treatment is directed towards aggressive reduction of LDL-C and non-HDL-C levels and reducing other cardiovascular risk factors.

 

Some mutations in Apo A-I are associated with low HDL-C levels and hereditary amyloidosis and are the second most frequent cause of familial amyloidosis (6,8). Note that HDL-C levels are not always decreased in patients with familial amyloidosis secondary to Apo A-I mutations. The N-terminal fragment of the mutated protein is found in the amyloid fragments.

 

Tangier Disease

 

Tangier disease is due to mutations in the gene that codes for ATP-Binding Cassette transporter A1 (ABCA1) and is inherited in an autosomal co-dominant manner (9,10).  Fredrickson first reported this condition in two patients who hailed from Tangier Island in the Chesapeake Bay, for which the disorder is named. ABCA1 facilitates efflux of intracellular cholesterol from peripheral cells to lipid poor A1, the key first step of reverse cholesterol transport (11). As such, this disorder is characterized by severe deficiency of HDL-C (HDL-C <5 mg/dL) and the presence of only thepreß-1 HDL fraction of HDL (10). The poorly lipidated Apo A-I is rapidly catabolized by the kidney. These patients also demonstrate moderate hypertriglyceridemia and low LDL-C levels (10). The decrease in LDL-C is likely due to absence of the transfer of cholesterol from HDL to LDL. Studies have also suggested that an increase in LDL uptake by the liver also occurs (12). The increase in triglycerides may be due to the failure of HDL to provide co-factors that increase lipoprotein lipase activity. Additionally, ABCA1 deficiency in the liver increases triglyceride secretion and hepatic angiopoietin-like protein 3 secretion which could inhibit lipoprotein lipase activity leading to an increase in triglycerides (12,13).

 

Since ABCA1 deficiency impairs free cholesterol efflux from cells, there is accumulation of cholesterol esters in many tissues throughout the body (10). Classically, patients present with hepatosplenomegaly and enlarged yellow-orange hyperplastic tonsils, however, a wide spectrum of phenotypic manifestations is now appreciated with considerable variability in terms of clinical severity and organ involvement (9,10). Peripheral neuropathies are also a common complication and may be relapsing-remitting or chronic progressive (9,10). Tangier disease patients appear to have an increased risk of premature ASCVD, though not as pronounced as those with familial hypoalphalipoproteinemia (3,9,14). When the non-HDL-C levels are greater than 70mg/dL patients with Tangier disease are at higher risk of ASCVD whereas when the non-HDL-C levels are less than 70mg/dL ASCVD is low (9). Less common complications include corneal opacities and hematological manifestations such as thrombocytopenia and hemolytic anemia (9,10).

 

Individuals who are heterozygous for ABCA1 mutations have HDL-C levels that are variable but approximately 50% of normal with normal levels of preß-1 HDL but a deficiency of large α-1 and α-2 HDL particles (10). Cholesterol efflux capacity in heterozygotes has been reported as ~50% of normal. A mutation in one ABCA1 allele has been associated with increased risk of ASCVD in some studies and with no increase in ASCVD risk in other studies (15-20). Different mutations in ABCA1 result in varying HDL-C levels and phenotypes, which might explain the difference in ASCVD risk (21).

 

While Tangier patients manifest characteristically low HDL-C and Apo A-I, this lipid/lipoprotein phenotype is not adequate to make the diagnosis. ABCA1 gene sequence analysis is the preferred test to make the diagnosis of Tangier disease (10). Alternatively, non-denaturing two-dimensional electrophoresis followed by anti-apo A-I immunoblotting demonstrates only preβ1-HDL.

 

Currently, there is no specific treatment for Tangier disease (10). In fact, HDL-C raising therapies such as niacin and fibrates have proven ineffective in patients with this condition (22). Even HDL infusion was not beneficial (23). The major clinical issue in Tangier patients is disabling neuropathy; however, there is no effective intervention to manage this complication (10). Aggressive LDL-C lowering and treatment of other risk factors for atherosclerosis is recommended (10).

 

LCAT Deficiency  

 

LCAT is an enzyme that is bound primarily to HDL, with some also found on LDL (24,25). It facilitates cholesterol esterification by transferring a fatty acid from phosphatidyl choline to cholesterol (24,25). The hydrophobic cholesteryl esters are then sequestered in the core of the lipoprotein particles. LCAT is critical in the maturation of HDL particles. LCAT deficiency is an autosomal co-dominant disorder that manifests as either familial LCAT deficiency (FLD) or fish-eye disease in homozygotes (FED) (24,25). In FLD, mutations in LCAT lead to the inability of LCAT to esterify cholesterol in both HDL and LDL, whereas in FED, mutations in LCAT lead to the inability of LCAT to esterify cholesterol in HDL but the ability of LCAT to esterify cholesterol in LDL is preserved (24,25). Patients with FLD have virtually no cholesterol esters in the circulation while patients with FED have subnormal levels of cholesterol esters carried in apo B containing lipoproteins (24,25). Heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes.  

 

Individuals with FLD develop corneal opacities (“fish eye”), normochromic hemolytic anemia (due to cholesterol enrichment of red blood cell membranes), mild thrombocytopenia, and proteinuric end stage renal disease, which is the major cause of morbidity and mortality (24,25). The corneal opacities begin early in life and some patients may need corneal transplants. The rate of development of renal disease is variable but in a large cohort renal failure occurred at a median age of 46 years (26). Patients with FED generally only manifest corneal opacities (24,25).

 

The lipid and lipoprotein profile in patients with FLD usually demonstrates low HDL-C levels (frequently <10 mg/dL) (24,27). In one cohort patients with FED tend to have higher HDL-C levels but in a large systematic review HDL-C levels were similar in patients with FLD and FED (24,27). LDL-C levels tend to be low in FLD and FED while triglyceride levels are increased (24,27). Lipoprotein X (Lp-X) particles are present in patients with FLD but not in patients with FED (24). Lp-X is a multilamellar vesicle with an aqueous core. It is primarily composed of free cholesterol and phospholipid with very little protein (albumin in the core and apolipoprotein C on the surface) and cholesteryl ester.

 

Given the association of Lp-X and kidney disease only with FLD (and not FED) and animal studies demonstrating the nephrotoxicity of Lp-X, it is likely that increased levels of Lp-X results in renal dysfunction in patients with FLD (25,28). Lp-X particles accumulate in the mesangial cells in the glomerulus and are thought to induce inflammation and breakdown of the basement membrane leading to proteinuria. It is notable that after renal transplantation in patients with FLD there is recurrence of renal damage in the transplanted kidney (26).

 

It is unclear as to whether LCAT deficiency is associated with an increased risk of ASCVD (25,29). Atherosclerosis imaging studies have yielded divergent data and the number of patients with FLD or FED studied is limited (25,29). In one study carriers of FLD mutations (i.e., heterozygotes) had a decrease in ASCVD while carriers of FED mutations had an increase in ASCVD (30). This may have been due to higher LDL-C levels in the carriers of FED mutations (30).

 

Current management of FLD focuses on managing the renal dysfunction. The associated kidney disease is traditionally managed with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and a low-fat diet (25). Whether lipid lowering drugs are beneficial is unknown. Possible future therapies include enzyme replacement therapy with recombinant human LCAT, liver-directed LCAT gene therapy, small peptide or molecule activators of LCAT, and HDL mimetics (31,32). Infusions of recombinant human LCAT has improved the anemia and most parameters of renal function in a patient with FLD (33). Administration of CER-001, an apolipoprotein A1 (apoA-1)–containing HDL mimetic, has been shown to have beneficial effects on kidney and eye disease in a patient with LCAT deficiency (34).  

 

Approach to the Patient with Low HDL-C Levels

 

When encountering a patient with very low HDL-C levels it is important to first determine if this is a new abnormality or has been present for a long time. If prior HDL-C levels are normal, this excludes a primary monogenic etiology. If the decrease in HDL-C is new, one should consider the possibility of very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, paraproteinemia, anabolic steroids, or a paradoxical response to PPAR agonists. Marked hypertriglyceridemia can also lead to very low HDL-C levels. 

 

In a patient with long-standing very low HDL-C levels without an identifiable secondary cause, one should consider a monogenic etiology. To evaluate potential monogenic causes, a detailed family history, with attention to HDL-C levels, is important. Obtaining lipid levels from relatives is very helpful. A focused physical examination, with particular attention to the skin, eyes, tonsils, and spleen may point to a specific monogenic disorder. Plasma apo A-I levels should be obtained. Individuals with familial hypoalphalipoproteinemia deficiency have undetectable plasma apo A-I. Patients with Tangier disease demonstrate very low apo A-I levels (<5 mg/dL). LCAT deficiency is associated with apo A-I levels that are low but substantially higher than the other monogenic etiologies. Patients with LCAT deficiency also have a higher ratio of free: total cholesterol in plasma and measurement of plasma free (unesterified) cholesterol can be helpful. Two-dimensional gel electrophoresis of plasma followed by immunoblotting with antibodies specific for apo A-I separates lipid-poor preß-HDL from lipid-rich–HDL and can be helpful in differentiating these disorders. Genetic analysis is indicated when a monogenic disorder is suspected.

 

HIGH HDL-C CONDITIONS (HYPERALPHALIPOPROTEINEMIA)

 

An elevated concentration of apo A-I and apo A-II is called hyperalphalipoproteinemia (HALP). HALP is classified as moderate (HDL-C levels between 80 and 100 mg/dL) or severe (HDL-C levels > 100 mg/dL). While it is well recognized that high HDL-C levels are associated with a decrease in ASCVD it should be noted that very high HDL-C levels are paradoxically associated with an increase in ASCVD (35,36).

 

HALP is a heterogeneous condition caused by a variety of genetic and secondary conditions (for example ethanol abuse, primary biliary cirrhosis, multiple lipomatosis, emphysema, exercise, and certain drugs such as estrogens). In many individuals, the very high HDL-C levels have a polygenic origin (5,37). Given the focus of this chapter, monogenic causes of HALP will be reviewed. Monogenic HALP includes CETP deficiency, hepatic lipase deficiency, endothelial lipase deficiency, and loss of function mutations in SRB1. Despite epidemiology that demonstrates an inverse relationship between HDL-C and ASCVD risk, some forms of familial HALP are paradoxically associated with increased cardiovascular risk.

 

HALP is generally identified incidentally after routine assessment of a lipid profile as it is not usually associated with any signs or symptoms. Generally, patients are asymptomatic and no medical therapy is required.

 

Cholesterol Ester Transfer Protein (CETP) Deficiency

 

CETP transfers cholesteryl esters from HDL particles to triglyceride rich lipoproteins and LDL in exchange for triglycerides (11). Individuals who are homozygous for CETP variants have very high HDL-C levels (>100mg/dL) while heterozygotes have moderately increased HDL-C levels (38-41). LDL-C and apo B levels may be normal or modestly decreased. The increase in HDL cholesterol are largely due to the accumulation of cholesterol esters (39). The decrease in LDL-C is due to the failure of cholesterol ester transport from HDL to apo B containing lipoproteins. There is a predominance of small LDL particles. Individuals who are heterozygotes for CETP mutations show modestly elevated HDL-C levels (38,39). In Japanese individuals with HDL-C levels > 100mg/dL 67% were demonstrated to have CETP gene mutations (42). CETP deficiency is the most important and frequent cause of HALP in Japan. CETP deficiency is common in other Asian populations but is relatively rare in other ethnic groups (39). Despite extensive studies the effect of CETP variants on the risk of ASCVD is uncertain (38-40,43). A variety of studies have indicated that a decrease in LDL-C and non-HDL-C levels (i.e. pro-atherogenic lipoproteins) rather than an increase in HDL-C induced by CETP variants underlies a potential beneficial effect on ASCVD (44).  

 

Endothelial Lipase (EL) Deficiency

 

Endothelial lipase (EL) is encoded by the LIPG gene and hydrolyzes phospholipids on HDL resulting in smaller HDL particles that are more rapidly metabolized (11). Genetic variants in LIPG have been identified Iin individuals with elevated HDL-C levels (38,39). As one would predict large HDL particles enriched in phospholipids are observed in individuals deficient in EL (39). Whether variants in LIPG leading to decreased EL activity and increased HDL-C levels reduces ASCVD risk is uncertain (38-40).

 

Hepatic Lipase (HL) Deficiency

 

Hepatic lipase (HL) is encoded by the LIPC gene and mediates the hydrolysis of triglycerides and phospholipids in intermediate density lipoproteins (IDL) and LDL leading to smaller particles (IDL is converted to LDL; LDL is converted from large LDL to small LDL) (11). It also mediates the hydrolysis of triglycerides and phospholipids in HDL resulting in smaller HDL particles (11). Several case reports of families with elevated HDL-C levels (HALP) caused by a genetically defined HL deficiency have been described (39,40). HL deficiency may also be associated with elevated triglycerides and cholesterol with increased intermediate density lipoproteins (IDL) (40,45). Several HL deficient individuals had premature ASCVD likely due to increased levels of apo B containing lipoproteins (40,45). Heterozygotes do not appear to have discrete lipoprotein abnormalities (45).

 

Scavenger Receptor Class B Type I (SR-BI)

 

Scavenger receptor class B type I (SR-BI) is encoded by the SCARB1 gene and facilitates the selective uptake of the cholesterol esters from HDL into the liver, adrenal, ovary, and testes (11). In macrophages and other cells, SR-B1 facilitates the efflux of cholesterol from the cell to HDL particles (11). SR-B1 deficient mice have an increase in atherosclerosis despite elevated HDL-C levels (46). Mutations in SCARB1 associated with decreased SR-B1 have been observed in individuals with high HDL-C levels (47-49). Heterozygotes have intermediate elevations of HDL-C between wild-type and homozygous individuals. Studies have suggested that some but not all mutations in SCARB1 result in an increased risk of ASCVD despite increased HDL-C levels (40,49). A decrease in adrenal function has been reported in some individuals with SCARB1 mutations likely due to a reduced ability of SR-B1 to facilitate cholesterol uptake into the adrenal glands (48,50). Abnormalities in platelet function have also been observed in some patients (50).

 

ACKNOWLEDGEMENTS

 

This work was supported by grants from the Northern California Institute for Research and Education.

 

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Pharmacological Causes of Hyperprolactinemia

ABSTRACT

 

Hyperprolactinemia represents a multifaceted endocrine disorder with both physiological and pathological causes. The increased use of anti-psychotic and anti-depressant medications has increased the role pharmaceutical agents play in inducing hyperprolactinemia, being the most frequent cause of hyperprolactinemia in clinical practice. This has particularly impacted females, who demonstrate a higher susceptibility to drug-induced hyperprolactinemia. Of these medications, anti-psychotics, neuroleptic-like medications, anti-depressants, and histamine receptor type 2 antagonists, emerge as the most prominent culprits. Furthermore, opioids, some anti-hypertensive agents, proton pump inhibitors, estrogens, and other less potent hyperprolactinemia-inducing medications are recognized as potential contributors to drug-induced hyperprolactinemia. Many herbal medicines are reported as lactogenic, but their ability to cause hyperprolactinemia remains unclear. This review endeavors to elucidate the intricate mechanisms underlying the induction of hyperprolactinemia by pharmacological agents. We have included available data on the prevalence and extent of drug-induced changes in prolactin levels. We have also included data on herbal agents. We have highlighted where controversial data are identified. Although a detailed exploration of how these medications impact prolactin regulation is beyond the scope of this chapter, this review aims to deepen our understanding of the interplay between pharmacological agents and their effects on prolactin levels, contributing to valuable insights, refined therapeutic approaches, and better patient care.

 

INTRODUCTION

 

The most common cause of consistently high prolactin levels is drug-induced hyperprolactinemia. The overall incidence is higher in women compared to men. Drug-induced causes tripled during the 20-year follow-up period, reflecting the increased prevalence of psychoactive drug use (1). Several drugs have been reported to induce hyperprolactinemia, either by inhibiting dopamine receptors or their actions or by directly stimulating prolactin secretion (2).

 

High levels of prolactin can be attributed to various physiological factors, such as pregnancy and breast-feeding, while minor increases in prolactin levels may also occur during ovulation, after sexual intercourse, during periods of stress, exercise, after food intake, or in association with irritation of the chest wall and breast stimulation. Pathological causes can be related to hypothalamo-pituitary disorders or non-hypothalamo-pituitary disorders. Hypothalamo-pituitary disorders include prolactin-secreting pituitary tumors (including lactotroph tumors, mammo-somatotroph tumors, mature pluri-hormonal PIT1-lineage tumors, immature PIT1-lineage tumors, acidophil stem cell tumors, multi-hormonal pituitary tumors, mixed somatotroph, and lactotroph tumors, and pluri-hormonal tumors) (3); hypothalamic and pituitary stalk compression or damage (non-prolactin-secreting pituitary adenomas, craniopharyngiomas, meningiomas, germinomas, granulomas, metastasis, Rathke cleft cysts, hypophysitis, radiation, surgery, and trauma); infiltrative pituitary disorders; pituitary hyperplasia (McCune-Albright, Carney complex, X-LAG). Other causes include primary hypothyroidism; adrenal insufficiency; systemic diseases such as chronic renal failure and liver cirrhosis; polycystic ovary syndrome; neurogenic causes (chest wall trauma or surgery, herpes zoster); seizures; untreated severe phenylketonuria; pseudocyesis (false pregnancy); autoimmune diseases (lupus, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, Behcet’s disease, polymyositis); cancers (breast, ovarian, colon, hepatocellular) (4). During the diagnostic process of hyperprolactinemia, it is crucial to consider the possibility of macroprolactinoma and the ‘hook’ effect, although the latter is usually not relevant in drug-induced cases (5).

 

Drug-induced hyperprolactinemia is often characterized by prolactin levels ranging from 25 to 100 ng/mL (530-2130 mIU/L). However, certain medications including metoclopramide, risperidone, amisulpride, and phenothiazines can lead to prolactin levels surpassing 200 ng/mL (4255 mIU/L) (6). On similar doses of prolactin-raising anti-psychotics, women with chronic use are more likely to develop hyperprolactinemia than men, reaching significantly higher prolactin levels, with mean levels of 50 ng/mL (1065 mIU/L) (7,8). Younger age was associated with higher prolactin levels in women, but not in men (8,9). Route of drug administration is important, with prolactin levels returning to normal after cessation of the drug: within 2-3 weeks after stopping oral treatment, but no sooner than 6 months after discontinuation of intramuscular depot administration (10).

 

This chapter will encompass a comprehensive discussion of all pharmacological causes as well as some alternative factors contributing to changes in prolactin levels especially hyperprolactinemia.

 

EPIDEMIOLOGY

 

Drug-induced hyperprolactinemia is the most common cause of consistently high prolactin levels. A retrospective follow-up study conducted in Scotland, involving 32,289 hyperprolactinemic individuals from 1993 to 2013, concluded that within the non-pregnancy-related group, the most prominent cause was drug-induced hyperprolactinemia (45.9%), followed by pituitary disorders (25.6%), macroprolactinoma (7.5%), and hypothyroidism (6.1%). Nevertheless, 15% of cases were deemed idiopathic. The overall incidence was higher in women aged 25-44 years old compared to men (1). Female predominance is reported in other studies with a female: male ratio of 5.9:1 and the mean age at diagnosis of hyperprolactinemia is 40 (range 14–85) years (2,11).The position of hyperprolactinemia as a side effect of medications has been assessed in a French Pharmacovigilance Database from 1985 to 2000, which reported 159 cases of hyperprolactinemia out of 182,836 adverse drug reactions (11). The rates of hyperprolactinemia related to therapeutic drug classes were recorded as 31% associated with anti-psychotics, 28% with neuroleptic-like drugs (medications with a similar mechanism of actions as neuroleptics, but used for different purposes, for example movement disorders or anti-emetics), 26% with anti-depressants, 5% with histamine receptor type 2 (H2-receptor) antagonists, and 10% with other drugs.

 

PROLACTIN CONTROL MECHANISMS

 

Prolactin is a polypeptide primarily produced in the anterior pituitary gland, with secondary production occurring in other tissues such as the gonads, mammary gland, endometrium, prostate, lymphocytes, hematopoietic cells, skin, brain, retina, inner ear cochlea, decidua, pancreas, liver, endothelium, and adipose tissue (12–14). In breast and prostate cancer, prolactin has even been proposed as a tumor marker (15,16). Prolactin acts through prolactin receptors (PRLR), which belong to the family of cytokine receptors associated with the non-receptor tyrosine Janus kinase 2. PRLR can activate the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway, MAPK (mitogen-activated protein kinase), PI3 (phosphoinositide 3-kinase), Src kinase, as well as the Nek3 / Vav2 / Rac1 serine / threonine kinase pathway (17). There are different isoforms of this receptor: a long isoform, intermediate isoform, 2 short isoforms S1a and S1b which are formed by alternative splicing and partial deletion of exons 10 and 11, and soluble PRLR (18). These different isoforms are expressed in different tissues, mostly studied in rats. The long isoform is mainly expressed in the adrenal glands, kidneys, mammary glands, small intestine, bile ducts, choroid plexus, and pancreas whereas the short isoform is in the liver and ovaries (19). Prolactin possesses nearly 300 functions apart from lactation including neuroprotection and neurogenesis, offspring recognition by both parents, adipose and weight homeostasis, islet functions, immune regulation, angiogenesis, osmoregulation, and mitogenesis (20).

 

The secretion of prolactin produced by lactotroph cells in the anterior pituitary gland has a circadian rhythm with higher levels during sleep and lower levels during wakefulness (21). Even though pulsatility frequency does not significantly change over 24 hours, the amplitude of pulses is higher during night and day sleep, while wakefulness is associated with an immediate offset of active secretion. Prolactin is lower during the rapid eye movement stage of sleep (22).

 

The synthesis and secretion of prolactin is under the complex control of peptides, steroid hormones, and neurotransmitters, which can act as inhibitory or stimulatory factors, either by a direct effect on lactotroph cells or by indirect pathways through inhibition of dopaminergic tracts, and are widely studied in mammals (2). Dopamine plays a crucial role in inhibiting prolactin secretion. Dopamine can bind the five types of dopamine receptors (G-protein coupled receptors): DRD1, DRD2, DRD3, DRD4 and DRD5, while lactotroph cells express mainly D2 receptors. Dopamine can reach the pituitary via three pathways (Figure 1): through the tuberoinfundibular dopaminergic (TIDA) system, the tuberohypophysial tract (THDA), and the periventricular hypophyseal (PHDA) dopaminergic neurons (23). TIDA neurons originate from the rostral arcuate nucleus of the hypothalamus and release dopamine into the perivascular spaces of the medial eminence and through long portal vessels dopamine reaches the anterior pituitary gland. The THDA neurons originate in the rostral arcuate nucleus and project into the medial and posterior pituitary lobes and release dopamine at these sites. From THDA tract dopamine then reaches lactotroph cells through the short portal vessels (24). PHDA neurons originate in the periventricular nucleus and axons terminate in the intermediate lobe and dopamine release follows the same direction as from the THDA neurons. Prolactin-inhibiting neurons are considered to be a functional unit working synchronously (23). The binding of dopamine to D2 receptors on the plasma membrane of lactotroph cells inhibits prolactin protein, PRL gene transcription, as well as lactotroph proliferation (24). The release of prolactin through exocytosis of prolactin secretory granules is influenced by dopamine through various pathways. Specifically, D2 receptors are coupled with pertussis toxin-sensitive G proteins, which subsequently inhibit adenylate cyclase activity, resulting in decreased levels of cyclic adenosine monophosphate (cAMP) (25).

 

Additionally, the activation of potassium (K+) channels occurs, leading to a reduction in voltage-gated calcium (Ca2+) currents and inhibition of inositol phosphate production. Collectively, these intracellular signaling events culminate in a decrease in the concentration of free calcium ions (Ca2+) resulting in membrane hyperpolarization, ultimately inhibiting the exocytosis of prolactin from its granules (26). The inhibition of PRL gene transcription occurs when D2 receptors are activated, leading to the inhibition of MAPK or protein kinase C pathways. This activation results in a reduction of phosphorylation events on Ets family transcription factors. These transcription factors play a crucial role in the stimulatory responses of thyrotropin-releasing hormone (TRH), insulin, and epidermal growth factor (EGF) on prolactin expression. Moreover, the Ets family transcription factors interact with the PIT1 protein, which is essential for cAMP-mediated PRL gene expression (27). Dopamine exerts anti-mitogenic effects by activating D2 receptors through multiple pathways. These include the inhibition of MAPK (mitogen-activated protein kinase) signaling, protein kinase A signaling, and stimulation of phospholipase D activity. Additionally, dopamine engages a pertussis toxin-insensitive pathway, activates the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, and inhibits the AKT/protein kinase B pathway (28–31).

 

In addition to the dopaminergic inhibitory system, the γ-aminobutyric acid (GABA)-ergic tuberoinfundibular system, culminating in the median eminence, exhibits inhibitory properties, albeit of lesser potency compared to the dopaminergic system, while also having a role in prolactin modulation. GABA-B receptors are discernible both within the anterior pituitary gland, contributing to the maintenance of low prolactin levels, and in TIDA neurons which can be powerfully inhibited by GABA via hyperpolarization, consequently contributing to an elevation in prolactin levels (32,33).

 

Prolactin itself has two negative feedback effects: through ``short-loop feedback regulation`` it enhances the activity of TIDA neurons, where both long and short forms of the PRLR are expressed, with the long isoform being predominant in the arcuate and periventricular nuclei, regulating tyrosine hydroxylase (a rate-limiting enzyme in dopamine synthesis) leading to increase of dopamine release, which inhibits prolactin, as well as autocrine inhibition (34,35). Several other local factors influence prolactin release within pituitary gland as shown in Figure 1.

Figure 1. Prolactin – Central Nervous System Regulation.

 

Other prolactin inhibitory factors include somatostatin, acetylcholine, endothelins, gastrin, and growth hormone, while stimulatory factors include thyrotropin-releasing hormone (TRH) (as seen in primary hypothyroidism), angiotensin II, vasopressin, oxytocin, VIP, galanin, and estrogen.

 

Experiments conducted on rats to elucidate the relationship between the adrenergic system and the regulation of prolactin secretion have focused on stimulating or inhibiting α and β adrenergic receptors. Functional hyperprolactinemia is a complex hormonal interplay of stress-induced neuroendocrine changes involving the dopamine, serotonin and adrenergic systems (36,37). Evidence suggests that the mediobasal hypothalamus and preoptic-anterior hypothalamus harbor the primary adrenoreceptors (38,39). Injecting the α2 agonist clonidine into the mediobasal hypothalamus resulted in a dose-dependent increase in prolactin secretion. This effect was counteracted by the blockade of idazoxan (α2 antagonist). Similarly, the stimulation of prolactin release was induced by isoprenaline (β agonist) and notably attenuated by the β antagonist propranolol. The β2 agonist salbutamol also exhibited efficacy in stimulating prolactin secretion. Conversely, adrenergic agonists such as noradrenaline (mixed α and β), phenylephrine (α1), and tyramine (sympathomimetic) in the mediobasal hypothalamus, failed to elicit an effect on prolactin secretion.

 

Within the preoptic anterior hypothalamus, noradrenaline and adrenaline were found to stimulate prolactin secretion (40). However, the administration of the α1 agonist phenylephrine failed to stimulate prolactin, indirectly suggesting that the stimulatory effect of noradrenaline in the preoptic anterior hypothalamus is likely due to its action at α2 sites. α2 agonism has been shown to reduce the function of tuberoinfundibular dopaminergic neurons leading to increase prolactin production and secretion (41). Consequently, it was inferred that the activation of α2 and β adrenoceptors in the mediobasal hypothalamus and α2 adrenoceptors in the preoptic-anterior hypothalamus, proximal to prolactin-regulating neurons, leads to heightened prolactin secretion, while the action of α1 in the mediobasal hypothalamus may be inhibitory (42).

 

Cholinergic activation may have opposite roles in rodents and humans. Cholinergic agonists suppress prolactin release induced by morphine in rats, suggesting that the central cholinergic system has an inhibitory effect on the prolactin release triggered by morphine or β-endorphine, but this cholinergic inhibition does not occur through catecholaminergic neurons (43). Conversely, in humans, cholinomimetic drugs can increase prolactin levels associated with raised plasma β-endorphin, suggesting a stimulatory interplay of cholinergic factors and endogenous opioids on prolactin levels (44), although circulating opioids may not directly relate to central levels.

 

TIDA neurons express estradiol and progesterone receptors. Estradiol action leads to reduced secretion of dopamine into the portal blood system and mediates a prolactin surge. Progesterone, in addition, suppresses dopamine release being responsible for the plateau phase of the surge (23). Estrogen specifically affects prolactin synthesis by influencing lactotroph cell sensitivity, expression of pituitary dopamine receptor downregulation, and the expression of the prolactin receptor gene (2,34). Ghrelin, a hormone involved in metabolic balance, directly stimulates prolactin secretion at the pituitary level (45).

 

Tachykinins (substance P, neurokinins A, and B, neuropeptide K, neuropeptide ϒ) can act directly on the lactotroph cell and indirectly within the hypothalamus or posterior pituitary. They have a multifaceted impact on prolactin secretion, with both stimulatory and inhibitory effects. They can stimulate prolactin secretion by stimulating and potentiating the release of oxytocin, vasopressin, TRH, VIP, serotonin and glutamate, and by inhibiting GABA. Tachykinins through paracrine actions can directly increase prolactin within the anterior pituitary. They can also increase dopamine but the overall effect is prolactin elevation. Under specific circumstances, the stimulation of dopamine release can be prominent leading to a decrease in prolactin (46). Endogenous opioids are involved in regulating prolactin secretion, particularly during stressful situations, by reducing the activity of tuberoinfundibular dopaminergic neurons mediated by μ-, κ‑, and δ- opioid receptors, resulting in increased prolactin release (47). Prolonged nicotine exposure has been associated with desensitization of dopamine receptors, diminished dopamine turnover, and a decrease in their abundance within the nigrostriatal pathways (48). These alterations have been suggested to contribute to a diminished prolactin response to opiate blockade observed in individuals who smoke. Similar to opioids, histamine has been shown to induce prolactin production predominantly through inhibiting dopaminergic and stimulating serotoninergic and vasopressin-ergic neurons (49).

 

Serotoninergic pathways originating from the dorsal raphe nucleus play a physiological role in mediating nocturnal surges and suckling-induced prolactin rises through a serotonin interaction via serotonin type 1 and 2 receptors (5-hydroxytryptamine receptors, 5HT1, and 5HT2). 5-HT could either release a PRL-releasing factor or inhibit dopamine release. The paraventricular nucleus, where serotoninergic pathways terminate, contains postsynaptic serotonin 5-HT1A, 5-HT2, and 5-HT2C receptor subtypes, and possibly 5-HT3 receptors (50). It was shown that the prolactin-releasing effect of serotonin probably occurs mostly via 5-HT1C / 2 receptors because ritanserin (an elective 5-HT1C / 2 receptor antagonist) opposed this effect (51). Serotonin stimulation of prolactin–releasing factor (PRF) neurons in the paraventricular nucleus leads to PRF release (like VIP and oxytocin) mediating hyperprolactinemia. Moreover, serotoninergic stimulation of GABAergic neurons in the tuberoinfundibular-GABA system has been shown to inhibit TIDA cells which contain 5-HT1A receptors, therefore inhibiting dopamine synthesis/release resulting in increased prolactin secretion (52).

 

Oxytocin, through the posterior pituitary and vasoactive intestinal peptide (VIP) in the anterior pituitary, play significant roles in enhancing PRL gene transcription and modulating dopamine inhibition. Animal studies suggest a potential mediation of VIP by oxytocin to stimulate prolactin secretion (53).

 

The extensive hormonal regulation of prolactin renders it susceptible to various disturbances caused by different classes of medications.

 

CLINICAL CHARACTERISTICS

 

Persistent hyperprolactinemia is associated with disturbances of the gonadal axis leading to interruptions of gonadotrophin-releasing hormone pulsatility and inhibition of luteinizing hormone and follicle-stimulating hormone release (54). Clinical manifestations attributed to hyperprolactinemia predominantly stem from the suppression of the gonadal axis. In premenopausal women, a spectrum of menstrual cycle dysfunctions is observed, spanning from luteal phase shortening to complete amenorrhea, often correlating with elevated prolactin levels. Secondary amenorrhea can be due to hyperprolactinemia in up 30% of patients, and up to 75% of patients with amenorrhea and galactorrhea (55). Beyond these effects, an array of hypoestrogenic indicators may manifest, including symptoms like vaginal dryness, diminished libido, and decreased energy levels. Galactorrhea can be present in up to 80% of females (55,56). In men, the impact of hyperprolactinemia is manifested through a decrease in libido, ranging from diminished sexual desire to oligospermia or even azoospermia attributed to hypogonadotropic hypogonadism. Notably, erectile dysfunction may arise, primarily attributed to the direct inhibitory influence of dopamine, and can be potentially reversed through the administration of dopamine agonists (57). Gynecomastia, on the contrary, is a manifestation of secondary hypogonadism rather than elevated prolactin levels, whereas galactorrhea is rare in men (21).

 

In both genders infertility can be observed, with diminished bone mineral density. In females, bone mineral density is significantly decreased in women with amenorrhea and increases during treatment and menstrual cycle restoration (58). Additionally, in cases where hyperprolactinemia is attributed to a mass, accompanying clinical indications may encompass headaches, visual field disturbances, cranial nerve palsies, and hypopituitarism. Notably, these manifestations may be the only clinical features in post-menopausal women (21,59).

