ABSTRACT

Incidence rates of thyroid cancer have increased substantially worldwide in the past several decades. Thus, diseases of the thyroid gland and their treatment remain one of the most interesting and dynamic areas of study in medicine. This chapter presents a clear and concise description of current thought and practice concerning the surgical treatment of thyroid diseases. Sections within this chapter include 1) normal and abnormal anatomy and embryology of the thyroid and surrounding neck structures; 2) indications for operation of benign lesions of this gland; 3) diagnosis of thyroid nodules, stressing the use of fine needle aspiration with cytologic analysis; 4) preparation for operation and care of patients with Graves’ disease; 5) surgical approaches for treatment of the different types of thyroid cancer; 6) operative techniques for thyroidectomy including descriptions of standard open, minimally invasive, endoscopic, robotic and transoral approaches; 7) complications of thyroidectomy and their treatment; and 8) developmental abnormalities of the thyroid and their treatment. This chapter offers information for physicians and endocrinologists, as well as for surgeons. For complete coverage of this and related areas in Endocrinology, visit our FREE web-book, www,thyroidmanager.org

HISTORY

The extirpation of the thyroid gland…typifies, perhaps better than any operation, the supreme triumph of the surgeon’s art…. A feat which today can be accomplished by any competent operator without danger of mishap and which was conceived more than one thousand years ago…. There are operations today more delicate and perhaps more difficult…. But is there any operative problem propounded so long ago and attacked by so many…which has yielded results as bountiful and so adequate? Dr. William S. Halsted, 1920

Modern thyroid surgery, as we know it today, began in the 1860s in Vienna with the school of Billroth (1). The mortality associated with thyroidectomy was high, recurrent laryngeal nerve injuries were common, and tetany was thought to be caused by “hysteria.” The parathyroid glands in humans were not discovered until 1880 by Sandstrom (2), and the fact that hypocalcemia was the definitive cause of tetany was not wholly accepted until several decades into the twentieth century. Kocher, a master thyroid surgeon who operated in the late nineteenth and early twentieth centuries in Bern, practiced meticulous surgical technique and greatly reduced the mortality and operative morbidity of thyroidectomy for goiter (3). He described “cachexia strumipriva” in patients years after thyroidectomy (Fig. 1). Kocher recognized that this dreaded syndrome developed only in patients who had total thyroidectomy. As a result, he stopped performing total resection of the thyroid. We now know that cachexia strumipriva was surgical hypothyroidism. Kocher received the Nobel Prize for his contributions to thyroid surgery and for this very important contribution, which proved beyond a doubt the physiologic importance of the thyroid gland.

By 1920, advances in thyroid surgery had reached the point that Halsted referred to this operation as a “feat which today can be accomplished by any competent operator without danger of mishap” (1). Unfortunately, decades later, complications still occur. In the best of hands, however, thyroid surgery can be performed today with a mortality that varies little from the risk of general anesthesia alone, as well as with low morbidity. To obtain such enviable results, however, surgeons must have a thorough understanding of the pathophysiology of thyroid disorders; be versed in the preoperative and postoperative care of patients; have a clear knowledge of the anatomy of the neck region; and, finally, use an unhurried, careful, and meticulous operative technique.

IMPORTANT SURGICAL ANATOMY

The thyroid (which means “shield”) gland is composed of two lobes connected by an isthmus that lies on the trachea approximately at the level of the second tracheal ring (Figs. 2 and 3). The gland is enveloped by the deep cervical fascia and is attached firmly to the trachea by the ligament of Berry. Each lobe resides in a bed between the trachea and larynx medially and the carotid sheath and sternocleidomastoid muscles laterally. The strap muscles are anterior to the thyroid lobes, and the parathyroid glands and recurrent laryngeal nerves are associated with the posterior surface of each lobe. A pyramidal lobe is often present. This structure is a long, narrow projection of thyroid tissue extending upward from the isthmus and lying on the surface of the thyroid cartilage. It represents a vestige of the embryonic thyroglossal duct, and it often becomes palpable in cases of thyroiditis or Graves’ disease. The normal thyroid varies in size in different parts of the world, depending on the iodine content in the diet. In the United States it weighs approximately 15 grams.

VASCULAR SUPPLY

The thyroid has an abundant blood supply (Figs. 2 and 3). The arterial supply to each thyroid lobe is two-fold. The superior thyroid artery arises from the external carotid artery on each side and descends several centimeters in the neck to reach the upper pole of each thyroid lobe, where it branches. The inferior thyroid artery, each of which arises from the thyrocervical trunk of the subclavian artery, crosses beneath the carotid sheath and enters the lower or midpart of each thyroid lobe. The thyroidea ima is sometimes present; it arises from the arch of the aorta and enters the thyroid in the midline. A venous plexus forms under the thyroid capsule. Each lobe is drained by the superior thyroid vein at the upper pole, which flows into the internal jugular vein; and by the middle thyroid vein at the middle part of the lobe, which enters either the internal ­jugular or the innominate vein. Arising from each lower pole is the inferior thyroid vein, which drains directly into the innominate vein.

NERVES

The relationship of the thyroid gland to the recurrent laryngeal nerve and to the external branch of the superior laryngeal nerve is of major surgical significance because damage to these nerves leads to disability in phonation and/or to difficulty breathing (4). Both nerves are branches of the vagus nerve.

Injury to the external branch of the superior laryngeal nerve leads to difficulty in singing and projection of the voice. Injury to one recurrent laryngeal nerve may lead to hoarseness of the voice, aspiration, and difficulty breathing. Bilateral recurrent laryngeal nerve injury is much more serious and often leads to the need for a tracheostomy. These injuries will be discussed in greater detail later in this chapter under “Postoperative Complications.”

Recurrent Laryngeal Nerve

The right recurrent laryngeal nerve arises from the vagus nerve, loops posteriorly around the subclavian artery, and ascends behind the right lobe of the thyroid (Fig. 4a). It enters the larynx behind the cricothyroid muscle and the inferior cornu of the thyroid cartilage and innervates all the intrinsic laryngeal muscles except the cricothyroid. The left recurrent laryngeal nerve comes from the left vagus nerve, loops posteriorly around the arch of the aorta, and ascends in the tracheoesophageal groove posterior to the left lobe of the thyroid, where it enters the larynx and innervates the musculature in a similar fashion as the right nerve. Several factors make the recurrent laryngeal nerve vulnerable to injury, ­especially in the hands of inexperienced surgeons (4,6)

1. The presence of a nonrecurrent laryngeal nerve (Fig. 4b). Nonrecurrent nerves occur more often on the right side (0.6%) than on the left (0.04%) (5). They are associated with vascular anomalies such as an aberrant takeoff of the right subclavian artery from the descending aorta (on the right) or a right-sided aortic arch (on the left). In these abnormal positions, each nerve is at greater risk of being divided.

2. Proximity of the recurrent nerve to the thyroid gland. The recurrent nerve is not always in the tracheoesophageal groove where it is expected to be. It can often be posterior or anterior to this position or may even be surrounded by thyroid parenchyma. Thus, the nerve is vulnerable to injury if it is not visualized and traced up to the larynx during thyroidectomy.
3. Relationship of the recurrent nerve to the inferior thyroid artery. The nerve often passes anterior, posterior, or through the branches of the inferior thyroid artery. Medial traction of the lobe often lifts the nerve ante­riorly, thereby making it more vulnerable. Likewise, ­ligation of the inferior thyroid artery, practiced by many surgeons, may be dangerous if the nerve is not identified first.
4. Deformities from large thyroid nodules (6). In the presence of large nodules the laryngeal nerves may not be in their “correct” anatomic location but may be found even anterior to the thyroid (Fig. 5). Once more, there is no substitute for identification of the nerve in a gentle and careful manner.

External Branch of the Superior Laryngeal Nerve

On each side, the external branch of the superior laryngeal nerve innervates the cricothyroid muscle. In most cases, this nerve lies close to the vascular pedicle of the superior poles of the thyroid lobe which requires that the vessels be ligated with care to avoid injury to it (Fig. 6) (7). In 21% of cases, the nerve is intimately associated with the superior thyroid vessels. In some patients the external branch of the superior laryngeal nerve lies on the anterior surface of the thyroid lobe, making the possibility of damage during thyroidectomy even greater (8). In only 15% of patients is the superior laryngeal nerve sufficiently distant from the superior pole vessels to be protected from manipulation by the surgeon. Unfortunately, many surgeons do not attempt to identify this nerve before ligation of the upper pole vessels of the thyroid (9, 9a).

PARATHYROID GLANDS

The parathyroids are small glands that secrete parathyroid hormone, the major hormone that controls serum calcium homeostasis in humans. Usually four glands are present, two on each side, but three to six glands have been found. Each gland normally weighs 30 to 40 mg, but they may be heavier if more fat is present. Because of their small size, their delicate blood supply, and their usual anatomic position adjacent to the thyroid gland, these structures are at risk of being ­accidentally removed, traumatized, or devascularized during thyroidectomy (100.

The upper parathyroid glands arise embryologically from the fourth pharyngeal pouch (Figs. 7 and 8). They descend only slightly during embryologic development, and their position in adult life remains quite constant. This gland is usually found adjacent to the posterior surface of the middle part of the thyroid lobe, often just anterior to the recurrent laryngeal nerve as it enters the larynx.

The lower parathyroid glands arise from the third pharyngeal pouch, along with the thymus; hence, they often descend with the thymus. Because they travel so far in embryologic life, they have a wide range of distribution in adults, from just beneath the mandible to the anterior mediastinum (see Fig. 8) (11). Usually, however, these glands are found on the lateral or posterior surface of the lower part of the thyroid gland or within several centimeters of the lower thyroid pole within the thymic tongue.

Parathyroid glands can be recognized by their tan appearance; their small vascular pedicle; the fact that they bleed freely when a biopsy is performed, as opposed to fatty tissue; and their darkening color of hematoma formation when they are traumatized. With experience, one becomes much more adept at recognizing these very important structures and in differentiating them from either lymph nodes or fat. Frozen section examination during surgery can be helpful in their identification.

LYMPHATICS

A practical description of the lymphatic drainage of the thyroid gland for the thyroid surgeon has been proposed by Taylor (12). The results of his studies, which are clinically very relevant to the lymphatic spread of thyroid carcinoma, are summarized in the following.

Central Compartment of the Neck

1. The most constant site to which dye goes when injected into the thyroid is the trachea, the wall of which contains a rich network of lymphatics. This fact probably accounts for the frequency with which the trachea is involved by thyroid carcinoma, especially when it is anaplastic.
2. A chain of lymph nodes lies in the groove between the trachea and the esophagus (Level 6, Fig. 8).
3. Lymph can always be shown to drain toward the mediastinum and to the nodes intimately associated with the thymus (Level 7, Fig. 8).
4. One or more nodes lying above the isthmus, and therefore in front of the larynx, are sometimes involved. These nodes have been called the Delphian nodes (named for the oracle of Delphi) because it has been said that if palpable, they are diagnostic of carcinoma. However, this clinical sign is often misleading.
5. A bilateral central lymph node dissection, called a level 6 dissection (See Fig. 8) clears out all these lymph nodes from one carotid artery to the other carotid artery and down into the superior mediastinum as far as possible (12a).