 

PSYCHOTROPIC MEDICATIONS

 

Anti-psychotics and neuroleptic-like drugs are psychotropic medications which primarily exert their anti-psychotic effects through the blockade of DRD2 and D4 receptors in the mesolimbic area. Newer classes of anti-psychotics block 5HT2 and sometimes noradrenergic α1 or α2 receptors (4,35,60). Blockade of D2 receptors in the hypothalamic tuberoinfundibular system and lactotroph cells results in disinhibition of prolactin secretion leading to hyperprolactinemia, being the most common drugs known to induce hyperprolactinemia (61) (Figure 2). On the contrary, strong binding to D2 receptors can extend the half-life of dopamine by approximately 50%. This effect is achieved through two primary mechanisms: direct blockade of the dopamine transporter (DAT) and antagonism of D2 autoreceptors. These processes collectively result in reduced reuptake of dopamine, prolonging its presence in the synaptic area and further stimulating an upregulation of receptors. However, it is important to note that chronic use of anti-psychotics can lead to the reversal of upregulation of DAT (mRNA and protein), potentially contributing to treatment resistance and potentially lower prolactin elevations in the long term. Nonetheless, it is worth mentioning that anti-psychotics typically exhibit a lower affinity for dopamine transporter blockade compared to selective DAT blockers such as nomifensine (62).

Figure 2. Mechanisms of drug-induced hyperprolactinemia with selected examples (adopted from La Torre et al. (2). In addition to opiates, cholinomimetics, PPIs and smoking indirectly also stimulate the opioid receptors. PPIs, Protein pump inhibitors; TCAs, tricyclic anti-depressants; MAO, monoamine oxidase; SSRIs, selective serotonin reuptake inhibitors; SNRIs, serotonin-noradrenaline reuptake inhibitors.

 

The potency of anti-psychotics and neuroleptic-like medications to induce a rise in prolactin levels varies (Table 1). The level of prolactin increase depends on the anti-psychotic drug (different affinity and selectivity for dopamine receptors; blood-brain barrier penetrating capability, degree of serotoninergic inhibition), the dose administered, and the patient's age and sex (34,35). Lastly, polymorphisms in genes related to dopamine receptors (such as DRD1, DRD2, DRD3) (63), dopamine transporters (SLC6A3), and dopamine-metabolizing enzymes (such as monoamine oxidase and catechol-O-methyltransferase) have been associated with individual variations in response to anti-psychotic treatment and the development of side effects, including hyperprolactinemia (64).

 

Although direct evidence establishing the involvement of adrenergic receptors in hyperprolactinemia caused by antipsychotic and antidepressant medications remains unproven, indirect indications, as elucidated in Figure 1, suggest the potential implication of these receptors. It is plausible that adrenergic receptors might play a partial role in the hyperprolactinemia induced by these medications.

           

Table 1. Medications and Their Ability to Cause Hyperprolactinemia

Cluster Name

Subclass mechanism of action

Medications

Prolactin increment

Frequency of prolactin increment (61,65)

Anti-psychotics

First generation anti-psychotics

Antagonize/block dopamine receptors, especially D2 receptors. Can block α1 adrenergic receptors.

Butaperazine

UP to 2-3-fold normal range with dozes 60 mg/daily. Higher in women (66)

High

Chlorpromazine

Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment) (67)

Moderate/ High

 

Flupenthixol

Up to 2-3-fold during the first month, and normalization in the next few months (68)

High

Fluphenazine

Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment (67). Up to 40-foldof the upper end of the normal range (69)

High

 

 

Haloperidol

Up to 9-fold at the beginning of treatment (3-fold in long-term treatment) (70)

High

Loxapine

Up to 3-foldof the upper end of the normal range in women (66)

Moderate

Perphenazine

Up to 40-fold of the upper end of the normal range (69)

Moderate

Pimozide

?

Moderate

Prochlorperazine

?

?

Promazine

Up to 4-fold of the upper end of the normal range (69)

 

Thiordiazine

Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment) (67)

High

Thiothixene

Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment (71)

Moderate/ High

 

Trifluoperazine

?

Moderate

Veralipride

Up to 10 time increment, transient (72)

High

Zuclopenthixole

?

?

Second generation anti-psychotics

Dopamine receptors blockade especially D2 receptors, serotonin (5-HT) receptor blockade, glutamate modulation, can antagonize α1 or α2 adrenergic receptors and histamine receptors.

Amisulpiride

 

Up to 10-fold at the beginning of treatment and remained elevated during treatment but lower levels (68)

Case reports

Aripiprazole

Reduce prolactin levels (73)

Case reports / No effect/ Reduced prolactin

Asenapine

Up to 2-fold increment and rarely with higher doses up to 4-fold (35)

Low, Moderate

 

 

Brexpiprazole

Mild increment (74)

 

Low

Clozapine

Mild (up to 2-fold) and transient (75)

 

Case reports or No effect

Iloperidone

Mild increment, transient (76)

Case reports or No effect

Levosulpiride

Up to 15-fold normal range (77)

Case reports / Moderate for galactorrhea (78)

 

Lurasidone

Up to 10-fold normal range (79)/ no effect (80)

Case reports or No effect

Molindone

?

Moderate

 

Olanzapine

Mild (up to 2-fold) and transient (75)

Low

 

Paliperidone

2-10-fold for depot formulations (81)

High

Perospirone

None (82)

None/ Case reports

Quetiapine

Mild and transient (75)

Low

 

Risperidone

2-10-fold

High (83)

 

Sertindole

Mild and transient (75)

?

Sulpiride

Up to 6-7-fold from baseline, dose dependent effect (84)

High

 

Thiethylperazine

?

?

Ziprasidone

Up to 4-fold from baseline and transient (35,75)

Low

 

Neuroleptic-like medications

 

Block D2 receptors

Domperidone

Up to 10-fold (85,86)

High

Droperidol

Significant increment after 10 minutes of administration, with peak at 20 minutes (87)

?

Metoclopramide

Up to 15-fold (2)

High

 

Anti-depressants

TCAs

Block the reuptake of both serotonin and noradrenaline.

Amitriptyline

2-fold increment on dosage 200/300mg (88)

Low

Amoxapine

3,5-fold to baseline (89)

High

Clomipramine

Up to 3-fold increment from baseline (90)

High

Desipramine

Just above the normal limit with 100 mg oral administration (91)

Low, Controversial

Imipramine

Up to 4-fold normal range (69)

Controversial

Nortriptyline

2-fold in the first 2 weeks in one patient (88)

None or Low

SSRI

Block the reuptake of serotonin.

Citalopram/

Escitalopram

Up to 3-fold increment (52)

None or Low (rare reports), Controversial data

Fluoxetine

Fluvoxamine

Paroxetine

Sertraline

SNRI

Block the reuptake of both serotonin and noradrenaline.

Duloxetine

Up to 2-fold normal range (92)

Case reports

Milnacipran

Not increased risk of hyperprolactinemia (93)

None

Venlafaxine

Up to 2-fold normal range, dose related (94)

Case reports

MAO inhibitors

Inhibit the enzyme. Monoamine oxidase, which breaks down serotonin, noradrenaline, and dopamine, though increasing their levels.

Clorgyline

Up to 2-fold from baseline (95)

Low

Pargyline

Up to 3-fold from baseline (95)

Low

Phenelzine

Unclear elevation, galactorrhea (96)

Low/ Case reports

Atypical anti-depressants

Inhibit noradrenaline and dopamine reuptake.

Bupropion

No significant change (80)

Case reports

Increases the release of both serotonin and noradrenaline.

Mirtazapine

No significant change (80)

Case reports

Serotonin modulators

Modulate serotonin receptors in the brain to enhance serotonin transmission.

Indoramine

 (97)

Case report

Nefazodone

Mild increment from baseline only at acute administration (98)

None/ Case reports

Trazodone

Up to1.5-fold from baseline (99)

None, Low

Vortioxetine

Up to 2-fold elevation (100)

Case reports

Selective noradrenaline reuptake inhibitor

Inhibit reuptake of norephinephrine.

Reboxetine

Up to 2-fold from baseline (101)

Case reports

NMDA receptor antagonist

Block NMDA receptors though influencing glutamate neurotransmission.

Esketamine

?

None

Gastric acid reducers

H2 receptor antagonists

H2 receptor antagonists.

Cimetidine

Up to 3-fold after 400 mg IV infusion (102)

Low

Ranitidine

Mild increment only in high IV doses (103)

Low

Protein pump inhibitors (PPIs)

Inhibit the activity of the proton pump (H+/K+ ATPase) in the stomach's parietal cells.

Esomeprazole

?

Case reports or No effect

Lansoprazole

4-fold increment from baseline (104)

Omeprazole

No significant change (105)

Pantoprazole

No significant change (106)

Rabeprazole

No significant change (107)

Opioids
 

They activate opioid receptors. Main types of opioid receptors: mu (μ), delta (δ), and kappa (κ).

Apomorphine

By acting as dopamine agonist it lowers prolactin (108)

None

Heroin

Elevated in addiction (within normal range) compared to healthy control or during abstinence (109)

Moderate in addicted patients that have values over 25 ng/mL

Methadone

Mild increment, transient increases for several hours following the administration (110)

?

Morphine

Up to 2-fold increment from baseline (111)

High

Antihypertensives
 

It decreases the release of noradrenaline.

Methyldopa

3-4-fold (65) up to 40-fold normal range (69)

Moderate

Inhibit the storage of neurotransmitters like noradrenaline and serotonin in nerve cells, though decreasing their release.

Reserpine

2.5-fold increment from baseline (112) Up to 40-fold normal range (69)

High

Block calcium channels in cardiac and smooth muscle cells.

Verapamil

2-fold (113)

Low

Estrogens
 

By using as contraceptives they suppress sexual axis.

Estradiol infusion

3-4-fold, dose-dependent, way of administration is important (oral and IV) (114)

Low

 

Estradiol withdrawal

?

 

Gonadotropins and GNRH agonists
 

Same as endogenous components, used for fertility induction.

hCG

Up to 4-fold increment, transient (115)

High

hMG

Up to 2.7-fold increment from baseline, transient (116)

High, Transient

GnRH agonist.

Leuprolide acetate

1.5-fold higher prolactin in compared to hMG alone, transient (117)

High

Other drugs

Benzodiazepines

Enhances the effects of GABA in the brain.

Diazepam

Mild, dose-dependent (118)

Controversial

Anxiolytics

Serotonin receptor agonist.

Buspirone

2-fold (119)

Case report or No effect

α-2 adrenergic agonist.

Clonidine

?

Case reports

Anticonvulsant

Block sodium channels in nerve cells.

Carbamazepine

Less than 2-fold in sleep entrained (120,121)

?

Phenytoin

Controversial, it can also lower prolactin levels (122)

?

Enhances the effects of GABA in the brain.

Phenobarbital

Controversial (123)

  

Valproic Acid

Controversial, it can also lower prolactin (124)

Case reports

Mood stabilizer

Decrease dopamine release and glutamate, increase GABA inhibition.

Lithium Carbonate

Controversial, no effect (183)

None

Antimigraine medication

Calcium channel blocker.

Flunarizine

Mild increment, up to 1.5-fold from baseline (125)

Case reports

Weight loss medications

Increase the release of serotonin and inhibit its reuptake.

Fenfluramine

Mild increment within normal range in previews non-hyperprolactinemic patients (126)

High

Inhibit the reuptake of serotonin, noradrenaline, and dopamine.

Sibutramine

4-fold (2,127)

Case report

Anticholinesterase inhibitors

Reversible acetylcholinesterase inhibitor.

Physostigmine salicylate

Less than 100 ng/mL (44).

Low

Prokinetic medication

Stimulate serotonin receptors in the gut.

Cisapride

High increment (up to 200 ng/dL) but in co-administration of other drug inducing hyperprolactinemia (128)

Case reports

Antihistaminic with sedative and antiemetic properties

Block histamine receptors.

Promethazine

?

?

Central Nervous System Stimulants

Increase the release and reduce the reuptake of noradrenaline and dopamine in the brain.

Amphetamine

Mild, only during withdrawal (129)

?

Methylphenidate

No effect (130)

Case reports/ No effect

ADHD medication

α -2 adrenergic agonist.

Guanfascine

Controversial, it can also lower prolactin (131)

Case reports

Decongestant

Sympathomimetic amine, predominantly α-1 agonist

Pseudoephedrine

Lower prolactin levels (132)

Case reports

Rheumatoid arthritis medications

Reduce inflammation, modify immune response.

Bucillamine

Mild increment within normal range (133)

Case report

Penicillamine

?

Case reports

Osteoporosis medication

Monoclonal antibody that inhibit the receptor activator of nuclear factor kappa-B ligand (RANKL).

Denosumab

?

Case reports

Substance of abuse

Blocks the reuptake of noradrenaline, dopamine, and serotonin in the brain.

Cocaine

Decrease prolactin levels (134)

Mild increment only during withdrawal (129)

Case reports

Increases the release and inhibits the reuptake of serotonin and to some extent, dopamine and noradrenaline.

Ecstasy

Mild or no effect (135)

?

Stimulates nicotinic acetylcholine receptors, leading to the release of neurotransmitters like dopamine and noradrenaline.

Smoking

Mild increment, transient (136)

Moderate

Anti-HIV medications

Protease inhibitors that prevent the cleavage of viral proteins and thereby inhibiting viral replication.

Ritonavir / Saquinavir

Mild (137)

Case reports

Radiotherapy

Use of high-energy radiation to damage the DNA within the targeted cells.

Intracranial radiotherapy

?

Moderate (138)
             

Frequency of increase to abnormal prolactin levels with chronic use: high: >50%; moderate: 25 to 50%; low: <25%; none or low: case reports. The effect may be dose-dependent. Drugs marked with blue have controversial data or decrease prolactin levels as explained in the table. Where we could not identify reliable data for the parameters in the table we added a question mark. *First-generation anti-psychotics, non-selective dopamine receptors antagonists. **Second-generation anti-psychotics.

 

Anti-Psychotics

 

Anti-psychotics are traditionally classified as first- and second-generation, but more recently a new classification taxonomy has been developed by McCutcheon et al. to express different receptor affinity of different anti-psychotics. Due to the impossibility to include in this new classification all drugs that cause hyperprolactinemia, we have used the old classification (Table 1) (139,140).

 

The first-generation anti-psychotics are typically associated with more severe hyperprolactinemia (2-3-fold increment), whereas second-generation drugs have lower D2 affinity and stronger blockade of 5HT2A receptors leading to milder prolactin elevations (1-2-fold), except risperidone, paliperidone, and amisulpiride. Amisulpiride has the greatest potential to cause hyperprolactinemia of all anti-psychotics (4).

 

The first-generation anti-psychotics, such as fluphenazine and haloperidol, act as non-selective dopamine receptors antagonists (2,10). The therapeutic effects on psychotic symptoms occur through D2 and D4 receptor binding in the mesolimbic area, while side effects are mediated by D2 blockade in the striatal area (linked to extrapyramidal effects) and in the hypothalamic infundibular system (linked to hyperprolactinemia) in more than 50% of patients. A clinical trial involving 69 patients examined the effects of various anti-psychotic medications on prolactin levels, including chlorpromazine, depot haloperidol, fluphenazine, zuclopenthixol, sulpiride, pimozide, droperidol, and flupenthixol. The study found a significant elevation in prolactin levels only in females, with a mean level of 1106 mIU/L (52 ng/mL) compared to the normal range of <480 mIU/L (22.6 ng/mL). In males, the mean prolactin levels were within the normal range, which may be attributed to the significantly lower total daily dose of chlorpromazine used in males (199.0-220.1 mg/day) compared to females (384.4-302.48 mg/day, P<0.05) (7).

 

Second-generation anti-psychotics with lower D2 affinity led to milder prolactin elevations (1-2-fold), except for paliperidone, risperidone, and amisulpiride whose effect on prolactin is similar to the first-generation neuroleptics. Chlorpromazine, loxapine, olanzapine and quetiapine have variable effects on prolactin secretion, while aripiprazole, clozapine, iloperidone, lurasidone have little or no effect on prolactin secretion (35).

 

An important factor contributing to variations in the induction of hyperprolactinemia by different anti-psychotic medications is the blood-brain barrier. Permeability glycoprotein transporter (P-gp), coded by the ABCB1 gene, is expressed in various tissues including in the cells of the blood-brain barrier. P-gp plays a role in actively transporting hydrophobic drugs with a molecular weight greater than 400 Da out of the brain, thus protecting the brain from these medications; therefore, this protein can change drug bioavailability (141,142).

 

The affinity of risperidone, paliperidone, and amisulpiride (prolactin rises up to 10-fold with these drugs) for P-gp is approximately twice that of olanzapine and chlorpromazine (prolactin rise is up to 3-fold with these drugs), and four times greater than haloperidol and clozapine (prolactin rise can be high initially but usually reduces with time) (143). The higher affinity of risperidone, paliperidone, and amisulpiride to P-gp could, among other mechanisms, partly explain the greater induction of hyperprolactinemia by these drugs, as P-gp does not allow them to enter the brain via the blood-brain barrier. Therefore, the portal circulation of the anterior pituitary delivers a somewhat higher concentration of these drugs to the lactotrophs, which are located outside the blood-brain barrier, to inhibit the D2 receptors (144).

 

Aripiprazole can act as a partial agonist at D2 receptors and display partial agonist activity at 5HT1A receptors, while also acting as an antagonist at 5HT2A receptors. Antagonism at these receptors can help to normalize prolactin levels since 5HT2A receptor activation has been associated with increased prolactin release. That is why it is considered a prolactin secretion modulator (145). Its role in prolactin levels has been investigated in a study involving both retrospective and prospective components (146). The retrospective part of the study included 30 patients undergoing risperidone treatment, when it was observed that after 6 months of treatment, prolactin levels remained high although somewhat lower than at the start of observation. In the prospective part of the study, 30 other patients were divided into two groups: one group receiving risperidone alone at a daily dosage of 2-4 mg and the other group receiving a combination of risperidone and aripiprazole at a daily dosage of 5-10 mg. The group receiving adjunctive aripiprazole exhibited significantly lower serum prolactin levels compared to the risperidone-only group at weeks1 (914±743 vs 1567±1009 mU/L), 2 (750±705 vs 1317±836 mUI/L) and 6 (658±590 vs 1557±882 mUI/L). Notably, during aripiprazole treatment, prolactin levels at weeks 1, 2, and 6 were significantly lower than at baseline (P< 0.05) (at baseline patients were treated with risperidone as monotherapy), suggesting that aripiprazole may effectively alleviate risperidone-induced hyperprolactinemia. Similar findings supporting the role of aripiprazole in reducing prolactin levels have been reported in other studies (147). Combination therapy presents a promising therapeutic approach for adjunctive treatment or for transitioning from risperidone to mitigate hyperprolactinemia (146).

 

More recently, a new medication SEP-363856, a trace amine-associated receptor 1 (TAAR1) and 5HT1A agonist, has been developed to treat schizophrenia. Its mechanism of action is not based on D2 antagonism, and has a favorable effectiveness and tolerability profile, without causing hyperprolactinemia (148). This category of medication serves as compelling evidence for the significant involvement of dopamine receptors in drug-induced hyperprolactinemia, and it is a future viable therapeutic choice for patients experiencing adverse effects associated with hyperprolactinemia.

 

DRUG-INDUCED HYPERPROLACTINEMIA IN PEDIATRIC PATIENTS

 

Anti-psychotic medications have been found to induce hyperprolactinemia in the pediatric population as well as in adults. In a trial involving 35 children and adolescents with early-onset psychosis, primarily diagnosed with childhood-onset schizophrenia or psychotic disorder not otherwise specified, prolactin levels were measured after a 3-week washout period, as well as after 6 weeks of treatment with haloperidol, olanzapine, and clozapine (149). Following the 6-week treatment period, haloperidol (9 of 10 patients – mean age 13.4 years) and olanzapine (7 of 10 patients– mean age 15.9 years) resulted in prolactin levels above the upper limit of normal. The mean increase was 5.2-fold for haloperidol and 2.4-fold for olanzapine. The prolactin response did not show statistically significant differences between females and males treated with haloperidol and olanzapine. Clozapine (22 patients, mean age 14.7) caused a small but significant rise in females (1.2-fold) but levels remained in the normal range for all patients. There was no rise in males. Why this difference between females and males is only on clozapine remains unclear and difficult to explain. However, in a study involving 36 girls aged 8-17 years, mean prolactin levels were higher in girls compared to boys, with the most significant increase occurring around the age of 13y, correlating with menarche. A highly significant correlation was found between increases in plasma prolactin and estradiol levels between the ages of 11 and 13 years. Girls with long menstrual cycles (>28 days) between the ages of 14 and 16 years had higher prolactin levels (p<0.05) (150). Even though the mean age of patients on clozapine was above 13 years, we do not possess information on the duration of the menstrual cycle of those girls, as if it is longer, the physiologic estrogenization because of the longer menstrual cycle can impact the range of prolactin elevation. In any case, the population sample size was relatively small to draw definitive conclusions and to provide answers why this happens only in clozapine patients (149).The authors concluded that the prolactin response in male children and adolescents treated with haloperidol or olanzapine was significantly higher than that observed in adult males. However, the prolactin response in female children and adolescents after haloperidol treatment did not differ significantly from that of adult females in similar studies, possibly due to the adult similarity of estrogen status seen in female adolescents (149). Aripiprazole in another study showed a lesser prolactin increase than olanzapine, quetiapine, and risperidone, similar to the adult population (151).

 

In another clinical trial involving 396 children and adolescents (aged 14.0 ± 3.1 years), the impact of anti-psychotic medications on prolactin levels was studied. The medications involved risperidone, olanzapine, quetiapine, and aripiprazole. Risperidone caused the highest incidence of hyperprolactinemia (93.5%) and had the highest peak prolactin levels (median = 56.1 ng/mL) followed in order by olanzapine, quetiapine, and aripiprazole. Menstrual disturbances were the most prevalent side effect (28.0%), particularly with risperidone (35.4%). Notably, severe hyperprolactinemia was associated with decreased libido, erectile dysfunction, and galactorrhea (152).

 

In conclusion, a comprehensive meta-analysis comprising 32 randomized controlled trials with a total of 4643 participants, with an average age of 13 years, has demonstrated that risperidone, paliperidone, and olanzapine are associated with a significant increase in prolactin levels among children and adolescents. Conversely, aripiprazole is linked to a notable decrease in prolactin levels in this age group. It is worth noting that haloperidol was not included in these studies, resulting in an absence of evidence regarding its prolactin-related effects in this population (153).

 

These findings underscore that haloperidol, risperidone, paliperidone and olanzapine are potent inducers of hyperprolactinemia in children and adolescents, mirroring observations in the adult population. A comprehensive listing of medications associated with hyperprolactinemia in children can be found in Table2.

 

Table 2. Drugs Reported to Induce Hyperprolactinemia in Children and Adolescents

Medication class

 

High

>50 percent of patients

Moderate

25-50 percent of patients

Low

<25 percent of patients

Case reports

Anti-psychotics, first-generation 'typical'

Fluphenazine (154)

Haloperidol (149,155)

 

Chlorpromazine (156)

Loxapine (157)

Pimozide (158,159)

 

 

Anti-psychotics, second-generation 'atypical'

Paliperidone (160,161)

Risperidone (152,155,162–164)

 

Asenapine (165)

Molindone (166)

Olanzapine (149,152)

Lurasidone (167,168)

Ziprasidone (169)

Quetiapine (152,162)

 

 

Clozapine (149)

Aripiprazole* (152,170)

Amisulpride (171)

Brexpiprazole (172)

Anti-depressants

Clomipramine (173)

 

 

Desipramine (174)

Bupropion (175)

Citalopram (176)

Escitalopram (177)

Fluoxetine (178)

Sertraline (179)

Duloxetine (177)

Paroxetine (180)

Venlafaxine (181)

Anti-emetics and gastrointestinal medications

Metoclopramide (182–184)

Domperidone (185,186)

 

 

 

Omeprazole (187)

Lansoprazole (187)

Cisapride

Others

Fenfluramine (188)

 

 

Estrogens (189)

Triptorelin (190)

Clonidine (191)

Methylphenidate (181)

Guanfacine (181)

Valproic acid (181)

Penicillamine (181)

*Aripiprazole is a partial agonist at the type 2 dopamine receptor and display partial agonist activity at the type 1A serotonin receptor (5HT1A) and antagonist at 5HT2A receptor. It can be used in combination with other psychotropic medications to reduce prolactin levels. Aripiprazole itself can sometimes cause mild hyperprolactinemia (192).

 

CLINICAL MANAGEMENT OF ANTI-PSYCHOTIC-INDUCED HYPERPROLACTINEMIA

 

Drug-induced hyperprolactinemia should be considered in the differential diagnosis of elevations of prolactin levels, sometimes greater than 200 µg/L (4260 mIU/L). Particularly when serum prolactin levels exceed 80-100 µg/L (1700-2130 mIU/L), pituitary magnetic resonance imaging (MRI) should be performed to rule out the presence of any underlying pituitary or hypothalamic masses that may contribute to hyperprolactinemia (193). According to the guidelines from the Endocrine Society, in symptomatic patients suspected of having drug-induced hyperprolactinemia, it is recommended the first test to diagnose drug-induced hyperprolactinemia is the discontinuation of the medication for 3 days or switch to an alternative drug (e.g. a prolactin-sparing anti-psychotic (e.g. aripiprazole), or an anti-psychotic with lower dopamine antagonist potency (Table 1) followed by retesting of serum prolactin levels.

 

However, any discontinuation or substitution of anti-psychotic agents should be done in consultation with the patient's psychiatric physician. If discontinuation is not possible or if the onset of hyperprolactinemia does not coincide with therapy initiation, obtaining a pituitary MRI is recommended (despite prolactin levels) to differentiate between medication-induced hyperprolactinemia and hyperprolactinemia caused by a pituitary or hypothalamic mass (194).

 

Before initiating treatment with an anti-psychotic medication, it is advised that clinicians inquire about the patient's previous treatment experience, sexual dysfunction, menstrual history (including irregularities and menopausal status), as well as any history of galactorrhea. Additionally, obtaining a baseline prolactin level is recommended before starting treatment (195). This pre-treatment screening for hyperprolactinemia can help determine whether subsequently elevated prolactin levels are due to medication-induced factors (196) and make the diagnosis easier without the need to perform further imaging.

 

While treatment of hyperprolactinemia in patients receiving anti-psychotics may not always be necessary, in cases where clinical hypogonadism is evident, several options are available (193). The initial step is to discontinue the drug if clinically feasible. If discontinuation is not possible, switching to a similar anti-psychotic that does not cause hyperprolactinemia is suggested. If neither of these options is feasible, cautious administration of a dopamine agonist may be considered in consultation with the patient's physician (194). It is worth noting that these interventions only result in the normalization of prolactin levels in approximately half of the patients receiving anti-psychotics, and careful psychiatric monitoring is required due to the possibility of psychosis exacerbation with dopamine agonists (193).

 

For patients with long-term hypogonadism demonstrated by hypogonadal symptoms or low bone mass, the use of estrogen or testosterone replacement is recommended. In rare instances where patients receiving anti-psychotics also present with a pituitary tumor, treatment options primarily revolve around tumor-specific interventions, considering especially optic chiasm compression. When a non-functioning tumor is suspected, the above options to eliminate the drug-induced component of hyperprolactinemia should be considered, with supervised short-term cessation of the medication to clarify drug-induced or tumor-derived hyperprolactinemia (193).

 

In addition to the interventions mentioned earlier, addressing fertility concerns in patients with drug-induced hyperprolactinemia may require further measures. Normalization of prolactin levels through medication adjustment or dopamine agonist therapy can often lead to the restoration of fertility. In situations where fertility is not spontaneously regained, fertility treatment with gonadotrophins or assisted reproductive procedures may be necessary.

 

NEUROLEPTIC-LIKE MEDICATIONS

 

Metoclopramide and domperidone are anti-emetic and gastrointestinal motility agents known also as neuroleptic-like medications which can increase pituitary prolactin secretion and breast milk production by a dopamine antagonistic action (Figure 1).

 

Metoclopramide is a central and peripheral D2 receptor antagonist (197). Its administration is followed by an acute increase in prolactin levels up to 15-fold above the baseline that persists in chronic administration of the drug (2). Even though the majority of patients treated with metoclopramide develop hyperprolactinemia (Table 1), related symptoms such as amenorrhea, galactorrhea, gynecomastia, and impotence remain unclear (2).

 

Domperidone is a peripheral D2 antagonist (it does not cross the blood-brain barrier) used for treating intestinal motility disorders, especially for the prevention of gastrointestinal discomfort with dopaminergic treatment in Parkinson's disease (198), as it antagonizes the D2 receptors in the upper gastrointestinal tract. It also reaches the D2 receptors on lactotroph cells inducing hyperprolactinemia, and can be used for stimulating lactation, for example in women with preterm infants, or an adoptive parent (199,200), and even in transgender women who wish to breastfeed (201,202). It can cause a 10-fold elevation in prolactin levels with normalization of prolactin levels after three days (85,86). Neuroleptic-like medications are summarized in Tables 1 and 3.

 

Table 3. H2 Receptor Antagonists, Opioids, Anti-Hypertensives, PPIs, Estrogens, and Other Drugs and Their Ability to Cause Hyperprolactinemia.

Medication class

High

>50% of patients

Moderate

25-50% of patients

Low

<25% of patients

Case reports

Anti-emetic and gastrointestinal

Domperidone

Metoclopramide

 

Prochlorperazine

 

Esomeprazole

Omeprazole

Lansoprazole

Cisapride

H2-receptor antagonists

 

 

Cimetidine

Ranitidine

 

Anti-hypertensives

 

Methyldopa

Verapamil

 

Others

Fenfluramine

Opioids

 

Estrogens

Protease inhibitors

Cocaine Bucillamine

Clonidine

Methylphenidate

Guanfascine

Valproic Acid

Penicillamine

 

ANTI-DEPRESSANTS

 

Anti-depressants can be classified based on their structure and mechanism of action into tricyclic anti-depressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), monoamine oxidase (MAO) inhibitors, atypical anti-depressants, serotonin modulators, selective noradrenaline reuptake inhibitor, and NMDA (N-Methyl-D-Aspartate) receptor antagonists (203). Data on their ability to cause hyperprolactinemia are controversial, as described below. The two main mechanisms, both related to elevated serotoninergic tonus, explain how anti-depressants can induce hyperprolactinemia by indirect modulation of prolactin release by serotonin and serotonin stimulation of GABAergic neurons (50) Adrenergic receptors involvement in their ability to cause hyperprolactinemia remains unclear. (Figure 1).

 

Data on the incidence of hyperprolactinemia with anti-depressant medications, especially SSRIs, MAO inhibitors, and some TCAs, suggest that they can cause modest and generally asymptomatic hyperprolactinemia (4,193). Their ability to cause hyperprolactinemia is summarized in Table 1.

 

TCAs

 

TCAs, such as amitriptyline, desipramine, clomipramine, and amoxapine, can induce sustained mild hyperprolactinemia (2) (Table 1). They manifest their mechanism of action by blocking the reuptake of noradrenaline and serotonin, through increasing serotoninergic tonus, leading to hyperprolactinemia. Amitriptyline's ability to cause hyperprolactinemia seems to be dose-dependent. A dosage of 150-250 mg/day to 5 patients showed no effect on prolactin levels after 3-7 weeks (204), whereas a dosage of 200-300 mg/day caused a 2-fold increment of prolactin levels in two of nine chronic treated patients (88). In 13 patients with depression taking amitriptyline or desipramine, prolactin levels were studied after intravenous injection of tryptophan (serotonin precursor). It was observed that tryptophan-induced prolactin elevation was significantly increased compared with a preceding placebo period (205). A similar tryptophan test was performed with clomipramine 20 mg vs placebo in 6 normal subjects. Levels of prolactin increased after tryptophan infusion in pretreated patients with clomipramine (206),suggesting serotonin involvement in this hyperprolactinemia.

 

The effect of the traditional TCA imipramine on serum prolactin levels is controversial. A five-week double-blind study on patients with depression taking imipramine did not show any significant change in serum prolactin (207). However, another study in young healthy men showed that imipramine’s effect on prolactin is dose-dependent (usually therapeutic dose is 50-150 mg): oral administration of 100 mg, but not of 40 mg, led to a consistent rise in prolactin levels after 3 hours of administration, but the rise was mild (maximum 25.85 ng/mL (550 mUI/L) (91).

 

Data regarding nortriptyline's ability to induce sustained hyperprolactinemia are lacking. Anyway, over a 4-6 week treatment of 8 patients with nortriptyline up to 150 mg/daily, no difference was found between placebo and the medication group. In only one patient was observed a transitional 2-fold elevation in the first 2 weeks (88).

 

Amoxapine, which is an anti-depressant with neuroleptic properties as well, is found to increase prolactin levels in 10 patients approximately 3.5-fold compared to baseline and more than desipramine in 12 patients, where almost no difference with baseline was observed (89). The proposed mechanism of hyperprolactinemia involves the blockade of the D2 receptor in tuberoinfundibular neurons or the anterior pituitary gland (2).

 

SSRIs

 

SSRIs enhance serotonin activity via inhibition of neuronal serotonin reuptake. This could be the most prominent mechanism leading to a prolactin elevation. A review of 13 case reports showed prolactin levels between 28 and 60 ng/mL (595 and 1276 mUI/L) (52). In a French study, 27 of 159 cases (17%) had SSRI-induced hyperprolactinemia with sertraline being the most prominent, followed by fluoxetine, paroxetine and fluvoxamine. Only citalopram was found not to increase prolactin levels significantly (208). However, in another study fluoxetine, paroxetine, and fluvoxamine were found again to elevate prolactin levels, but they also found citalopram to induce hyperprolactinemia. In this study duloxetine, milnacipran, and sertraline (which was the most prominent in the previous study) were not associated with an increased risk of hyperprolactinemia (93). Fluoxetine-induced hyperprolactinemia was found in patients with major depression (4.5% of men and 22.2% of women) following 12 weeks of fluoxetine treatment (209).Differences in the ability to cause hyperprolactinemia can be attributed to the variations in the affinity of the SSRIs for dopamine, histamine, and GABA receptors.

 

The Nurses’ Health Study and its follow-up study assessed anti-depressant use and circulating prolactin levels in 610 women (including 267 anti-depressant users) with two measurements of prolactin an average of 11 years apart (210). In this study, mean prolactin levels were similar among SSRI users (13.2 µg/L, (280 mUI/L), 95% CI 12.2-14.4), users of other classes of anti-depressants (12.7 µg/L (270 mUI/L), 95% CI 11.0-14.6), and non-users (13.1µg/L, (278 mUI/L), 95% CI 12.8-13.4) (210). However, the duration and dosage of anti-depressant use at the time of prolactin sampling had not been assessed, as the participants had only responded as current anti-depressant users/non-users on a questionnaire.

 

MAO Inhibitors

 

Monoamine oxidase is an enzyme responsible for breaking down neurotransmitters such as serotonin, noradrenaline, and dopamine in the brain. Inhibition of this enzyme is expected to increase the levels of all those neurotransmitters. Even though increased dopamine is suspected to be related to lower prolactin levels, probably the serotonin increment prevails and that is why this class of medications is related to hyperprolactinemia; or dopamine and serotonin increment neutralize each other and no difference in prolactin levels is seen.

 

MAO inhibitors with serotoninergic activity (pargyline and cordyline) can cause modest and generally asymptomatic hyperprolactinemia (2,61). Phenelzine was observed to persistently increase prolactin levels in 4 of11 patients, which returned to normal during a placebo week and rose again in all 4 patients after treatment restart (88).

 

Atypical Anti-Depressants

 

Mirtazapine's mechanism of action is different from other anti-depressants, as it does not inhibit the reuptake of serotonin or noradrenaline, but it increase the release of serotonin and noradrenaline (211). In any case, prolactin levels did not show any difference in 8 healthy male subjects pre- and post-mirtazapine 15 mg oral administration (212).

 

Serotonin Modulators

 

Trazodone acts through dual inhibition of serotonin reuptake and serotonin type 2 receptors, coupled with antagonism of histamine and α-1-adrenergic receptors (213). In 12 patients with depression, 150 mg of trazodone for 3 weeks caused significantly higher prolactin levels after 12 hours, 1 week, and 2 weeks of treatment compared to baseline. Higher levels were after the first week 15,3+/-8,5 ng/mL (325 +/- 180 mUI/L) compared to baseline 9,1 +/- 5,6 ng/mL (194+/-119 mUI/L) (99).

 

Selective Noradrenaline Reuptake Inhibitor

 

Reboxetine is a selective noradrenaline reuptake inhibitor that was shown to increase prolactin in healthy men after acute administration, but this effect can be reversed if reboxetine is simultaneously administered with α2-blocker mirtazapine, suggesting a role of α2-receptors in the enhancement of prolactin release after reboxetine (214). In any case, conflicting data are present even with this drug, with one other study showing no difference between pre-treatment and after-treatment prolactin levels in patients taking up to 8 mg reboxetine for 4 weeks in 17 patients (215). This discrepancy can be due to the limited number of patients or other neuroendocrine mechanisms still less explored.

 

SNRI medications are described to cause only mild and rare elevations on prolactin levels, whereas (94), esketamine (NMDA receptor antagonist)is not described to cause hyperprolactinemia.

 

Summary

 

In summary, controversial data are available on anti-depressant-induced hyperprolactinemia. Routine monitoring of prolactin levels in patients taking anti-depressants is not recommended unless symptoms related to prolactin increase (in premenopausal women: menstrual cycle dysfunction leading to amenorrhea, oligomenorrhoea, anovulatory cycles, low libido, and energy; in men: erectile dysfunction, decreased energy and libido, decreased muscle mass, decreased body hair; and for both of them osteopenia, galactorrhea and infertility) occur (50,193). If the prolactin serum level is elevated (> 25 µg/L (531 mUI/L), in these patients a differential diagnosis is needed. The proposed approach is to withdraw the anti-depressant drug slowly over 2 weeks and replace it with another anti-depressant less likely to cause hyperprolactinemia, then reassess symptoms and prolactin levels after 2-4 weeks (193). If the serum prolactin remains elevated, other causes of hyperprolactinemia should be addressed by an endocrinologist. Another approach is to perform a pituitary MRI if the replacement of the anti-depressant is difficult to manage.

 

GASTRIC ACID REDUCERS

 

Histamine-Receptor Inhibitors

 

Histamine, a CNS neurotransmitter, binds to both H1 and H2 receptors. It can stimulate prolactin secretion via H1 receptors by inhibiting the dopaminergic system. On the contrary, histamine can also inhibit prolactin secretion via H2 receptors using a non-dopaminergic mechanism involving β-endorphin, vasoactive intestinal peptide, vasopressin, or TRH (216), all of which act as prolactin-releasing factors (49) (Figure 1).

 

In the French Pharmacovigilance Study, H2 receptor antagonists were found to contribute to 5% of drug-induced hyperprolactinemia, primarily with ranitidine (odds ratio = 4.43; 95% CI: 1.82–10.8) (11). Other studies have shown that H2-receptor antagonists such as cimetidine and ranitidine can elevate prolactin levels (217,218). Specifically, cimetidine caused a three-fold increase in prolactin levels after a 400 mg IV infusion, although this effect was not observed with oral administration of 800 mg cimetidine in healthy individuals (102) (Table 1,3).

 

Interestingly, it has been observed that systemic administration of the H2 agonist impromidine does not prevent cimetidine-induced hyperprolactinemia. In contrast, pre-administration of benzodiazepines or GABA lowered the prolactin response. This suggests that cimetidine-induced hyperprolactinemia may be mediated through neurotransmitters in the GABA-ergic system (219).

 

Proton-Pump Inhibitors

 

Proton-pump inhibitors (PPIs) have been recently reviewed regarding the risk of hyperprolactinemia and related sexual disorders observed with long-term use (220). The exact mechanism by which PPIs increase prolactin levels is not fully understood; possible explanations include inhibition of dopamine receptors, interference with other dopamine receptors, involvement of the serotoninergic pathway, modulation of the opioid pathway, and a potential role in decreasing prolactin clearance (220) (Figure 2). In addition, PPIs can increase gastrin levels in chronic use especially in females using high doses (221). As gastrin can act as prolactin-inhibitory factor (108), this antagonistic effect may explain the relatively mild hyperprolactinemia occurring with this class of drugs. Esomeprazole has a mild inhibitory effect on CYP3A4, which leads to decreased metabolism of estrogen, thereby increasing serum estrogen levels which can stimulate the production of prolactin (222). Gynecomastia, impotence, irregular menses, and galactorrhea have been described with PPI use. Hyperprolactinemia occurred less often than sexual disorders, and most cases of hyperprolactinemia were reported with omeprazole, esomeprazole, and lansoprazole use (e.g. 4-fold increment with lansoprazole) (104) (Table 1,3). Pantoprazole and rabeprazole were only sporadically associated with hyperprolactinemia. The authors assert that the occurrence of sexual dysfunction in individuals using PPIs, despite having normal prolactin levels, may be attributed to the development of low vitamin B12 levels, hypomagnesaemia and iron deficiency resulting from PPI usage. These nutritional deficiencies have been implicated in the manifestation of sexual disorders in other studies (220,223,224).

 

OPIOIDS

 

Endogenous opioids, morphine, and related drugs, activate ε-, μ-, κ- and δ- opioid receptors in the hypothalamus, modulating pituitary hormone secretion. They do not possess direct effects on pituitary cells (225,226). They inhibit the gonadal axis via ε receptors (GnRH suppression) and stimulate prolactin production by reducing the activity of tuberoinfundibular dopaminergic neurons via μ, κ and δ opioid receptors – mostly μ receptors as the μ receptor opioid antagonist naloxone prior to morphine and methadone use prevents opioid-induced hyperprolactinemia (227). Indirect stimulation of prolactin release can be mediated by stimulating prolactin-releasing factors production (the serotoninergic pathways discussed above) (228) (Figure 2). Hyperprolactinemia can then lead to additional gonadal axis suppression. In addition, opioids can modulate the corticotroph axis via κ and δ opioid receptors and the somatotroph axis via μ, κ and δ opioid receptors (226). Additionally, opioids manifest negative effects on bone health through direct inhibition of osteoblasts by opioids, gonadal axis suppression, altered mental status, and other comorbidities (chronic conditions, smoking, alcohol use) (229).

 

Acute intravenous or intra-ventricular administration of endogenous opioids leads to a rapid plasma prolactin increase in a dose-dependent manner (44,225). Chronic use of opioids effects on prolactin can vary: oral opioids for chronic pain increase prolactin, but morphine administered intrathecally for chronic non-cancer pain had no effect on prolactin (226). Hyperprolactinemia induced by opioids can be symptomatic: painful gynecomastia, galactorrhea, and hypogonadism have been reported in chronic opioid users. These can be alleviated with discontinuation or reduction of opioid dose, and sometimes dopamine agonists such as bromocriptine (226). Methadone induces a transient increase in prolactin levels, whereas chronic methadone users have normal basal prolactin levels (110), (Table 1, 3).

 

On the contrary, in a study involving six patients with hyperprolactinemia and amenorrhea, the use of naltrexone, an opioid antagonist, was investigated to determine if blocking endogenous opioids could improve the sexual axis. On the first day of naltrexone administration, significant increases were observed in the mean concentration of luteinizing hormone (LH), LH pulse amplitude, and estradiol levels compared to the control day. This indicated a prompt partial reactivation of the hypothalamic-pituitary-gonadal axis as a result of naltrexone, leading to heightened gonadotrophin levels and subsequent release of estradiol. However, it was found that the effect of opioid antagonism did not result in a sustained increase in estradiol secretion with chronic treatment. Additionally, prolactin levels continued to increase over time (mean prolactin level 255 ± 121 microgram/L), despite the initial improvement in the gonadal axis. This study demonstrated that although prolactin-induced suppression of the gonadal axis can be reversed to some extent by acute opioid antagonism, it is not an effective treatment for revitalizing the gonadal axis in the long term. Possible explanations for this lack of sustained effect include desensitization of the hypothalamic-pituitary unit for the effects of opioid receptor blockade and other disruptors of the axis, which may counteract the positive effects of opioid antagonism (230,231).

 

For endocrinopathies caused by opioids, including hyperprolactinemia, potential management choices include reducing or discontinuing opioid usage whenever feasible and exploring alternative pain relief therapies for chronic pain situations. Hormonal replacement therapy can be considered for hypogonadism and hypoadrenalism (226).

 

ANTIHYPERTENSIVES

 

Some antihypertensive medications including α-methyldopa, verapamil, labetalol, and reserpine, have been associated with hyperprolactinemia. This phenomenon is attributed mainly to the potential inhibition of dopaminergic pathways, highlighting the complex interplay between anti-hypertensive therapy and endocrine function. Other mechanisms are drug-specific and will be explained below.

 

α -methyldopa is an α-adrenergic inhibitor that leads to the suppression of monoamine synthesis, including noradrenaline, dopamine, and serotonin, which likely contributes to its anti-hypertensive effect. It causes hyperprolactinemia through the inhibition of dopamine synthesis by competitive inhibition of DOPA decarboxylase which transforms L-dopa into dopamine (61) (Figure 2). Long-term treatment resulted in elevated basal prolactin levels (3-4-fold), while a single dose of 750-1000mg reaches a peak of high prolactin level after 4-6 hours of administration (232). Gynecomastia is the most common endocrine side effect.

 

Calcium channel blockers are other drugs studied for their potential to cause hyperprolactinemia. The dihydropyridine class was found to have no effects on prolactin levels. Whereas, from the non-dihydropyridine class, which mainly blocks L-type of calcium channel receptors in the heart, only verapamil was found to cause 2-fold persistent hyperprolactinemia (and galactorrhea),while drug discontinuation reversed hyperprolactinemia in all patients (113). A clinical trial suggested that verapamil acts by reducing dopamine release in the tuberoinfundibular pathway through calcium influx inhibition (Figure 2), possibly by N-calcium channels which are known to be involved in the regulation of dopamine release and other neurotransmitters (233).

 

Labetalol is an α- and β-adrenoceptor blocker anti-hypertensive that has been reported to increase prolactin levels when administered intravenously, but not when administered orally (100 or 200 mg) as labetalol cross the blood-brain-barrier only in negligible amounts. The increase in prolactin release caused by intravenous labetalol is not readily explained by its interference with adrenergic receptors. The exact mechanism underlying this effect of the drug is currently unclear, but it is possible that labetalol's ability to block dopamine activity (anti-dopaminergic activity) might be involved in this response (234) (Figure 2). Pre-treatment with levodopa and carbidopa can prevent prolactin response after labetalol (235), suggesting dopamine pathway involvement suppression inside the blood-brain-barrier.

 

Reserpine is a rauwolfia alkaloid previously used for the treatment of hypertension as well as psychosis, schizophrenia, and tardive dyskinesia; it reduces dopamine by inhibiting their hypothalamic storage in secretory granules (236), and by blockade of vesicular monoamine transporter type 2 in monoamine neurons (237), leading to hyperprolactinemia. Prolactin levels are higher during treatment with reserpine than 6 weeks after discontinuation of the drug. Increased incidence of gynecomastia and breast cancer has also been reported among patients on anti-hypertensive therapy with reserpine (236). The ability of anti-hypertensives to cause hyperprolactinemia is summarized in Tables 1 and 3.

 

ESTROGENS

 

Estrogens stimulate prolactin secretion by several mechanisms: They bind to specific intracellular lactotroph cells receptors, though enhancing prolactin gene transcription and synthesis (238). They also inhibit tuberoinfundibular dopamine synthesis, stimulate lactotroph cell hyperplasia, downregulate dopamine receptor expression, and modify lactotroph responsiveness to other regulators (23,239) (Figure 2). Estrogen-induced hyperprolactinemia is dependent on the degree of estrogenization. Higher levels of estrogens in pregnancy and during ovulation increase prolactin levels with the last, contributing to a higher normal range of prolactin in pre-menopausal women.

 

Studies documenting the incidence of hyperprolactinemia showed that women on oral contraceptives were reported to have higher prolactin levels by 12% to 30% (240,241). Some, but not all, studies suggest that there is a dose-dependent effect (4). No increase in basal prolactin levels is reported during therapy with modern contraceptives with lower amounts of estrogen (242)or estrogen plus cyproterone acetate alone (243).

 

In transgender patients, estradiol or ethinyl estradiol treatment, the prolactin level rise was dependent on the dose of estrogen, duration of exposure, and alteration of SHBG levels. Estradiol infusion at levels above 10,000 pg/mL for as short as 6-7 hours significantly elevated prolactin levels by 3- to 4-fold, whereas ethinyl estradiol 2 mg/day for 1 month did not consistently elevate prolactin in all patients, which can be due to its ability to increase SHBG binding and maintaining free portion in the normal range (114) (Table 1, 3).

 

For women on post-menopausal hormone replacement therapies over 2.5 years, serum prolactin measured were within the normal range (244). In another study on 75 women, who were randomly assigned to three groups: control (receiving placebo), transdermal hormonal replacement (biphasic 17β-estradiol and progesterone, natural hormones), and oral ethinyl-estradiol and desogestrel, prolactin levels significantly increased in the oral group, but not in the transdermal group. There was a significant difference in hormone levels: in the oral group, estradiol levels increased five times and estrone levels eleven times. In the transdermal group, estrone and estradiol levels were increased three times (245).

 

GONADOTROPHINS AND GNRH AGONISTS

 

In addition to the known prolactin function in lactation, several studies have suggested other benefits of prolactin in oocyte development, formation of corpus luteum and its survival, steroidogenesis and implantation (246). In natural cycles there is a transient increase in late follicular phase of prolactin, but this increment is higher in stimulated cycles (246). In a cohort study were included 79 patients; 60 individuals underwent in vitro fertilization, 14 received clomiphene citrate treatment, and five patients with premature ovarian failure were administered estradiol. During the course of human menopausal gonadotrophin (hMG) treatment, a notable increase in both serum estradiol and prolactin concentrations were observed from early to late follicular days (P < 0.01). Specifically, prolactin levels increased from an initial mean value of 367±38 mIU/L (17.25±1.8 ng/mL) to 991±84 mIU/L (46.6±4 ng/mL) (Table 1). Bromocriptine effectively mitigated the increase in prolactin levels but was associated with a significant elevation in estradiol levels (P < 0.05) because prolactin itself works as a controller of estradiol increment. Clomiphene treatment led to a significant increase in serum estradiol levels (P < 0.01) but a significant decrease in serum prolactin concentrations during the late follicular phase (P < 0.01), indicating disruption of the estradiol-prolactin feedback mechanism. Among patients with premature ovarian insufficiency, serum prolactin concentrations increased concomitantly with rising serum of estradiol concentrations (after estradiol administration). Additionally, it was observed that the presence of prolactin significantly reduced estradiol production by granulosa cells (P < 0.05) (116).

 

An increment of prolactin levels is found even after hCG administration with a maximum prolactin level of 93.2 ng/mL; 1983 mIU/L (115).Notably, knowing that prolactin is a stress hormone, during assisted procedures it is increased, but this is a transitory increment without consequences in fertility outcome (247).

 

Not only gonadotrophins but also GnRH agonists are widely used during invitro fertilization to maintain a controlled and synchronized ovarian stimulation. Use of leuprolide acetate (GnRH agonist) concomitantly with hMG, resulted in higher prolactin and estradiol levels in comparison with patients receiving only hMG (prolactin 24.2 vs 16.8 ng/mL; 515 vs 358 mIU/L) (117). In another randomized study, along protocol with 0.1 mg subcutaneous triptorelin starting from day 10 of the preceding stimulation cycle and short protocol, where 0.1 mg subcutaneous triptorelin is given in the stimulating cycle, were compared. Prolactin levels were measured at 9 am in the first day of hCG administration. The long protocol correlated with higher prolactin levels (31.3 ± 16.9 vs 23.7 ± 11 ng/mL; 666 ± 359 vs 504 ± 234 mIU/L) (248).

 

In children, GnRH agonists are used in precocious puberty (CPP)as well as growth hormone deficiency (GHD)who do not properly respond to exogenous growth hormone treatment. In a study involving 119 children with CPP and 93 with GHD, treated with triptorelin or leuprolide, prolactin levels were measured before and every six months for 6 years for CPP group and for 2 years for GHD group. Moreover, prolactin levels were checked after 6 and 12 months of treatment withdrawal. In this study was concluded that even though prolactin levels were higher in triptorelin treated patients (only 3.8% developed hyperprolactinemia in triptorelin group which was solved after withdrawal – baseline 12.5 ± 3.7 ng/mL (266 ± 79 mIU/L) to max 45.6 ± 4.5 ng/mL; 970 ± 96 mIL/L), no significant difference was found in prolactin in basal condition and during GNRH agonist treatment in CPP and GHD (190)(Table 2).

 

OTHER DRUGS

 

A lot of other drugs have been reported to cause mild (less than 2-fold increment) increases in prolactin levels. A synthesized visualization of these mechanisms is shown in Figure 2.

 

The acute administration of buspirone, an anxiolytic medication, was investigated in a study involving 8 healthy volunteers. The findings revealed an increase in plasma prolactin levels across all participants compared to the baseline levels observed in 8 control subjects. During the study, blood samples were collected at 30-minute intervals over a duration of 2 hours. The zenith of prolactin levels was observed between minutes 90 and 120 for all individuals, with the maximum elevation reaching 37 ng/mL (787 mIU/L) (249). It is noteworthy that the augmentation of prolactin is believed to exhibit a dose-dependent relationship. Furthermore, it was observed that chronic usage of buspirone did not lead to significant alterations in prolactin levels, indicating a potential adaptation to the acute changes induced by the medication. The underlying mechanism responsible for this phenomenon is posited to involve both serotoninergic and dopaminergic implications (119).

 

Carbamazepine, a widely used anticonvulsant, was examined in a cohort comprising 4 patients with complex partial seizures undergoing chronic carbamazepine treatment (200 mg administered three times daily). Blood samples were collected at intervals of 2 hours. Additionally, a group of 5 patients with untreated epileptic seizures participated, wherein a thyrotrophin-releasing hormone (TRH) stimulation test was performed both prior to and 35-50 days post the administration of 200 mg carbamazepine three times daily. Blood samples were obtained 10, 30, and 60 minutes following intravenous injection of 200µg TRH. Furthermore, 4 normal volunteer subjects were included in the study. On the first day, a placebo was administered, followed by the administration of 400 mg carbamazepine at 8 AM on the second day. Blood samples were collected at baseline on both days and subsequently at hourly intervals until 4 PM. After a span of two weeks, a nocturnal study was conducted, spanning from 6 PM to 6 AM. The investigation revealed that there were no discernible alterations in spontaneous prolactin release or TRH-stimulated prolactin levels. However, a slight increase in sleep-entrained prolactin values was observed, while retaining the secretory circadian rhythm. Given that the release of prolactin during sleep is largely attributed to serotoninergic activity, it is plausible that the modest increment (less than 2-fold) may implicate serotoninergic modulation (working as a serotonin-releasing factor and reuptake inhibitor) facilitated by carbamazepine (120,121).

 

Sympathomimetic amines fenfluramine and sibutramine, formerly used for appetite suppression due to their stimulatory effect on the synaptic concentration of serotonin, have been shown to induce hyperprolactinemia as a result of increased serotoninergic activity and postsynaptic stimulation of 5HT2Areceptors. In a case report, after starting sibutramine, a 38-year-old female patient developed hyperprolactinemia (prolactin levels 46 and 89.6 ng/mL (978 and 1906 mUI/L) with amenorrhea and galactorrhea. Discontinuation of sibutramine, confirmed by a sella MRI, led to rapid normalization of prolactin levels within 15 days, and symptoms resolved during a 90-day follow-up (2,127).

 

Cholinomimetic drugs have been reported controversially in the literature regarding their ability to cause hyperprolactinemia. However, in collaborative studies from the National Institute of Mental Health and the University of California, San Diego, three separate experiments were conducted involving volunteers of different genders and ages. In the first experiment, nine volunteers received physostigmine salicylate at 33 µg/kg, while in the second experiment, eleven male volunteers were given 22 µg/kg of physostigmine salicylate. The third experiment involved six volunteers receiving 3 mg of arecoline hydrobromide. Placebo saline was administered in all experiments as well. It was shown that intravenous injection of physostigmine or arecoline can elevate prolactin correlating with raised β-endorphin levels in the blood. Prolactin elevation was less than 100 ng/mL (2127 mUI/L). Cholinergic activation in the hypothalamus, particularly focusing on β-endorphin, might help in explaining how peptides modify primary neurochemical effects on hormone regulation in the hypothalamus and pituitary (44).

 

Bucillamine, an analogue of D-penicillamine used as an antirheumatic drug in Japan, has been reported to induce hyperprolactinemia (109 ng/mL (2319 mUI/L)) after 30 months of treatment start, associated with gynecomastia and galactorrhea in one case report. The mechanism remains unclear (133).

 

‘Ecstasy’ (MDMA) was shown to increase prolactin secretion in rhesus monkeys by stimulating serotonin release and by direct-acting as a 5HT2A agonist (250); In nine studies, five of them observed an increase in prolactin levels due to the intervention. However, in the remaining studies, there was no significant change in prolactin levels, and these unresponsive results tended to occur when a lower dose of the intervention was used on average. This suggests a potential relationship between the dosage of the intervention and its effect on prolactin levels (135).

 

Smoking, particularly the consumption of high-nicotine cigarettes, has been associated with a significant acute elevation in prolactin levels, ranging from 50% to 78% above the baseline, within 6 minutes after smoking. These elevated levels persist for approximately 42 minutes and return to baseline within 120 minutes of initiating smoking (136). The underlying mechanism probably involves the stimulation of rapid prolactin release through the augmentation of endogenous opioids, which subsequently inhibits dopamine release (251). However, prolonged nicotine exposure leads to desensitization of dopamine receptors, and lowers dopamine turnover (48) probably contributing to hyperprolactinemia. It has been hypothesized that the increased incidence of osteopenia and osteoporosis could be at least partly related to this effect (252).

 

Recently, an association has been reported between HIV-1 protease inhibitors and the adverse effect of galactorrhea and hyperprolactinemia in four HIV-1 infected women treated with indinavir, nelfinavir, ritonavir, or saquinavir. The cause of this unexpected toxicity could be attributed to several possible mechanisms: 1) Protease inhibitors may enhance the stimulatory effects of prolactin due to their inhibition of the cytochrome P450 system, leading to longer half-life of prolactin; 2) opportunistic infections in AIDS patients may induce cytokine-driven prolactin production by pituitary or immune cells; 3) protease inhibitors might exert direct endocrine effects on the pituitary or hypothalamus (253,254). To explore mechanisms of hyperprolactinemia induced by protease inhibitors, experiments were conducted using rat pituitary cells and hypothalamic neuronal endings. The results showed that both ritonavir and saquinavir could directly stimulate prolactin secretion, while not affecting dopamine release. This suggests that these protease inhibitors might interact with specific mammalian proteins in the anterior pituitary involved in prolactin secretion, leading to the observed galactorrhea and hyperprolactinemic effect (137).

 

Regarding chemotherapy and immunosuppression, there are some controversial data on the effect of chemotherapy and immunosuppression on prolactin levels, as significant prolactin increases are not frequent and usually mild (2). Prolactin and growth hormone have been involved as part of a cytokine system in the recovery of the immune response after chemotherapy and bone marrow transplantation (255). In a study of 20 breast cancer patients undergoing high-dose chemotherapy and autologous stem-cell transplantation, plasma prolactin levels increased within and 30 days after transplant, yet still remaining within the normal range. The use of antiemetic drugs further raised prolactin levels. Patients in continuous complete remission after transplantation exhibited higher prolactin levels, while elevated prolactin did not impact disease-free survival, suggesting potential for further research into post-transplant immune response (256).

 

Radiotherapy for intracranial germ cell tumors was shown to induce hyperprolactinemia with a prevalence of 35.3% (138).

 

Other drug inducing hyperprolactinemia are described in Table 1.

 

DRUGS REPORTED TO DECREASE PROLACTIN LEVELS OR HAVE AN EQUIVOCAL EFFECT

 

Several medications, beyond the established treatments like cabergoline, bromocriptine, carbidopa and levodopa, have reported effects on reducing prolactin levels. For instance, pseudoephedrine, an α-adrenergic stimulant primarily affecting α1 receptors, shares structural similarities with amphetamine and moderately stimulating dopamine release in the brain by acting on D2 receptors in the pituitary, consequently lowering prolactin levels. Studies have indicated pseudoephedrine's potential to decrease milk production, at least partly attributed to its effect on prolactin levels through dopaminergic actions in the pituitary (132). Moreover, indirect evidence suggests that α-1 receptors stimulation leads to decreased prolactin levels (41).

 

Amphetamine was seen to produce a poor prolactin suppressant effect in either normal- or hyperprolactinemic subjects. The proposed mechanism of prolactin lowering potential is due to their ability to stimulate the release of dopamine (257). However, during the withdrawal period of cocaine use, hyperprolactinemia has been observed, probably due to a decrease in dopamine levels, leading to dysregulation in the dopamine system and increased prolactin. Moreover, during withdrawal, prolactin can be secreted as a stress hormone (129).

 

Guanafascine, an α2 adrenergic agonist, used to treat ADHD, has been shown to decrease prolactin levels. In a longitudinal study spanning three years involving 15 patients diagnosed with hyperprolactinemia, the noteworthy suppressive impact of guanfacine on prolactin levels suggests potential involvement of hypothalamic or extrahypothalamic adrenergic pathways in the intricate regulation of prolactin secretion (131). Even though α2 stimulation has been shown to increase prolactin levels in rats, this is not fully understood in humans making the explanation in this case confusing (42).

 

The impact of benzodiazepines (BDZ) on prolactin secretion is a subject of debate. Research findings have yielded conflicting results. Some studies conducted on both non-epileptic patients and healthy volunteers have not detected significant modifications in prolactin levels following BDZ treatment (258). A study on 30 adolescent patients with schizophrenia with gradually increasing doses of diazepam to a maximum of 100-400 mg/day, with 4 weeks of treatment, showed that only doses higher than 250 mg/day give a significant but mild increase in prolactin levels. Proposed mechanisms are inhibition of TIDA neurons by activation of the GABA system, or activation of the endorphin-ergic system leading to hyperprolactinemia (118). On the contrary, diazepam was found to suppress the secretion of prolactin in vitro through one of two mechanisms: it either strengthens the direct inhibitory action of GABA on prolactin release, or it hinders a benzodiazepine-sensitive Ca2+-calmodulin dependent protein kinase at micromolar concentrations leading to a reduction of prolactin secretion (259).

 

Moreover, phenytoin, an anticonvulsant impeding sodium channels in nerve cells, have generated conflicting data regarding their impact on prolactin levels. In animal studies, phenytoin showcased a rapid decline in both prolactin release and mRNA concentrations, functioning as a partial T3 agonist by binding to T3 nuclear receptors (260). However, clinical observations revealed elevated resting levels of prolactin in phenytoin-treated patients compared to untreated counterparts. Remarkably, responses to metoclopramide and bromocriptine remained unaltered, indicating a limited effect of phenytoin on the D2 receptors present on lactotrophs (261). Even the conclusions drawn from these findings remain contentious. Evidence suggests that phenytoin treatment may enhance the growth hormone response to levodopa, implying a phenytoin-induced dopaminergic activity at the hypothalamic-pituitary level (122). More specifically, it is postulated that phenytoin might enhance dopamine receptor sensitivity by inhibiting the Ca2+ calmodulin complex. This effect could contribute to reduced prolactin secretion (122). On the contrary, other studies have not demonstrated any notable alterations in prolactin levels due to phenytoin administration (262). The discordant outcomes surrounding phenytoin's impact on prolactin levels underscore the complexity of its effects and necessitate further investigation for conclusive insights into its mechanisms of action.

 

In another comprehensive study involving 126 subjects, both males and females, with generalized or partial epilepsy receiving phenobarbital as monotherapy or in combination with phenytoin or benzodiazepines, a distinct pattern emerged. Specifically, the administration of phenobarbital, either alone or in combination, resulted in elevated prolactin levels, but this elevation was found to be statistically significant only in the male participants. Notably, knowing that an epileptic attack itself can cause hyperprolactinemia, those data remain confusing. The proposed mechanism in this study is phenobarbital interaction with GABA receptors, leading to increased prolactin levels (263). However other studies do not show any change in prolactin levels (123).

 

Valproic acid, an anticonvulsant working as a central stimulant of GABAergic neurons, has demonstrated the ability to reduce prolactin basal levels as well as TRH-stimulated prolactin levels. This is indirect proof of the synergically acting of GABA neurons with dopaminergic tracts (124). However, in another study, no effect of valproic acid on prolactin levels was noticed during the night (264).

 

Lithium carbonate, a pharmaceutical agent employed as a mood stabilizer, has undergone investigation in different studies, as it decreases dopamine release and glutamate, and increases inhibitory GABA (265). One of them encompassed a longitudinal examination involving 9 patients diagnosed with bipolar disorder. The focus of this study was the assessment of plasma prolactin levels before and 12 hours after the evening administration of lithium. Evaluations were conducted on days 1, 6, 8, 13, 30, 60, and 90. Notably, this investigation yielded no discernible correlation between lithium concentration and prolactin levels, and no statistically significant alterations in prolactin levels were observed. The second part of this study adopted a cross-sectional design, involving 26 patients with an established history of long-term lithium treatment spanning durations of 3 months to 20 years. A comparative analysis revealed that prolactin levels, measured at 9 AM following a one-hour period of rest, did not demonstrate elevation in comparison to 16 controls. It is noteworthy that in both studies, lithium concentrations ranged from 0.4 to 1.4 mmol/L (normal range 0.5-1.2 mmol/L) (266). Additionally, the administration of lithium did not exert an impact on the plasma prolactin response to thyrotrophin-releasing hormone (TRH) stimulation compared to pre-treatment levels (267). The combined findings from these investigations provide compelling evidence that lithium does not contribute to hyperprolactinemia, thereby distinguishing it from medications with such an effect.

Cocaine has been shown to decrease prolactin levels beginning at 30-min following cocaine administration reaching statistical significance at the 90- and 120-minute time points (134).