Lateral Compartment of the Neck

A constant group of nodes lies along the jugular vein on each side of the neck (Levels 2, 3, and 4). The lymph glands found in the supraclavicular fossae or more laterally in the neck (Level 5) may also be involved in more extensive spread of malignant disease from the thyroid gland (12a). It should not be forgotten that the thoracic duct on the left side of the neck, a lymph vessel of considerable size, arches up out of the mediastinum and passes forward and laterally to drain into the left subclavian vein or the internal jugular vein near their junction. If the thoracic duct is damaged, the wound is likely to fill with lymph; in such cases, the duct should always be sought and ligated. A wound that discharges lymph postoperatively should always raise suspicion of damage to the thoracic duct or a major tributary. A lateral lymph node dissection encompasses removal of these lateral lymph nodes (Fig. 9a). Rarely, the submental nodes (Level 1) are involved by metastatic thyroid cancer as well.

INDICATIONS FOR THYROIDECTOMY

Thyroidectomy is usually performed for the following ­reasons:
1. As therapy for some individuals with thyrotoxicosis, both those with Graves’ disease and others with hot nodules
2. To establish a definitive diagnosis of a mass within the thyroid gland, especially when cytologic analysis after fine-needle aspiration (FNA) is either nondiagnostic, equivocal, or indeterminate
3. To treat benign and malignant thyroid tumors
4. To alleviate pressure symptoms or respiratory difficulties associated with a benign or malignant process
5. To remove an unsightly goiter (Figs. 9b and 9c)
6. To remove large substernal goiters, especially when they cause respiratory difficulties

SOLITARY THYROID NODULES

Solitary thyroid nodules are found in 4% to 9% of patients by clinical examination, and in 22% or more of patients by ultrasound in the United States; most are benign (13). Therefore, rather than operating on every patient with a thyroid nodule, the physician or surgeon should select patients for surgery who are at high risk for thyroid cancer. Furthermore, each surgeon must know the complications of thyroidectomy and be able to either perform a proper operation for thyroid cancer in a safe and effective manner or refer the patient to a center where it can be done.

LOW-DOSE EXTERNAL IRRADIATION OF THE HEAD AND NECK

A history of low-dose external irradiation of the head or neck (less than 1500 to 2000 rads) is probably the most important historical fact that can be obtained in a patient with a thyroid nodule because it indicates that cancer of the thyroid, usually papillary cancer, is more likely (in up to 35% of cases), even if the gland is multinodular (14,15). Low-dose irradiation and its implications are discussed elsewhere (15a). Fortunately, treatments of low-dose radiation for benign conditions--thymic enlargement, tonsils, and acne-- have long been discontinued. However, patients who had this therapy in infancy or childhood are still seen and are still at a greater risk of cancer (15b).

HIGH-DOSE EXTERNAL IRRADIATION THERAPY

High-dose external irradiation therapy (more than 2000 rads), does not confer safety from thyroid carcinoma, as was previously thought (16). Rather, an increased prevalence of thyroid carcinoma, usually papillary cancer, has been found, ­particularly in patients with Hodgkin’s disease and other lymphomas who received upper mantle irradiation that included the thyroid gland (15b). Usually, a dose of approximately 4000 to 5000 rads was given. Both benign and malignant thyroid nodules have been recognized, now that these persons survive for longer periods (17). If a thyroid mass appears, it should be treated aggressively. These patients should also be observed carefully for the development of hypothyroidism.

RISK OF IONIZING RADIATION

Children exposed to ionizing radiation in the area of the Chernobyl nuclear accident have been shown to have at least a 30-fold increase in papillary thyroid cancer (18). This cancer may also be more aggressive than the usual papillary carcinoma and demonstrated more local invasion and lymph node metastases. It is thought to be the result of exposure to iodine isotopes that were inhaled or that entered the food chain. The mechanism of radiation-induced thyroid cancer is thought to be caused primarily by chromosomal rearrangements such as RET/PTC (19) and less commonly to BRAF mutations (19a, 19b).

DIAGNOSIS OF THYROID NODULES

Ultrasound examinations are used very commonly as screening procedures for thyroid nodules or as exams after a thyroid nodule has been palpated to look for multicentricity, invasion, etc. When suspicious thyroid nodules are detected, the sonographer should examine the central and lateral neck areas for suspicious lymph nodes which suggest metastatic disease.

Diagnostic modalities such as nuclear scans had been used widely in the past, but currently they have been superseded by a fine needle aspiration (FNA) of the mass with cytologic analysis (Fig. 10). In the hands of a good thyroid ­cytologist, more than 90% of nodules can be categorized histologically. Approximately 60% to 70% are found to be compatible with a colloid (benign) nodule. Fifteen percent to 30% demonstrate sheets of follicular cells with little or no colloid (an indeterminate lesion). An indeterminate lesion can be classified further as a follicular lesion of undetermined significance (FLUS) or as a possible follicular neoplasm (Table 1). Five percent to 10% of FNA’s are malignant, and less than 10% are nondiagnostic. In order to improve the diagnostic ability of FNA, researchers are adding biomarkers to the cytologic analyses (20, 21). A system for reporting thyroid cytopathology with the potential risk of malignancy, called the Bethesda system, is shown in Table 1 (20a).

TABLE 1. Implied Risk of Malignancy and Recommended Clinical Management

TABLE 1. Implied Risk of Malignancy and Recommended Clinical Management
Diagnostic category	Risk of malignancy (%)	Usual management
Nondiagnostic or unsatisfactory	1-4	Repeat FNA with ultrasound guidance
Benign	0-3	Clinical follow-up
Atypia of undetermined significance or follicular lesion of undetermined significance	˜5-15	Repeat FNA (or operate)
Follicular neoplasm or suspicious for a follicular neoplasm	15-30	Surgical lobectomy
Suspicious for malignancy	60-75	Near-total thyroidectomy or surgical lobectomy
Malignant	97-99	Near-total or total thyroidectomy

Adapted from: Cibas ES and Ali SZ: The Bethesda System for Reporting Thyroid Cytology. Thyroid 19:1159-1165, 2009 (20a).

As shown in Table 1, all patients who have malignant cytologic results should be operated upon. False positive results are rare. Patients with a “suspicious of malignancy” cytologic diagnosis should also be operated upon and are also likely to have a malignant lesion. Patients with the cytologic diagnosis of a follicular neoplasm or suspicion of a follicular neoplasm should also be operated upon, for up to 30% of these tumors prove to be carcinoma. When atypia of undetermined significance or a follicular lesion of undetermined significance (FLUS) is reported, some clinicians recommend a repeat FNA several months later (Table 1). However, others recommend operation, since up to 15% of these FLUS lesions also prove at operation to be malignant. In studies of follicular lesions, experiments are being conducted to determine whether the use of molecular markers such as BRAF, RAS, RET/PTC, PAX8-PPAR, or Galectin 3 will aid in differentiating benign from malignant lesions (21a). In another recent study of 265 indeterminate nodules, classified by FNA and then operated upon, 85 (32%) proved to be carcinomas. Using a diagnostic test that measures the expression of 167 genes, investigators were able to identify 78 of the 85 carcinomas as suspicious and to recognize most of the other lesions as benign (21b). Thus, in the future, perhaps these or similar tests will become routine and will reduce the number of operations currently performed for these indeterminate lesions which are ultimately found to be benign. An excellent review of the value of molecular diagnosis of FNA specimens for indeterminate nodules was written by Keutgen and associates (21c).

When the diagnosis of colloid nodule is made cytologically, the patient should be observed and not operated on unless tracheal compression or a substernal goiter is present, or unless the patient desires the benign mass to be removed. Finally, if an inadequate specimen is obtained, FNA with cytologic examination should be repeated. Usually one waits several months between needle biopsies.

Especially with small, nonpalpable masses, and probably in all cases, it is wise to perform FNA under sonographic guidance to be certain that the needle is in the correct location. Furthermore, when performing an FNA on a suspicious lymph node in the neck, one should measure thyroglobulin in the specimen as well as perform a cytologic analysis. The presence of thyroglobulin is diagnostic of metastatic thyroid cancer even if the cytology is non-diagnostic. FNA with cytologic assessment is the most powerful tool in our armamentarium for the diagnosis of a thyroid nodule.

In summary, the algorithm for the diagnosis of a thyroid nodule with isotope scintigraphy and ultrasonography as initial steps has been replaced in most hospitals by emphasizing the importance of early cytologic examination using fine needle aspirate (FNA) (Fig. 10). Far fewer isotope scans are being done because carcinomas represent only 5% to 10% of all cold nodules. This test is usually reserved for diagnosis of a “hot” nodule.

 

 

PREPARATION FOR SURGERY

Most patients undergoing a thyroid operation are euthyroid and require no specific preoperative preparation related to their thyroid gland. Determination of serum calcium and parathyroid hormone (PTH) levels is often helpful, and endoscopic or indirect laryngoscopy should definitely be performed in those who are hoarse and in others who have had a prior thyroid, parathyroid, or cervical disc operation in order to detect the possibility of a recurrent laryngeal nerve injury. Evaluation of vocal cord movement by a transcutaneous ultrasound technique has recently been described (21d). An increasing number of surgeons propose that all patients should have their vocal cords examined prior to undergoing thyroidectomy in order to rule out a vocal cord paralysis that is not clinically apparent. Certainly, if a surgeon is planning to report rates of recurrent laryngeal nerve injury postoperatively, both preoperative and postoperative vocal cord examination is important.

HYPOTHYROIDISM

Modest hypothyroidism is of little concern when treating a surgical patient; however, severe hypothyroidism can be a significant risk factor. Severe hypothyroidism can be diagnosed clinically by myxedema, as well as by slowness of affect, speech, and reflexes (22). Circulating thyroxine and triiodothyronine values are low. The serum thyroid-stimulating hormone (TSH) level is high in all cases of hypothyroidism that are not caused by pituitary insufficiency, and it is the best test of thyroid function. In the presence of severe hypothyroidism, both the morbidity and the mortality of surgery are increased as a result of the effects of both the anesthesia and the operation. Such patients have a higher incidence of ­perioperative hypotension, cardiovascular problems, gastrointestinal hypomotility, prolonged anesthetic recovery, and neuropsychiatric disturbances. They metabolize drugs slowly and are very sensitive to all medications. Therefore, when severe myxedema is present, it is preferable to defer elective ­surgery until a euthyroid state is achieved.

If urgent surgery is necessary, it should not be postponed simply for repletion of thyroid hormone. Endocrine consultation is imperative, and an excellent anesthesiologist is mandatory for success. In most cases, intravenous thyroxine can be started preoperatively and continued thereafter. In general, small doses of thyroxine are initially given to patients who are severely hypothyroid, and then the dose is gradually increased.

HYPERTHYROIDISM

In the United States, most patients with thyrotoxicosis have Graves’ disease. Furthermore, in the U.S., about 90% of all patients with Graves’ disease are treated with radioiodine therapy. Young patients, those with very large goiters, some pregnant women, and those with thyroid nodules or severe ­ophthalmopathy are commonly operated upon. Radioiodine therapy can make the ophthalmopathy worse in some cases.

Persons with Graves’ disease or other thyrotoxic states should be treated preoperatively to restore a euthyroid state and to prevent thyroid storm, a severe accentuation of the symptoms and signs of hyperthyroidism that can occur during or after surgery. Thyroid storm results in severe tachycardia or cardiac arrhythmias, fever, disorientation, coma, and even death. In the early days of thyroid surgery, operations on the toxic gland were among the most dangerous surgical procedures because of the common occurrence of severe bleeding, as well as all the symptoms and signs of thyroid storm. Now, with proper preoperative preparation, operations on the thyroid gland in Graves’ disease can be performed with about the same degree of safety as operations for other thyroid conditions (23).

In mild cases of Graves’ disease with thyrotoxicosis, iodine therapy alone has been used for preoperative preparation, although we do not recommend this approach (22). Lugol’s solution or a saturated solution of potassium iodide is given for 8 to 10 days. Although only several drops per day are needed to block the release of thyroxine from the toxic thyroid gland, it is our practice to administer two drops two or three times daily. This medication is taken in milk or orange juice to make it more palatable. Iodine therapy suppresses thyroid hormone release only in Graves’ disease and should not be given to patients with toxic nodular goiter.

Most of our patients with Graves’ disease are treated initially with the antithyroid drugs propylthiouracil (PTU) or methimazole (Tapazole) until they approach a euthyroid state. Then iodine is added to the regimen for 8 to 10 days before surgery. The iodine decreases the vascularity and increases the firmness of the gland. Sometimes thyroxine is added to this regimen to prevent hypothyroidism and to decrease the size of the gland. Beta-adrenergic blockers such as propranolol (Inderal) have increased the safety of thyroidectomy for patients with Graves’ disease (23). We use them commonly with antithyroid drugs to block adrenergic receptors and ameliorate the major signs of Graves’ disease by decreasing the patient’s pulse rate and eliminating the tremor. Some surgeons recommend preoperative use of propranolol alone or with iodine (24). These regimens, they believe, shorten the preparation time of patients with Graves’ disease for surgery and make the operation easier because the thyroid gland is smaller and less friable than it would otherwise be. We do not favor these regimens for routine preparation because they do not appear to offer the same degree of safety as do preoperative programs that restore a euthyroid state before surgery. Instances of fever and tachycardia have been reported in persons with Graves’ disease who were taking only propranolol. We have used propranolol therapy alone or with iodine without difficulty in some patients who are allergic to antithyroid medications. In such patients it is essential to continue the propranolol for several weeks postoperatively. Remember that they are still in a thyrotoxic state immediately after surgery, although the peripheral manifestations of their disease have been blocked.

The major advantages and disadvantages of radioiodine vs. thyroidectomy as definitive treatment of Graves’ disease are listed in Table 2. In our patients we have never had a death from thyroidectomy for Graves’ disease in over 45 years. Surgical resection involves subtotal, near total thyroidectomy (Fig. 11), or lobectomy with contralateral subtotal or near total lobectomy (Dunhill procedure). Previously we left 2 to 2.5 grams of thyroid in the neck. However, this resulted in a recurrence rate of approximately 12% at about the 10 year followup (25). Hence, we leave very small thyroid remnants and treat the patients with thyroxine replacement. Especially in children and adolescents, one should consider a total thyroidectomy or leaving a very small amount of tissue because the incidence of recurrence of thyrotoxicosis appears to be greater in this young group. Finally, when operating for severe ophthalmopathy, we try to perform near-total or total thyroidectomy, for improvement in the eyes may occur after this procedure. When operating on the thyroid, and especially in young patients with a benign condition, the surgeon should be very careful to avoid permanent hypoparathyroidism and nerve injury. These complications will be discussed later in this chapter.

The major benefits of thyroidectomy appear to be the removal of nodules if they are present, the speed with which normalization of thyroid function is achieved, possible improvement in the eyes, and possibly a lower rate of hypothyroidism than is seen after radioiodine therapy.

(Basic Surgery, 4th ed. St. Louis, Quality Medical Publishing, 1993, pp 162–195)

TABLE 2. Ablative Treatment of Graves’ Disease with Thyrotoxicosis

Method	Dose or extent of surgery	Onset of responses	Complications	Remarks
Surgery	Subtotal excision of gland (leaving about 1-2 g remnant or less)	Immediate	Mortality: <1% Permanent hypothyroidism: 20-30% or greater Recurrent hyperthyroidism: <15% Vocal cord paralysis: ~1% Hypoparathyroidism: ~1%	Applicable in young patients and pregnant women
Radioiodine 5-10 mCi		Several weeks to months	Permanent hypoparathyroidism: 50%-70% or more, often with delayed onset; multiple treatments sometimes necessary; recurrence possible	Avoid in children or pregnant women

 

 

SURGICAL APPROACH TO THYROID NODULES

Colloid Nodule(s)

If a colloid nodule is diagnosed on FNA, there is no urgency to operate in most cases. Patients often are rebiopsied in the future to reduce the chance of error and are followed in 6 to 12 months with repeat ultrasound. Respiratory compromise, substernal goiter, rapid growth, and pain are reasons for operation. Some patients desire a thyroidectomy to get over the problem of further evaluation or wish to have surgery to rid themselves of an unsightly mass. Thyroid lobectomy, subtotal thyroidectomy, or near total or total thyroidectomy can be done for a goiter and each approach has advocates.A “toxic nodule” can occur and can be cured by enucleation since it is rarely a carcinoma and since normal thyroid function might follow this approach. Otherwise, a thyroid lobectomy is appropriate.

Follicular (indeterminate) Nodules

The surgical treatment of a nodule which is diagnosed on FNA as either a follicular lesion of undetermined significance (FLUS) or a follicular neoplasm is more controversial. The problem is that the pathologist rarely can tell which nodules are benign and which are malignant on frozen section. He/She cannot tell which are follicular adenomas and which are follicular carcinomas, for example, at the time of operation. This usually requires careful evaluation of many sections for capsular invasion or vascular invasion on permanent sections. Of course, at operation, lymph nodes can be biopsied, but in most small masses of this type they are negative.

These difficulties should be discussed with the patient preoperatively and usually he or she will guide the surgeon. There are two possible operative courses: a lobectomy or a near-total or total thyroidectomy. The American Thyroid Association guidelines recommend a thyroid lobectomy in such an instance and to await the final diagnosis (21a). This is the choice of most patients. However, if the lesion turns out to be a carcinoma on permanent pathologic analysis, a second operation is necessary, with a second anesthesia, etc., and may be more difficult because of adhesions. Furthermore, many patients are already on thyroid hormone or require thyroid hormone replacement therapy after only a thyroid lobectomy. Thus, some patients choose to have a near-total or total thyroidectomy at the first operation, especially if they have bilateral nodules. But the wise surgeon will have this discussion preoperatively and do what the patient wants, since there is no correct answer.

Irradiated Patients

Patients who received low-dose (less than 2000 rads) or high-dose external irradiation or who were exposed to excessive ionizing radiation are at increased risk of developing single or multiple nodules of the thyroid, both benign and malignant (15b). There is a greater chance of malignancy than in the non-irradiated gland. For single nodules, FNA analysis is performed and the decision as to whether or not to operate is determined by the result of the cytology. Multiple nodules in such a patient present more of a diagnostic problem since it is difficult to evaluate each nodule preoperatively and benign and malignant nodules often are found in the same gland. In such a patient, a decision to operate is likely. When an operation is performed in a patient with a radiation history and a suspicious nodule, we are more inclined to perform a near-total or total thyroidectomy rather than a lobectomy. This procedure removes all of the nodules and also removes all potentially damaged thyroid tissue.

SURGICAL APPROACH TO THYROID CANCER

PAPILLARY CARCINOMA

It is estimated that 62,450 new cases of thyroid cancer will be diagnosed in 2015, with 3 or 4 times the number of cases occurring in women when compared to men (26c). Thyroid cancer is the fastest increasing cancer in both men and women. Since 2004, incidence rates have been rising 6.6% per year in women and 5.5% per year in men. An estimated 1950 deaths from thyroid cancer are expected in 2015. The death rates of women and men have increased slightly from 2004 to present as well (25a).

Approximately 80% to 85% of all thyroid cancers are papillary cancer. The surgical treatment of papillary cancer is best divided into two groups based on the size, clinical characteristics, and aggressiveness of these lesions.

Treatment of Minimal or Micro-Papillary Carcinoma

The term minimal or micro papillary carcinoma refers to a small papillary cancer, less than 1 cm in diameter, that demonstrates no local invasiveness through the thyroid capsule, that is not associated with lymph node metastases, and that is often found in a young person as an occult lesion when thyroidectomy has been performed for another benign condition. In such instances, especially when the cancer is unicentric and smaller than 5 mm, lobectomy is sufficient and reoperations are unnecessary. Thyroid hormone is given to suppress serum TSH levels, and the patient is monitored at regular intervals (21a).

In recent studies from Japan (25b), between 1993 and 2011, 1235 patients with low risk papillary micro carcinomas were followed and not operated upon. Patients were operated upon if the lesions grew to 12 mm in size or if they developed lymph nodes suggestive of malignancy. One hundred ninety one of the 1235 patients underwent surgery after some period of observation. During this follow up period, none of the 1235 patients showed distant metastases or died of papillary cancer. Only a small number showed progression or developed clinical disease. This study demonstrates that many micro carcinomas (less than 10 mm in diameter) may be safely actively observed and not operated upon. They grow very slowly or not at all and were harmless. These studies add evidence for not performing FNA on most lesions of the thyroid that are less than 10 mm in diameter and suggest that a less aggressive treatment may be appropriate for these small papillary cancers.

Standard Treatment of Most Papillary Carcinomas

Most papillary carcinomas are neither minimal nor occult. These tumors are known to be microscopically multicentric in up to 80% of patients; they are also known occasionally to invade locally into the trachea or esophagus, to metastasize commonly to lymph nodes and later to the lungs and other tissues, and to recur clinically in the other thyroid lobe in 7% to 18% of patients if treated only by thyroid lobectomy (26a, 26b).

Most surgeons have treated papillary cancer that is not a micro-papillary lesion by near-total or total thyroidectomy (see Fig. 11), with appropriate central and lateral neck dissection when nodes are involved. The so-called cherry-picking operations, which remove only the enlarged lymph nodes, should not be performed. Rather, when lymph nodes with tumor are found in the lateral triangle, a modified radical neck dissection should be performed (Fig. 9a) (27). At the conclusion of a modified radical neck dissection, the lymph node–bearing tissue from the lateral neck is removed, whereas the carotid artery, jugular vein, phrenic nerve, sympathetic ganglia, brachial plexus, and spinal accessory nerve are spared and left in place. Sensory nerves--the posterior occipital, and greater auricular nerves--should be retained as well. On the left side, care should be exercised not to injure the thoracic duct. Prophylactic neck dissection of the lateral triangle should not be performed for papillary cancer; such dissections should be done only when enlarged nodes with tumor are found. For clarity and uniformity of reporting, the location of lymph nodes in the neck and upper mediastinum has been defined as shown in Fig. 8. Central lymph nodes (level VI) are frequently involved with metastases from ipsilateral thyroid cancers, as are levels III, IV, and V which are removed in most lateral neck dissections. Level II nodes may be involved as well and often require removal.

Should Prophylactic Central (Level 6) Lymph Node Dissections be Performed?

There is agreement that therapeutic central and lateral lymph node dissections should be performed at the time of total thyroidectomy when lymph nodes are suspicious or proved to harbor cancer by sonographic appearance or by FNA analyses preoperatively or when suspicious lymph nodes are found at operation. Prophylactic lateral lymph node dissections were common in the past, but have been abandoned for several decades or longer (12a). Delbridge and his group and others have proposed that unilateral or bilateral prophylactic central lymph node dissections (level 6 dissections) with parathyroid autotransplantation be performed in all cases of papillary thyroid cancer at the time of total thyroidectomy (12a, 27a). This, they state, might decrease mortality from thyroid cancer, would greatly decrease recurrence of cancer, and would further clarify who needs radioiodine therapy postoperatively. Some studies by very experienced surgeons demonstrate no increase in hypoparathyroidism or recurrent laryngeal nerve injuries after this procedure, while other equally competent surgeons have found an increase in permanent hypoparathyroidism (27b, 27c). We and others have not practiced routine bilateral central lymph node dissections prophylactically, but have generally reserved unilateral dissection for instances in which lymph nodes are clearly involved with tumor (27d). However, recently we have performed some prophylactic unilateral central lymph node dissections when a large carcinoma is present, as well as in some children.