 

Those medications are mentioned in Table 1. Their mechanism of altering prolactin levels is summarized in Figure 2.

 

HERBAL MEDICINES AFFECTING PROLACTIN LEVELS

 

In Table 4 is list of herbal medicines has been used traditionally to stimulate lactation (268). However, firm scientific evidence that they actually induce hyperprolactinemia is scarce.

Table 4. Lactogenic Herbs (268)

Family name

Species name

Common name

Amaryllidaceae

Allium sativum

Garlic

Annonaceae

Xylopia aethiopica

 

African Pepper or Ethiopian Pepper

Asclepiadaceae

Secamoneafzelii

 

-

Costaceae

 

Costusafer

 

African Ginger

Euphorbiaceae

 

Euphorbia hirta

 

Asthma Plant or Tawa-Tawa

Euphorbia thymifolia

 

Petty Spurge

Hymenocardiaacida

 

African Almond or Honeytree

Plagiostylesafricana

 

-

Ricinus communis

 

Castor Bean Plant

Leguminosae

 

Tamarindus indica

 

Tamarind

Acacia nicolita

 

-

Desmodiumadscendens

 

-

Malvaceae

 

Hibiscus sabdariffa

 

Roselle or Red Sorrel

Gossypium herbaceum

 

Cotton Plant

Moraceae

 

 Milicia excelsa

 

African Teak or Iroko

Ficus species

Ficus or Fig trees

Musaceae

 

Musa paradisiaca

 

Plantain

Ranunculaceae

 

Nigella sativa

 

Black Cumin or Black Seed

 

Actaea (Cimiciguga) racemose

Black Cohosh

Solanaceae

 

Solanum torvum

 

Turkey Berry or Devil's Fig

Verbanaceae

 

Lippia multiflora

 

Bush Tea or False Green Tea

Zingiberaceae

 

Aframomummelegueta

 

Grains of Paradise or Alligator Pepper

Fabaceae

Trifolium pratense

Red Clover

Trigonella foenum-graecum

 

Fenugreek

Apiaceae

Foeniculum vulgare

Fennel

Some herbs are known to decrease prolactin levels. For example, chaste tree (Vitex agnus-castus) decrease prolactin levels by activating to D2-receptors and suppressing prolactin release, as shown in in vitro experiments on lactotroph cell cultures and in in vivo animal experiments (269). Another herb, Mucuna pruriens, which is a natural source of l-dihydroxyphenylalanine (a dopamine precursor) is found to decrease prolactin levels in humans (270). Vitamin B6 (pyridoxine), by acting as a coenzyme in dopamine synthesis and aspartame, a sweetener metabolized in phenylalanine (dopamine precursor), have been shown to interfere with milk production by reducing prolactin levels (271). Ashgawanda (Withania somnifera) is found to decrease prolactin levels up to 12% (272). Moreover, oral zinc is found to decrease prolactin levels below the normal range in all 17 subjects with normal prolactin levels, in scenario of increased zinc levels in the blood (273).

 

While none of the mentioned herbs are currently established within clinical guidelines for specifically lactogenic or prolactin-reducing purposes, ongoing research and anecdotal evidence suggest potential roles for these botanicals as adjunctive therapies.

 

CONCLUSION

 

In summary, this review underscores the significant role of drug-induced hyperprolactinemia in causing higher prolactin levels and provides detailed insights into how pharmaceutical agents contribute to this effect. However, understanding the complex mechanisms behind drug-induced hyperprolactinemia is still a work in progress. More research is needed to delve deeper into these mechanisms and gain better insights. These efforts will contribute to refining treatment strategies and improving patient care.

 

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Familial Hypercholesterolemia

ABSTRACT

 

Familial hypercholesterolemia (FH) is a prevalent, autosomal co-dominant disorder of lipid metabolism that results in elevated low-density lipoprotein cholesterol (LDL-C) levels and premature atherosclerosis. Screening for and identifying heterozygous FH in childhood is critical, given its high prevalence and asymptomatic presentation. Furthermore, treatment of FH in childhood is effective at lowering LDL-C levels and has the potential to reduce atherosclerotic cardiovascular disease (ASCVD) events in adulthood. Selective screening based on family history had previously been recommended to identify children and adolescents with FH or other lipid disorders. However, studies indicated that many individuals with heterozygous FH were missed with this approach, and therefore in 2011 the National Heart, Lung, and Blood Institute Expert Panel recommended universal screening of children and adolescents between ages 9 and 11 years and again at ages 17 to 21 years, in addition to selective screening, in order to identify pediatric individuals with heterozygous FH. This approach was affirmed in the 2018 American College of Cardiology and the American Heart Association (ACC/AHA) Cholesterol Guidelines, along with endorsing cascade screening as another reasonable approach to identifying children with FH. Once FH is diagnosed, prompt treatment with lifestyle modification should be initiated. When lifestyle interventions are not sufficient, pharmacotherapy using statins has been shown to be effective at lowering LDL-C, generally safe in short and medium- term studies, and may be beneficial at reducing ASCVD events. Other medications can be useful at lowering LDL-C in conjunction with statin therapy, although generally statins are sufficient in young patients. Homozygous FH is a rare disorder manifesting as extremely high LDL-C and ASCVD in childhood, requiring aggressive multimodal management. Overall, studies are needed to determine the optimal timing and intensity of statin therapy, and to better understand long-term safety and ASCVD outcomes in adulthood for lipid-lowering pharmacotherapy initiated in pediatric patients with heterozygous FH.

 

INTRODUCTION

 

Familial hypercholesterolemia (FH), as classically described, is the most common single gene disorder of lipoprotein metabolism and causes severely elevated low-density lipoprotein cholesterol (LDL-C) levels. The prevalence of FH is 1 in 200 to 1 in 300 individuals of different ethnicities (1,2), and it is strongly associated with premature coronary artery disease (CAD) (3). Data from observational studies suggest that untreated FH is associated with ~90-fold increase in mortality due to atherosclerotic cardiovascular disease (ASCVD) in young adults (4). Since early treatment may significantly reduce CAD-related morbidity and mortality in individuals with heterozygous FH (5), early identification and intervention during childhood may greatly improve outcomes in adulthood.

 

EPIDEMIOLOGY

 

The prevalence of FH varies substantially, depending upon the criteria used to define the disorder and the ancestry of the population.  Previously, the prevalence had been described as 1 in 500 individuals based on early work by Drs. Brown and Goldstein (6,7).  More recent analysis of white European populations, which tend to be less ethnically and racially diverse than the US, show higher prevalence rates (8,9).  An analysis using a modified version of the Dutch Lipid Clinic (DLC) criteria applied to participants in the 1999 to 2012 National Health and Education National Surveys (NHANES), a nationally representative survey of the US population, suggest FH affects 1 in 250 US adults (1). The prevalence of FH in US children and adolescents is not as well characterized, although presumably it is similar. Some estimate that as few as 10% of individuals with FH have been identified in the US.

 

PATHOPHYSIOLOGY AND GENETICS

 

FH was initially defined by Brown and Goldstein as a disorder or defect in the LDL receptor (LDL-R) (3). More recently, the description of FH has been expanded and used to describe any defects in LDL-C processing and/or signaling that may lead to a phenotype characteristic of FH (10). FH may be more common and complicated than previously thought, with many different genetic variants leading to pathogenesis. Overall, nine genes are causative for autosomal forms of FH, and up to 50 polymorphic loci contribute to polygenic susceptibility to elevated LDL- cholesterol levels (11).  Some etiologies for the FH phenotype include defects in apoB100 lipoprotein, the major atherogenic lipoprotein component of LDL-C, as well as gain of function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes the degradation of the LDLR, resulting in reduced LDL-C clearance (7). Gene mutations in LDL-R, the apolipoprotein B gene, or in PCSK9 occur in approximately 93, 5, and 2 percent of individuals with a phenotype consistent with FH, respectively (12). The associated impairment in function of these receptors or proteins results in overall reduced clearance of LDL particles from the circulation and elevation in plasma LDL-C. There is also increased uptake of modified LDL by macrophage scavenger receptors, resulting in lipid accumulation in macrophages and foam cell formation, a precursor to atherosclerotic plaque development (13). Thus, the typical lipid profile of FH is characterized by elevated LDL-C (as high as 300 mg/dL) with subsequently increased total cholesterol (TC) levels; in general, triglycerides are normal, and high-density lipoprotein (HDL-C) can be low or normal. FH has an autosomal dominant inheritance pattern which results in hypercholesterolemia and early ASCVD events.

 

 

There are various genetic mutations that can cause FH, which can either be monogenic or polygenic in nature. See Table 1 below for a list of genes that are associated with FH.

 

Table 1. Genes Associated with FH

Monogenic

Autosomal Dominant

LDLR

APOB

PCSK9

APOE*

Autosomal Recessive

LDLRAP1

LIPA (Lysosomal acid lipase deficiency**)

ABCG5 and ABCG8 (Sitosterolemia**)

Polygenic

Genetic variants associated with FH which together can impact LDL-C levels

*Specific mutations or candidate regions associated with FH

**Refer to specific Endotext sections on lysosomal acid lipase deficiency and sitosterolemia for more information

 

In general, homozygotes for mutations in the LDL-R gene are more adversely affected than heterozygotes, reflecting a “gene dosing effect” of inheritance.  Unless there is consanguinity in a family in which heterozygous FH is present, homozygous FH is less prevalent and may affect as many as 1 in 160,000–300,000 individuals (14).  Additionally, the homozygous FH phenotype can be seen in compound heterozygotes which can occur in offspring of unrelated parents due to a different disease-causing mutation on each allele. The severity of the FH phenotype does not necessarily depend upon the presence of true homozygosity or compound heterozygosity inheritance; rather it is determined by the degree of disturbance in LDL metabolism.  For additional information on the genetics as well as pathophysiology, refer to “Familial Hypercholesterolemia: Genes and Beyond” by Warden et al., www.endotext.org (15).

 

FH PHENOTYPE

 

Clinical Symptoms

 

HOMOZYGOUS FH

 

Due to the excessively high plasma LDL-C levels in homozygous FH, cholesterol deposits are common in the tendons (xanthomas) and eyelids (xanthelasmas), and generally appear by one year of age. Tendon xanthomata are most common in the Achilles tendons and dorsum of the hands but can occur at other sites. Tuberous xanthomata typically occur over extensor surfaces such as the knee and elbow.  Planar xanthomas may occur on the palms of the hands and soles of the feet and are often painful.  Xanthelasmas are cholesterol-filled, soft, yellow plaques that usually appear on the medial aspects of the eyelids. Corneal arcus is a white or grey ring around the cornea (16).

 

HETEROZYGOUS FH

 

In addition to increased serum cholesterol and risk for premature coronary artery disease (see below), patients with heterozygous FH may have tendon xanthomas and corneal arcus that appear after the age of 20 years (16).

 

LDL-C Levels

 

In clinical practice, there is not a universal LDL-C threshold that determines a diagnosis of FH. Generally, the level of LDL-C that warrants further evaluation depends upon the age of the patient and whether additional family members have known hypercholesterolemia and/or early ASCVD. As suggested by recent guidelines (7,17), children and adolescents with a negative or unknown family history and LDL-C level persistently ≥190 mg/dL (4.9 mmol/L) suggests FH; in patients with a positive family history of hypercholesterolemia or early ASCVD, an LDL-C level of ≥160 mg/dL(4.1 mmol/L) is consistent with FH (see Table 2).

 

Table 2. Acceptable, Borderline, and High Lipid Levels for Children and Adolescents

Lipid

Low (mg/dl)

Acceptable (mg/dl

Borderline-High (mg/dl)

High (mg/dl)

TC

 

<170

170-199

>200

LDL-C

 

<110

110-129

>130

Non-HDL-C

 

<120

120-144

>145

Triglycerides

0-9 years

10-19 years

 

 

 

 

<75

<90

 

75-99

90-129

 

>100

>130

HDL-C

< 40

>45

 

 

Adapted from the NCEP expert panel on cholesterol levels in children (14)

 

Cardiovascular Disease

 

Cardiovascular risk in FH patients is determined by both the LDL-C concentration and by other traditional risk factors. Homozygotes have early-onset atherosclerosis, including myocardial infarction, in the first decade of life (reported as early as age two years), and are at increased risk for CAD-related mortality in the first and second decades (16).  Additionally, patients with homozygous FH can develop cholesterol and calcium deposits that can lead to aortic stenosis and occasionally to mitral regurgitation.

 

Heterozygotes are also at increased risk for early-onset CAD between the ages of 30-60 years (18). Children with heterozygous FH have thicker carotid intima-media thickness (cIMT), an anatomic measure of arterial thickness associated with atherosclerosis, compared to unaffected siblings and healthy controls (19,20). One study showed those treated with statin medications (HMG-CoA reductase inhibitors) at younger ages had less carotid atherosclerosis compared to the placebo group. Results from long-term studies of statins in children with FH are just emerging, and indicate that statin treatment during childhood may slow progression of cIMT and reduce the risk of CVD in adulthood (21).

 

SCREENING

 

Given the high prevalence of FH and the improved outcomes with early treatment, pediatric lipid screening has become very important for the detection of FH. However, the approach to lipid screening in childhood and adolescences has varied over the past decades and is somewhat controversial, with the US Preventative Services Task Force (USPSTF) recently concluding that that the current evidence is insufficient to assess the balance of benefits and harms of screening for lipid disorders in children and adolescents (22). Selective screening of young individuals with a family history of hypercholesterolemia and/or early CV events or patients at risk for atherosclerosis for other medical conditions has been recommended for several decades (23–25). However, screening individuals based only on family history may miss 30-50% of children with elevated LDL (24–26). Thus, the 2011 Expert Panel, supported by the more recent 2018 ACC/AHA Cholesterol Guidelines (27), recommended universal lipid screening, which involves screening in childhood at two time points, once between ages 9 and 11 years, and then again between ages 17 and 21 years (28). Universal screening is recommended in those not already selectively screened based on family history or personal risk factors (Table 3).

 

Selective screening for FH involves obtaining a fasting or non-fasting lipid panel in individuals ages 2 to 21 years with:

  1. Family history of early atherosclerosis or high cholesterol.
  2. Relatives of individuals with identified FH.

 

Lipid testing should also be performed in the presence of risk factors or medical diagnoses that increase risk for CVD (including hypertension, current cigarette smoking, body mass index ≥ 85th percentile, diabetes mellitus type I and II, chronic kidney disease/end-stage renal disease, chronic inflammatory diseases, human immunodeficiency virus infection, and nephrotic syndrome) (28).

 

Universal screening involves obtaining either a fasting lipid profile or a non-fasting non-HDL, (calculated by subtracting HDL from TC) in childhood at two time points:

  1. Between ages 9-11 years.
  2. Between ages 17-21 years.

 

Table 3. Screening for Hypercholesterolemia

Approach

Age in Years

Population

Selective

2-21

Family history of early atherosclerosis or high cholesterol

Presence of risk factors or medical conditions that increased early CVD risk*

Universal

9-11 and 17-21

All

*Selective screening is indicated in individuals with hypertension, current cigarette smoking, body mass index ≥ 85thpercentile, diabetes mellitus type I and II, chronic kidney disease/end-stage renal disease, chronic inflammatory diseases, human immunodeficiency virus infection, and nephrotic syndrome

 

DIAGNOSIS

 

The diagnosis of FH can be made clinically and through genetic testing; genotype needs to be interpreted in the context of phenotype. For heterozygous FH, the clinical diagnosis is made based on the presence of high levels of total and LDL cholesterol in combination with one or more of following (17):

 

  1. Family history of hypercholesterolemia (especially in children) or known FH.
  2. History of premature CAD in the patient or in family members.
  3. Physical examination findings of abnormal deposition of cholesterol in extravascular tissues (e.g., tendon xanthoma), although these rarely occur in childhood.

 

There are several clinical scoring systems used to diagnose FH, and these vary based on the weight given for each diagnostic criteria (11).  In general, clinical diagnosis of homozygous FH can be made in individuals with the following criteria (14):

 

  1. Untreated LDL-C >500mg/dL (>13 mmol/L) or treated LDL-C ≥300 mg/dL (>8 mmol/L), AND
  2. Cutaneous or tendon xanthoma before age 10 years, OR
  3. Elevated LDL-C levels consistent with heterozygous FH in both parents.

 

There are various disease states or other factors that can increase LDL-C levels and should be considered when diagnosing FH.  Some of these include the following:

 

  • Obesity
  • Hypothyroidism
  • Diabetes mellitus
  • Nephrotic syndrome
  • Chronic renal failure
  • Cholestasis
  • Biliary atresia
  • Hepatitis
  • Biliary cirrhosis
  • HIV infection/AIDS
  • Various drugs/medications
  • Alcohol
  • Pregnancy
  • Very low carbohydrate ketogenic diets

 

Genetic Testing

 

Identifiable gene defects in LDLR, APOB, or PCSK9 have been identified in 60 to 80% of individuals with a heterozygote FH phenotype. Genetic testing has not routinely been performed in the clinical setting due to concerns about cost to the patient and because it was not likely to alter management, given that treatment decisions were usually based on LDL-C levels. However, FH genetic testing has recently been recommended to become the standard of care for patients with definite or probable FH, and the rationale for such testing includes the following: 1) facilitation of definitive diagnosis of FH lowers the concern for other secondary causes of high LDL-C; 2) pathogenic variants correlate with higher cardiovascular risk, which indicates the potential need for more aggressive lipid lowering; 3) increase in initiation of and adherence to therapy; and 4) cascade testing of at-risk relatives (29). Overall, the clinical significance of normal or moderately elevated LDL-C levels in the setting of a genetic defect in the LDLR or other possibly pathogenic defects is unknown.

 

TREATMENT

 

The guidelines for initiating treatment in patients with the FH phenotype are based on age, severity of LDL-C elevation, as well as family and medical histories. Lifestyle therapy is recommended for all children and adolescents with LDL-C levels ≥ 130 mg/dL. If lifestyle intervention is insufficient, medications can be considered in children beginning at age 10 years, or as early as age 8 in high-risk patients and in the presence of a very high-risk family history. For healthy children and adolescents ages 10-21 years, lifestyle therapy should be provided to those with an LDL-C ≥ 130 mg/dL, and medication should be initiated if LDL-C remains ≥ 190 mg/dL despite 6 or more months of lifestyle modification. If there is a family history of early atherosclerotic disease, then medication should be started in individuals with LDL-C levels ≥ 160 mg/dL who do not respond sufficiently to lifestyle modification. If an individual has a high-risk medical condition, as noted above, medication can be considered for those with a persistently elevated LDL-C ≥ 130 mg/dL. In general, the goal of treatment is to maintain an LDL-C level ≤ 130 mg/dL or ≥ 50% reduction in LDL concentration; lower ranges may be considered in high-risk patients. Medications should be initiated in all patients with homozygous FH at the time of diagnosis, regardless of age, and additional treatments should also be considered.

 

Lifestyle Treatment

 

The mainstay of treatment for pediatric lipid disorders is lifestyle modification. A low saturated fat diet, without trans-fat, and high in fruits and vegetables, is the recommended diet for lowering LDL-C. This dietary approach has been shown to be both safe and beneficial in the general pediatric population (28,30,31).  Additionally, nutritional and physical activity interventions have been shown to lower LDL-C and improve CVD risk factors in children with obesity in meta-analyses (32). Despite this, in adults with FH, lifestyle modifications have been shown to only lower LDL-C modestly (33).  Furthermore, the effect of physical activity on LDL-C levels has not been well studied in children with FH.

 

Pharmacotherapy

 

STATINS  

 

The majority of patients with FH are treated with medications, and statins are the recommended first line pharmacotherapy. In a Cochrane meta-analysis of pediatric patients with FH, statins were shown to lower LDL-C by 32% (34). Furthermore, more intensive statin therapy in high doses has been shown to lower LDL-C even more significantly, by up to 50% (35,36).  Follow-up data from a statin trial in pediatric Dutch patients suggest efficacy and safety, as well as decreased atherosclerosis compared to the subjects’ parents (37). Most recently, the use of statins in children with FH have shown reduced CVD risk in adulthood for these patients compared to their untreated parents with FH, after a 20 year follow-up (21). Although this study was not a controlled or placebo study, it suggests that long-term statin use in childhood may prevent ASCVD compared to not treating.

 

Several different formulations of statin therapy are available and approved by the US Food and Drug Administration (FDA) for use in children. Treatment is initiated at a low dose (generally 5-20mg depending on the statin potency), which is given once a day, often at night. If needed, the dose is increased to meet the goals of therapy. Side effects with statins are rare, but include myopathy, new-onset type 2 diabetes mellitus (reported in adult primary prevention statin trials), and hepatic enzyme elevation. In pediatric clinical trials, rates of side effects with statin therapy were low and adherence to statin therapy was generally good (38). Side effects of statins are more likely at higher doses and in patients taking other medications, particularly cyclosporine, azole antifungal agents, and other medications and foods (such as grapefruit) that impact the cytochrome P450 system. Adolescent females should be counseled about the possibility of drug teratogenicity and appropriate contraceptive methods while receiving statin therapy. Additionally, providers should be aware that oral contraceptive pills can increase lipid levels.

 

The NHLBI guidelines recommend the following baseline laboratory evaluation when initiating statin therapy (28):

 

  • Fasting lipid profile.
  • Serum creatine kinase (CK).
  • Hepatic enzymes (i.e., serum alanine aminotransferase [ALT] and aspartate aminotransferase [AST]).

 

Screening for type 2 diabetes is also reasonable prior to starting statins. Fasting lipid profiles are repeated at four weeks after the initiation of statin therapy to titrate dose and are repeated every six months in patients on stable therapy. Liver function tests, CK, and hemoglobin A1C should be obtained if signs of adverse effects arise, and may be obtained at regular intervals, for example after dose changes based on best clinical judgement. Ongoing monitoring of growth, other measures of general and cardiovascular health, and review for the presence of additional ASCVD risk factors, such as smoking exposure, should also occur at each visit.

 

BILE ACID BINDING RESINS

 

Although bile acid binding resins or bile acid sequestrants have been shown to lower LDL-C by ~10-20% in pediatric trials (39,40), they are often difficult to tolerate given their unpalatability and associated adverse effects (such as bloating and constipation) (41). For these reasons, bile acid binding resins are used relatively infrequently. However, they may be useful in combination with a statin for patients who fail to meet target LDL-C levels (42). The sequestrants are not absorbed systemically, remain in the intestines, and are excreted along with bile containing cholesterol. Therefore, they are considered to be very safe. They can be used in patients who prefer to avoid statins, although they may not lower LDL-C sufficiently to achieve goal levels.

 

CHOLESTEROL ABSORPTION INHIBITORS (EZETIMIBE)

 

Ezetimibe is a lipid-lowering mediation that inhibits absorption of cholesterol and plant sterols in the intestines. This agent can be useful in pediatric patients with FH who are not able to reach LDL-C treatment goals on high-intensity statin therapy. Ezetimibe further lowers serum LDL-C and in adults has been shown to improve cardiovascular outcomes without altering the side effect profile (43–45). Specifically in the pediatric population, ezetimibe has been shown to be safe and effective at lowering LDL-C by up to almost 30%, even when used as monotherapy (44,46,47)

 

PCSK9 INHIBITORS

 

PCSK9 inhibitors (evolocumab and alirocumab) are human monoclonal antibodies that bind to PCSK9 and promote plasma LDL cholesterol clearance. In Europe, evolocumab is approved in adolescents (≥12 years old) with homozygous FH. In the US, alirocumab is approved only for use in adult patients, and evolocumab is approved for use in adults with heterozygous FH and in homozygous FH, ages 13 and older, who have not responded to other LDL-C lowering therapies. Overall, PCSK9 inhibitors appear to have a good safety and side effect profile in adults (48).  These medications have been shown to be very effective, reducing LDL-C by more than 15% in patients with homozygous FH and by 35% in patients with heterozygous FH (49–51). The main disadvantage of PCSK9 inhibitors is that they require injection for administration; cost is also a concern.

 

Inclisiran is another medical therapy that targets PCSK9 synthesis through a different mechanism.  It is a small interfering RNA molecule that triggers the breakdown of messenger RNA coding for the PCSK9 protein. This medication has been recently approved for clinical use and has been shown to be safe and effective in adult patients with heterozygous FH (49). Clinical trials of inclisiran are currently underway in adolescent patients with heterozygous and homozygous FH (50).

 

EMERGING MEDICAL THERAPIES

 

There are several promising therapies that aim to reduce LDL-C through different approaches.  These include Bempedoic Acid, Lomitapide, and Evinacumab.  Bempedoic acid blocks the cholesterol biosynthetic pathway upstream of HMG-CoA reductase through inhibition of adenosine triphosphate citrate lyase. This therapeutic agent has been shown to be effective in treating statin-resistant hypercholesterolemia and in reducing ASCVD in adults (51,52). Bempedoic acid is currently being studied in children with heterozygous FH.

 

Microsomal triglyceride transfer protein (MTP) plays an essential role in the formation of apoB-containing lipoproteins and has been shown to be inhibited by Lomitapide (53). This drug can reduce LDL-C by approximately 58% and has been approved for use in adults with homozygous FH.  So far, Lomitapide has been observed to be safe and effective in pediatric patients with homozygous FH (54).

 

Evinacumab is a human monoclonal antibody that targets angiopoietin-like 3 (ANGPTL3), which results in the reduction of LDL-C levels via an LDL-receptor independent mechanism (55–57).  This drug has been shown to significantly reduce LDL-C in patients with homozygous FH who show little to no LDL-receptor activity and who have had poor response to other treatments (58). Additionally, patients with heterozygous FH have also shown improvements in LDL-C by 50% reduction (59).  Evinacumab was approved in early 2021 by the FDA for the treatment of homozygous FH patients starting at age 12 years (60).

 

Lipoprotein Apheresis

 

Although statin therapy has been shown to be effective in reducing LDL and prolonging life expectancy in patients with homozygous FH (61), medical treatment alone may not be adequate to achieve recommended treatment goals. Therefore, it is suggested that lipoprotein apheresis (LA) be initiated in patients with homozygous FH as young as 2 years. The efficacy is dependent upon the type of apheresis but can reduce LDL-C by as much as 45 to 80%. Studies in pediatric patients are limited, but there is some evidence suggesting that LA therapy is safe and effective in children with homozygous FH (62).

 

SUMMARY

 

FH is an autosomal dominant disorder of LDL metabolism that affects 1 in 200 to 300 individuals. Screening involving lipid measurements, family and medical history, and physical examination is needed to identify affected individuals; cascade screening can be helpful. Lifestyle modification is the first-line therapy for hyperlipidemia in pediatric patients but is usually not sufficient to achieve goal LDL levels. Available evidence suggests that treatment with lipid-lowering pharmacotherapy, such as statins, is effective and generally safe in the short and medium-term.  However, further studies are needed to determine the long-term safety and efficacy in preventing ASCVD of lipid lowering medication in pediatric patients with FH.

 

REFERENCES

 

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  36. Avis HJ, Hutten BA, Gagné C, et al. Efficacy and safety of rosuvastatin therapy for children with familial hypercholesterolemia. J Am Coll Cardiol. 2010;55(11):1121-1126. doi:10.1016/j.jacc.2009.10.042
  37. Kusters DM, Avis HJ, Groot E, Wijburg FA, Kastelein JJP, Wiegman A. Ten-year follow-up after initiation of statin therapy in children with familial hypercholesterolemia. JAMA Internet. Published online September 10, 2014. http://www.ncbi.nlm.nih.gov/pubmed/25203086
  38. Braamskamp MJAM, Kusters DM, Avis HJ, Smets EMA, Wijburg FA, Kastelein JJP. Long-term statin treatment in children with familial hypercholesterolemia: more insight into tolerability and adherence. Paediatr Drugs Internet. Published online 2015. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4372689&tool=pmcentrez&rendertype=abstract
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  45. van der Graaf A, Cuffie-Jackson C, Vissers MN, Trip MD, Gagné C, Shi G, Veltri E, Avis HJ, Kastelein JJ. Efficacy and safety of coadministration of ezetimibe and simvastatin in adolescents with heterozygous familial hypercholesterolemia. J Am Coll Cardiol. 2008 Oct 21;52(17):1421-9
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Use of Lipid Lowering Medications in Youth

ABSTRACT

 

The first comprehensive pediatric dyslipidemia guidelines were published by the National Cholesterol Education Program’s Expert Panel on Blood Cholesterol Levels in Children and Adolescents in 1992 and were updated by the American Academy of Pediatrics (AAP) in 1998. In 2008 the AAP issued an updated clinical report detailing recommendations for screening and evaluation of cholesterol levels in children and adolescents as well as prevention and treatment strategies. Since the publication of the first guidelines, rates of pediatric obesity have significantly increased, resulting in a concomitant increase in dyslipidemia. Recently, options for pharmacotherapeutic interventions in pediatric patients have expanded with new FDA approved indications of several lipid lowering medications, as well as additional safety and efficacy data. In 2011, The National Heart Lung and Blood Institute (NHLBI) published its comprehensive report Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents. As with previous guidelines, lifestyle modifications with an emphasis on a heart-healthy diet and daily moderate to vigorous exercise remain an integral part of treatment for pediatric lipid disorders; however, the recommendations for patients requiring management with pharmacotherapy have changed, and will be the focus of this discussion.

 

INTRODUCTION

 

The diagnosis, treatment, and monitoring of dyslipidemia in youth has undergone significant transformations in recent years.  As detailed by the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) and Bogalusa Heart studies, dyslipidemia plays a vital role in both the initiation, as well as the progression of atherosclerotic lesions in children and adolescents (1-3). Because of their role in premature cardiovascular disease, control of dyslipidemia provides clinicians with an opportunity for reducing morbidity and mortality.  Observational data from individuals with genetic mutations that lower atherogenic cholesterol, low-density lipoprotein cholesterol (LDL-C) and non-high-density lipoprotein cholesterol (non-HDL-C), over a lifetime are associated with fewer events and longer life expectancy (4, 5). While these observations are very encouraging, it is not known if achieving the same level of lipid lowering with medications over decades will offer the same protective effects as observed in individuals with life-long lower cholesterol secondary to a genetic mutation (6). Table 1 provides a comparison across the evolution of guideline recommendations for the initiation of pharmacologic intervention with the goal of balancing risk and benefit (7-11). Table 2 details the risk factors and risk conditions described in the NHLBI guidelines (9).

 

Table 1. Comparison of Recommendations for Treatment

Guidelines

NCEP, AAP – 1992 & 1998

AAP – 2008

NHLBI, AAP – 2011

Pharmacologic Treatment Initiation Parameters*

•

Age > 10 years with LDL-C

•

Age ≥ 8 years with LDL-C

•

Ages 10-21 years with LDL-C

 

o

≥ 190 mg/dL

 

o

≥ 190 mg/dL

 

o

≥ 190 mg/dL

 

o

> 160 mg/dL in addition to a positive family history of premature CVD or presence of at least 2 CVD risk factors in the child/adolescent

 

o

≥ 160 mg/dL in addition to a positive family history of premature CVD or presence of risk factors

 

o

160-189 mg/dL in addition to a positive family history of premature CVD or presence of 1 high level risk factor/condition or presence of 2 moderate level risk factors/conditions

 

 

  

 

  

 

  

 

 

  

 

o

≥ 130 mg/dL in addition to presence of diabetes mellitus

 

  

 

 

    

 

  

 

  

 

 

    

•

Age < 8 years with LDL-C:

 

o

130-159 mg/dL in addition to the presence of 2 high level risk factors/conditions or 1 high level and at least 2 moderate level risk factors/conditions

 

 

    

 

o

≥ 500 mg/dL

 

 

 

    

 

    

 

  

 

 

    

 

    

 

  

 

 

    

 

    

•

Age < 10 years with severe hyperlipidemia or high-risk conditions associated with serious morbidity

 

 

    

 

    

 
 

 

    

 

    

 

*After an adequate trial of diet and lifestyle management.

 

    

 

    

•

Ages 8-9 years with LDL-C levels consistently ≥ 190 mg/dL in addition to a positive family history OR presence of risk factors

 

    

 

    

 

 

    

 

    

 

 

 

 

 

 

 

 

Pharmacologic Medication Recommendations

•

Bile acid sequestrants

•

Bile acid sequestrants

•

Statins

 

    

•

Cholesterol absorption inhibitors

 

  

 

 

 

 

•

Statins

 

 

 

 

 

Table 2. NHLBI Risk Factors and Risk Conditions (9)

NHLBI, AAP 2011 Guidelines: Risk Factors and Risk Conditions

High Level Risk Factors

•       Hypertension requiring drug therapy

•       Tobacco use

•       BMI ≥ 97th percentile

•       High risk conditions

Moderate Level Risk Factors

•       Hypertension not requiring drug therapy

•       BMI ≥ 95th percentile, < 97th percentile

•       HDL < 40 mg/dL

•       Moderate risk conditions

High Risk Conditions

•       T1DM and T2DM

•       CKD, ESRD, post-renal transplant

•       Post-orthotopic heart transplant

•       Kawasaki disease with current aneurysms

Moderate Risk Conditions

•       Kawasaki disease with regressed aneurysms

•       Chronic inflammatory disease

•       HIV infection

•       Nephrotic syndrome

 

PHARMACOTHERAPEUTIC TREATMENT IN YOUTH

 

The treatment of youth with lipid lowering medications presents some unique challenges and consideration due to their developmental stage, the possibility of extended durations of treatment, and the potential use of concurrent medications that may be counter-productive by increasing lipid levels. As patients progress into adolescence it is particularly important for the patient to understand not only the need for their lipid lowering therapies, but also the consequences of non-compliance. Counseling regarding pharmacotherapy should begin at an early age with developmentally appropriate explanations and expand as patients mature. During adolescence when patients are developing their independence counseling that addresses how to integrate their therapy into their own social norms is important for achieving compliance to both pharmacotherapy as well as lifestyle modifications. Additionally, the cost of therapy significantly impacts compliance and should be factored into therapy decisions especially as youth transition into adulthood and may be faced with changes in insurance coverage. It is also critical to continually readdress the correct use of medications as patients are likely to be on multiple therapies and adolescents will begin some their own medication management as they mature. Regular monitoring for adverse events and side effects of therapy is essential as youth will have a greater lifetime exposure compared to adults and long-term data is generally limited.