Finally, surgeons with limited experience probably should not perform total or near-total thyroidectomy unless capable of doing so with a low incidence of recurrent laryngeal nerve injuries and permanent hypoparathyroidism, because these complications are serious. Otherwise, it may be advisable to refer such patients to a major medical center where such expertise is available.

Radioiodine Therapy

In the past, radioiodine therapy with 131I was commonly used in order to ablate any remaining normal thyroid remnant that was present in the thyroid bed after near-total or total thyroidectomy or to treat local or distant metastatic thyroid cancer (28, 28a). In order to prepare for RAI therapy, patients are placed on a low iodine diet for two to three weeks prior to treatment. Furthermore, in order to increase TSH levels to high values, either L-thyroxine is stopped for three weeks or injections of genetically engineered TSH (Thyrogen) are given for two days without stopping thyroxine. Then the radioiodine is given. More recently, there has been a trend to use radioiodine more sparingly in low risk patients with small tumors because this treatment has not been shown to definitively decrease mortality in such individuals. Radioiodine is commonly recommended postoperatively in patients with high risk papillary cancer and in all patients with metastatic disease, gross extrathyroidal extension, or when tumors are greater than 4 cm. It is recommended for selected patients with tumors 1 cm to 4 cm in diameter and others with lymph node metastases, but is optional in low risk patients with tumors less than 1 cm in diameter (21a).

Controversy remains in this area. Many reports indicate decreased recurrences after RAI is given to patients with tumors 1 cm or larger and without known metastases. Also, RAI ablation using low does (30 mCi) carries minimal risk, makes postoperative scans and the use of thyroglobulin (TG) determinations more effective and reliable, and simplifies follow-up. In higher doses, both short-term and long-term complications have been associated with radioactive iodine therapy. These are discussed elsewhere in these chapters.

If all or a substantial part of a lobe of normal thyroid remains after the first operation and radio-iodine therapy is to be given for treatment of metastases, this can be performed effectively after completion thyroidectomy has been performed. Usually, reoperative completion thyroidectomy is done and then the radioiodine is given.

Total Thyroidectomy versus thyroid Lobectomy? Controversies

Because randomized prospective studies have not been performed, controversy still exists over the proper treatment of papillary cancer in some patients. Most clinicians now accept that patients with this disease can be separated into different risk groups according to a set of prognostic factors. Using the AGES (29), AMES (30), or MACIS (31) criteria, which evaluate risk by age, distant metastases, extent of local involvement, and size (MACIS adds completeness of excision), approximately 80% of patients fall into a low-risk group. Treatment of this low-risk group is most controversial, perhaps because the cure rate is so good, certainly in the high 90% range. Should a lobectomy be done, or is bilateral thyroid resection more beneficial?

Low-risk Papillary Cancer

Hay and associates studied 1685 patients treated at the Mayo Clinic between 1940 and 1991; the mean follow-up period was 18 years (32). Of the total, 98% had complete tumor resection and 38% had initial nodal involvement. Twelve percent had unilateral lobectomy, whereas 88% had bilateral lobar resection; total thyroidectomy was done in 18%; while near-total thyroidectomy was performed in 60%. Cause-specific mortality at 30 years was 2%, and distant metastases occurred in 3%. These indices did not differ between the surgical groups; however, local recurrence and nodal metastases in the lobectomy group (14% and 19%, respectively) were significantly higher than the 2% and 6% rates seen after near-total or total thyroidectomy. This study is excellent. Although no differences in mortality were reported, a three-fold increase in tumor recurrence rates in the thyroid bed and lymph nodes was reported in the lobectomy group. In addition, this study recognizes patients’ anxiety about tumor recurrence, and their strong desire to face an operation only once and to be cured of their disease. If the operation can be done safely with low morbidity, this study supports the use of near-total or total thyroidectomy for patients with low-risk papillary cancer.

More recently, studies from Japan have shown excellent results (99% cause specific survival) for low-risk patients for papillary thyroid cancer treated by less than a total or near total thyroidectomy. This study and others like it are causing a reexamination of how low-risk papillary cancer is treated in the U.S.

High-risk Papillary Cancer

For high-risk patients, it is agreed that bilateral thyroid resection improves survival (29) and reduces recurrence rates (33) when compared with unilateral resection.

The Authors’ Series

In a retrospective study at the University of Chicago, total or near-total thyroidectomy has been practiced and most patients also received radioiodine ablation or treatment with radioiodine as indicated (34). In general, our studies (34, 35) and those of Mazzaferri and Jhiang (36) have demonstrated a decrease in mortality and in recurrence after near-total or total thyroidectomy followed by radioiodine ablation or therapy, when compared with lesser operations in papillary cancers 1 cm or greater.

Our series of patients have now been followed for a mean time of 27 years (36a). Predictors of death were increasing age, metastases, and advancing stages of disease. The mean time following diagnosis until recurrence of disease was 8.1 years and mean time of death was 9 years. However, 4% of recurrences and 17% of deaths were recognized only after a mean of 20 years, which emphasizes the importance of long-term followup of patients with papillary cancer.

FOLLICULAR CARCINOMA

True follicular carcinomas are far less common than papillary cancer. Remember that the “follicular variant” of papillary cancer should be classified and treated as a papillary carcinoma. Patients with follicular carcinoma are usually older than those with papillary cancer, and females predominate. Microscopically, the diagnosis of follicular cancer is made when vascular and/ or capsular invasion is present. Tumor multicentricity and lymph node metastases are far less common than in papillary carcinoma. Metastatic spread of tumor often occurs by hematogenous dissemination to the lungs, bones, and other peripheral tissues.

A follicular cancer that demonstrates only microinvasion of the capsule has a very good prognosis (37). In this situation, ipsilateral lobectomy is probably sufficient. However, for patients with follicular cancer that demonstrates gross capsular invasion or vascular invasion, the ideal operation is similar to that for papillary cancer, although the rationale for its performance differs. Near-total or total thyroidectomy should be performed not because of multicentricity but rather to facilitate later treatment of metastatic disease with radioiodine (36). Remnants of normal thyroid in the neck are ablated by radioiodine, and if peripheral metastases are detected (Fig. 12), they should be treated with high-dose radioiodine therapy. Although lymph node metastases in the lateral region of the neck are not ­commonly found, a modified radical neck dissection should be performed if metastatic nodes are identified.

Finally, regardless of the operation, patients with papillary or follicular cancer are usually treated for life with levothyroxine therapy in sufficient doses to suppress TSH to the appropriate level (36). Care should be taken to not cause cardiac or other problems from thyrotoxicosis, however. Recent studies have questioned whether TSH suppression is necessary in low-risk patients.

HÜRTHLE CELL TUMORS AND CANCER

Hürthle cell (oncocytic) tumors are thought to be variants of follicular neoplasms, but others regard them as a totally separate disease entity (38a). They are more difficult to treat than the usual follicular neoplasms, however, for several reasons (38): 1) the incidence of carcinoma varies from 5.3% to 62% in different clinical series; 2) benign-appearing tumors later metastasize in up to 2.5% of patients; and 3) Hürthle cell cancers are far less likely to concentrate radioiodine than are the usual follicular carcinomas, which makes treatment of metastatic disease particularly difficult.

The difficulty in diagnosing Hurtle cell cancers and differentiating them from benign lesions is shown in the following study. Of 54 patients with Hürthle cell tumors whom we treated, four had grossly malignant lesions (38). But during a mean follow-up period of 8.4 years, three additional Hürthle cell tumors were recognized as malignant after metastases were discovered. Thus, 7 of 54 (13%) of our patients who had a Hürthle cell tumor had Hürthle cell carcinoma. One of the seven patients with Hürthle cell cancer died of widespread metastases after 35 years, and the other six were currently free of disease.

In a more recent study, the size of the tumor was the major factor in determining whether or not a Hurthle cell neoplasm was malignant (38b). Overall, 20% of the tumors were malignant, but those less than 2 cm were always benign. Tumors 4 cm or larger had a greater than 50% chance of malignancy, and all tumors greater than 6 cm were universally cancers. Finally, overexpression of Cyclin D1 and D3 may help predict malignant behavior in fine needle aspirates suspicious for malignant behavior (38c).

We believe that treatment of these lesions should be individualized (38, 39). Total thyroid ablation is appropriate for frankly malignant Hürthle cell cancers, for all Hürthle cell tumors in patients who received low-dose childhood irradiation, for patients with associated papillary or follicular carcinomas, for all large tumors, certainly for those greater than 2 cm in diameter, and for patients whose tumors exhibit partial capsular invasion. On the other hand, single, well-encapsulated, benign-appearing Hürthle cell tumors that are small may be treated by lobectomy and careful follow-up because the chance that they will later exhibit malignant behavior is low (2.5% in our series and 1.5% among patients described in the literature) (38). Nuclear DNA analysis may aid the surgeon in recognizing tumors that are potentially aggressive, because such tumors usually demonstrate aneuploidy (40). Furthermore, increased genetic abnormalities have been shown in Hürthle cell carcinomas when compared with Hürthle cell adenomas (41).

In a review of follicular cancers at the University of Chicago, the overall mortality rate was 16%, twice that of papillary carcinomas (39). However, in non–Hürthle cell follicular cancers the mortality was 12%, whereas in Hürthle cell cancers it was 24%. This demonstrates the difficulty in treating metastatic disease which cannot be resected in the Hurthle group, because radioiodine therapy is almost always ineffective. These data are similar to those found in a recent large database study in which the overall death rate was approximately 18% for patients with Hurthle cell cancer and 11% for other well-differentiated thyroid cancers (41a).

ANAPLASTIC CARCINOMA

Anaplastic thyroid carcinoma remains one of the most aggressive of all cancers in humans. It makes up 1.3% to 9.8% of all thyroid cancers globally (1.7% of cancers in the U.S.) (40a, 40b, 40c). This tumor often arises from a differentiated thyroid cancer or from a prior goiter. It grows very rapidly, and systemic symptoms are common. Survival for most patients is measured in months. Median survival is 5 to 6 months, and one year survival is approximately 20%. The previously so-called small cell type is now known to be a lymphoma and is most often treated by a combination of external radiation and chemotherapy. The large cell type may be manifested as a solitary thyroid nodule early in its clinical course. If it is operated on at that time, near-total or total thyroidectomy should be performed, with appropriate central and lateral neck dissection. However, anaplastic cancer is almost always advanced when the patient is first evaluated. Widespread metastases to lymph nodes or to the lungs are common. Be sure to check vocal cord function preoperatively, for unilateral vocal cord paralysis is frequent.

With advanced disease, surgical cure is unlikely no matter how aggressively it is pursued. In particularly advanced cases, diagnosis by needle biopsy or by small open biopsy may be all that is appropriate. Sometimes the isthmus must be divided to relieve tracheal compression, or a tracheostomy might be beneficial. Most treatment, however, has been by external radiation therapy, chemotherapy, or both. Hyperfractionated external radiation therapy that uses several treatments per day has some enthusiasts, but complications may be high (42). Radioiodine treatment is almost always ineffective because tumor uptake is absent. Although some success has been observed with doxorubicin, prolonged remissions are rarely achieved, and multidrug regimens, especially with Paclitaxel or Cisplatin, and combinations of chemotherapy with radiation therapy are being tried (43). Although remissions do occur, cures have rarely been achieved in advanced cases. New experimental drugs (44) including monoclonal antibodies, kinase inhibitors, antiangiogenic drugs, and others are being tried because results of conventional therapy have been so dismal (40c). A phase 2 trial of efatutazone (alpha PPAR-gamma agonist) with paclitaxel versus efatutazone alone for patients with advanced anaplastic cancer is being conducted by the NIH (40d). American Thyroid Association guidelines for management of patients with anaplastic cancer including ethical considerations have recently been published (44a).

MEDULLARY THYROID CARCINOMA

Medullary thyroid carcinoma accounts for 5% to 8% of all thyroid cancers. It is a C-cell, calcitonin-producing tumor that contains amyloid or an amyloid-like substance. In addition to calcitonin, it may elaborate or secrete other peptides and amines such as carcinoembryonic antigen, serotonin, neurotensin, and a high-molecular-weight adrenocorticotropic hormone-like peptide. These substances may result in a carcinoid-like syndrome with diarrhea and Cushing’s syndrome, especially when widely metastatic tumor is present. Most medullary cancer of the thyroid is sporadic (about 70% to 80%), but it can also be transmitted in a familial pattern in 20% to 30% of cases. In the familial form, this tumor or its precursor, C-cell hyperplasia, occurs as a part of the multiple endocrine neoplasia type 2A (MEN2A) and type 2B (MEN2B) syndromes (45) (Table 3; Figs. 13 and 14); or, rarely, as part of the familial medullary thyroid cancer syndrome. MEN2A makes up approximately 95% of all hereditary cases and MEN2B approximately 5%. The MEN2 syndromes are transmitted as an autosomal-dominant trait, so 50% of the offspring would be expected to have this disease. Mutations of the RET oncogene on chromosome 10 (10 of 11.2) have been found to be the cause of the MEN2 syndromes (46). These defects are germ-line mutations and can therefore be found in blood samples. All patients with medullary thyroid carcinoma should probably be screened for hyperparathyroidism and pheochromocytoma (47). However, the risk of these two disease states accompanying the medullary cancer in MEN 2A is greatest in patients with a 630 or 634 RET mutation (50c). If a pheochromocytoma (or its precursor, adrenal medullary hyperplasia) is present, this should be operated on first because it has the greatest immediate risk to the patient. Family members, especially children, of a patient with medullary cancer of the thyroid should also be screened for medullary cancer of the thyroid if a patient has MEN2 with a RET oncogene mutation, but also if the tumor is bilateral or if C-cell hyperplasia is present. Genetic testing for RET mutations has largely replaced screening by calcitonin in family members. However, calcitonin and CEA measurements are still useful for evaluating patients with a thyroid mass when FNA analysis raises the possibility of medullary thyroid cancer.

The degree of clinical aggressiveness of a medullary cancer corresponds with the mutation of RET which is found (50d). A list of all known mutations with corresponding phenotype and risk profile can be found in the American Thyroid Association guidelines (50d). The most aggressive tumors called “highest risk, HST” are found in patients with the MEN2B and those with a RET codon M918T mutation. The “high risk, H” includes patients with RET codon C634 mutations, and the “moderate risk, MOD” category includes patients with RET codon mutations other than M918T and C634. In all of the hereditary cases (MEN2A, MEN2B, and familial MTC) germline mutations are found. However, about 50% of sporadic MTC have somatic RET mutations (only in the tumor). A RET codon M918T mutation found in a sporadic medullary cancer, for example, portends an aggressive clinical course and a poor prognosis, as well.

TABLE 3. Diseases Included in the MEN2 Syndromes
MEN2A	MEN2B
Medullary carcinoma	Medullary carcinoma
Pheochromocytoma	Pheochromocytoma Hyperparathyroidism-unlikely Ganglioneuroma phenotype
Hyperparathyroidism
MEN = Multiple endocrine neoplasia

Medullary cancer spreads to the lymph nodes of the neck and mediastinum, and disseminates to the lungs, bone, liver, and elsewhere. The tumor is relatively radioresistant, does not take up radioiodine, and is not responsive to thyroid hormone suppression. Hence, an aggressive surgical approach is mandatory. The operation of choice for medullary cancer is total thyroidectomy coupled with aggressive resections of central and lateral lymph nodes, as well as mediastinal lymph nodes (46). Careful and extensive modified radical neck dissections are required. Reoperations for metastatic tumor were rarely considered to be rewarding until the work of Tisell and Jansson (48). Their work, and that of others (49), demonstrated that 25% to 35% of patients with ­elevated circulating calcitonin levels could be rendered eucalcitoninemic after extensive, meticulous, reoperative neck dissection under magnification to remove all the tiny lymph nodes. In other patients, computed tomography (CT) and magnetic resonance imaging (MRI) have localized some sites of tumor recurrence, whereas octreotide and meta-iodobenzylguanidine scanning have sometimes been helpful. Positron emission tomography combined with computerized tomography (PET-CT) have been successful in many patients (49a). Laparoscopic evaluation of the liver is helpful before a reoperation since small metastatic lesions on its surface can sometimes be identified.

Cure is best in young children who are found by genetic screening to have a mutated RET oncogene associated with the MEN2A syndrome. One hopes to operate on them when C-cell hyperplasia is present and before medullary cancer has started (46a). Patients with MEN2A syndrome have a better prognosis than do those with sporadic tumor (45). Patients with MEN2B syndrome have the most aggressive tumors and rarely survive to middle age. Thus, in recent years, children 5 years of age or younger who are found by genetic screening to have MEN2A with a 634 RET mutation have received prophylactic total thyroidectomy to prevent the development of medullary cancer. In children with MEN2B, screening for RET oncogene mutation should be done soon after birth because this cancer develops at a younger age than does MEN2A. With a mutated RET oncogene in MEN2B, total thyroidectomy should be done as early as possible within the first year of life in an experienced tertiary care setting. In MEN2B patients or infants and children older than one year, a prophylactic Level VI neck dissection should be considered as well since metastatic disease has been sometimes found in these very young children (50c). In all of these infants and children, preservation of parathyroid function and recurrent nerve integrity is of the highest priority (49). With these ­prophylactic operations, cures are possible. Careful genetic counseling is highly recommended.

Long-term studies of medullary cancer from the Mayo Clinic group have shown that in patients without initial distant metastatic involvement and with complete resection of their medullary cancer, the 20-year survival rate, free of distant metastatic lesions, was 81% (50). Overall 10- and 20-year survival rates were 63% and 44%, respectively. Thus, early diagnosis and complete initial resection of tumor are very important. A number of new therapies using tyrosine kinase inhibition are now being evaluated for metastatic disease (50a). Screening and treatment for pheochromocytoma and hyperparathyroidism in children with MEN syndromes are discussed elsewhere.

An excellent review of medullary cancer has been written by Pacini and colleagues (50b). In 2009, a very comprehensive set of guidelines for diagnosis, screening, surgical and medical treatment and followup of MEN2A and 2B, their pheochromocytomas and hyperparathyroidism as well as for sporadic medullary cancer has been published (50c).   A recent paper, “Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma” is excellent and is highly recommended (50d).

OPERATIVE TECHNIQUE FOR THYROIDECTOMY

While some groups have utilized local anesthesia with superficial cervical block, essentially all of our patients receive general anesthesia. The following description of thyroidectomy is used by many surgeons. The patient is placed in a supine position with the neck extended. A low collar ­incision is made and carried down through the subcutaneous tissue and platysma muscle (Fig. 15A). Currently, small incisions are the rule unless a large goiter is present. Superior and inferior subplatysmal flaps are developed, and the strap muscles are divided vertically in the midline and retracted laterally (Fig. 15B).

The thyroid isthmus is often divided early in the course of the operation. The thyroid lobe is rotated medially. The middle thyroid vein is ligated (Fig. 15C). The superior pole of the thyroid is dissected free, and care is taken to identify and preserve the external branch of the superior laryngeal nerve (see Fig. 6). The superior pole vessels are ligated adjacent to the thyroid lobe, rather than cephalad to it, to prevent damage to this nerve (Fig. 15D). This nerve can be visualized in over 90% of patients if it is carefully dissected (51). The inferior thyroid artery and recurrent laryngeal nerve are identified (Fig. 15E). To preserve blood supply to the parathyroid glands, the inferior thyroid artery should not be ligated laterally as a single trunk; rather, its branches should be ligated individually on the capsule of the lobe after they have supplied the parathyroid glands (Fig. 15F). The parathyroid glands are identified, and an attempt is made to leave each with an adequate blood supply while moving the gland off the thyroid lobe. Any parathyroid gland that appears to be devascularized can be placed in saline and later minced and implanted into the sternocleidomastoid muscle after a frozen section biopsy confirms that it is, in fact, a parathyroid gland. Care is taken to identify the recurrent laryngeal nerve and to gently follow along its course if a total lobectomy is to be done (Fig. 15G). The nerve is gently unroofed from surrounding tissue, with care taken to avoid trauma to it. The nerve is in greatest danger near the junction of the trachea with the larynx, where it is adjacent to the thyroid gland. Once the nerve and parathyroid glands have been identified and preserved, the thyroid lobe can be removed from its tracheal attachments by dividing the ligament of Berry (Fig. 15G). The contralateral thyroid lobe is removed in a similar manner when total thyroidectomy is performed. A near-total thyroidectomy means that a very small amount of thyroid tissue is left on the contralateral side to protect the parathyroid glands and recurrent nerve. Careful hemostasis and visualization of all important anatomic structures are mandatory for success. Some surgeons utilize the harmonic scalpel or electrothermal bipolar vessel sealing system and believe that they decrease the time of operation. However, one must be careful not to cause thermal damage (51a).

 

When closing, some surgeons do not tightly approximate the strap muscles in the midline; this allows drainage of blood superficially and thus prevents a hematoma in the closed deep space. Furthermore, one can obtain better cosmesis by not approximating the platysmal muscle. Rather, the dermis is approximated by interrupted 4-0 sutures, and the epithelial edges are approximated with a running subcuticular 5-0 absorbable suture. Sterile paper tapes (Steri-strips) are then applied and left in place for approximately one to two weeks. When it is needed, a small suction catheter is inserted through a small stab wound; it is generally removed within 12 hours.

SUBTOTAL THYROIDECTOMY

Bilateral subtotal or near total lobectomy is the operation which is often used for Graves’ disease. An alternative operation, which is equally good, is lobectomy on one side and subtotal or near total lobectomy on the other side (Dunhill procedure). Once more, the parathyroid glands and recurrent nerves should be identified and preserved. Great care should be taken to not damage the recurrent laryngeal nerve when cutting across or suturing the thyroid lobe. At the end of the operation, 1 or 2 grams or less of thyroid tissue is usually left in place. The aim is to achieve an euthyroid state without a high recurrence of hyperthyroidism. When the operation is performed for severe ophthalmopathy, however, near-total or total thyroidectomy is performed.

After thyroidectomy, even if a modified neck dissection is done for carcinoma, many patients can be safely discharged on the first postoperative day. Others are kept longer if the need arises. Some surgeons do not think that it is safe to discharge a patient on the day of surgery because of the risks of bleeding or hypocalcemia; however, same-day discharge is being practiced at some centers, usually after lobectomy (52).