 

As with all pharmacotherapy careful consideration should be given to potential drug interactions including those that may increase lipid levels. It is not uncommon for adolescent patients to be prescribed medications which have the potential to negatively impact lipid levels such as systemic steroids or oral contraceptive pills. Each patient case must be evaluated on an individual basis to determine the risk and benefit of prescribing medications which negatively alter lipid levels for patients also utilizing lipid lowering therapies. It should be noted there is significant risk for adolescent females should they become pregnant while taking lipid lowering medications as some have demonstrated a negative impact on fetal development. Adolescent females should be counselled regarding pregnancy and methods of contraception should be discussed. The US Medical Eligibility Criteria for Contraceptive Use compiled by the CDC details several contraceptive methods where the benefits generally outweigh any theoretical or proven risk for patients with hyperlipidemias (12).

 

HMG-CoA REDUCTASE INHIBITORS

 

HMG-CoA reductase inhibitors, or statins, are recommended as first line treatment of youth with severe dyslipidemia who fail non-pharmacologic interventions (i.e., diet and lifestyle modification) (8-11). Statins first debuted in clinical practice in 1987 with the FDA’s approval of lovastatin. At present, there are seven HMG-CoA reductase inhibitors with FDA approval, at varying dosages, for youth with heterozygous familial hypercholesterolemia. Lovastatin, simvastatin, atorvastatin and fluvastatin are approved for children 10 years of age and older. Pitavastatin and pravastatin are approved starting at 8 years of age and rosuvastatin is indicated in children as early as age 6 (13-21). Table 3 provides a summary of HMG-CoA reductase inhibitors, pediatric approval and indications, recommended dosing ranges, comments on dosing, and supporting clinical trials (13-21).

 

Table 3. HMG-CoA Reductase Inhibitors

Medication

Pediatric Approvals & Indications

Dosing

Comments

Supporting

Clinical Trials

Atorvastatin

Age 10-17
Heterozygous familial hypercholesterolemia

10-20 mg/day

May be titrated at ≥ 4-week intervals

McCrindle, et al (22)

Fluvastatin

Age 10-16
Heterozygous familial hypercholesterolemia

20-80 mg/day

May be titrated at ≥ 6-week intervals

van der Graaf, et al (23)

Lovastatin

Age 10-17
Heterozygous familial hypercholesterolemia

10-40 mg/day

Initiated at 20 mg/day for ≥20% LDL reduction, may be titrated at ≥ 4-week intervals

Clauss, et al (24)
Lambert, et al (25)
Stein, et al (26)

Pravastatin

Age 8 and older
Heterozygous familial hypercholesterolemia

20-40 mg/day

Age 8-13: 20 mg/day
Age 14-18: 40 mg/day

Knipscheer, et al (27)
Wiegman, et al (28)
Rodenburg, et al (29)

Rosuvastatin

Age 6 and older
Heterozygous familial hypercholesterolemia

5-20 mg/day

May be titrated at ≥ 4-week intervals

Avis, et al (30)

Simvastatin

Age 10-17
Heterozygous familial hypercholesterolemia

10-40 mg/day

May be titrated at ≥ 4-week intervals

de Jongh, et al (31)
de Jongh, et al (32)

Pitavastatin

Age 8 and older
Heterozygous familial hypercholesterolemia

1-4 mg/day

May be titrated at ≥ 4-week intervals

Ferrari, et al (20)

The above are approved as an adjunct to a diet that is low in cholesterol and saturated fat.  The above agents are approved for both males and females (females must be at least one-year post-menarche) if, despite an adequate diet and other non-pharmacologic measures, the following are present: LDL-C ≥ 190 mg/dL or LDL-C ≥ 160 mg/dL and the patient has a family history of premature cardiovascular disease or two or more cardiovascular disease risk factors.
Abbreviations: mg=milligrams, LDL=low density lipoprotein

 

As Table 4 outlines, statin therapies have demonstrated variable efficacy involving clinical trials of youth (21-32). With the longest half-life, rosuvastatin is the most potent statin, followed by atorvastatin (33). Given this knowledge, if pediatric patients who are initiated on statin therapy are having trouble meeting LDL-C goals, consideration should be given to switching to rosuvastatin or atorvastatin given potency prior to exploring second-line therapy options. Simvastatin is a moderately potent statin at clinically tolerable maximum doses of 40 mg/day (33-35). Lovastatin, pravastatin, and fluvastatin, respectively, are the least potent statins (34, 35). As many studies have demonstrated, reduced potency can be compensated by an increase in the amount of statin given; however, dose escalation is often associated with an increased occurrence of adverse events (21-35). As a result, selection of a specific statin therapy should be individualized and capable of reaching treatment goals. Equally important, consideration should be given to the prevalence and severity of reported side effects (36).

 

Table 4. Statin Therapy Results

Study

Medication

Dose

Results

LDL-C

HDL-C

TC

TG

McCrindle, et al (22)

Atorvastatin

10-20 mg/day

-40%

+6%

-30%

-13%

van der Graaf, et al (23)

Fluvastatin

80 mg/day

-34%

+5%

-27%

-5%

Clauss, et al (24)

Lovastatin

40 mg/day

-27%

+3%

-22%

-23%

Lambert, et al (25)

Lovastatin

10 mg/day

-21%

+9%

-17%

-18%

Lambert, et al (25)

Lovastatin

20 mg/day

-24%

+2%

-19%

+9%

Lambert, et al (25)

Lovastatin

30 mg/day

-27%

+11%

-21%

+3%

Lambert, et al (25)

Lovastatin

40 mg/day

-36%

+3%

-29%

-9%

Stein, et al (26)

Lovastatin

10 mg/day

-17%

+4%

-13%

+4%

Stein, et al (26)

Lovastatin

20 mg/day

-24%

+4%

-19%

+8%

Stein, et al (26)

Lovastatin

40 mg/day

-27%

+5%

-21%

+6%

Knipscheer, et al (27)

Pravastatin

5 mg/day

-23%

+4%

-18%

+2%

Knipscheer, et al (27)

Pravastatin

10 mg/day

-24%

+6%

-17%

+7%

Knipscheer, et al (27)

Pravastatin

20 mg/day

-33%

+11%

-25%

+3%

Rodenburg, et al (29)

Pravastatin

20 mg/day or 40 mg/day

-29%

+3%

-23%

-2%

Wiegman, et al (28)

Pravastatin

20-40 mg/day

-24%

+6%

-19%

-17%

Avis, et al (30)

Rosuvastatin

5 mg/day

-38%

+4%

-30%

-13%

Avis, et al (30)

Rosuvastatin

10 mg/day

-45%

+10%

-34%

-15%

Avis, et al (30)

Rosuvastatin

20 mg/day

-50%

+9%

-39%

-16%

de Jongh, et al (31)

Simvastatin

10-40 mg/day

-41%

+3%

-31%

-9%

de Jongh, et al (32)

Simvastatin

40 mg/day

-40%

+5%

-30%

-17%

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report.  Pediatrics.  2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

 

Although long term studies evaluating the safety and efficacy of lipid-lowering medications in youth are lacking, results of short term observational and randomized controlled trials are encouraging.  For example, atorvastatin was found to be well tolerated with no statistically significant differences in adverse events reported for either the treatment or placebo groups (22). Additionally, the percentage of patients with abnormal laboratory results was similar for both groups; the only noted difference was an increased percentage of patients with elevated triglycerides in the placebo group. Treatment with atorvastatin resulted in no significant difference in sexual development as assessed by Tanner staging. Van der Graaf and colleagues found that in youth treated with fluvastatin, 58 (68.2%) reported non-serious adverse events. Only four were believed to be drug related. Treatment with fluvastatin resulted in no abnormalities in hormone levels or sexual maturation (23). 

 

In their study of lovastatin, Clauss and colleagues reported no clinically significant alterations in vital signs; growth; hormone levels including luteinizing hormone, follicle-stimulating hormone, dehydroepiandosterone sulfate, estradiol, and cortisol; menstrual cycle length; liver function tests; or muscle function tests (24). Lambert and colleagues also found lovastatin generally well tolerated with no serious clinical adverse effects noted (25). While increased, levels of aspartate aminotransferase did not exceed two times the upper limit of normal and alanine aminotransferase did not display significant changes in study participants. Creatine kinase was elevated, greater than three times the upper limit of normal, in three patients. All subjects remained asymptomatic and the elevated creatine kinase levels resolved spontaneously while no adjustment was need in their medication. Assessment of growth and sexual maturation, by Tanner staging and estimation of testicular volumes, in youth treated with lovastatin found no significant differences between the treatment groups and placebo at either 24 or 48 weeks (26). While the authors reported no significant change in serum hormone levels or biochemical parameters of nutrition, they noted that the study was under powered to detect statistically significant changes in these safety parameters.

 

Use of pravastatin has been shown to have minimal adverse events dispersed evenly between the active drug recipients and those who received placebo (27). Plasma thyroid-stimulating hormone, adrenocorticotropic hormone, cortisol, creatine phosphokinase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, and alkaline phosphatase levels failed to show significant changes from baseline in all treatment groups. Rodenburg and colleagues also evaluated the safety of pravastatin based on annual or biannual evaluation of plasma creatine phosphokinase levels, liver enzymes, sex steroids, gonadotropins, and hormones of the pituitary-adrenal axis (29). Height, weight, age at menarche, Tanner staging, and testicular volume were recorded at baseline and either annually or biannually. Two subjects demonstrated elevated creatine phosphokinase levels which returned to normal without adjustments in therapy, and were presumed to be due to extreme physical exercise. No occurrence of myalgia was associated with elevation in levels of creatinine phosphokinase.  None of the subjects discontinued therapy due to adverse events or laboratory abnormalities.  Similarly, Wiegman and colleagues examined the safety of pravastatin evaluated at baseline, one year, and two years via multiple variables including: sex steroids, endocrine function parameters, height, weight, body surface area, Tanner staging, menarche or testicular volume, alanine aminotransferase, aspartate aminotransferase, and creatine phosphokinase (28). All safety parameters demonstrated no statistically significant differences between active drug recipients verses placebo in changes from baseline.

 

The safety of rosuvastatin was assessed by Avis and colleagues.  Laboratory monitoring including liver enzymes and creatine kinase levels, and markers of growth and development, such as Tanner staging (30). Two serious adverse events were reported including blurred vision in one patient in the placebo group and a vesicular rash progressing to cellulitis in one patient taking rosuvastatin 20 mg. Transaminase levels either remained normal or normalized without permanent discontinuation of treatment. While elevations in creatine kinase and reports of myalgia did occur, symptoms and creatine kinase levels normalized without permanent discontinuation of therapy. Normal progression of height, weight, and sexual development were observed. 

 

The safety of simvastatin during short term therapy has also been reported. Levels of alanine aminotransferase, aspartate aminotransferase, creatine kinase, and physical examination all demonstrated no significant differences between simvastatin treated and placebo participants (31). de Jongh and colleagues evaluated the safety of simvastatin in a second trial by monitoring adverse events as well as changes in alanine aminotransferase, aspartate aminotransferase, and creatine kinase levels (32). Of note, none of the differences reported in events or laboratory values between simvastatin recipients and placebo reached statistical significance. Additionally, there were no statistically significant differences documented for height, body mass index, cortisol levels, testicular size and testosterone levels, menstrual cycle and estradiol levels, and Tanner staging.  Dehydroepiandrosterone sulfate levels demonstrated a statistically significant decrease in the simvastatin group compared to placebo. 

 

Braamskamp and colleagues investigated the efficacy and safety of pitavastatin in pediatric patients diagnosed with hyperlipidemia. 106 patients were enrolled in the study, ages 6-17, for a 12-week period (37). Patients were randomly assigned to 4 different groups categorized by dose 1 mg, 2 mg, 4 mg, or placebo (37). The results showed a reduction in all 3 dose groups in comparison to placebo, the 1 mg group showed a 23.5% reduction in LDL-C, the 2 mg group showed a 30.1% reduction, and the 4 mg group showed a 39.3% reduction (37). There was also a 52-week extension period where patients assigned to the 1 mg group were up-titrated to a maximum dose of 4 mg to try and achieve an LDL-C level of >110 mg/dL (37). There were no safety issues of concern throughout the study. The results indicated that pitavastatin is safe and efficacious for use in pediatric patients, 6-17 years of age, and it was well-tolerated (37).

 

Long-term data regarding the impact of statin therapy on growth, development, and reduction of cardiac risk are limited, particularly for high intensity statin therapy. Recently, Kusters and colleagues evaluated the safety of statin therapy in children and adolescents with familial hypercholesterolemia after 10 years of treatment comparing laboratory safety markers as well as growth and maturation in untreated siblings (36). Only three patients discontinued therapy due to adverse events. Safety parameters such as aspartate aminotransferase, alanine aminotransferase, creatine kinase, estimated glomerular filtration rate, c-reactive protein, and age of menarche did not differ between treated patients and siblings, demonstrating safety over the 10-year treatment period. The authors do note; however, that the study was underpowered to detect the occurrence of rare events (36).

 

When initiating HMG-CoA reductase inhibitor therapy, as with any new medication therapy, it is imperative for clinicians to establish an accurate baseline, monitor for new symptoms, and counsel both patients and family members regarding potential adverse events. Females should be informed about the need to avoid pregnancy and breastfeeding while using statins. Statins may be taken with or without meals, but are commonly given with the evening meal or at bedtime as this has the potential to improve LDL-C reduction (38). As a major substrate of P450, such as CYP3A4, there are multiple drug interactions associated with statin therapy.  Grapefruit juice has gained considerable notoriety as a potential food interaction; however, it should be noted that more than a quart of grapefruit juice would have to be consumed to increase serum statin levels. Of more concern is the possible interaction of gemfibrozil and statins, which should either be avoided as it can increase the toxicity of HMG-CoA reductase inhibitors. Macrolides and antifungal azoles are classes of drugs commonly prescribed to children, and they should be avoided as often as possible as they increase serum statin levels leading to potentially enhanced myopathic effects. Additionally, patients should avoid herbal products and nutraceuticals, such as red yeast rice, which may further enhance adverse effects.

 

BILE ACID SEQUESTRANTS

 

Bile acid sequestrants, or bile acid binding resins, present an additional treatment option for youth with severe dyslipidemia. Bile acid sequestrants represent one of the oldest classes of medications available to treat dyslipidemia and were the only medication recommended in the 1992 NCEP Pediatric Panel Report, at a time when no data were available for statin use in youth (4). While no longer a recommended first-line therapy, bile acid sequestrants do have potential as a treatment option either alone or in combination with a statin (9, 10). At present, colesevelam is the only bile acid sequestrant with FDA approval for youth age 10 years and older with heterozygous familial hypercholesterolemia (39, 40). Despite the lack of FDA approval, both colestipol and cholestyramine have been studied in pediatric patients (41-49).

 

A number of clinical trials have evaluated the bile acid sequestrants in pediatric patients with heterozygous familial hypercholesterolemia and other forms of severe dyslipidemia. While palatability and tolerance remain potential barriers to effective therapy, in general, bile acid sequestrants have demonstrate significant reductions in both total cholesterol and LDL-cholesterol in study subjects (40, 43, 44, 48, 49). Table 5 provides a summary of bile acid sequestrants, pediatric approval and indications, recommended dosing ranges, comments on therapy, and supporting clinical trials. As outlined in Table 6, studies demonstrated some variability in efficacy for the available bile acid sequestrants (40, 43, 44, 48, 49).

 

Table 5. Bile Acid Sequestrants

Medication

Pediatric Approvals & Indications

Dosing

Comments

Clinical Trials

Colesevelam

Age 10-17
Heterozygous familial hypercholesterolemia

1.875 g twice daily or 3.75 g daily

May be used as monotherapy or in combination with a statin

Stein, et al (40)

Colestipol

(Note: Not FDA Approved)
Age 7-12
Primary hypercholesterolemia

5 g twice daily, 10 g daily, or
125-500 mg/kg/day

N/A

McCrindle, et al (43)
Tonstad, et al (44)

(Note: Not FDA Approved)
Age ≥12
Primary hypercholesterolemia

10-15 g/day

N/A

Cholestyramine

(Note: Not FDA Approved)
Age 6-12
Hypercholesterolemia adjunct

240 mg/kg/day divided three times daily before meals

Initiate at 2-4 g twice daily

McCrindle, et al (48)
Tonstad, et al (49)

(Note: Not FDA Approved)
Age ≥12

8 g/day divided twice daily before meals

N/A

Colesevelam is approved as an adjunct to a diet that is low in cholesterol and saturated fat.  Colesevelam is approved for both males and females (females must be at least one-year post-menarche) if, despite an adequate diet and other non-pharmacologic measures, the following are present: LDL-C ≥ 190 mg/dL or LDL-C ≥ 160 mg/dL and the patient has a family history of premature cardiovascular disease or two or more cardiovascular disease risk factors.
Abbreviations: g=grams, mg=milligrams, kg=kilograms, N/A=not applicable.

 

Table 6. Bile Acid Sequestrant Results

Study

Medication

Dose

Results

LDL-C

HDL-C

TC

TG

Stein, et al (40)

Colesevelam

1.875 g/day

-6%

+5%

-3%

+6%

Stein, et al (40)

Colesevelam

3.75 g/day

-13%

+8%

-7%

+5%

McCrindle, et al (43)

Colestipol

10 g/day

-10%

+2%

-7%

+12%

McCrindle, et al (43)

Colestipol & Pravastatin

Colestipol: 5 g/day
Pravastatin: 10 mg/day

-17%

+4%

-13%

+8%

Tonstad, et al (44)

Colestipol

2-12 g/day

-20%

-7%

-17%

-13%

McCrindle, et al (48)

Cholestyramine

8 g/day

-10% to
 -15%

+2% to +4%

-7% to
 -11%

+6% to +9%

Tonstad, et al (49)

Cholestyramine

8 g/day

-17%

+8%

-12%

N/A

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report.  Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: g=grams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

 

Stein and colleagues assessed the safety of colesevelam at weeks 8-26 during an open-label study from.  All subjects received colesevelam 3.75 grams per day in addition to a statin (40). Safety was measured via adverse events, vital signs and physical exam, laboratory monitoring, and Tanner staging.  The most common adverse events related to use of colesevelam were gastrointestinal, including diarrhea, nausea, vomiting, and abdominal pain. It is important to note that no choking or difficulty swallowing were reported with the use of colesevelam. Vital signs, physical exams, laboratory monitoring, and Tanner staging remained the same or progressed as expected throughout the study period. 

 

McCrindle and colleagues evaluated conventional high-dose colestipol versus a combination of low-dose colestipol plus pravastatin, but did not cite safety as an endpoint for their study (40). The researchers did conduct safety monitoring in the form of laboratory tests, physical evaluation, and adverse event reporting. Significant deviations from baseline were noted for alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase at various time intervals with different medication regimens; however, the authors noted that when compared to reference values, none of the laboratory results were considered abnormal. Study participants in the two medication regimens did not significantly vary in weight gain, height changes, or body mass index. While the majority of patients experienced no adverse events, gastrointestinal symptoms such as constipation, gas or bloating, or stomach ache were more commonly reported by the patients taking the high-dose colestipol. The authors found similar suboptimal compliance with both regimens as determined by medication counts at the end of each study period. Tonstad and colleagues also assessed the tolerability of colestipol granules by monitoring side effects as well as by having subjects’ complete subjective evaluations (44). Side effects associated with colestipol included constipation, dyspepsia, flatulence, nausea, reduction in appetite, and abdominal pain. The subjective evaluations indicated that only 21% of patients liked the taste of the colestipol; however, of those who had previously taken bile acid binding resin, 86% preferred the taste of the newer orange flavored granules. Thirty seven percent of subjects also reported that they frequently forgot to take the medication, while 44% intentionally eliminated the medication from their routine on special occasions or during trips. 

 

The acceptability and compliance of cholestyramine has been studied (48). Eighty two percent (82%) of children preferred the pill formulation of cholestyramine compared to 16% who preferred the powder. Two percent of children in the study preferred neither form of the medication. Compliance was significantly impacted by medication formulation with patients taking the pill form reporting 61% compliance while those on the powder formulation were only 50% compliant. Compliance increased by at least 25% for 42% of patients when they switched to the pill formulation. Tonstad and colleagues assessed the safety of cholestyramine by measuring height velocity, erythrocyte folate, total plasma homocysteine, serum fat-soluble vitamins, and side effects (49). Weight and mean height velocity standard deviation scores were not statistically significant between treatment and placebo groups during the study. The cholestyramine active treatment group demonstrated decreased vitamin D levels and increased homocysteine levels. Differences in erythrocyte folate were not significant between the active treatment and placebo groups. Reported adverse events included intestinal obstruction, abdominal pain, nausea, and loose stools. Unpalatability was a common reason participants withdrew from the study.

 

As demonstrated by the previous studies, while bile acid sequestrants do present an effective therapy option, their side effect profile, issues of tolerability and drug interactions with statins make their use clinically challenging. It is generally recommended that all concurrent medications be given either one hour before or four hours after bile acid sequestrants to prevent decreased absorption of the additional therapies (41, 46). Use of bile acid sequestrants is generally limited to patients optimized on statin therapy who require additional therapy to achieve goal or those that cannot tolerate statins.  Data on long-term safety, however, are generally lacking. It should also be noted that bile acid sequestrants can increase triglyceride levels and should not be used in patients with increased triglyceride levels.

 

FIBRIC ACID DERIVATIVES

 

Experience with fibric acid derivatives in youth is limited. Currently there are no fibric acid derivatives with FDA approval for use in pediatric patients. Both fenofibrate and gemfibrozil are available in the United Sates, but lack pediatric data on safety, efficacy, and dosing (50, 51).  While there is very limited information on the use of bezafibrate in youth, the product is not available in the United States (52). It should be noted that fibric acid derivatives have the potential to increase the incidence in adverse events, such as rhabdomyolysis, when used with statins (45, 50, 51). However, the use of fibrates should be considered and can be beneficial in pediatric patients who also have triglyceride abnormalities (TG levels > 500 mg/dL) (9, 53).

 

NIACIN

 

Niacin provides a potential adjunct therapeutic option for youth with severe dyslipidemia who have not achieved their lipid goal. Extended-release niacin is the only formulation that has FDA-approval for use in children > 16 years of age (54). Despite a lack of FDA approval for ages younger than 16, limited efficacy and safety data are published for the use of niacin in children 10 years of age as older as adjunct therapy (54). Table 7 summarizes data on recommended dosing ranges, comments on dose adjustments, and references supporting clinical trials. 

Colletti and colleagues conducted a retrospective review to evaluate the efficacy and adverse effect profile of niacin for children with severe hypercholesterolemia (54). The effects on serum lipid profiles are detailed in Table 8.  Adverse effects were common, affecting 76% of children, and similar to those reported for adults including: flushing, abdominal pain, vomiting, headache, and elevated liver enzymes. Due to the high prevalence of adverse effects, use of niacin should be limited to patients not achieving goal with other therapies or those who cannot tolerate alternative adjunctive options. As with fibrates, niacin can also be considered for the purposes of treating pediatric patients who are concurrently diagnosed with hypertriglyceridemia (9).

 

Table 7. Niacin

Medication

Pediatric Approvals & Indications

Dosing

Comments

Clinical Trials

Niacin

Extended release: >16 years of age

Note: Immediate release is not FDA Approved:
Age ≥ 10
Adjunct therapy

Initial: 100-250 mg/day
(Max: 10 mg/kg/day)
divided three times daily with meals

May titrate weekly by 100 mg/day or every 2-3 weeks by 250 mg/day

Colletti, et al (54)

Abbreviations: mg=milligrams, kg=kilograms.

 

Table 8. Niacin

Study

Medication

Dose

Results

Colletti, et al (54)

Niacin

500-2,250 mg/day

LDL-C

HDL-C

TC

TG

-17%

+4%

-13%

+13%

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report.  Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

 

EZETIMIBE

 

Ezetimibe is FDA approved for adolescents 10 years of age and older with FH (53, 55), and it presents a potential therapy option either as monotherapy or when synergistically paired with an HMG-CoA reductase inhibitor (56-58). Due to its favorable tolerability, it has become the most frequently used second-line agent (59). Table 9 summarizes data on recommended dosing ranges and references supporting clinical trials while Table 10 details efficacy of therapy. 

Tolerability of ezetimibe was prospectively evaluated by Yeste and colleagues via a combination of biochemical markers and adverse event reports (56). No change was seen in hemogram, transaminases, creatinine, calcium, phosphorus, and vitamins A and E for any of the 17 patients. Additionally, there were no reports of adverse events during the study period. Clauss and colleagues retrospectively evaluated ezetimibe; therefore, safety parameters were less defined, but included intermittent measurement of liver enzymes, occasional CK levels, and adverse event reports (57). There were no reported abnormalities in liver enzymes for study participants. Ultimately, one patient was discontinued from ezetimibe therapy for asymptomatic elevated CK levels, later determined to be likely unrelated to therapy.

 

Table 9. Ezetimibe

Medication

Pediatric Approvals & Indications

Dosing

Comments

Clinical Trials

Ezetimibe

Age ≥10
Homozygous familial hypercholesterolemia

10 mg/day

N/A

Yeste, et al (56)
Clauss, et al (57)
van der Graaf, et al (58)

Abbreviations: mg=milligrams, N/A=not applicable.

 

Table 10. Ezetimibe

Study

Medication

Dose

Results

Yeste, et al (56)

Ezetimibe

10 mg/day

            LDL-C

HDL-C

TC

TG

PH

-42%

N/A

-31%

N/A

FH

-30%

-15%

-26%

N/A

Clauss, et al (57)

Ezetimibe

10 mg/day

            LDL-C

HDL-C

TC

TG

FH

-28%

N/A

-22%

N/A

FCHL

N/A

-13%

N/A

van der Graaf, et al (58)

Ezetimibe
&
Simvastatin

Ezetimibe: 10 mg/day
Simvastatin: 10-40 mg/day

            LDL-C

HDL-C

TC

TG

              -49%

+7%

-38%

-17%
                 

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report.  Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides, PH=polygenic hypercholesterolemia, FH=familial hypercholesterolemia, FCHL=familial combined hyperlipidemia.

 

Van der Graaf and colleagues assessed the safety of combination therapy with ezetimibe and simvastatin based on reported adverse events as well as laboratory monitoring and clinical examination (58). After 53 weeks, 71% of study participants reported some types of treatment-emergent adverse events. Of those events reported, only influenza, nasopharyngitis, and headache occurred in greater than 5% of participants. Consecutive transaminase elevations of at least three times the upper limit of normal were reported in 6 participants; however, all values resolved with interruption or discontinuation of therapy.  Elevations in creatine phosphokinase occurred infrequently and were not associated with myalgia at levels greater than three times the upper limit of normal.  Height, weight, and sexual maturation were not significantly impacted by therapy. Ezetimibe affords flexibility in administration time with the ability to administer it without regard to meals or time of day (45). HMG-CoA reductase inhibitors have the risk of increasing myopathy and elevation in hepatic transaminases, but are generally considered a safe combination with ezetimibe.

 

OMEGA-3 FISH OILS

 

Omega-3 fish oils are a class of therapy for which there is significantly limited data in youth. To date the FDA approved formulations of omega-3 fatty acid lack a pediatric indication. High dose omega-3 fatty acid supplementation was evaluated by de Ferranti and colleagues, but ultimately the authors found no statistically significant improvement when subjects were compared to placebo (60). Chahal and colleagues similarly found no significant impact on hypertriglyceridemia when treating pediatric patients with fish oil (61).

 

Khorshidi and associates performed a systematic review and meta-analysis of the effect of omega-3 supplementation on lipid profiles in children and adolescents. They found that omega-3 supplementation improved triglyceride levels in patients diagnosed with hypertriglyceridemia that were less than or equal to 13 years of age; however, there was no significant effect seen in HDL, LDL, or TC values (62). Omega-3 fish oils can be considered in those who have elevated triglyceride levels.

 

FAMILIAL HYPERCHOLESTEROLEMIA AND THERAPEUTIC ADVANCES

 

Familial hypercholesterolemia (FH) is a common, but often misdiagnosed inherited gene disorder (63). The most common gene mutation seen in FH is in the low-density lipoprotein receptor gene (LDLR) accounting for 85-90% of cases, followed by the apolipoprotein (ApoB) gene (5-15% of cases) and the proprotein convertase subtilisin kexin 9 (PCSK9) genes (1% of cases) (63, 64). Patients who are diagnosed with FH have abnormally elevated LDL levels from birth. FH is associated with a twenty-fold increased risk in premature cardiovascular disease and cardiovascular events (65). There are two different types of FH, heterozygous (HeFH) and homozygous (HoFH). Heterozygous patients have one mutated allele and are more commonly seen in practice, while homozygous patients have two mutated alleles and are very rare (66). Distinction between the two types is imperative because HoFH patients tend to be treatment resistant and carry a worse prognosis, if left untreated, these patients rarely make it past age 20 (66).

 

Lipid lowering in these patients can be quite challenging, especially HoFH patients. Most typical therapies for lipid lowering require functional LDL receptors, therefore given the gene mutations which often render the receptors inactive, modest reductions of 10-25% in HoFH patients are usually all that will be gained (67). HeFH patients tend to see higher rates of reduction (25-40%) (68). However, first line treatment for both forms of FH is still high-intensity statin therapy at moderate to high doses to be initiated as early as age 8 (69). All seven statins are FDA-approved for the treatment of FH and have been proven to slow down the progression of carotid intima-media thickness (63). Statins also reduce the incidence of cardiovascular events and cardiac death (63). Until recently, long term data on the use of statins in this patient population was not available, but in 2019, a 20-year follow-up study of statins in pediatric patients with FH was published (70). The results found that the incidence of cardiovascular events and death was much lower in patients treated with statins (70). If LDL-C goals are not being met with the use of statins alone, the next recommend agent is ezetimibe; however, this should not be used in patients younger than 10 years of age (59).

 

Ezetimibe is the second-line option to statins in these patients. It’s use in combination with statins has demonstrated a reduction of LDL-C levels below 135 in more than 90% of children with FH (71). In one of the only studies that assessed the coadministration of simvastatin and ezetimibe in children with HeFH, it was found to be safe, well tolerated and provided a higher LDL reduction (15%) compared with simvastatin alone in HeFH patients (72). However, when it was investigated as a monotherapy option for children with HeFH, it only produced LDL-C lowering of 27% (71, 73). It is more appropriate to use as an adjunct therapy in this population of patients.

 

Alternative medications that can be considered other second-line options are bile acid sequestrants (71). Colesevelam is safe to use, but limited to children >10 years of age (71). These drugs however have minimal LDL-C lowering effects, usually only seeing a 10-20% reduction, and more importantly are very poorly tolerated due to gastrointestinal side effects (71).

 

PCSK9 Inhibitors

 

Given the difficulty and importance of treating these patients, especially those with HoFH, there is a need for stronger lipid lowering options, which is where PCSK9 inhibitors come into play. These are a more recent class addition to the therapy options for managing FH, which help to reduce the degradation of LDL receptors and the removal of LDL-cholesterol (74). In a recent study assessing the efficacy/safety of lipid-lowering agents in patients with familial hypercholesterolemia, it was concluded that PCSK9 inhibitors were the most effective in lowering lipid levels (75). They have none of the same side effects as statins and produced similar CV benefits. Therefore, based on these conclusions, PCSK9 inhibitors are recommended as first-line agents in patients with hypercholesteremia that have intolerances or resistance to statins (75). There is currently one PCSK9 inhibitor approved for pediatric use. Repatha (evolocumab) was originally approved for use in patients 13 years of age and older (76), but the HAUSER-RCT study assessed the use of Repatha in patients ages 10-17 years of age for 24 weeks. It showed that the drug improved lipid levels (by approximately 38% in HeFH patients and 21-24% in HoFH patients) and was safe for use as the incidence of adverse events was similar in both the drug and placebo groups (71, 77). So now it is considered safe to use in pediatric patients 10 years of age and older. The HAUSER-RCT trial was then continued for another 80 weeks to further assess the safety and efficacy of Repatha (78). This new trial, HAUSER-OLE, further confirmed that the drug was safe and well-tolerated (78).