ALTERNATIVE TECHNIQUE OF THYROIDECTOMY

An alternative technique of thyroidectomy is practiced by some excellent surgeons and is used by the authors in some operations (6, 53). In this technique, the dissection is begun on the thyroid lobe and the parathyroids are moved laterally, as described previously. However, the recurrent laryngeal nerve is not dissected along its length, but rather small bites of tissue are carefully divided along the thyroid capsule until the nerve is encountered near the ligament of Berry. Proponents of this technique believe that visualization of the recurrent laryngeal nerve by its early dissection may lead to greater nerve damage; however, most surgeons feel that seeing the nerve and knowing its pathway is safer and facilitates the dissection in many instances.

MINIMALLY INVASIVE OPTIONS FOR THYROIDECTOMY

Over recent years, the development of ultrasonic shears and other electrothermal bipolar vessel sealing devices for hemostasis and small size endoscopes has allowed surgeons to perform thyroidectomies through much smaller incisions than using traditional techniques. Two different approaches have been taken to minimally invasive thyroidectomies. One technique, largely popularized in areas of the Far East such as Japan, China, and Korea, involves making incisions away from the neck in hidden areas such as in the axillae, chest, or the areola of the breast. The surgeon then creates a tunnel up to the neck where the thyroidectomy is performed with endoscopic instruments utilizing the endoscope for visualization. Approaches such as this are generally performed with low pressure insufflation and can completely avoid any incisions on the neck itself. Thus, the major advantage is a thyroidectomy without a neck scar (54-57). Most reports suggest significantly longer operative times, especially during a learning period. Perhaps most concerning to many American surgeons with these approaches is that if bleeding problems are encountered in the course of the thyroid dissection, a separate neck incision may need to be made to solve the problem. Additionally, recent reports have suggested the possibility that recurrent thyroid cancer can develop in the subcutaneous tunnel after the performance of an endoscopic thyroidectomy (57a). Such complications will need to be carefully evaluated before wide acceptance of this technique can be recommended in cases of malignancy.

An alternative technique, developed by Dr. Paolo Miccoli, more widely utilized in Europe and to a less extent in the United States, utilizes a smaller incision than usual that is placed in the conventional location in the neck (58, 59). In general, a 1.5 to 2.0 cm incision is made in a conventional location in the neck and after the strap muscles are retracted from the thyroid gland, a 5 mm 30 degree endoscope is introduced into the incision. The scope is utilized to visualize the tissue along the lateral aspect of the thyroid gland and especially for the superior pole vessels. Usually after the superior and lateral aspects of the thyroid gland have been dissected free, the parathyroid glands and recurrent nerve are visualized and then the thyroid lobe is delivered through the neck incision. The remainder of the operation is performed in the conventional manner through the small cervical incision.

Several authors in the United States have reported good results in small series with this video-assisted approach (60, 61, 61a). A significant benefit of this approach is that the incision is in the usual location so that if any bleeding results in difficulty with visualization during the procedure, the incision can be enlarged and a conventional thyroidectomy can readily be completed. Most authors have found this approach to be similar to conventional thyroidectomy in operative time, although the small neck incision does limit the size of the thyroid gland that could be resected utilizing this technique (62-64).

Recently Miccoli and his group have shown that minimally invasive video-assisted thyroidectomy and conventional thyroidectomy have the same rate of hypoparathyroidism and recurrent laryngeal nerve injury following operations for papillary thyroid cancer (64a). Furthermore, no differences in outcome were noted at five years or in exposure to radioiodine therapy, suggesting the same degree of completeness of resection by both techniques. They believe that minimally invasive video-assisted thyroidectomy is a valid option to treat low and intermediate risk papillary thyroid cancer. In the United States, minimally invasive video-assisted thyroidectomy is offered in few specialized centers for selected patients with small thyroid nodules (usually less than 3 cm) and without evidence of thyroiditis. Except in the hands of surgeons very experienced in the technique, it should not be utilized for the treatment of most thyroid cancers. An excellent review of these minimally invasive techniques was written by Grogan and Duh (63a).

Robotic Transaxillary Thyroid Surgery

Using the da Vinci robot, several groups, largely from Korea, have developed a transaxillary approach to thyroidectomy (64b, 64c, 64d). An incision between 5 and 10 cm is made unilaterally or in both axillae and the dissection progresses from the axilla to the neck with performance of a lobectomy or total thyroidectomy. In malignant cases, a neck dissection can be performed as well (64c). In their hands, robotic surgery can be performed with low complications, but the procedure takes longer than open surgery. There is a prolonged learning curve. Furthermore, use of the robot is expensive. While reimbursement in Korea was higher for robotic surgery than for conventional thyroidectomy, in the U.S. reimbursement was the same for both procedures and the costs were higher for robotic thyroidectomy. The obvious advantage is cosmetic, since no scar is placed in the neck.

Initially there was considerable enthusiasm for robotic thyroidectomy in the U.S. However, because of increased costs and probably increased complications in American patients, who are generally larger than Korean counterparts, Intuitive Surgical, the company that makes the robots, decided it could no longer support the use of its robots for thyroid surgery. In 2010 to 2011 in the National Cancer Database, 224 patients with thyroid cancer underwent robotic thyroidectomy and close to 58,000 patients underwent open surgery (64e). More recently, fewer robotic thyroidectomy operations are being done in the U.S. and this procedure is limited to only a few institutions. In Korea, on the other hand, this procedure remains very popular.

Transoral Thyroidectomy

Experiments have been conducted to determine whether or not it is possible and safe to perform a thyroidectomy through the floor of the mouth (64f). Once more, the objective is to eliminate a scar in the neck. A number of investigators have moved this procedure to clinical practice. Both endoscopic and robotic techniques have been described. A summary of the experience in 2014 and the complications associated with these techniques are reviewed by Clark et al (64g).

POSTOPERATIVE COMPLICATIONS

Many authors have reported large series of thyroidectomies with no deaths. In other reports, mortality does not differ greatly from that from anesthesia alone. However, each patient should be evaluated for comorbidities, for it has been shown that elderly patients are more likely to suffer postoperative complications and longer hospitalizations than their younger counterparts following thyroidectomy (64h). Five major complications are associated with thyroid surgery: 1) thyroid storm, 2) wound hemorrhage, 3) wound infection, 4) recurrent laryngeal nerve injury, and 5) hypoparathyroidism.

Thyroid Storm

Thyroid storm reflects an exacerbation of a thyrotoxic state; it is seen most often in Graves’ disease, but it occurs less commonly in patients with toxic adenoma or toxic multinodular goiter. Clinical manifestations and management of thyroid storm are discussed elsewhere in this text.

Wound Hemorrhage

Wound hemorrhage with hematoma is an uncommon complication reported in 0.3% to 1.0% of patients in most large series. However, it is a well-recognized and potentially lethal complication (52). A small hematoma deep to the strap muscles can compress the trachea and cause respiratory distress. A small suction drain placed in the wound is not usually adequate for decompression, especially if bleeding occurs from an arterial vessel. Swelling of the neck and bulging of the wound can be quickly followed by respiratory impairment.

Wound hemorrhage with hematoma is an emergency situation, especially if any respiratory compromise is present. Treatment consists of immediately opening the wound and evacuating the clot, even at the bedside. Pressure should be applied with a sterile sponge and the patient returned to the operating room. Later, the bleeding vessel can be ligated in a careful and more leisurely manner under optimal sterile conditions with good lighting in the operating room. The urgency of treating this condition as soon as it is recognized cannot be overemphasized, especially if respiratory compromise is present.

Injury to the Recurrent Laryngeal Nerve

Injuries to the recurrent laryngeal nerve occur in 1% to 2% of thyroid operations when performed by experienced neck surgeons, and at a higher prevalence when thyroidectomy is done by a less experienced surgeon. They occur more commonly when thyroidectomy is performed for malignant disease, especially if a total thyroidectomy is done. Sometimes the nerve is purposely sacrificed if it runs into an aggressive thyroid cancer. Nerve injuries can be unilateral or bilateral and temporary or permanent, and they can be deliberate or accidental. Loss of function can be caused by transection, ligation, clamping, traction, or handling of the nerve. Tumor invasion can also involve the nerve. Occasionally, vocal cord impairment occurs as a result of pressure from the balloon of an endotracheal tube on the recurrent nerve as it enters the larynx. In unilateral recurrent nerve injuries, the voice becomes husky because the vocal cords do not approximate one another. Shortness of breath and aspiration of liquids sometimes occur as well. Most nerve injuries are temporary and vocal cord function returns within several months; it certainly returns within 9 to 12 months if it is to return at all. If no function returns by that time, the voice can be improved by operative means. The choice is insertion of a piece of Silastic to move the paralyzed cord to the midline; this procedure is called a laryngoplasty. Early in the course of management of a patient with hoarseness or aspiration, the affected vocal cord can be injected with various substances to move it to the midline and to alleviate or improve these symptoms.

Bilateral recurrent laryngeal nerve damage is much more serious, because both vocal cords may assume a medial or paramedian position and cause airway obstruction and difficulty with respiratory toilet. Most often, tracheostomy is required. In the authors’ experience, permanent injuries to the recurrent laryngeal nerve are best avoided by identifying and carefully tracing the path of the recurrent nerve. Accidental transection occurs most often at the level of the upper two tracheal rings, where the nerve closely approximates the thyroid lobe in the area of Berry’s ligament. If recognized, many believe that the transected nerve should be reapproximated by microsurgical techniques, although this is controversial. A number of procedures to later reinnervate the laryngeal muscles have been performed with improvement of the voice in unilateral nerve injuries, but with only limited success when a bilateral nerve injury has occurred (65).

Injury to the external branch of the superior laryngeal nerve may occur when the upper pole vessels are divided (Fig. 6) if the nerve is not visualized (9). This injury results in impairment of function of the ipsilateral cricothyroid muscle, a fine tuner of the vocal cord. This injury causes an inability to forcefully project one’s voice or to sing high notes because of loss of function of the cricothyroid muscle. Often, this disability improves during the first few months after surgery.

Recurrent Laryngeal Nerve Monitoring

Many surgeons have sought to try to further diminish the low incidence of recurrent laryngeal nerve (RLN) injury by use of nerve monitoring devices during surgery. Although several devices have been utilized, all have in common some means of detecting vocal cord movement when the RLN or the ipsilateral vagus nerve is stimulated. Many small series have been reported in the literature assessing the potential benefits of monitoring to decrease the incidence of nerve injury (65a, 65b). Given the low incidence of RLN injury, it is not surprising that no study has shown a statistically significant decrease in RLN injury when using a nerve monitor. The largest series in the literature by Dralle reported on a multi-institutional German study of 29,998 nerves at risk in thyroidectomy (65c). Even with a study this large, no statistically significant decrease in rates of RLN injury could be shown with nerve monitoring. Despite this, the use of nerve monitoring has become more popular.

Among the problems of nerve monitoring technology are that the devices can malfunction, often because of endotracheal tube misplacement, so that the surgeon cannot depend on the device to always identify the nerve. Proponents of nerve monitoring suggest that the technology is helpful even if a statistically significant decrease in the rate of RLN cannot be shown. Goretzki and his group, for example, have published data that when operating upon bilateral thyroid disease, if nerve monitoring suggests a nerve injury on the first side, they have modified or restricted the resection of the contralateral thyroid lobe (65d). In this way, they have decreased or eliminated the incidence of bilateral RLN injuries. This is very important.

Many authors have suggested that RLN monitors may be most helpful in difficult reoperative cases when significant scar tissue is encountered, and have limited their use to such cases. This has not generally been shown to be the case. Most authors have advocated routine use (67a). Nerve monitoring technology in thyroid surgery should never take the place of meticulous dissection. Surgeons may choose to use the technology, but the data do not support the suggestion that nerve monitors allow thyroid surgery to be performed in a safer fashion than that by a good surgeon utilizing careful technique. An excellent international guide to the use of electrophysiologic RLN monitoring during thyroid and parathyroid surgery has been published (65f). Furthermore, a review of the medical, legal, and ethical aspects of RLN monitoring has been written by Angelos (65e).

A recent advance is continuous intraoperative recurrent laryngeal monitoring, done by continuously stimulating the ipsilateral vagus nerve. It has been shown that a decrease in signal amplitude and an increase in signal latency often occur before some recurrent laryngeal nerves are permanently damaged. In a recent experimental study, it was shown that modification of the surgical maneuver by the operator (such as release of traction) led to recovery of the EMG changes and aversion of the impending recurrent nerve injury (65G). This technique shows great promise for helping the surgeon intraoperatively, for it signals that the nerve is in danger and that the surgeon should stop doing whatever is causing the problem. A note of caution is appropriate, however. Terris and associates recently described two serious adverse events in nine patients who underwent continuous vagal nerve monitoring—hemodynamic instability and reversible vagal neuropraxia—both attributable to the monitoring apparatus (65h). They feel that this technique may cause harm and they have stopped its routine use. Clearly, further clarification and study must be done, but surgeons should use caution until it’s safety has been proved.

HYPOPARATHYROIDISM

Postoperative hypoparathyroidism can be temporary or permanent. The incidence of permanent hypoparathyroidism has been reported to be as high as 20% when total thyroidectomy and radical neck dissection are performed, and as low as 0.9% for subtotal thyroidectomy. Other excellent neck surgeons have reported a lower incidence of permanent hypoparathyroidism, even about one percent following total thyroidectomy (66). Postoperative hypoparathyroidism is rarely the result of inadvertent removal of all of the parathyroid glands but is more commonly caused by disruption of their delicate blood supply. Devascularization can be minimized during thyroid lobectomy by dissecting close to the thyroid capsule, by carefully ligating the branches of the inferior thyroid artery on the thyroid capsule distal to their supply of the parathyroid glands (rather than ligating the inferior thyroid artery as a single trunk), and by treating the parathyroids with great care. If a parathyroid gland is recognized to be ischemic or nonviable during surgery, it can be autotransplanted often after identification by frozen section. The gland is minced into 1 to 2 mm cubes and placed into a pocket(s) in the sternocleidomastoid muscle.

Postoperative hypoparathyroidism results in hypocalcemia and hyperphosphatemia; it is manifested by circumoral numbness, tingling of the fingers and toes, and intense anxiety occurring soon after surgery. Chvostek’s sign appears early, and carpopedal spasm can occur. Symptoms develop in most patients when the serum calcium level is less than 7.5 to 8 mg/dL. Parathyroid hormone is low or absent in most cases of permanent hyopoparathyroidism.

Patients who have had a thyroid lobectomy rarely develop significant hypocalcemia postoperatively since two contralateral parathyroid glands are left intact. Many of these patients may be discharged on the day of operation if they are otherwise satisfactory. However, patients who have had a total or near total thyroidectomy for cancer or for Graves’ disease are at greater risk of a low calcium and are generally observed in the hospital postoperatively. We have found the one hour postoperative PTH level to be equivalent to the parathyroid hormone level drawn the following morning. A value of 15 pg/ml or greater at one hour is very reassuring and is rarely associated with symptomatic postoperative hypocalcemia or with permanent hypoparathyroidism. A central lymph node dissection makes transient hypocalcemia more likely.

As well as one hour PTH, we measure the serum calcium and parathyroid hormone levels approximately 12 hours after operation and thereafter. Most patients are able to leave the hospital on the morning after surgery if they are asymptomatic and their serum calcium level is 7.8 mg/dL or above. Oral calcium pills are used liberally. Patients with severe symptomatic hypocalcemia are treated in the hospital with 1 g (10 mL) of 10% calcium gluconate infused intravenously over several minutes. Then, if necessary, 5 g of this calcium solution is placed in each 500 mL intravenous bottle to run continuously, starting with approximately 30 mL/hour. Oral calcium, usually as calcium carbonate (1250 mg to 2500 mg four times per day), should be started. Each 1250 mg pill of calcium carbonate contains 500 mg of calcium. With this treatment regimen most patients become asymptomatic. The intravenous therapy is tapered and stopped as soon as possible, and the patient is sent home and told to take oral calcium pills. This condition is referred to as transient or temporary hypocalcemia or transient hypoparathyroidism. Serum phosphorus determinations are helpful to rule out bone hunger in which both calcium and phosphorus levels are low, while PTH may be normal.

Management of persistent severe hypocalcemia requires the addition of a vitamin D preparation to facilitate the absorption of oral calcium. We prefer the use of 1,25-­dihydroxyvitamin D (Calcitriol) because it is the active metabolite of vitamin D and has a more rapid action than regular vitamin D. Calcitriol 0.5 mcg with oral calcium carbonate therapy is given four times daily for the first several days, then this priming dose of vitamin D is reduced. The usual maintenance dose for most patients with permanent hypoparathyroidism is Calcitriol 0.25 to 0.5 mcg once daily, along with calcium carbonate, 500 mg Ca ²⁺ once or twice daily, although some patients require larger doses. When high doses of vitamins are used, serum calcium levels must be monitored carefully after discharge, and the dosage of the medications is adjusted promptly to prevent hypercalcemia as well as hypocalcemia. Finally, the serum parathyroid hormone level should be analyzed periodically to determine whether permanent hypoparathyroidism is truly present, because the authors and others have seen cases of postoperative tetany, perhaps caused by “bone hunger,” that later resolved completely. In such cases, circulating parathyroid hormone is normal and all therapy can be stopped. Remember that in bone hunger, both the serum calcium and phosphorus values are low, whereas in hypoparathyroidism, the serum calcium value is low but the phosphorus level is elevated. Permanent hypoparathyroidism is usually not diagnosed until at least six months have passed and parathyroid hormone remains low or absent.

McHenry has shown that the incidence of complications following thyroidectomy varies greatly (66a). In general, those surgeons with excellent training and a large experience with this operation have fewer complications, particularly following cancer procedures and reoperative surgery.

DEVELOPMENTAL ABNORMALITIES OF THE THYROID

To understand the different thyroid anomalies, it is important to briefly review normal development of this gland. The ­thyroid is embryologically an offshoot of the primitive alimentary tract, from which it later becomes separated (Figs. 16 and 17) (67-70). During the third to fourth week in utero, a median anlage of epithelium arises from the pharyngeal floor in the region of the foramen cecum of the tongue (i.e., at the junction of the anterior two thirds and the posterior third of the tongue). The main body of the thyroid, referred to as the median lobe or median thyroid component, follows the descent of the heart and great vessels and moves caudally into the neck from this origin. It divides into an isthmus and two lobes, and by 7 weeks it forms a “shield” over the front of the trachea and thyroid cartilage. It is joined by a pair of lateral thyroid lobes originating from the fourth and fifth branchial pouches (Fig. 17). From these lateral thyroid components, now commonly called the ultimobranchial bodies, C cells (parafollicular cells) enter the thyroid lobes. C cells contain and secrete calcitonin and are the cells that give rise to medullary carcinoma of the thyroid gland. Williams and associates have described cystic structures in the neck near the upper parathyroid glands in cases in which thyroid tissue was totally lingual in location (71). These cysts contained both cells staining for calcitonin and others staining for thyroglobulin. This study, they believe, offers evidence that the ultimobranchial body contributes both C cells and follicular cells to the thyroid gland of humans.

As the gland moves downward, it leaves behind a trace of epithelial cells known as the thyroglossal tract. From this structure both thyroglossal duct cysts and the pyramidal lobe of the thyroid develop. The mature thyroid gland may take on many different configurations depending on the embryologic development of the thyroid and its descent (Fig. 18).

THYROID ABNORMALITIES

The median thyroid anlage may, on rare occasions, fail to develop. The resultant athyrosis, or absence of the thyroid gland, is associated with cretinism. The anlage also may differentiate in locations other than the isthmus and lateral lobes. The most common developmental abnormality, if looked on as such, is the pyramidal lobe (Fig. 18), which has been reported to be present in as many as 80% of patients in whom the gland was surgically exposed. Usually, the pyramidal lobe is small; however, in Graves’ disease or in lymphocytic thyroiditis, it is often enlarged and is commonly clinically palpable. The pyramidal lobe generally lies in the midline but can arise from either lobe. Origin from the left lobe is more common than is origin from the right lobe (72).

THYROID HEMIAGENESIS

More than 100 cases have been reported in which only one lobe of the thyroid is present (73). The left lobe is absent in 80% of these patients. Often, the thyroid lobe that is present is enlarged, and both hyperthyroidism and hypothyroidism have been reported at times. Females are affected three times as often as males. Both benign and malignant nodules have been reported in this condition (74).

Other variations involving the median thyroid anlage represent an arrest in the usual descent of part or all of the thyroid-forming material along the normal pathway. Ectopic thyroid development can result in a lingual thyroid (Fig. 19) or in thyroid tissue in a suprahyoid, infrahyoid, or intratracheal location. Persistence of the thyroglossal duct as a sinus tract or as a cyst (called a thyroglossal duct cyst) is the most common of the clinically important anomalies of thyroid development (Fig. 20). Finally, the entire gland or part of it may descend more caudally; this results in thyroid tissue located in the superior mediastinum behind the sternum, adjacent to the aortic arch or between the aorta and pulmonary trunk, within the upper portion of the pericardium, and even within the interventricular septum of the heart. Most intrathoracic goiters, however, are not true anomalies, but rather are extensions of pathologic elements of a normally situated gland into the anterior or posterior mediastinum. Each of these abnormalities is discussed in greater depth.

ECTOPIC THYROID

Lingual Thyroid

A lingual thyroid is relatively rare and is estimated to occur in 1 in 3000 cases of thyroid disease. However, it represents the most common location for functioning ectopic thyroid tissue. Lingual thyroid tissue is associated with an absence of the normal cervical thyroid in 70% of cases. It occurs much more commonly in women than in men.

The diagnosis is usually made by the discovery of an incidental mass on the back of the tongue in an asymptomatic patient (Fig. 19). The mass may enlarge and cause dysphagia, dysphonia, dyspnea, or a sensation of choking (75). Hypothyroidism is often present and may cause the mass to enlarge and become symptomatic, but hyperthyroidism is very unusual. In women, symptomatic lingual thyroid glands develop during puberty or early adulthood in most cases. Buckman, in his review of 140 cases of symptomatic lingual thyroids in females, reported that 30% occurred in puberty, 55% between the ages of 18 and 40 years, 10% at menopause, and 5% in old age (76). He attributed this distribution to hormonal disturbances, which are more apparent in female subjects during puberty and may be precipitated by pregnancy. The incidence of malignancy in lingual thyroid glands is low (77). The diagnosis of a lingual thyroid should be suspected when a mass is detected in the region of the foramen cecum of the tongue, and it is definitively established by radioisotope scanning (see Fig. 19).

 

The usual treatment of this condition is thyroid hormone therapy to suppress the lingual thyroid and reduce its size. Only rarely is surgical excision necessary. Indications for extirpation include failure of suppressive therapy to reduce the size, ulceration, hemorrhage, and suspicion of malignancy (78). Autotransplantation of thyroid tissue has been tried on rare occasions when no other thyroid tissue is present, and it has apparently been successful. A lingual thyroid was reported in two brothers, which suggests that this condition may be inherited (79).