 

Praluent (Alirocumab) is another PCSK9 inhibitor that is available for treatment of FH, however it is not currently approved for use in pediatric patients (79). Nevertheless, there are studies currently assessing the safety and efficacy within this population. Bruckert and associates utilized Praluent and conducted an open-label phase 3 study specifically in pediatric patients (8-17 years of age) diagnosed with HoFH that were inadequately controlled (80). Patients received 75 or 150 mg of the drug based on weight (<50 or >50 kg, respectively) every 2 weeks for 12 weeks (80). The primary endpoint was percent change in LDL-C levels from week 0 to 12 (80). Interestingly, the results showed only a 4.1% decrease in LDL-C levels by week 12 (80). The secondary endpoints (assessing percent change LDL-C levels from baseline to weeks 24 and 48, changes in other lipid parameters from baseline to weeks 12, 24, 48, patients with a reduction of more than 15% in LDL-C levels at weeks 12, 24 and 48, and absolute change in LDL-C from baseline to weeks 12, 24, and 48) produced incredibly variable results (80). Overall, there were quite small changes in LDL-C levels observed in this study with mean reductions of LDL-C levels noted to range anywhere from ~33 to 52 mg/dL (80). More importantly, previous studies have shown that PCSK9 inhibitors are linked to a decrease in major coronary/vascular events and all-cause mortality, so although the results produced small values, the changes seen are still clinically significant based on these added benefits (80). It was also noted that this study produced similar results when compared to the ODYSSEY study which assessed the use of Praluent in adult patients with HoFH (80). The drug was deemed safe and there were no issues with tolerability (80). The study supports the use of Praluent as an adjunct therapy in HoFH patients already on first- and second-line therapies and not reaching their goal LDL-C levels (80).

 

Another study assessed the use of Praluent in pediatric patients diagnosed with HeFH. The ODYSSEY KIDS study was a phase 2 dose-finding study that enrolled pediatric patients anywhere from 8-17 years of age (81). Patients were split into 4 cohorts and dosed every 2 weeks. Dosing was determined by weight and the primary endpoint assessed percent change in LDL-C from baseline to week 8 (81). Praluent demonstrated the best reduction in LDL-C levels in the highest dosed cohorts and was well-tolerated. This study also supported the use of the drug (with further analysis) in patients who require adjunct therapy, there is a phase 3 trial planned to assess the doses from this study that resulted in the greatest reduction in LDL-C levels. Overall, it is important to note that HoFH patients are more likely to fail PCKS9 inhibitors (82). This is attributable to their mechanism of action.  This class of medication requires functional LDL receptors, and this is impaired or completely absent in HOFH patients (82). Therefore, effectiveness of PCSK9 inhibitors tends to be much higher in HeFH patients (82).

 

Leqvio (inclisiran) is also another PCSK9 inhibitor currently not approved for pediatric use (83). The mechanism of action of this drug differs from Repatha and Praluent. Leqvio is a small interfering RNA (siRNA) that utilizes the RNA interference mechanism to cause the catalytic breakdown of mRNA for PCSK9, thus stopping the translation of the protein (84). It also only requires administration twice yearly as opposed to biweekly (84). There are currently ongoing studies investigating the possibility of using Leqvio in pediatric patients. ORION-13 and ORION-16 are studies assessing the efficacy, safety and tolerability of Leqvio in pediatric patients diagnosed with HoFH and HeFH, respectively (84). They are two-part (1-year double blind, the other year open-label) phase 3 trials consisting of patients aged 12 to <18 years with FH (84). The primary endpoint is the percentage change in LDL-C from baseline to day 330 (84). Based on the results, this could be another drug option as adjunct therapy to consider for use.

 

Angiopoietin-Like Protein 3 (ANGPTL3)

 

Angiopoietin-like protein 3 (ANGPTL3) also presents a novel target of adjunctive therapy for patients with homozygous familial hypercholesterolemia that are not meeting LDL-C goals with first-line agents (85, 86).  Evkeeva (evinacumab-dgnb) is a monoclonal ANGPTL3 inhibitor that is FDA-approved specifically for the adjunctive treatment of homozygous familial hypercholesterolemia in patients 5 years of age and older (87).

 

Lomitapide

 

Lomitapide is another potential treatment option for patients with HoFH. This medication works differently from more conventional options. It binds to microsomal triglyceride transfer protein (MTP) and prevents the production of lipoproteins that contain apo-B (88). This causes a decrease in the production of very-low-density lipoprotein (VLDL) and chylomicrons. Since VLDL is converted into LDL, this mechanism ultimately causing a decrease in LDL-C levels (89). It is administered once daily at doses ranging from 5 to 60 mg (88). The side effect profile of lomitapide can be difficult for patients as it can cause severe gastrointestinal side effects (due to the decrease in absorption of fats in the intestines), most often diarrhea (89). But it is also associated with raised hepatic fats and enzymes (82). It is currently approved for adult use only, but it has become an option for use in pediatric patients through an expanded access program or a named patient basis (82). There was a case series done exploring the effect of lomitapide in 11 pediatric patients diagnosed with HoFH. It demonstrated that the drug was effective in reducing LDL-C with all 11 HoFH patients and showed a similar side effect profile to that seen in adult patients (82). GI complaints were moderated and did not cause any discontinuation of use (82). It also showed greater reduction in LDL-C levels at lower doses (82). The greatest benefit of lomitapide was associated with its ability to reduce or stop the need for lipoprotein apheresis in the patients incorporated in this case study (82). An interesting mention about lomitapide from the case series is that adult patient data shows that early intervention utilizing the drug showed a potential for increased life expectancy and a delay in the time to first major adverse cardiovascular event (82). There is also currently an ongoing phase 3, open label trial investigating the efficacy and safety of lomitapide in pediatric patients with HoFH, estimated completion date is April of 2024 (82).

 

Bempedoic Acid

 

Bempedoic acid is a new medication that exerts its effects very similarly to that of statins. It works in the same pathway as statins and targets cholesterol biosynthesis (90-92). It is administered however as a prodrug and converted to active drug only in the liver and not in the muscles (90-92). The other difference between the two classes is that bempedoic acid inhibits ATP-citrate lyase (ACL), while statins inhibit HMG CoA reductase (90-92). Due to the lack of activation in skeletal muscles, this drug is a promising alternative to patients unable to take statins due to muscle related symptoms (90-92). The medication is FDA approved for use in patients with HeFH and those with established cardiovascular disease (93). It has shown promising results in adult trials, but there are currently no published pediatric trials to date assessing the safety or efficacy of use of the drug (93). There does however appear to be a trial in development: “An Open-Label Study to Evaluate the Pharmacokinetics, Pharmacodynamics, and Safety of Bempedoic acid in Pediatric Patients with Heterozygous Familial Hypercholesterolemia.” The results are highly anticipated so that this can offer another promising drug class for use in patients intolerant or unable to meet their LDL-C goals.

 

FAMILIAL CHYLOMICRONEMIA SYNDROME

 

Familial chylomicronemia syndrome (FCS) is an incredibly rare autosomal recessive gene disorder (94). There is reduced or absent lipoprotein lipase activity causing disruption in chylomicron metabolism leading to severely elevated triglyceride levels resulting in acute recurrent pancreatitis (94). There is not however an increased risk of ASCVD with an FCS diagnosis (94). The best way to treat FCS is also often referred to as the most difficult as it requires patients to restrict dietary intake to <10-15% of daily calories (94). Other treatment options utilized are fibrates, omega-3 fatty acids and statins with variable responses, but the use of these medications is most commonly seen in patients who have multifactorial chylomicronemia syndrome (94). Given the difficulty of ensuring these patients maintain low levels of triglycerides, medications like volanesorsen are being examined (94, 95).

 

Volanesorsen

 

Volanesorsen is a second-generation 2’-O-methoxyethyl (2’-MOE) antisense therapeutic oligonucleotide. It works by inhibiting apoC3 thus lowering triglyceride plasma levels (94). When it binds to apoC3, this interrupts mRNA translation which consequently promotes triglyceride clearance/lowering of triglyceride plasma levels (94). The efficacy and safety of volanesorsen was assessed in the APPROACH study (96). It included 67 patients that were randomized to either weekly volanesorsen or placebo for 3 months (97). The results showed a 77% reduction in triglyceride plasma levels at the end of the study period and there was only 1 event of pancreatitis in the study group (97). The largest trial performed assessing the use of volanesorsen was the COMPASS trial (97). It included 114 patients who were randomized to either weekly injections of volanesorsen or placebo for a total of 26 weeks (97). The results showed that patients in the treatment group saw a reduction in triglyceride levels, chylomicron triglycerides, VLDL levels and apoC3 levels by more than 70% (97). There were also no occurrences reported of pancreatitis in any of the patients randomized to the volanesorsen group (93). In both trials, volanesorsen proved itself as a promising agent for treatment of hypertriglyceridemia in FCS patients (95-98). This drug is not approved for use in the US but is approved in other countries.

 

CONCLUSION

 

As noted in the 2011 NHLBI’s guidelines, available information regarding the treatment of youth with lipid disorders has greatly expanded. HMG-CoA reductase inhibitors, or statins, are now considered first-line pharmacologic treatment of children and adolescents with severe hypercholesterolemia who fail treatment with diet and exercise alone, although statins are only FDA approved for youth with familial hypercholesterolemia. Despite their ability to effectively reduced cholesterol levels, use of bile acid sequestrants continue to pose challenges for pediatric patients due to their unpalatability and are typically utilized as adjunctive therapy or for patients not able to tolerate statins. Fibric acid derivatives, as a class of medications, not only lack an FDA approved agent, but also continue to lack significant pediatric safety and efficacy data. Niacin, a potential adjunct therapy, lacks FDA approval for pediatric patients and is plagued by significant adverse effects, making it an unlikely therapeutic option for youth.  Ezetimibe provides clinicians with an alternative adjunct therapy option when synergistically paired with an HMG-CoA reductase inhibitor or used as monotherapy for patients intolerant to statins and bile acid sequestrants. Despite their inherit appeal and popularity amongst the lay public, omega-3 fish oils have failed to demonstrate statistically significant cholesterol lowering in pediatric and adolescent patients, but can be used to lower triglyceride levels. PCSK9 and ANGPTL3 inhibiting agents are promising novel treatment options in pediatric patients diagnosed with FH. While recent years have witnessed a dramatic increase in studies of lipid lowering medications in youth, the long-term safety and efficacy data continue to present an active focus of research.

 

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Ambiguous Genitalia in the Newborn

ABSTRACT 

 

Ambiguous genitalia in a newborn are the clinical sign of atypical sexual development of the external genitalia in utero. This condition is rare and can result from various underlying factors, including certain disorders with potentially severe consequences, such as cortisol deficiency due to congenital adrenal hyperplasia. Therefore, it is crucial to promptly determine etiology when ambiguity is observed. The formation of typical male or female external genitalia is a complex process involving a cascade of genetic and physiological events that begin with sex determination and progress through the differentiation of internal and external reproductive structures. When this process is disrupted and does not occur in the typical manner, it is referred to as a difference or disorder of sex development (DSD). Not all DSD cases present with ambiguous genitalia at birth; for example, complete androgen insensitivity syndrome does not, but all cases of ambiguous genitalia are the result of a DSD. This chapter focuses on genital ambiguity associated with DSD in newborns who have either a 46,XY or 46,XX chromosomal sex. However, DSD with genital ambiguity may also be observed in newborns with other combinations of sex chromosomes, such as 45,X/46,XY. The chapter offers a comprehensive overview of the evaluation and management of newborns with ambiguous genitalia. It emphasizes the importance of a structured medical assessment of the external genitalia to diagnose and determine the underlying cause of genital ambiguity. It includes tables for differential diagnosis and step-by-step workup algorithms to guide medical professionals in their evaluation. It also includes additional information on structured physical assessments of external genitalia and tables with normative values for hormonal measurements, which are recommended in the diagnostic process. As the etiology of genital ambiguity in newborns is diverse and can have significant implications for management, the authors stress that obtaining an accurate diagnosis through a professional medical workup is crucial. The chapter highlights the recommendation for newborns with ambiguous genitalia (DSD) to be cared for by highly specialized, interdisciplinary DSD teams. These teams are equipped with medical and psychosocial expertise, and specialized psychologists are available to support parents and caregivers. The chapter recognizes that having a child with ambiguous genitalia can be very stressful for parents. It underscores the importance of early education, access to expert care through DSD network teams, psychological support, shared decision-making, and promoting acceptance and inclusivity. By providing comprehensive support and guidance, families can better navigate the challenges and uncertainties they may encounter when caring for a newborn with ambiguous genitalia.

 

INTRODUCTION

 

Typical male and female development commences with the presence of the typical 46,XY or 46,XX chromosomes, which play a pivotal role in determining the indifferent gonads. Around the 6th week of gestation, these gonads follow pathways to develop into male-typical or female-typical gonads (Figure 1). This gonadal determination is intricately regulated by a complex interplay of multiple genes, which guide the male gonads to become testes and the female gonads to become ovaries.

 

Subsequently, the gonads initiate the production of various hormones, including anti-Müllerian hormone (AMH) and testosterone. These hormones are responsible for driving the differentiation of the embryologic Wolffian and Müllerian structures into sex-typical internal reproductive organs (Figure 1).

 

Figure 1. Typical male and female sex determination and differentiation occur during fetal life. The presence of the Y chromosome in the karyotype and the SRY gene (sex-determining region Y gene) are the primary determinants of male sex development. The SRY gene initiates a cascade that leads to the development of testes from bipotential gonads. Testosterone and Anti Mullerian Hormone (AMH) are secreted by testicular cells, which masculinize the Wolffian (mesonephric) duct and cause the regression of the Mullerian (paramesonephric) duct, respectively. Dihydrotestosterone (DHT), produced by the 5-alpha reduction of testosterone, plays a major role in male external genital development and prostate formation. The absence of the SRY gene on the Y chromosome is the primary factor for the induction of the female sex, and the development of the uterus, fallopian tubes, cervix and upper vagina from the Mullerian duct.

 

The development of external genitalia begins with a neutral anlage, including a genital tubercule, genital folds, and a urogenital sinus. These structures are subsequently differentiated into typical male external genitalia under the influence of androgens, particularly dihydrotestosterone (DHT). Conversely, for the development of typical female external genitalia, the absence of androgens is crucial to prevent virilization.

 

Therefore, ambiguous genitalia at birth can arise from either an excessive or insufficient androgen effect on the neutral external genital anlage in males or females, respectively (Figure 2).

Figure 2. External genital differentiation. The development of external genitalia begins with a bipotential anlage which includes genital tubercle, urethral fold, urethral groove, and genital swellings. In a typical 46, XY, androgen secretion from the testes triggers the fusion of urethral folds, allowing the enclosure of the urethral tube. This, together with the cells from the genital swelling, forms the shaft of the penis. Genital swellings fuse in the midline to allow formation of the scrotum and genital Tubercle expands to give rise to the glans penis. Female external genital development is ensured by the absence of testosterone, it is independent of ovarian endocrine activity. Urethral folds and genital swellings remain separate to form the labia minora and majora. Genital tubercle forms the clitoris.

 

CHARACTERISTICS OF TYPICAL MALE AND FEMALE EXTERNAL GENITALIA

 

A full-term male infant is typically expected to have bilateral testicles that are descended, complete formation of scrotal folds with midline fusion, and a typical-sized penis, which includes well-formed corporal bodies and a urethral meatus located at the tip. The average penile length for a full-term newborn is approximately 3.5 ± 0.4 cm, but this measurement can vary with gestational age (Table 1) (1, 2). If an infant presents with bilateral cryptorchidism, a bifid scrotum, hypospadias, or isolated penoscrotal hypospadias, further investigations for a DSD are recommended. However, it’s important to note that isolated micropenis, as long as both testes are descended and normal in size, is not typically considered a manifestation of ambiguous genitalia. Similarly, in males, distal hypospadias with no other atypical genital features usually does not indicate a DSD (3).

 

Table 1. Stretched Penile Length in Preterm and Term Newborns According to Gestational Age (2)

Gestational age (week)

Number of cases

Median (cm)

Mean (cm)

SD

Min (cm)

Max (cm)

26

30

1.9

1.9

0.32

1.1

2.5

27

31

2.1

2.0

0.28

1.1

2.6

28

32

2.0

1.9

0.31

1.3

2.9

29

29

2.3

2.3

0.39

1.3

2.9

30

31

2.4

2.4

0.32

1.6

3.0

31

33

2.4

2.4

0.38

1.8

3.3

32

32

2.9

2.6

0.44

2.0

3.2

33

28

2.9

2.8

0.45

1.9

3.4

34

30

2.9

2.8

0.43

2.0

3.6

35

29

3.1

2.9

0.61

2.0

4.1

36

31

3.1

3.0

0.50

2.2

4.0

37

29

3.1

3.0

0.47

2.2

4.1

38

44

3.1

3.1

0.54

2.1

4.5

39

82

3.2

3.2

0.55

2.0

4.2

40

65

3.5

3.4

0.53

2.2

4.4

41

29

3.6

3.5

0.47

2.6

4.3

 

A full-term female infant typically presents with bilateral separated labial folds, no palpable gonads, and distinct urethral and vaginal openings (Figure 2). The average clitoral length and width for a full-term infant girl are approximately 6.1 mm and 4.2 mm, respectively (1, 4). In cases where clitoromegaly (clitoral size ˃10 mm), labial fusion or palpable gonads are observed in otherwise typical female genitalia, further investigation for a DSD is warranted. It’s important to note that perceived clitoromegaly is not typically associated with an underlying DSD in the event that the newborn girl was born prematurely (1). Please be aware that perceived clitoromegaly or pseudo-clitoromegaly is often seen in preterm newborns, where the clitoral size falls within the normal range, but the labia majora cannot completely cover labia minora, creating a misleading impression of clitoromegaly. Genital edema may also be observed in sick preterm babies or cases of ovarian hyperstimulation, which can cause pseudo-clitoromegaly.

 

Table 2. Clitoral Length and Width in Preterm and Term Newborns According to Gestational Age (4)

Gestational age (week)

Number of cases

Measure

Mean (mm)

+1 SD (mm)

+2 SD (mm)

+3 SD (mm)

28-30

30

Width

3.28

3.87

4.46

5.05

Length

5.03

5.91

6.79

7.67

30-32

57

Width

3.32

3.68

4.04

4.4

Length

5.03

5.52

6.01

6.5

32-34

74

Width

3.41

3.77

4.13

4.49

Length

5.14

5.65

6.16

6.67

34-36

104

Width

3.69

4.17

4.65

5.13

Length

5.53

6.12

6.71

7.3

36-38

128

Width

4.08

4.5

4.92

5.34

Length

5.83

6.29

6.75

7.21

>38

187

Width

4.21

4.64

5.07

5.5

Length

6.11

6.49

6.87

7.25

 

GENERAL THOUGHTS ON AMBIGUOUS GENITALIA AT BIRTH

 

The reported incidence of ambiguous genitalia in newborns is approximately one in 4,500. It is essential that a newborn with ambiguous genitalia and their parents are promptly referred to a specialized center for evaluation. This evaluation should be carried out by a multi-disciplinary team specializing in DSD in accordance with international recommendations on diagnostic and therapeutic strategies. This team should provide holistic care and treatment guidance, collaborating closely with relevant subspecialists and peer support groups (5). Key subspecialties involved in DSD teams typically include psychology, pediatric endocrinology, urology, neonatology, gynecology, andrology, nursing, social work, genetics, and medical ethics. It's worth noting that certain aspects of DSD management remain contentious or uncertain, resulting in recent changes in clinical practice that are ongoing. Notably, genital surgery has been banned in several countries, and it is increasingly avoided in young children to protect their rights to an open future and the integrity of their bodies.

 

Consequently, we can anticipate a growing number of children growing up with atypical-looking genitalia, requiring a careful assessment of the psychological impact (6). Providing psychological support to affected families as a standard component of care from the outset is, therefore, of utmost importance. Issues related to gender that may arise in the context of ambiguous genitalia should be openly and thoughtfully discussed.

 

A comprehensive evaluation of ambiguous genitalia in a newborn is essential for understanding the underlying cause and potential consequences. On one hand, this evaluation can lead to the detection of concomitant, treatable disorders, such as adrenal insufficiency that requires cortisol replacement. Also, it is important to note that in up to 30% of children with a Difference/Disorder of Sex Development (DSD), other organ system disorders may be identified, necessitating medical care and qualifying as syndromic DSD (7, 8). On the other hand, identifying the underlying cause of ambiguous genitalia forms the basis for making an informed decision regarding sex registration at birth, if necessary and desired by the parents. Gender assignment/sex registration at birth should take into account long-term satisfaction with the sex of rearing, sexual function, and fertility potential (1, 9, 10). Ideally, it should result from a shared decision-making process involving the child's parents and the DSD network team. This decision should be based on the individual's phenotype/genotype, existing knowledge from literature, databases, and patient representative experience (11-13). This process may require time, as parents need to grasp the complexity of their child's unique biology in order to participate in this decision. As a first step, the overall health of the child should be assessed, and parents should be educated about typical sexual development before explanations regarding the underlying cause and its implications for their child's ambiguous genitalia are provided. Even if the exact details of the underlying cause remain unresolved, it is essential to communicate what is known and what remains uncertain to the parents and make plans for the family's future in the best interests of the child.

 

Several websites are available for patients and their families to exchange information, share coping strategies, and discuss decision-making processes that can enhance outcomes for newborns with ambiguous genitalia. Such resources can be found on websites supported by organizations like the Accord Alliance (www.accordalliance.org), DSD families (www.dsdfamilies.org), AIS DSD Support Group (www.aisdsd.org), and the CARES Foundation (www.caresfoundation.org). However, the most current information will always be available from the responsible DSD team, which will also be aware of country-specific support services.

 

GENETICS OF SEX DETERMINATION AND DIFFERENTIATION 

 

Sex development is a sequential process that involves the coordinated action of numerous genes and pathways. This process culminates in the development of functional gonads, differentiated internal sex organs, external genitalia, and typical secondary sexual characteristics that manifest after puberty. Prenatal sex development can be divided into two distinct processes: sex determination and sex differentiation (Figure 3). Sex determination is the initial step that occurs shortly after conception, approximately at 6-7 weeks gestation. During this phase, the undifferentiated gonads evolve into either testes or ovaries. By contrast, sex differentiation occurs subsequently and involves hormones produced after the formation of the sex-specific gonads. These hormones play a critical role in shaping the further development of the dependent embryonic structures, leading to the formation of male and female internal and external phenotypes.

 

Figure 3. The genes and transcription factors required for gonadal development. The formation of the bipotential gonad requires at least seven genes including NR5A1 (Nuclear Receptor Subfamily 5 Group A Member 1), WT1 (WilmsTumor1 Transcription Factor), LHX9 (LIM Homeobox 9), EMX2 (Empty Spiracles Homeobox 2), CBX2 (Chromobox 2), PBX1 (Pre-B-Cell Leukemia Transcription Factor 1) and GATA4 (GATA Binding Protein 4). Adapted from (14).

 

The Male Specific Pathway

 

In the male specific pathway, the expression of the SRY gene in pre-Sertoli cells serves as a pivotal switch that directs the fate of the gonads toward testicular development. SRY, in conjunction with NR5A1, activates the SOX9 signaling pathway, initiating testis development (Figure 3). Other important genes for the early testis development are NR0B1(Nuclear Receptor subfamily 0 group B member 1), AMH (Anti-Mullerian Hormone) and GATA4. AMH secreted by Sertoli cells, plays a crucial role in promoting the regression of Müllerian structures and supporting the development of Leydig cells in the testes. Subsequently, androgens produced by the testes facilitate the development of male internal organs (epididymis, vas deferens, seminal vesicle) and external genitalia (penis, scrotum). The production of sex steroids relies on an intact steroidogenic pathway, with all steroid hormones originating from cholesterol. The initial steps of sex steroid biosynthesis overlap with the synthesis of mineralo-corticosteroids and gluco-corticosteroids (Figure 4). It is important to note that certain steroid disorders, often referred to as congenital adrenal hyperplasia (CAH), can affect both adrenal and gonadal steroidogenesis or result in androgen excess or deficiency that may impact fetal genital development. INSL3, produced in Leydig cells, plays a role in guiding the descent of the testes from the abdomen to the scrotal folds.

 

The process of masculinization of initially sex-neutral, undifferentiated external genitalia begins around 8-9 weeks of gestation when the potent androgen DHT (dihydrotestosterone) is produced by the testes. DHT is crucial for the fusion of the urethral and labioscrotal folds, elongation of the genital tubercle, regression of the urogenital sinus, and the development of the prostate (15). The enzyme 5α-reductase is responsible for converting testosterone produced by fetal Leydig cells into DHT. Complete masculinization of the external genitalia through DHT is typically achieved by week 14 of gestation (16).

 

Figure 4. Steroidogenesis of the adrenals and gonads. Adrenal and gonadal steroidogenesis starting from cholesterol and showing key steroids and intermediates of the classical and alternative pathways is illustrated. Cortisol and androgen biosynthesis are under control of hypothalamic-pituitary-adrenal (HPA) axis in a negative feedback loop. Androgen synthesis occurs via classical or aberrant and alternative pathways (i.e., backdoor pathway and 11oxC19 androgen pathway). Aberrant or alternative pathways are particularly active whenever cortisol with or without aldosterone biosynthesis is impaired and androgenic precursors accumulate proximal to enzymatic block. Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, CYP21A2; 21α-hydroxylase, HSD3B2; 3β-hydroxy steroid dehydrogenase 2, CYP11B1; 11β-hydroxylase, CYP11B2; Aldosterone synthase, DOC; 11-deoxycorticosterone, DHEA; Dehydroepiandrosterone, A4; Androstenedione, T; Testosterone, CYP17A1; 17α-hydroxylase/17,20-lyase, b5; CYP19A1; Cytochrome P450 aromatase, Cytochrome b5, POR; P450 oxidoreductase, 17β-HSD; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SRDA1/2; Steroid 5α -reductase 1/2, 11oxC19; 11-oxygenated 19-carbon (11oxC19) steroids; Fdx; Ferredoxin, FdR; Ferredoxin reductase, 11βOHA; 11β-hydroxyandrostenedione, 11βOHT; 11β-hydroxytestosterone, Pdione; 5α-Pregnane- 17α-ol-3,20-dione, Pdiol; 5α-Pregnane- 3α,17α-diol 20-one, AKR1C2,4; Aldo-Keto Reductase Family 1 Member C2,4, DHT; Dihydrotestosterone, HSD17B5,3,6= 17β-hydroxysteroid dehydrogenase 5,3,6. Adapted from (17).

 

The Female Specific Pathway

 

By contrast, the female-specific pathway is initiated in the absence of SRY and requires the suppression of SOX9 (Figure 3). Activation of Rspo1/Wnt4/b-catenin and Foxl2 (Forkhead Box L2) signaling pathways leads to the formation of typical ovaries. The absence of AMH allows the Müllerian structures to proliferate, resulting in the formation of the typical fallopian tube, uterus, cervix and the upper part of the vagina. In the absence of both testosterone secretion and 5α-reductase activity, the sex-neutral external genitalia develop along the female pathway. Specifically, without DHT, the labioscrotal and urethral folds give rise to the labia majora and minora, respectively. The genital tubercle develops into a clitoris, and the urogenital sinus contributes to the formation of the urethral opening and anterior portion of the vagina (16).

 

Any disruption in the intricate genetic and hormonal processes involved can lead to atypical sex development and result in atypical external genitalia at birth. Figure 3 provides a summary of these processes and highlights some key genes and hormones essential for typical development.

 

DIFFERENCES/DISORDERS OF SEX DEVELOPMENT (DSD) 

 

The discordance between genetic, gonadal, or anatomic sex is commonly referred to as DSD. In addition to candidate gene testing, the new era of molecular diagnostic tools, including whole exome/genome sequencing has uncovered numerous novel molecular etiologies in recent years. Accurate molecular diagnosis aids in managing affected individuals and provides families with information concerning prognosis and the risk of recurrence.  This information is often sourced from literature and comprehensive data registries, such as the I-DSD (18-24). For clinical purposes, DSD (including ambiguous genitalia in newborns) is typically classified based on the affected individual's karyotype. The consensus statement addressing the approach and care of DSD (originally published in 2006 and updated in 2016) suggests the following broad classification for DSD: (A) Sex Chromosome DSD, (B) 46,XY DSD, and (C) 46,XX DSD (Table 3) (1, 20).

 

Table 3. DSD Classification According to the Chicago Consensus.

Sex Chromosome DSD

46,XY DSD

46,XX DSD

A. 45,X0 (Turner syndrome and variants)

A. Disorders of gonadal (testicular) development

Complete gonadal dysgenesis (Swyer syndrome) or partial gonadal dysgenesis (e.g., SRY,SOX9, NR5A1, WT1, DHHgene mutations etc)

Testicular regression (e.g., DHX37 gene mutation)

Ovo-Testicular DSD

A. Disorder of gonadal (ovarian) development

Ovo-Testicular DSD

Testicular DSD (SRY (+))

Gonadal dysgenesis

B. 47,XXY (Klinefelter syndrome and variants)

B. Disorders of testicular hormone production or action

Impaired testosterone production

LH receptor mutations

CAH (3βHSD2 deficiency, 17OHD, POR, StAR, CYP11A1 deficiency)

HSD17B3 deficiency

 

Impaired testosterone action

5α-reductase deficiency

Androgen insensitivity syndrome (Complete/partial)

 

Impaired AMH production or action

Persistent Mullerian Duct Syndrome (AMH and AMHR2gene mutations)

B. Androgen excess

Fetal

CAH (21-hydroxylase, 11β-hydroxylase, 3βHSD2 deficiency)

Glucocorticoid resistance

 

Feto-placental

Aromatase deficiency

POR deficiency

 

Maternal

Luteoma, hilar cell tumors, arrhenoblastoma, lipoid cell tumors, Krukenberg tumors, androgen producing adrenal tumors,

External androgen exposure

C. 45,X0/46,XY (Mixed gonadal dysgenesis, ovotesticular DSD)

C. Other

Syndromic

Smith-Lemli-Opitz, Cloacal anomaly, Aarskog, Robinow, Meckel, Joubert, Hand-Foot-Genital, popliteal pterygium, CHARGE, VATER/VACTERL, IMAGe etc.

 

Drugs

Flutamide, ketoconazole, progestagens

 

Endocrine disruptors

Phthalate, BPA, paraben

 

Cryptorchidism

INSL3, GREAT gene mutations etc.

 

Isolated hypospadias

MAMLD1, HOXA4 gene mutations etc.

C. Other

Syndromic

Cloacal anomaly etc.

 

Mullerian anomalies

Mayer-Rokitanski-Küster-Hauser, MURCS, McKusick-Kaufmnann syndrome and variants, MODY5

 

D. 46,XX/46,XY (chimeric, ovotesticular DSD)

 

 

Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, 3βHSD2; 3β-hydroxy steroid dehydrogenase 2, CYP17A1; 17α-hydroxylase/17,20-lyase deficiency, 17OHD; 17α-hydroxylase/17,20-lyase deficiency, POR; P450 oxidoreductase, HSD17B3; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SF1/NR5A1; steroidogenic factor 1.

 

The 46,XY Newborn With Ambiguous Genitalia

 

Ambiguous genitalia in a 46XY newborn can result from abnormal formation of the early fetal testes (testicular dysgenesis), reduced production of testosterone or dihydrotestosterone (5α-reductase deficiency), or the inability to respond to androgens (androgen insensitivity syndrome, or AIS) (Table 3). Depending on the extent of androgen production defect or resistance, affected newborns may exhibit a range of external genitalia phenotype, varying from those that appear typically female to those that appear typically male but with a small phallus, hypospadias, and a bifid scrotum, with or without palpable testes. Here we focus primarily on causes that lead to ambiguous genitalia. Broadly, four groups of underlying causes of 46, XY DSD with ambiguous genitalia can be identified: (a) partial gonadal dysgenesis, (b) partial testosterone biosynthetic defects, (c) partial 5α-reductase deficiency, and (d) partial androgen insensitivity syndrome or PAIS.

 

PARTIAL GONADAL DYSGENESIS

 

In cases of partial gonadal dysgenesis, it is presumed that a gene mutation leads to a partial abnormality in the development of the early urogenital ridge or gonadal anlage. This category of DSD can also involve mutations in genes such as SRY and SOX9, which are essential for the differentiation of bipotential gonads into testes (Figure 3). Partial gonadal dysgenesis is typically associated with incomplete masculinization of the external genitalia, along with varying degrees of maintenance of Wolffian ducts and inhibition of Müllerian ducts. For a summary of known genetic variations associated with gonadal dysgenesis, please refer to Table 4.

 

Leydig cell aplasia or hypoplasia represents a variation of gonadal dysgenesis, characterized by inadequate Leydig cell differentiation in the male gonad, resulting in impaired androgen production.  Mutations in the LH receptor gene (LHCGR) as well as polymorphisms have been associated with this condition (25). The phenotype associated with Leydig cell hypoplasia includes incomplete masculinization of the external genitalia, along with incomplete development and maintenance of the Wolffian ducts. 

 

 

Table 4. Monogenic Disorders of Gonadal Development/Differentiation and Associated DSD Presentations in Humans, Both 46,XY and 46,XX

Gene defect

Locus

Mode of transmission

Clinical presentation

Reference

 

 

 

DSD phenotype

Other

 

 

 

 

46, XX

46, XY

 

 

ARX

Xp22.13

XL

 

Testicular dysgenesis

Lissencephaly, epilepsy, intellectual disability temperature instability

(26)

ATRX

Xq13.3

XL

 

Testicular dysgenesis

Dysmorphic features, intellectual disability, α-thalassemia

(27)

CBX2

17q25

AR

Ovarian dysgenesis

Testicular dysgenesis

 

(28)

DHH

12q13.12

AR

 

Testicular dysgenesis

Minifascicular neuropathy

(29)

DMRT1/DMRT2                       

9p24.3

AD

Ovarian dysgenesis

Testicular dysgenesis

Hypotonia, developmental delay, impaired intellectual development

(30)

DHX37

12q24.31

AD

 

Testicular dysgenesis, testicular regression

 

(31)

EMX2

10q26.11  

 

Ovarian dysgenesis

Testicular dysgenesis

Intellectual disability, kidney agenesis

(32)

ESR2

14q23.2-q23.3

AD,?

Ovarian dysgenesis

Testicular dysgenesis

Dysmorphic features, eye abnormalities, anal atresia, rectovestibular fistula

(33)

FGFR2

10q26.13

AD

 

Testicular dysgenesis, ovotesticular

Craniosynostosis

(34)

GATA4 

8p23.1

AD

 

Testicular dysgenesis

Congenital heart defects (atrial septum defects, ventricular septum defects, tetralogy of Fallot), diaphragmatic hernia

(35)

HHAT

1q32.2

AR

 

Testicular dysgenesis

Nivelon-Nivelon-Mabille syndrome. Dwarfism, chondrodysplasia, narrow, bell-shaped thorax, micromelia, brachydactyly, microcephaly with cerebellar vermis hypoplasia, facial anomalies, hypoplastic irides and coloboma of the optic discs

(36)

LHX9

1q31.3

AD

 

Testicular dysgenesis

Limb anomalies

 

(37)

MAMLD1

Xq28

XL

 

Severe hypospadias

Myotubular myopathy

 

(38)

MAP3K1

5q11.2

AD

 

Testicular dysgenesis

 

(39)

NR0B1 (Duplications, inversion

and upstream deletion)

X21.3

XL

 

 

Testicular dysgenesis

Congenital adrenal hypoplasia, hypogonadotropic hypogonadism, cleft palate, intellectual disability

 

(40)

NR2F2

15q26.2

AD

Ovarian dysgenesis, ovotesticular DSD

 

Congenital heart disease, somatic anomalies including

blepharophimosis-ptosis-epicanthus

inversus syndrome

(41)

NR5A1

9q33

AD

Ovarian dysgenesis, ovotesticular DSD

Testicular dysgenesis

Adrenocortical insufficiency,

spermatogenic failure, primary ovarian insufficiency, asplenia, polysplenia

(42)

PPP2R3C

14q13.2

AR

 

Testicular dysgenesis

Dysmorphic facies, retinal dystrophy, and

myopathy.

 (43)

RSPO1

1p34.3

AR

Testicular/

ovotesticular DSD

 

Palmoplantar hyperkeratosis

 (44)

SART3

12q23.3

AR

 

Testicular dysgenesis

Intellectual disability, global developmental delay and a subset of brain anomalies

 

(45)

SOX3 (Upstream deletion and

Duplication)

Xq27.1

XL

 

Testicular/ovotesticular DSD

 

Intellectual disability, hypopituitarism

(46)

SOX8 (SNV, upstream

duplication, inversion)

16p13.3

AD

 

 

Testicular dysgenesis

Variants may contribute to male and female infertility, primary ovarian insufficiency.

(47)

SOX9 (SNV, upstream

duplication, inversion)

17q24.3

AD

 

Testicular/ovotesticular DSD

Testicular dysgenesis

Campomelic dysplasia, Cooks syndrome, Pierre Robin sequence

 

 (48)

SOX10 (Duplication)

22q13.1

AD

Testicular/ovotesticular DSD

 

Peripheral and central demyelination, Waardenburg syndrome type IV, and Hirschsprung disease

 (49)

SRY (SNV, deletion,

Translocation)

Yp11.3

XL, YL

Testicular/ovotesticular DSD

Testicular dysgenesis

 

 (50, 51)

 

TSPYL1

6q22.1

AR

 

Testicular dysgenesis

Sudden infant death syndrome

(52)

WT1

11p13

AD

Testicular/ovotesticular DSD

Testicular dysgenesis

Denys-Drash syndrome; Frasier syndrome; WAGR syndrome; Meacham syndrome, Nephrotic syndrome; Wilms tumor

(53, 54)

 

 

Abbreviations: AD; autosomal dominant, AR; autosomal recessive, XL; X-linked, YL; Y-linked

 

DEFICIENCY OF TESTOSTERONE BIOSYNTHESIS

 

The inability to produce testosterone stems from defects in the activity of any of the enzymes required for testosterone biosynthesis from cholesterol (Figure 4able 5). Identified defects encompass genetic variants in Steroidogenic Acute Regulatory Protein (StAR), P450 side-chain cleavage (CYP11A1), 3β-hydroxy steroid dehydrogenase 2 (HSD3B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and P450 oxidoreductase (POR) (59). In addition, apparent lyase deficiency due to variants in cytochrome b5 (CYB5) and variants in AKR1C2/4 have been very rarely described (60-62). Moreover, 17β-hydroxysteroid dehydrogenase deficiency (also known as 17β–hydroxysteroid oxidoreductase or 17-ketosteroid reductase, HSD17B3) is another cause of 46,XY DSD (63). Furthermore, variants in steroidogenic factor 1 (SF1/NR5A1), which regulates the transcription of several genes involved in steroidogenesis, may lead to ambiguous genitalia. Notably, variants in HSD3B2, POR, and NR5A1 can result in ambiguous genitalia in both 46,XY and 46,XX newborns (64-66).

 

Similar to partial gonadal dysgenesis, a partial testosterone biosynthesis defect leads to ambiguous external genitalia and variable degrees of Wolffian duct development. However, unlike partial gonadal dysgenesis, Müllerian ducts are not retained in 46,XY newborns with partial testosterone biosynthesis defects due to intact Sertoli cell function and normal AMH production.

 

Table 5. Adrenal and/or Gonadal Causes of Impaired Sex Steroid Biosynthesis Associated with Ambiguous Genitalia in 46,XX and 46,XY Individuals

Disorder

Gene/ OMIM

Adrenal Insufficiency

46,XY Gonadal Phenotype

(Testosterone Deficiency)

46,XX Gonadal Phenotype

(E2 Deficiency)

Fertility

Other Features

 

Lipoid congenital adrenal hyperplasia (LCAH)

StAR

201710

YES

Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia

Non-classic form: normal or NK

Classic: primary or secondary ovarian insufficiency (POI)

Non-classic: NK or normal

Classic:Absent in 46,XY; variable in 46,XX

 

P450 side chain cleavage syndrome (CAH)

CYP11A1

118485

YES

Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia

Non-classic form: normal

Classic: primary or secondary ovarian insufficiency (POI)

Non-classic: NK or normal

Reported in 46,XX

 

3β-hydroxysteroid dehydrogenase II deficiency (CAH)

HSD3B2

201810

YES

46,XY DSD, gonadal insufficiency; ambiguous genitalia

Non-classic form: normal, but premature adrenarche

46,XX DSD with atypical genital development; gonadal insufficiency; ambiguous genitalia

Non-classic form: normal, but premature adrenarche

Absent in 46,XY; reported in 46,XX

 

21-hydroxylase deficiency (CAH)

CYP21A2

201910

YES

Classic form: normal

Non-classic form: normal

46,XX DSD with atypical genital development;

Non-classic form: premature adrenarche, virilization, PCO

Normal in both 46,XX and 46,XY, if treated

Cave: Testicular adrenal rest tumor (m>>f)

CAH-X (when combined with Ehlers-Danlos syndrome with contiguous gene variants)

11-hydroxylase deficiency (CAH)

CYP11B1

202010

YES

Classic form: normal

Non-classic form: normal

46,XX DSD with atypical genital development;

Non-classic form: premature adrenarche, virilization, PCO

Normal in both 46,XX and 46,XY, if treated

Hypertension

Combined 17-hydroxylase, 17,20 lyase deficiency (CAH)

CYP17A1

202110

Rare

46,XY DSD, gonadal insufficiency; atypical genital development

Lack of pubertal development, POI

Possible in 46,XX with assisted fertility measures

Hypertension and hypokalemic alkalosis (not seen with isolated lyase deficiency)

P450 oxidoreductase deficiency (CAH)

POR

124015

201750

Variable

Mild to severe 46,XY DSD, gonadal insufficiency; atypical genital development

46,XX DSD with atypical genital development or

premature adrenarche, virilisation, POI, PCO

Reported

Maternal virilization during pregnancy; Antley-Bixler skeletal malformation syndrome; changes in drug metabolism

Cytochrome b5 deficiency

CYB5A

613218

NO

46,XY DSD; atypical genital development

NK

NK

Methemoglobinemia

17β-hydroxysteroid dehydrogenase III deficiency / 17-ketosteroid reductase deficiency

HSD17B3

264300

NO

46,XY DSD; atypical genital development; progressive virilization and gynecomastia at puberty

Normal

Decreased or absent in 46,XY

 

5α-reductase II deficiency

SRD5A2

607306

NO

46,XY DSD; atypical genital development; progressive virilisation and gynecomastia at puberty

Normal

Impaired in 46,XY

 

3α-hydroxysteroid dehydrogenase deficiency

AKR1C2/4

600450

600451

NO

46,XY DSD; gonadal insufficiency; atypical genital development

Normal

NK

 

Aromatase deficiency

CYP19A1

107910

NO

Normal

46,XX DSD with variable degree of virilisation at birth (ambiguous genitalia), gonadal insufficiency, POI

Impaired in 46,XX

Overgrowth and metabolic anomalies in males

Steroidogenic factor 1

NR5A1/

SF1

184757

Rare

Mild to severe 46,XY DSD; gonadal insufficiency – very variable; ambiguous genitalia

POI or normal; rarely 46,XX DSD with atypical genital development

Mostly impaired in 46,XY; variable in 46,XX

 

Abbreviations: NK; not known, POI; primary ovarian insufficiency, PCO; polycystic ovary.

 

5-ALPHA-REDUCTASE-2 DEFICIENCY (SRD5A2 VARIANTS)

 

Deficiency of the 5α-reductase enzyme arises from variants in the steroid 5α-reductase type 2 (SRD5A2) gene, which can show single point mutations to complete gene deletions. Newborns affected by this condition possess functioning Leydig and Sertoli cells, but due to their inability to convert testosterone to DHT, they may exhibit varying degrees of under-masculinized external genitalia, including genital ambiguity at birth in some cases (16, 67-69). The Müllerian ducts in affected individuals regress as expected, due to normal Sertoli cell function and AMH production. During puberty, significant virilization becomes possible in affected individuals, and the testes are capable of supporting spermatogenesis, as DHT is not required for germ cell maturation. Therefore, fertility is attainable in less severely affected individuals (70-72) or with the use of intrauterine insemination. Recent evidence also suggests that SRD5A2 activity may be even influenced by genetic polymorphisms of SRD5A2 (50).

 

GENETIC DEFECTS OF ANDROGEN ACTION

 

Complete or partial androgen insensitivity syndromes (CAIS/PAIS) are caused by genetic mutations affecting androgen receptor (AR) function (73, 74), and serum testosterone levels are typically elevated. Internationally, incidence of AIS is reported in 1 in 20,400 live born 46,XY infants, with CAIS occurring at a higher rate than PAIS (75).

 

The human androgen receptor (AR) is encoded by a single gene (AR) composed of 8 exons located in the q11-12 region of the X chromosome. The AR gene mutation database includes hundreds of AR variants that lead in varying degrees of atypical sex differentiation of 46,XY fetuses, also classified as AIS type I (76-78). A small number of complete AR gene deletions have been reported, as well as deletions that start at exons 2, 3 or 4 and extend to the terminus of the gene. In addition, a limited number of mutations resulting from premature terminations, base deletions and terminations have been identified. However, the most common type of AR gene mutation results from base substitutions (79). When mutations result in partial inactivation of AR activity (PAIS), the phenotypic variability in under-masculinization can be substantial and depends on the residual activity of the AR. In some cases, this can lead to ambiguous genitalia in newborns with a 46,XY chromosomal sex.

 

Unlike in CAIS, the underlying genetic defect in PAIS is only found in the AR gene in about 40% of cases (80). However, the clinical and biochemical phenotype is similar, and in vitro functional tests demonstrate androgen resistance in all of them. Thus, individuals with AIS without AR mutations are classified as AIS type II (73). In cases of AIS type II without AR variants, largely unidentified regulators or cofactors of the AR are responsible for the impaired AR signaling, as revealed by an AR-dependent bioassay using genital skin fibroblasts and the targeted apolipoprotein D as a biomarker (81). Given that AR activity can be regulated at various levels, the potential mechanisms of AIS type II are diverse.Recently, altered DNA methylation of the AR promoter has been observed in some individuals with PAIS (82). Additionally, mutations in the Disheveled associated activator of morphogenesis 2 gene (DAAM2) that impair nuclear actin assembly at AR have been associated with AIS type II (83).

 

Overall, individuals with PAIS experience variable degrees of end-organ unresponsiveness to androgens, resulting in varying degrees of Wolffian duct development and external genital ambiguity. Within families with the same AR mutation, phenotypic variability can occur due to variable degrees of insensitivity to androgens (84, 85). Consequently, solely relying on genetic information, it is challenging to predict whether a newborn with PAIS will identify as male, intersex, or female in later life and respond to future testosterone therapy. Similarly, genetic counselling is challenging.

 

The 46,XX Newborn With Ambiguous Genitalia

 

Ambiguous genitalia in a chromosomal 46,XX fetus invariably results from androgen excess during fetal development, and the degree of virilization (Figure 2) often provides clues about the timing and severity of the underlying disorder.

 

Similar to a 46,XY fetus, a 46,XX fetus may experience partial gonadal dysgenesis or develop testicular or ovo-testicular DSD due to pathogenic variants in genes involved in early sex determination (Table 3 and 4). Ovo-testicular DSD occurs when both ovarian and testicular tissue develop in the same individual (1). Most newborns with ovo-testicular DSD possess 46,XX chromosomes and present with ambiguous genitalia, although some affected individuals have 46,XY chromosomes or 46,XX/46,XY mosaicism. Just like in 46,XY DSD, the degree of testicular development determines the extent of masculinization/virilization of the external genitalia, Wolffian duct development, and Müllerian duct regression in affected newborns (79).  A summary of known genetic variations is given in Table 4.

 

In the majority of cases, ambiguous genitalia in a 46,XX fetus with typical ovarian organogenesis can be attributed to excessive androgen production/exposure early in utero, originating either from the fetus itself (Table 3 and Table 5) or from the mother or environment.

 

ABNORMAL FETAL ANDROGEN PRODUCTION BY CONGENITAL ADRENAL HYPERPLASIA (CAH)

 

The term congenital adrenal hyperplasia (CAH) encompasses several adrenal disorders, each linked to a mutation in one of the enzymes necessary for the biosynthesis of cortisol from cholesterol (86) (Figure 4 and Table 5). These abnormalities lead to increased ACTH secretion by the pituitary gland, which, in turn, results in the increased secretion of cortisol precursors, including adrenal androgens.

 

Deficiency of steroid acute regulatory protein (StAR) causes congenital lipoid adrenal hyperplasia, characterized by salt loss and a lack of cortisol, androgen, and estrogen secretion. Genetic 46,XX infants affected by StAR deficiency exhibit typical female external genitalia, while genetic 46,XY are born with ambiguous genitalia or may experience sex reversal in cases of severe loss-of-function variants. A similar phenotype is also observed with variants in CYP11A1.

 

Deficiency in 17α-hydroxylase/17,20-lyase leads to hypertension due to the hypersecretion of corticosterone and impaired androgen secretion. Similar to congenital lipoid adrenal hyperplasia, 46,XX fetus affected by 17α-hydroxylase/17, 20-lyase deficiency (CYP17A1) develop typical female external genitalia, while 46,XY fetus present with ambiguity.

 

Deficiency of 3β-hydroxysteroid dehydrogenase (HSD3B2) results in salt loss and impaired androgen synthesis. Affected 46,XX females may or may not exhibit minimal genital masculinization, whereas all genetic 46,XY males are variably under-masculinized (87).

 

CAH due to 11β-hydroxylase deficiency (CYP11B1) and 21-hydroxylase deficiency (CYP21A2) results in the most significant masculinization/virilization of external genitalia in 46,XX fetus compared to all other types of CAH. Additionally, 11β-hydroxylase deficiency can lead to hypertension in individuals of either sex in later life (88, 89).

 

21-hydroxylase deficiency CAH, caused by variants in CYP21A2, represents the most common form (accounting for more than 90% of cases) of CAH and is the most frequent genetic cause of DSD with ambiguous genitalia at birth in 46,XX newborns (59, 63). Cortisol deficiency necessitates replacement therapy in all classic forms of CAH. In the milder (simple-virilizing) presentation of classical 21-hydroxylase deficiency, salt loss is typically not an issue, whereas in the more severe (salt-wasting) form of classical 21-hydroxylase deficiency, salt-loss occurs and may pose a risk to the newborn shortly after birth if not treated with both mineralocorticoids and glucocorticoids (86, 90, 91).

 

The pathological mechanisms of 21-hydroxylase deficiency leading to 46,XX virilization in utero have been well described (92). It involves androgen excess due to lack of 21-hydroxylase activity during the critical time window around 6-12 weeks of gestation when the external genitalia are formed, and adrenal androgen production should be redirected to cortisol production to protect the female external genital anlage. In 46,XX CAH (Table 5), the resulting adrenal androgen excess exceeds the androgen metabolizing capacities of the fetal-placental unit, and alternative pathways of androgen production (Figure 4) are also activated, contributing to the overall androgen excess. Figure 5 illustrates in a scheme how the fetal adrenals, liver and placenta cooperate under normal conditions to metabolize fetal adrenal androgens into estrogens.

 

Figure 5. Steroid metabolic pathways in the fetal-placental-maternal unit, also known as the fetal-placental unit. The main function of the FZ of the fetal adrenal is the production of DHEA-S from cholesterol, which is transported to the placenta, desulfated to DHEA, and sequentially metabolized by placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), 17β-hydroxysteroid dehydrogenases (17βHSD) and cytochrome P450 aromatase (CYP19A1), to androstenedione (A4), testosterone (T) and estradiol (E2), respectively. E2 is then transported from the placenta into the maternal circulation. A4 is additionally also a substrate for placental CYP19A1, producing estrone (E1). Fetal adrenal DHEA-S is also metabolized by the fetal liver to 16α-hydroxy-DHEA-S by cytochrome P450 family 3 subfamily A member 7 (CYP3A7), which is metabolized to estriol (E3), the estrogen marker of pregnancy. Abbreviations: PREG, pregnenolone; 17OHPREG, 17α-hydroxypregnenolone; PROG, progesterone; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone-sulfate; CYP11A1, cytochrome P450 cholesterol side chain cleavage; StAR, steroidogenic acute regulatory protein; CYP17A1, cytochrome P450 17α-hydroxylase/17,20-lyase; SULT2A1, sulfotransferase; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP19A1, cytochrome P450 aromatase; HSD17B, 17β-hydroxysteroid dehydrogenase; HSD3B1, 3β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; STS, sulfatase.

Figure 5. Steroid metabolic pathways in the fetal-placental-maternal unit, also known as the fetal-placental unit. The main function of the FZ of the fetal adrenal is the production of DHEA-S from cholesterol, which is transported to the placenta, desulfated to DHEA, and sequentially metabolized by placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), 17β-hydroxysteroid dehydrogenases (17βHSD) and cytochrome P450 aromatase (CYP19A1), to androstenedione (A4), testosterone (T) and estradiol (E2), respectively. E2 is then transported from the placenta into the maternal circulation. A4 is additionally also a substrate for placental CYP19A1, producing estrone (E1). Fetal adrenal DHEA-S is also metabolized by the fetal liver to 16α-hydroxy-DHEA-S by cytochrome P450 family 3 subfamily A member 7 (CYP3A7), which is metabolized to estriol (E3), the estrogen marker of pregnancy. Abbreviations: PREG, pregnenolone; 17OHPREG, 17α-hydroxypregnenolone; PROG, progesterone; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone-sulfate; CYP11A1, cytochrome P450 cholesterol side chain cleavage; StAR, steroidogenic acute regulatory protein; CYP17A1, cytochrome P450 17α-hydroxylase/17,20-lyase; SULT2A1, sulfotransferase; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP19A1, cytochrome P450 aromatase; HSD17B, 17β-hydroxysteroid dehydrogenase; HSD3B1, 3β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; STS, sulfatase.

 

FETAL AND PLACENTAL AROMATASE DEFICIENCY

 

During fetal development, the adrenals produce substantial amounts of DHEA/S. These are either converted to 16-hydroxy-DHEA/S in the fetal liver and then transported or directly transferred to the placenta, where they are converted into estrogens (Figure 5). In case of an aromatase enzyme deficiency in the fetus (caused by variants in the CYP19A1 gene), androgen precursors accumulate and lead to virilization of 46,XX fetuses, typically leading to ambiguous genitalia at birth (93). Androgen excess due to fetal aromatase deficiency may also induce maternal virilization during pregnancy, as indicated by a deepening of the voice, oily skin and hair, and excessive hair growth (hirsutism).

 

EXCESS MATERNAL ANDROGEN PRODUCTION

 

Excessive androgen production can negatively impact fertility due to anovulation, therefore cases of maternal androgen production during pregnancy are exceedingly rare. Nonetheless, androgens originating from the mother can travers the placenta and lead to the masculinization of a female fetus. These maternal androgens usually originate from the ovaries or adrenal glands. Androgen-producing ovarian tumors include hilar cell tumors, arrhenoblastomas/androblastomas, lipoid cell tumors, and Krukenberg tumors. In contrast, pregnancy luteomas, rare non-neoplastic ovarian lesions occurring during pregnancy, are believed to be caused by hormonal effects related to pregnancy. Although exceptionally rare, androgen-secreting tumors of the adrenals can also occur during pregnancy.

 

DRUGS ADMINISTERED TO THE MOTHER DURING GESTATION

 

In 1958, Wilkins et al. reported that certain synthetic progestins administered to pregnant women, such as 17α-ethinyl-19-nortestosterone, can masculinize the external genitalia of female fetuses (94). Similarly, diethylstilbesterol (DHE), a nonsteroidal synthetic estrogen, has been linked with urogenital anomalies in both 46,XX and 46,XY fetuses (95, 96). More recently, numerous studies have indicated that fetal exposure to various endocrine disrupting chemicals (EDCs) such as pesticides, fungicides, herbicides, and plasticizers may adversely affect genital development (97-99).

 

Syndromes Associated with Ambiguous Genitalia

 

As previously mentioned in sections of 46,XX and 46,XY DSD, ambiguous genitalia can be associated with syndromes characterized by multiple congenital malformations, as summarized in Table 4. These associations arise because many of the transcription factors involved in sex development and differentiation also have roles in other aspects of development (Table 4).

 

For example, mutations of the WT-1 gene (11p3) can lead to several syndromes, including WAGR, Denys-Drash or Frasier syndromes (70-72). Mutations in SOX-9 (17q24-25) can result in campomelic dysplasia (100).

 

Mutations in DMRT1/DMRT2 (9p24.3), EMX2 (10q25.3q26.13), ATRX (Xq13.3) and WNT-4 (1p35) are often associated with developmental delay (101, 102). SF-1 (9q33) and DAX-1 (Xp21.3) play roles in the early formation of the adrenals, anterior pituitary, and parts of the hypothalamus.

 

Consequently, mutations can lead to abnormalities of these organs, in addition to those of the urogenital system. Abnormal development of the lower abdominal wall with pubic diastasis can result in urogenital anomalies observed in the VA(C)TER(L) and CHARGE syndromes, as well as bladder and cloacal exstrophy (103).

 

These examples underscore the complexities of the anomalies that may be associated with ambiguous genitalia in newborns and emphasize the importance of conducting a thorough physical examination and involving an experienced DSD team when faced with a child presenting ambiguous genitalia.

 

WORKUP OF NEWBORNS WITH AMBIGUOUS GENITALIA

 

The etiology of genital ambiguity in newborns is diverse and can have significant implications for management. Therefore, obtaining an accurate diagnosis through a medical workup is crucial. Some investigations are urgent, such as ruling out or confirming and treating potentially life-threatening adrenal insufficiency with mineralocorticoid and glucocorticoid replacement therapy in children with underlying CAH. Additionally, associated organ anomalies may require immediate attention to ensure the child’s survival, such as cardiac, pulmonary, or kidney malfunctions.

 

When the newborn is shown to be capable of excreting urine and stool without problems, and adrenal and other organ functions are found to be intact, investigations to understand genital ambiguity become primarily a psychosocial “emergency” for the family and caregivers. As a result, psychological support and holistic care by a DSD team as early as possible are essential to guide the parents and make decisions in the child’s best long-term interest. Obtaining an accurate diagnosis of the underlying cause of genital ambiguity early on is necessary for decision-making processes regarded to sex registration, hormonal and surgical treatments, and outcomes such as potential fertility and risk of gonadal malignancy. Therefore, it is recommended practice that a thorough workup should be performed for every child born with ambiguous genitalia, and guidelines for such assessments are available (22, 104-107).

 

Overall Assessment of the Child with Ambiguous Genitalia

 

Figure 6 provides an overview of the initial workup for a newborn with ambiguous genitalia. Several aspects are considered, including pregnancy and family history, as well as postnatal adaptation (such as feeding, weight gain, alertness, excretion).  In some cases, suspicion of genital ambiguity may arise during pregnancy through ultrasound surveillance. In such cases, first contact and information through a DSD team member (e.g., neonatologist, geneticist, pediatric endocrinologist) together with the DSD psychologist is recommended to guide the parents through pregnancy and alert the future care team early. Prenatal workup of ambiguous genitalia is generally not necessary if the fetus is otherwise developing normally without other organ anomalies that require attention. It is important to note that a fetus with CAH does not suffer from mineralocorticoid and cortisol deficiency in utero, as these hormones become essential only after birth (Figure 4). Genetic workup of a child with ambiguous genitalia during the prenatal period may only be recommended when there is a previous index case with a diagnosis, or when parental genetic data inform an abnormality, and when genetic counseling of the parents has led to a personalized action plan.

 

When taking the medical history of a newborn with ambiguous genitalia, asking specific questions can help guide the diagnostic workup and management. Figure 6 provides a list of important questions to consider. For example, androgenic drugs used in pregnancy may explain the virilization of a 46, XX newborns. Maternal virilization during pregnancy, for example, voice change and hirsutism may be important for the diagnoses of maternal virilizing tumors, P450 oxidoreductase (POR) deficiency, or placental aromatase deficiency. Questions about parental consanguinity are important, because many of the causes of ambiguous genitalia are recessively inherited. History of previously unexplained neonatal deaths, ambiguous genitalia, infertility or genital surgery in the family is important for identifying potential genetic etiologies.

 

In addition to the medical history, every newborn with ambiguous genitalia should undergo a comprehensive physical examination, with a special focus on signs of adrenal insufficiency (dehydration, lethargy, poor feeding) and any dysmorphic features (Figure 6). This examination may guide the diagnostic process.

 

Figure 6. Algorithm for initial workup of the child with ambiguous genitalia at birth.
Abbreviations: SDSST; Standard dose synthetic ACTH (synacthen) stimulation test, HCG; Human chorionic gonadotropin, WES; Whole exome sequencing, WGS; Whole genome sequencing, EMS; External masculinization score, EGS; External genital score, QF-PCR; quantitative fluorescence polymerase chain reaction, SRY FISH; SRY fluorescent in situ hybridization.

 

Physical Examination of the External Genital Area

 

Systematic assessment of the external genital area in newborns with ambiguous genitalia is crucial, and it should be carefully documented. Over the years, different scales and scoring systems have been used to evaluate the external genitalia. These assessments help provide a standardized way to document the physical characteristics, which is essential for comparing data over time and between different healthcare providers and patients. The use of these scales and scoring systems aids in follow-up and tracking of changes in the external genitalia.

 

Historically, the Prader or Quigley scales were commonly used for this purpose (108, 109) (Figure 7). Later, the external masculinization score (EMS) was introduced as a more comprehensive assessment tool (104) (Table 6). Nowadays, the external genital score (EGS) is recommended for evaluating and documenting the external genitalia (110) (Table 7). These tools provide a structured way to assess and record the physical findings, making it easier to monitor and communicate about the patient's condition.

 

The careful examination of the external genitalia in newborns with ambiguous genitalia provides valuable non-invasive, indirect information about prenatal exposure and response to DHT, and about the timing of this exposure. For instance, androgen exposure must have occurred in the first trimester to cause labial fusion.

 

During the genital exam, it’s important to measure the stretched phallic length, assess the quality of the corpora (erectile tissues), inspect of the labia, labio-scrotal folds, or scrotum, and document the position of the urethral opening (and vaginal opening, if applicable). Furthermore, the presence and location of palpable gonads should also be recorded. Any asymmetry in the gonads and external genitalia should be noted. Asymmetry in internal and/or external genital structures may suggest mixed gonadal dysgenesis or ovo-testicular DSD. All these observations can be documented using the external genital score (EGS), which provides a standardized way to assess and record the physical characteristics of the external genitalia (Table 7). In addition, the anogenital distance (AGD), the distance between the anus and the base of the genitalia, can be helpful in determining the extent of androgen exposure during fetal development, as a higher ratio is generally consistent with increased androgen exposure (111) (Table 8). In older infants, the anogenital index (AGI), which is the body weight standardized index of AGD [AGI/weight (mm/kg)], may be used for assessments to account for changes of the external genitalia through growth and postnatal development (111).

 

Overall, a thorough examination and documentation of the external genitalia provide important clinical information that can guide the diagnosis and management of newborns with ambiguous genitalia.

 

Figure 7. Prader and Quigley Scales for visual grading of external genital virilization. In Stage 3 a single urogenital orifice can be observed.

 

 

Table 6. External Masculinization Score (EMS)

External Masculinization Score (EMS).

Scrotal fusion

Micropenis

Urethral meatus

Right gonad

Left gonad

3

Yes

No

Normal

 

 

2

 

 

Distal

 

 

1.5

 

 

 

Labioscrotal

Labioscrotal

1

 

 

Mid

Inguinal

Inguinal

0.5

 

 

 

Abdominal

Abdominal

0

No

Yes

Proximal

Absent

Absent

An EMS score <11 out of 12 needs further clinical investigation for DSD in 46,XY.

 

Table 7. External Genital Score (EGS)

External genital score (EGS)

Labio-scrotal fusion

Genital tubercle size (mm)

Urethral meatus

Right gonad

Left gonad

3

Fused

≥31

Typical male

 

 

2.5

 

26-30

Coronal glandular

 

 

2

 

 

 Penile

 

 

1.5

Posterior fusion

21-25

Peno-scrotal

Labio-scrotal

Labio-scrotal

1

 

10-20

Scrotal

 Inguino-scrotal

Inguino-scrotal

0.5

    

Perineal

Inguinal

Inguinal

0

Unfused

≤ 10

Typical female

Impalpable

Impalpable

Median EGS (10th to 90th centile) in males < 28 weeks gestation is 10 (8.6-11.5); in males 28-32 weeks 11.5 (9.2-12); in males 33-36 weeks 11.5 (10.5-12) and in full-term males 12 (10.5-12). In all female babies, EGS is 0 (0-0).

 

Table 8. Reference Ranges for Anogenital Distance and Anogenital Index in Term Neonates

 

5th

10th

25th

50th

75th

90th

95th

Males

 

 

 

 

 

 

 

AGD (mm)

18.47

19.44

21.00

22.68

25.20

27.80

29.08

AGI (mm/kg)

5.59

5.83

6.30

6.93

7.62

8.22

8.57

Females

 

 

 

 

 

 

 

AGD (mm)

9.40

9.85

10.55

11.65

12.60

13.45

14.10

AGI (mm/kg)

3.04

3.15

3.38

3.59

3.80

4.00

4.19

Anogenital index (AGI): the body weight standardized index of AGD [AGI=AGD/weight (mm/kg)] and found that using AGI as parameter has a better correlation with age.

 

Basic Auxiliary Investigations in the First Days

 

In the first few days of life, basic investigations are essential to evaluate adrenal and gonadal development and function in otherwise healthy newborns with ambiguous genitalia. Some key aspects to address include serum electrolytes and glucose levels. They should be monitored daily as cortisol deficiency can manifest as hypoglycemia in newborns affected by CAH. Body weight and feeding characteristics should also be monitored as excessive weight loss may indicate dehydration and electrolyte disturbances.

 

KARYOTYPE

 

Besides careful clinical evaluation, first-line investigation of an individual with DSD involves confirming the chromosomal sex using quantitative fluorescence polymerase chain reaction (QF-PCR) and karyotype (22). QF-PCR detects a series of markers on the sex chromosomes and has a turn-around time of about 1–2 days. This technique has largely replaced fluorescence in situ hybridization (FISH) methods. The analysis of karyotype is a cytogenetic technique (involving G-banding) and is crucial for the initial classification of a newborn with ambiguous genitalia (Table 3). Some centers have replaced karyotyping by array-comparative genomic hybridization (aCGH) or SNP array, with faster turn-around times of 5–10 days. But these techniques will not detect structural chromosomal rearrangements and may be less effective at detecting sex chromosome mosaicism.

 

HORMONE STUDIES 

 

Detailed hormone studies may be indicated including serum gonadotropins (LH, FSH), androgens and androgen precursors (17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone), adrenal steroids and hormones (cortisol, aldosterone, and their precursors; ACTH, renin), and anti-Müllerian hormone (AMH) (1, 24).

 

Care must be taken when interpreting the results in premature babies, in whom these studies may need to be repeated at a later age and special normative value may apply. Even in term babies the timepoint and the clinical context (mode of delivery, drug effects, actual stress level etc.) need to be considered when evaluating hormonal data immediately after birth. Hormonal data change massively during delivery (e.g., surge) and within days thereafter. Additionally, it's essential to be aware that normative values for hormone levels can vary depending on the laboratory and the methods used for testing. The interpretation of hormonal data should be done by experienced healthcare providers and, if necessary, in consultation with a specialist in DSD.

 

IMAGING STUDIES

 

Ultrasound imaging of the abdominal-pelvic organs is a valuable diagnostic tool in the evaluation of newborns with ambiguous genitalia. It provides important insights into the internal reproductive and urinary structures, helping to determine the presence and characteristics of gonads, Müllerian structures (e.g., the uterus), and any associated abnormalities of the urinary tract. It can be performed at the first visit of a child with ambiguous genitalia. Of course, this investigation is not easy and may only be performed by an experienced ultra-sonographer to reveal meaningful results. Ultrasound can also detect abnormalities of the urinary tract (kidney, ureters, bladder) that sometimes occur in conjunction with genital ambiguity. Interestingly, data show that a child with a uterus and a female internal system that presents with ambiguous genitalia without gonads in the genital folds and has a 46,XX karyotype, will have in 80% of cases a diagnosis of a 46,XX CAH (most likely 21-hydroxylase deficiency). The information needed to come to this conclusion in a newborn with ambiguous genitalia can be available within 48 hours in institutions with an experienced DSD team.

 

In a second step, usually after months or years, additional imaging studies with MRI can be helpful in identifying both the type and extent of internal sex organ development in more details. An MRI may reveal further details on Müllerian structures (uterus remanent, fallopian tubes, upper portion of the vagina) than an ultrasound and can also help for localizing abdominal gonads. MRI may also be used for surveillance of the gonad at risk for cancer.

 

A genitogram had been used in the past (often early) to visualize the urinary tract, and to determine its position in relation to the vagina or vagino-utricular pouch but is no longer performed in most institutions.

 

However, in follow-up, cystoscopy and vaginoscopy may be used. These investigations are invasive procedures requiring general anesthesia, and therefore need to have a clear indication. Often, they are performed after complications such as urinary tract infections, to better understand the anatomy and its consequences, and in preparation for a surgical procedure (112).

 

Overall, the choice of imaging studies and procedures should be guided by the individual's clinical presentation, diagnosis, and the specific clinical questions that need to be addressed. In many cases, a multi-disciplinary DSD team collaborates to determine the most appropriate imaging approach to provide a comprehensive evaluation and guide treatment decisions.

 

GENETIC STUDIES

 

The rapid advancement of genetic technologies, particularly massive parallel sequencing methods like DSD panels, whole exome sequencing (WES), and whole genome sequencing (WGS), has significantly expanded our understanding of the genes involved in sex development. As a result, genetic workup has become an integral part of the recommended routine evaluation for individuals with DSD and thus ambiguous genitalia at birth (8, 22, 24, 113, 114).

 

While making a precise genetic diagnosis is still a challenge in some cases, the use of advanced genetic techniques has improved the diagnostic yield. Currently, a clear molecular-level diagnosis can be established in approximately half of individuals with 46,XY DSD who undergo genetic workup. However, as genetic technologies continue to evolve, and our understanding of sex development genes expands, it is likely that more cases, including those involving individuals with ambiguous genitalia at birth, will receive accurate and valid diagnoses. Therefore, re-review of genetically unsolved cases by a DSD team and genetic re-analysis by DSD-specialized teams is recommended to achieve a final diagnosis in as many as possible.

 

In summary, the integration of genetic workup, in combination with ongoing advancements in technology and genetic knowledge, is expected to enhance our ability to provide individuals with DSD, including those with ambiguous genitalia at birth, with more precise and comprehensive diagnoses in the future. This progress will lead to better-informed clinical decisions and management strategies for newborns with ambiguous genitalia.

 

DIFFERENTIAL DIAGNOSIS

 

Generally, the result from the karyotype plays a major role in the differential diagnosis and workup of DSD and thus ambiguous genitalia (Table 3). In most cases, the chromosomes in newborns with ambiguous genitalia will be either 46,XX or 46,XY. In rare instances, chromosomal aberration such as 45,X/46,XY or 46,XX/46,XY mosaicism may be the underlying cause explaining the phenotype. Additionally, measurements of specific hormones, such as serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex steroids, AMH, inhibin B at birth and at follow-up, in the so-called mini-puberty of infancy period, can provide valuable information for the differential diagnosis of DSD and ambiguous genitalia.

 

In the following we provide two flowcharts and give some general information for the step-by-step workup of newborns with ambiguous genitalia and either 46,XX or 46,XY chromosomes.

 

46, XX DSD

 

A 46, XX karyotype in a newborn with ambiguous genitalia indicates that the child is a genetic female who was exposed to excessive amounts of androgens during fetal life. A flowchart for differential diagnosis is given in Figure 8.

 

Marked elevation of plasma 17-hydroxypregnenolone, 17-hydroxyprogesterone, 21-deoxycortisol and androstenedione, along with male-typical levels of testosterone, are characteristic of 21-hydroxylase deficiency (CYP21A2). High values of corticosterone and 11-deoxycortisol, along with elevated androgens, indicate 11β-hydroxylase deficiency (biallelic mutations in CYP11B1). In 3beta-HSD deficiency (biallelic mutations in HSD3B2), calculation of ratios of delta-5 (pregnenolone, 17OH-pregnenolone, DHEA) over delta-4 (progesterone, 17OH-progesterone, androstenedione) steroid metabolites (see Figure 4) will point towards the genetic defect.

 

Overall, comprehensive steroid profiling from blood or urine using chromatographic, mass spectrometric methods allow the diagnosis of most steroidogenic defects causing androgen excess CAH in 46,XX newborns (115-117) (see also Table 5). In addition, they have led to the description of novel alternate pathways to produce active androgens in the intermediate metabolism through the backdoor or 11-oxygenated pathways, especially in disorders such as 21-hydroxylase or POR deficiencies, thereby enhancing our understanding and diagnostic possibilities (115, 117, 118)(Figure 4). However, these methods are not (yet) widely available, and the interpretation of these data is not trivial. In addition, instruments and methods vary from lab to lab. Thus, method validation is tedious and comparison between labs difficult so far (119-121).

 

When excess maternal androgen production is the underlying cause for masculinization of a female fetus, the source of these steroids is eliminated postnatally. Thus, the various steroids studied in affected newborns will be in the female-typical range despite masculinization of the external genitalia. Then, detailed examination of the mother may provide the explanation.

 

Likewise, in cases of mild POR deficiency or aromatase deficiency, in which the feto-placental unit was not able to metabolize the fetal androgens and led to ambiguous genitalia, the problem will not (always) be visible in the newborn’s steroid metabolome, and the diagnosis may only be made by genetic testing. If not, only in later life, when aromatase activity is needed for estrogen production in puberty, or when ovarian cysts hint an underlying problem, the diagnosis may be made (Figure 8).

 

In individuals with ovo-testicular DSD and 46,XX chromosomes, masculinization arises from androgens secreted by the testicular portion of the differentiated gonads. Androgen production is similar to that produced by testes in unaffected males except that the amount is usually smaller. The degree of masculinization of the genitalia is thus linked to the amount of functioning testicular tissue, which can be determined by a serum AMH measurement (Table 9) (122).

 

Very low values of AMH and female-typical values for LH/FSH (Tables 9 and 10) are expected in 46,XX newborns with masculinized/virilized genitalia that is attributed to CAH or excess maternal androgen production during gestation (122, 123). AMH is higher in newborns with ovo-testicular DSD, due to Sertoli cell development in the testicular portion of their gonads where AMH is mainly produced.

 

On occasion, translocation of the pseudo-autosomal part of the Y chromosome along with a mutated SRY gene to an X chromosome occurs. The result is partial masculinization of the genitalia in a 46,XX newborn. With maturity, the phenotype of affected individuals closely resembles that of boys and men with Klinefelter syndrome.

 

Figure 8. 46,XX DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; CAH: Congenital adrenal hyperplasia; 21OHD: 21α-Hydroxylase deficiency; 11β-OHD: 11β-Hydroxylase deficiency; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; PORD: P450 oxidoreductase deficiency.

 

 

Table 9. Normal Values for AMH (ng/mL)* in Fetuses, Newborns and Infants Under 1 Year (122)

 

Female

Male

 

n

Median

5-95th percentile

n

Median

5-95th percentile

Fetal blood

25

<0.4

<0.4-0.5

23

44.4

13.4-86.2

Cord blood

53

<0.4

<0.4-1.1

48

30.7

7.4-610.7

1-30 days

70

<0.4

<0.4-1.3

73

57.2

23.8-124.0

31-120 days

31

0.8

<0.4-6.4

39

88.4

46.8-173.0

4-12 months

13

1.0

<0.4-2.7

53

124.7

67.4-197.0

* AMH/MIS enzyme immunoassay kit cat 2368, Immunotech-Beckman Coulter, France

 

Table 10. LH and FSH Values (means ± SD) From Female and Male Newborns. Modified from (123)

 

 

Female

Male

Age (days)

Hormone

n

Median

Mean±SD

n

Median

Mean±SD

1-5

FSH (IU/L)

31

1.80

2.00 ± 1.37

30

0.85

0.96 ± 0.60

LH (IU/L)

0.20

0.48 ± 0.66

0.20

0.39 ± 0.48

6-10

FSH (IU/L)

17

1.40

2.44 ± 2.52

15

1.40

2.91 ± 4.38

LH (IU/L)

0.30

0.45 ± 0.33

0.30

0.45 ± 0.33

11-15

FSH (IU/L)

8

8.95

8.16 ± 4.27

17

3.00

3.71 ± 2.69

LH (IU/L)

1.60

1.58 ± 1.28

2.90

3.55 ± 2.84

16-20

FSH (IU/L)

6

1.90

1.62 ± 1.05

14

2.15

2.63 ± 1.45

LH (IU/L)

0.35

1.03 ± 1.39

3.65

4.13 ± 2.76

21-25

FSH (IU/L)

3

3.90

7.07 ± 5.92

7

2.10

2.50 ± 1.51

LH (IU/L)

0.50

0.46 ± 0.25

2.70

2.86 ± 1.51

26-28

FSH (IU/L)

8

6.15

9.74 ± 9.89

8

2.40

2.25 ± 0.81

LH (IU/L)

2.80

2.75 ± 2.39

1.40

2.22 ± 2.37

Abbreviations: LH, luteinizing hormone; FSH, follicle-stimulating hormone.

 

46, XY DSD

 

A 46, XY karyotype reveals that one is dealing with a genetic male who was under-masculinized during fetal development. A flowchart for differential diagnosis is given in Figure 9.

 

Laboratory findings of normal or elevated testosterone and DHT with normal AMH (Figure 9) indicate a diagnosis of AIS. If testosterone levels are normal but DHT levels are low, a diagnosis of steroid 5α-reductase deficiency can be made. Low levels of testosterone and DHT, along with marked elevation of some androgen precursors, indicate a deficiency of one of the enzymes required for androgen biosynthesis (Figure 9). For example, if the elevated precursors include androstenedione and 17-hydroxyprogesterone, then the defective enzyme is 17-ketosteroid reductase (HSD17B3). In all cases of testosterone biosynthetic defects, AMH levels are similar to those observed in healthy 46,XY male infants with male-typical external genitalia (Figure 9). With early defects of steroidogenesis that affect both adrenal and gonadal function, it is though important to evaluate adrenal function, as it might be impaired with deficiencies of STAR, CYP11A1, HSD3B2, CYP17A1 and POR (Table 5, Figure 9).

 

Finally, when all androgens and their precursors are below normal levels, one is dealing with gonadal dysgenesis or 46,XY ovo-testicular DSD. In these cases, AMH values should also be low. In contrast, for babies affected by Leydig cell hypoplasia, androgens and their precursors are low, while AMH values should be in the normal male range.

 

Figure 9. 46,XY DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; AMH: Anti-Müllerian hormone; T: Testosterone; N: Normal; PMDS: Persistent Müllerian duct syndrome; AIS: Androgen insensitivity syndrome; SRD5A2: 5α-reductase A2; HCG: Human chorionic gonadotropin; DHT: Dihydrotestosterone; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; CYP17A1: 17α-Hydroxylase/17-20 lyase; POR: P450 oxidoreductase; StAR: Steroidogenic acute regulatory protein; CYP11A1; Cholesterol side-chain cleavage enzyme; 17β-HSD3: 17β-Hydroxysteroid dehydrogenase type 3; AKR1C2/4: Aldo-keto reductase family 1 member C2/4; CYB5: Cytochrome b5.

 

Testing Early Postnatal Gonadal Function and Mini-Puberty

 

In cases of ambiguous genitalia where a diagnosis may not be reached through first line investigations, and where a gonadal malfunction is suspected, functional testing and/or re-analysis of gonadal function at the timepoint of mini-puberty may be helpful, especially also with respect to having information when making considerations towards sex of registration (e.g., in utero effect of sex hormones on the brain) and prediction of later pubertal development and fertility potential.

 

Mini-puberty is the time window at day 30-100 after birth, when the hypothalamic-pituitary-gonadal (HPG) axis is re-activated for a short time frame in infancy, before going to inactivity until puberty. Little is known about the purpose of mini-puberty, but as the HPG axis is active and stimulates the gonads, it is a time window of opportunity to assess gonadal function without the necessity to stimulate with human chorionic gonadotrophin (hCG).

 

As a principle, in mini-puberty, the typical ovaries of a 46,XX newborn show little functional activity, while the typical testes of a 46,XY newborn are quite (re)active in testosterone (T), DHT, AMH and inhibin B production. While androgens are produced in the Leydig cells of the testes, AMH and inhibin B originate from the Sertoli cells. Table 11 gives an overview of the hormonal values observed during normal mini-puberty in healthy 46,XX and 46,XY infants (124).

 

Table 11. Evaluation of Reproductive Hormones During Mini-Puberty in Infants. Normative Values of Different Serum Hormones in 46,XX Female and 46,XY Male Infants. Modified from  (124)

 

 

Females

Males

 

Post-natal time (months)

n

GMean

2.5-97.5th percentile

n

GMean

2.5-97.5th percentile

LH (IU/L)

2-3.5

432

<LOD

<LOD-0.98

581

1.71

0.62-4.08

3.5-5

110

<LOD

<LOD-1.25

166

1.40

0.54-3.32

FSH (IU/L)

2-3.5

435

3.98

1.23-17.4

578

1.19

0.41- 3.02

3.5-5

111

3.93

1.30-17.7

165

1.11

0.42-2.68

Testosterone (RIA,nmol/L)

2-3.5

74

<LOD

<LOD-0.40

592

3.04

0.69-7.60

3.5-5

14

<LOD

<LOD-NA

168

1.97

<LOD-7.01

Testosterone

(LC-MS/MS, nmol/L)

2-3.5

165

<LOD

<LOD-0.21

251

4.75

1.35- 11.3

3.5-5

125

<LOD

<LOD-0.17

175

2.25

0.32- 9.65

Estradiol (pmol/L)

2-3.5

455

29

<LOD-79

571

<LOD

<LOD-47

3.5-5

113

31

<LOD-98

160

<LOD

<LOD-49

SHBG (nmol/L)

2-3.5

427

133

67-264

579

135

66-268

3.5-5

110

141

72-282

162

143

71-262

Inhibin B (pg/mL)

2-3.5

423

62

<LOD-184

571

379

229-631

3.5-5

106

67

<LOD-174

158

379

222-662

AMH (pmol/L)

2-3.5

339

11

<LOD-49

48

1013

425- 1810

3.5-5

67

15

2-46

12

1183

797-NA

Abbreviations: LOD, Limit of detection; GMean, geometric mean; LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone; RIA, radioimmunoassay; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NA, not available.

 

HCG Testing of Gonadal Function in a DSD Newborn

 

The human chorionic gonadotrophin (hCG) test can assess whether in a newborn with ambiguous genitalia, functioning Leydig cells are present. The test may therefore be useful for diagnosing testosterone biosynthesis defects such as HSD17B3 or SRD5A2 deficiency (Table 12). Several protocols for the hCG test exist, but all baseline serum samples are taken for T, DHT and precursors. Then, one to three intramuscular injections of high dose hCG (500-1500 IU) are given at 24 hours interval, and finally repeat stimulated samples are taken for androgen measurements after 72 or 24 hours, respectively, after the last injection. The following Table 12 shows how results of the test may be interpreted in various DSD conditions that may lead to ambiguous genitalia (125, 126).  

 

Table 12. Interpretation of hCG Stimulation Test Results (125, 126)

Indication

Consideration

Abnormal result

Testosterone biosynthesis defects

T before and after hCG administration

T increase < 100-150 ng/dL (3.5-5 nmol/L)

5α-Reductase deficiency

T/DHT ratio after hCG administration

≥ 8.5 for minipuberty,

≥ 10 for prepuberty,

≥ 17 for puberty,

> 20 in genetically proven cases using LC-MS/MS

17β-HSD3 deficiency

T/A4 ratio after hCG administration

< 0.8

AIS

T, SHBG before and after hCG administration

T increases but SHBG does not change significantly

Abbreviations: T, testosterone; DHT, dihydrotestosterone; HCG, human chorionic gonadotropin; A4, androstenedione; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 17β-HSD3 deficiency, 17β-hydroxysteroid dehydrogenase type 3 deficiency; AIS, androgen insensitivity syndrome

 

RISK OF MALIGNANCY OF THE DSD GONAD

 

Individuals with certain disorders of sex development (DSD), especially those involving Y chromosome material, have an increased risk of developing gonadal tumors. These tumors are mainly gonadal germ cell tumors (GGCTs), which occur in about 15 % of all DSD cases, with the risk varying depending on the age and specific underlying diagnosis. Gonadal stromal tumors and smooth muscle leiomyomas/hamartomas are less common and are found in only about 0.9% of DSD cases (127). Risk factors for development of gonadal malignancy in DSD are shown in Figure 10. Several factors contribute to the increased risk of gonadal malignancy in individuals with DSD, including genetic factors, the presence of Y chromosome material, and the type of DSD. Therefore, ongoing monitoring and appropriate management are essential to minimize this risk. While gonadectomy (surgical removal of the gonads) is not recommended for every case of 46,XY DSD, it's crucial to consider the risk of malignant tumors in the decision-making process (1, 69). The consensus statement on the management of DSD provides guidelines and recommendations regarding the timing of gonadectomy for specific patients. These recommendations aim to balance the potential benefits of gonadectomy with the associated risks and individual patient needs.

 

In individuals with DSDs, germ cells typically undergo a process of apoptosis (programmed cell death) as the body grows and develops. This process eliminates germ cells that are not needed for reproduction. However, in some cases, certain germ cells may not undergo the normal maturation process and remain in an immature state. These immature germ cells can continue to express embryonic markers such as octamer-binding transcription factor 3/4 (OCT3/4) encoded by POU5F1; stem cell factor (SCF), also known as c-KIT ligand (encoded by KITLG); and placental alkaline phosphatase (PLAP). These cells are susceptible to developing into cancerous lesions, starting as pre-invasive lesions (gonado-blastoma) or as precursors to invasive GGCT (dysgerminoma) (Figure 10).

 

DSD patients who have Y chromosome material in their gonadal karyotype are at risk of GGCTs regardless of the degree of testicular differentiation. Y chromosome material associated with gonadal tumors is GBY locus which is around the centromere of the Y chromosome and includes the gene for ¨testis-specific protein on the Y chromosome¨ (TSPY, located in Yp11.2). Other potentially related genes are SRY and DYZ3.

 

The risk of developing gonadal tumors in gonadal dysgenesis may also vary according to the underlying molecular etiology. Patients with genetic defects that lead to early blockage of gonadal development such as mutation or deletions of SRY or WT1 have a higher risk. The risk of developing gonadal tumors ranges from 40% to 60% in gonadal dysgenesis due to WT1 gene mutations (Frasier syndrome and Denys-Drash syndrome). Early bilateral gonadectomy is therefore recommended. Similarly, early bilateral gonadectomy is recommended in patients with complete or partial gonadal dysgenesis due to SRY gene mutations due to the 20% to 52.5% risk of gonadal tumor development. Gonadal dysgenesis due to MAP3K1 gene mutations is another indication for early gonadectomy (127). Gonadal tumor risk is also present, though somewhat lower in gonadal dysgenesis due to 45,X0/46,XY mosaicism. The exact time of malignancy development and the prevalence of tumors in other molecular etiologies require further research for a comprehensive understanding.

 

Histologically the risk of gonadal tumor development increases with gonadal immaturity. Fully developed gonads such as the testes, ovary or ovotestis have lower risk of malignancy than streak or dysgenetic testes (Figure 10). In ovo-testicular DSD, the risk of germ cell tumors is low (3%) (1). Newborns with PAIS are at high risk (50%) for developing gonadal tumors, and bilateral orchiopexy or gonadectomy is recommended at the time of diagnosis, if the testes are located in the abdomen (1, 128).

 

The risk of developing germ cell malignancy in newborns with CYP11A1, HSD3B2 or CYP17A1 deficiency is unknown. Newborns with 17-ketosteroid reductase deficiency have a fairly low risk for malignancy (1). Tumor risk in newborns with Leydig cell hypoplasia is unclear, and thus the recommendation for gonadectomy is not well established (1, 25, 129, 130).

 

Overall, it remains challenging to predict the personal risk of gonadal neoplasia (mostly GGCT) for a DSD individual, because it depends on many other factors besides genetics. Stimulating gonadotropins and gonadal hormones during mini-puberty and after puberty onset are also regarded as risk factors increasing gonadal tumor risk in patients with DSD. Similarly, gonads located intra-abdominal carry a higher tumor risk. The management of gonadal tumor risk in individuals with DSD is a complex issue that requires careful consideration and a personalized approach, taking into account the specific DSD subtype, genetic factors, and patient preferences. Regular monitoring and discussions with a healthcare team experienced in DSD management are essential to make informed decisions about gonadectomy. If gonads are located in the scrotum, then periodic examination of the testes may be advised as an alternative to gonadectomy, while an intra-abdominal gonad at risk may only be monitored by imaging methods such as ultrasound or MRI.

 

Figure 10. Risk of malignancy development in the DSD gonad.
Abbreviations: (OCT3/4); Octamer-binding transcription factor 3/4, TSPY; testis-specific protein on the Y chromosome, c-KITL; c-KIT ligand also called stem cell factor (SCF).

 

SEX REGISTRATION OF A NEWBORN

 

Assigning or registering the sex of a newborn with atypical external genitalia is a complex and important decision that requires careful consideration, consultation, and a patient-centered approach. The parents and DSD network team should not rush but take the time needed to come to a consensus in the best interest of the child in its environment. Not knowing whether the newborn is a girl or boy causes mostly a lot of distress and one is tempted to call it an emergency, but if there are no medical reasons to make it an emergency (like e.g., suspected adrenal insufficiency or additional organ anomalies), it is important to declare it accordingly and inform that the newborn is not at risk and actually does not care whether it is a male or female or intersex person at this moment. Thus, the stress of ambiguity of the newborn is actually at this moment “only” an issue of the parents and the care team who mostly wish to know ASAP whether the child can be assigned/registered a “correct” sex that might also fit later in life.

 

To consider sex registration, not only is it important to take into account the underlying cause and knowledge on the possible outcome of the specific, often unique condition, but also the family, cultural, and societal preconditions and aspects. Most societies have a binary sex registration obligation by legislation, but some societies have more recently introduced a third sex category besides male and female. Although this seems to make things easier, it has been discussed that such categorization in a third or other or unclassified group might also stigmatize a person in a society that is mostly binary. Thus, in real life practice, there is rumor that a third sex category is rarely chosen for a newborn with ambiguous genitalia by the parents and the DSD team. Overall, a personalized approach for every newborn with ambiguous genitalia involving the parents and the DSD network team is recommended following the shared-decision-making process (12, 13).

 

Most important is that the team around the newborn with ambiguous genitalia works together in the best interest of the child with the latest knowledge of the literature and expertise when taking the decision of sex registration, and still remains humble and open minded for the child’s future that may follow a different path leading to sex reassignment later in life. Therefore, continuous support of the family and the child by a DSD psychologist is extremely helpful to recognize early signs of gender dysphoria, inform the child about the condition in an age-appropriate manner and follow its will, if case sex reassignment is desired.

 

Generally, people believe that the sex chromosomes indicate a person’s “true sex” and  laws exist supporting this idea, to the detriment of some individuals affected by DSD (131). Scientifically speaking, it is clear that the majority of genes on the X chromosome do not influence sex development and differentiation, although the AR gene is necessary for phenotypic masculinization. Concerning the Y chromosome, only the SRY gene contributes to testicular formation. In fact, most of the genes required for sex development and differentiation are found on the autosomes (1). Therefore, chromosomes do not dictate the sex of rearing in newborns affected by a DSD and are only one piece of a big puzzle (Figure 3).

 

Although there is no overall recommendation for sex registration at birth for a child with ambiguous genitalia, some helpful information is available from larger patients’ cohorts with a specific DSD diagnosis in the literature.

 

Newborns presenting with 46,XY chromosomes and female external genitalia at birth (thus actually not with ambiguous genitalia) due to complete androgen insensitivity syndromes (CAIS), complete gonadal dysgenesis (Swyer syndrome), or other complete loss of testosterone biosynthesis live successfully when assigned a female sex of rearing. Female assignment in such cases is widely accepted by patients throughout their lives despite challenges that they may experience regarding sexual dysfunction and infertility (1, 132-134).

 

For newborns with ambiguous genitalia affected by partial gonadal dysgenesis, no general recommendation can be made. Fewer procedures were historically required for surgical feminization compared to surgical masculinization of the genitalia. However, the functional outcome in individuals who received feminizing or masculinizing procedures is less than optimal (135, 136). Additionally, rates of satisfaction with sex of rearing are similar for affected individuals whether raised male or female (1, 20, 137). Thus, the decision should not be made on the basis of “surgical possibilities”, and overall, such surgical procedures should be executed with reservation. Clearly, further studies are needed to elucidate why some individuals with partial gonadal dysgenesis experience gender dysphoria, while others do not.

 

Likewise, no general recommendation can be made for patients with ambiguous genitalia due to a partial androgen biosynthesis defect, (e.g., partial 3βHSD2, CYP17A1, POR, StAR, CYP11A1 deficiencies). But some studies suggest that the degree of masculinization of the external genitalia may reflect the degree of virilization of the brain and thus the gender preference of this person (138). This might be true for 46,XY newborns with ambiguous genitalia that are manifesting with severe hypospadias, but not without exceptions. For patients with variants in the SRD5A2 or HSD17B3genes recognized with ambiguous genitalia at birth (or even missed because manifesting with a female-typical external genitalia), there is quite a strong body of data recommending male sex registration at birth as this is the preferred sex of these persons when asked in later life (139, 140). Normal virilization at puberty, coupled with intact fertility potential, are strong factors for this recommendation at birth. If registered as female at birth and diagnosed later, gender dysphoria and desire of sex reassignment later in life is very likely.

 

Similar to partial gonadal dysgenesis, consensus regarding an optimal sex of registration for newborns affected by PAIS does not exist. Although earlier data showed that fewer procedures were usually required for surgical feminization, compared to surgical masculinization, in a person with ambiguous external genitalia in the past, when larger series of genital surgery were analyzed, it is unclear if the functional outcome was better among newborns who received feminizing procedures compared to those who received masculinizing procedures. Rates of satisfaction with sex of rearing are similar for individuals with PAIS raised female or male (132, 141). While reports exist of impaired sexual function in people with PAIS raised male, it is highly suspected that sexual functional outcomes in affected people raised female are similarly poor (137). Reports of sexual satisfaction for people with PAIS who have not received genitoplasty in infancy, or at a later age, are limited. However, more information on outcome of newborns with ambiguous genitalia who had no genital surgery, at least not in infancy and childhood, will hopefully soon be added to the medical literature as current developments recommend abstaining from genital surgery.

 

By contrast, in 46,XX newborns with ambiguous genitalia due to CAH, it is generally recommended to register them a priori in the female sex, even if they are severely virilized (142-144). Masculinization due to 21-hydroxylase deficiency does not impair the development of the ovaries or the Müllerian ducts and thus fertility potential. Limited information is available about the medical and psychosexual outcome of 46,XX newborns affected by 21-hydroxylase deficiency and raised male (145, 146), although these patients do tend to have an increased risk of gender dysphoria compared to their female counterparts (147). Thus, a female sex of rearing is typically the decision for 46,XX newborns affected by 21-hydroxylase deficiency (145). Female sex of rearing in 46,XX newborns with ambiguous genitalia is also recommended for conditions resulting from maternal overexposure to androgens and placental aromatase deficiency.

 

FINAL REMARKS TO OVERALL MANAGEMENT

 

Only recently has the understanding of, and reaction to, having a child with ambiguous genitalia received systematic study. For some parents and caregivers, feelings of isolation and concern over what the future may hold for their affected child in terms of stigmatization and sexual dysfunction are paramount (148). One potential approach to ameliorate this issue is to provide information and management options to the family early in pregnancy or as soon as the child is born. Guidance by a DSD network team is therefore extremely important.

 

Some research has been done to examine the effects of prenatal diagnosis and treatment. In females with 21-hydroxylase deficiency CAH, one of the most common causes of ambiguous genitalia, dexamethasone treatment of the pregnant mother has been reported to reduce virilization of an affected female fetus by 80-85% (149), but this approach is no longer offered or recommended in guidelines because of potential adverse effects of dexamethasone on the developing fetus. Before the discovery of methods to measure fetal DNA in maternal blood, which enabled specific diagnosis of sex and whether the fetus inherited and was affected by the mutation, controversies existed because 7 unaffected fetuses would be treated unnecessarily (1:4 chance of inherited mutation and 1:2 chance of male vs female), So, on the one hand 7 unaffected fetuses and the mother would be exposed to an unnecessary drug, while on the other hand, even in affected fetuses long-term outcome showed adverse effects on neurocognitive parameters (150). Today, noninvasive prenatal diagnosis using massive parallel sequencing of cell-free fetal DNA can be used to determine a specific diagnosis, such as CAH, as early as 6 weeks of gestation (149, 151). Even preimplantation diagnostics is available (152). These techniques could be used to look at other autosomal dominant or X-linked conditions that cause ambiguous genitalia, especially when the fetus is known to be at risk (151). Further studies need to be done on this subject, but for families who already have an affected child or know that they are carriers, this may be an option that needs to be discussed with a DSD geneticist.

 

Patients for whom prenatal diagnosis and/or treatment is not an option, prognosis and management has improved significantly over time, but many open questions and controversies still exist. Our studies of parents of children with DSD reveal that the appearance of atypical genitalia can result in significant stress and maladaptive parenting strategies. For example, mothers of children with ambiguous genitalia experience greater stress if their child has not received “corrective” genital surgery (153) and some parents believe such surgeries eliminate stigmatization that may arise due to their child’s ambiguous genitalia However, as their children mature past infancy, some parents realize that their child’s DSD has not been ameliorated by genitoplasty, and concerns for their son or daughter resurface (148). To illustrate, among parents studied by our group, caregivers of adolescents with DSD experienced increased stress as their child matured despite the fact that many had received earlier surgery (154). Additionally, parents of children reared male reported the most perceived child vulnerability for their sons (155),and their reported levels of depression were directly associated with more atypical genital appearance for their sons (156). Overall, parents report that while early genitoplasty seems to “fix” some of their concerns for their child with DSD, this “fix” is not long-lasting. Instead, what needs to be emphasized to families is how to support their child who is different, but not damaged. Family centered, interdisciplinary care, with open and clear communication between patients, parents, and caregivers has been shown to be essential for optimal quality of life (157). In addition, networks of families who have the personal experience of parenting children with ambiguous genitalia can be an invaluable resource (158). Reservation towards early interventions needs to be considered,keeping in mind to preserve the personal right of the child to take his/her own decision towards body integrity. Any intervention that can be delayed without harming the newborn with ambiguous genitalia should be postponed until personal consent can be given. Finally, referring parents to support groups and introducing them to other caregivers of children born with ambiguous genitalia is extremely important to optimize parents’ understanding and acceptance of their child’s condition. With increased understanding and acceptance, optimal growth and development for children born with ambiguous genitalia may be obtained.

 

In conclusion, this Chapter give some insight into the current diagnostic evaluation and care of the newborn with ambiguous genitalia. We underscore the importance of a multidisciplinary and compassionate approach to the care of newborns with ambiguous genitalia, acknowledging the complex medical, psychological, and emotional aspects involved in such cases. We highlight the critical role of specialized teams and support in ensuring the well-being of both the child and his/her family.

 

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