Suprahyoid and Infrahyoid Thyroid

In these cases, thyroid tissue is present in a midline position above or below the hyoid bone. Hypothyroidism with elevation of thyrotropin (TSH) secretion is commonly present because of the absence of a normal thyroid gland in most instances. An enlarging mass commonly occurs during infancy, childhood, or later life. Often, this mass is mistaken for a thyroglossal duct cyst, because it is usually located in the same anatomic position (80). If it is removed, all thyroid tissue may be ablated, a consequence that has definite physiologic as well as possible medicolegal implications. To prevent total thyroid ablation, it is recommended that an ultrasound examination be performed in all cases of thyroglossal duct cyst before its removal to be certain that a normal thyroid gland is present. Furthermore, before removing what appears to be a thyroglossal duct cyst, a prudent surgeon should be certain that no solid areas are present. If any doubt exists, the normal thyroid gland should be explored and ­palpated. Finally, if ectopic thyroid tissue rather than a thyroglossal duct cyst is encountered at surgery in an infant, its blood supply should be preserved; the ectopic gland divided vertically; and each half translocated laterally, deep to the strap muscles, where it is no longer manifested as a mass. If normal thyroid tissue is demonstrated to be present elsewhere or in the adult, it may be ­better to remove the ectopic tissue rather than transplant it, because carcinoma arising from these developmental abnormalities, although rare, has been reported.

THYROGLOSSAL DUCT CYSTS

Both cysts and fistulas can develop along the course of the thyroglossal duct (Fig. 20) (81). These cysts are the most common anomaly in thyroid development seen in clinical practice (82). Normally, the thyroglossal duct becomes obliterated early in embryonic life, but occasionally it persists as a cyst. Such lesions occur equally in males and females. They are seen at birth in about 25% of cases; most appear in early childhood; and the rest, about one third, become apparent only after age 30 years (83). Cysts usually appear in the midline or just off the midline between the isthmus of the thyroid and the hyoid bone. They commonly become repeatedly infected and may rupture spontaneously. When this complication occurs, a sinus tract or fistula persists. Removal of a thyroglossal cyst or fistula requires excision of the central part of the hyoid bone and dissection of the thyroglossal tract to the base of the tongue (the Sistrunk procedure) if recurrence is to be minimized. This procedure is necessary because the thyroglossal duct is intimately associated with the central part of the hyoid bone (Fig. 21). Recurrent cysts are very common if this operative procedure is not followed.

At least 115 cases of thyroid carcinoma have been reported to originate from the thyroglossal duct (82). Often, in such cases an association is noted with low-dose external irradiation of the head and neck in infancy or childhood. Almost all carcinomas have been papillary, and their prognosis is excellent. If a carcinoma is recognized, at the time of surgery the thyroid gland should be inspected for evidence of other tumor nodules, and the lateral lymph nodes should be sampled. Our practice and that of many others is to perform near-total or total thyroidectomy with appropriate nodal resection when a thyroglossal duct carcinoma is found and resected. In one series of 35 patients with papillary carcinoma arising in a thyroglossal duct cyst, the thyroid gland of 4 patients (11.4%) also contained papillary cancer (82). This operative ­procedure permits later radioiodine therapy as well.

In addition to papillary cancer, approximately 5% of all carcinomas arising from a thyroglossal duct cyst are squamous; rare cases of Hürthle cell and anaplastic cancer have also been reported. Finally, three families have been reported in which a total of 11 members had a thyroglossal duct cyst (84).

LATERAL ABERRANT THYROID

Small amounts of histologically normal thyroid tissue are occasionally found separate from the thyroid. If these tissue elements are near the thyroid, not in lymph nodes, and entirely normal histologically, it is possible that they represent developmental abnormalities. True lateral aberrant thyroid tissue or embryonic rests of thyroid tissue in the lymph nodes of the lateral neck region are very rare. Most agree that the overwhelming number of cases of what in the past was called “lateral aberrant thyroid” actually represented well-differentiated thyroid cancer metastatic to a cervical lymph node rather than an embryonic rest. In such cases, we favor near-total or total thyroidectomy with a modified radical neck dissection on the side of the lymph node, possibly followed by radioiodine therapy.

Several lateral thyroid masses have been reported that are said to be benign adenomas in lateral ectopic sites (85, 86). The authors of these studies suggest that they may develop ectopically because of failure of fusion of the lateral thyroid component with the median thyroid. However, before accepting this explanation, it is important to be certain that each of these lesions does not represent a well-differentiated metastasis that has totally replaced a lymph node and in which the primary thyroid carcinoma is small or even microscopic and was not recognized.

SUBSTERNAL GOITERS

Developmental abnormalities may lead to the finding of thyroid tissue in the mediastinum or, rarely, even within the tracheal or esophageal wall. However, most substernal goiters undoubtedly originate in the neck and then “fall” or are “swallowed” into the mediastinum and are not embryologically determined at all.

Substernal goiters have been reported to occur in 0.1% to 21% of patients in whom thyroidectomies were performed. This large variability is undoubtedly caused partly by a difference in classification among the authors, but it may also be caused by the incidence of endemic goiter. More recent series report an incidence of 2% or less (87).

Many substernal goiters are found on routine chest radiography in patients who are completely asymptomatic. Other patients may have dyspnea or dysphagia from tracheal or esophageal compression or displacement. Superior vena caval obstruction can occasionally occur with edema and cyanosis of the face, and venous engorgement of the arms and face (Fig. 22) (88). Most individuals with substernal goiters are euthyroid or hypothyroid; however, hyperthyroidism occurs as well. Although the goiters of Graves’ disease are rarely intrathoracic, single or multiple “hot” nodules may occur within an intrathoracic goiter and result in hyperthyroidism as part of a toxic nodular goiter.

Intrathoracic goiters are usually found in the anterior mediastinum and, less commonly, in the posterior mediastinum. In either instance the diagnosis is suggested if a goiter can be palpated in the neck and if it appears to continue below the sternum. Rarely, however, no thyroid enlargement in the cervical area is present, and instead of being in continuity, the intrathoracic component may be attached to the cervical thyroid only by a narrow bridge of thyroid or fibrous tissue. The diagnosis of an intrathoracic thyroid mass can be made by the use of a thyroid isotope scan; however, CT or MRI are usually more helpful. Regarding therapy, we generally agree with the recommendation made by Lahey and Swinton more than 50 years ago that goiters that are definitely intrathoracic should usually be removed if the patient is a good operative risk (89). Because of the cone-shaped anatomy of the upper thoracic outlet, once part of a thyroid goiter has passed into the superior mediastinum, it can increase its size only by descending further into the chest. Thus, delay in surgical management may lead to increased size of the lesion, a greater degree of symptoms, and perhaps a more difficult or hazardous operative procedure.

Substernal goiters should be operated on initially through a cervical incision, because the blood supply to the substernal thyroid almost always originates in the neck and can be readily controlled in this area. Only rarely does an intrathoracic goiter receive its blood supply from mediastinal vessels; however, such a finding favors a developmental cause. Thus, in most instances, good hemostasis can be obtained by control of the superior and inferior thyroid arteries in the neck. Thus, most substernal goiters can be removed through the neck, but in some cases a VATS procedure or a partial sternotomy may be necessary. The authors like to divide the isthmus and the upper pole vessels early in the dissection. The affected thyroid lobe is then carefully dissected along its capsule by blunt dissection into the superior mediastinum. While gentle traction is exerted from above, the mass is elevated by the surgeon’s ­fingers or blunt, curved clamps (Fig. 23). Usually these maneuvers suffice to permit extraction of a mass from the mediastinum and into the neck area. Any fluid-filled cysts may be aspirated to reduce the size of the mass and permit its egress through the thoracic outlet. However, piecemeal morcellation of the thyroid gland should not be practiced, because this occasionally has led to severe bleeding. Furthermore, rarely a substernal goiter has been found to contain carcinoma, and this technique violates all principles of cancer surgery.

With the use of this method, the great majority of substernal thyroid glands can be removed transcervically. If the thyroid gland cannot be easily extracted from the mediastinum, however, a partial or complete sternotomy should be performed. This procedure affords direct control of any mediastinal vessels and permits resection of the thyroid gland to be carried out safely.

As in all thyroid surgery, the recurrent laryngeal nerves must be preserved and treated with care. The parathyroid glands should be identified and preserved, and the inferior thyroid artery’s branches should be ligated close to the thyroid capsule to prevent ischemia of the parathyroid glands, which might result in hypoparathyroidism.

STRUMA OVARII

Ectopic development of thyroid tissue far from the neck area can also lead to difficulties in rare instances. Dermoid cysts or teratomas, which are uncommon ovarian germ cell tumors, occur in female subjects of all age groups. About 3% can be classified as an ovarian struma, because they contain functionally significant thyroid tissue or thyroid tissue occupying more than 50% of the volume of the tumor. Many more such tumors contain small amounts of thyroid tissue. Some strumae ovarii are associated with carcinoid-appearing tissue. These strumal-carcinoid tumors secrete or contain thyroid hormones as well as somatostatin, chromogranin, serotonin, glucagon, insulin, gastrin, or calcitonin (90). Some are associated with carcinoid syndromes.

Struma ovarii is sometimes manifested as an abdominal mass lesion, often with a peritoneal or pleural effusion that may be bloody. Most of these lesions synthesize and iodinate thyroglobulin poorly, and thus, despite growth of the mass, thyrotoxicosis does not develop. However, perhaps one fourth to one third of ovarian strumae are associated with thyrotoxicosis (91, 92). Many of these lesions may be contributing to autoimmune hyperthyroidism in response to a common stimulator such as thyroid-stimulating immunoglobulins. In other instances, the struma alone is clearly responsible for the thyrotoxicity. An elevated free thyroxine index or free T4, a suppressed TSH value, and uptake of radioiodine in a mass in the pelvis are the obvious prerequisites for making the diagnosis (93). Often, in ovarian struma, symptoms and findings of thyrotoxicosis are present in patients who have low uptake of radioiodine in their thyroid glands. Thus, a “high index of suspicion” is most important. Usually, operative resection of an ovarian tumor is indicated. After surgery, transient postoperative hypothyroidism and “thyroid storm” have occasionally been reported.

Benign thyroid adenomas in strumae are common, and about 5% manifest evidence of carcinoma (94). Usually, these lesions are resectable, but external radiation therapy and/or ¹³¹ I ablation has been advised after resection of the malignant tumors to avoid the tendency for late recurrence or metastatic disease, which has sometimes been fatal. Metastatic disease occurs in approximately 5% of these malignant tumors. It is best treated with ¹³¹ I therapy. Thyroidectomy is necessary in such cases before giving radioiodine therapy. Then, TSH should be suppressed with thyroxine as is done for thyroid cancer originating in the usual location.

STRUMA CORDIS

Functioning, apparently normal intracardiac thyroid tissue has been reported a few times and has been visualized by radioiodine imaging (95). The clinical finding is usually a right ventricular mass, and the diagnosis has typically been made after operative removal.

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ABSTRACT

The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the regulation of metabolic processes throughout the body. As at any factory, effective production depends on three key components - adequate raw material, efficient machinery, and appropriate controls. Iodine is the critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where it reduces H₂O₂, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to tyrosyls in Tg. H₂O₂ is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about 70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available for recycling within the gland. TSH is the stimulator that affects virtually every stage of thyroid hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply, either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can produce goiter and hypothyroidism. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.

 

INTRODUCTION

The thyroid contains two hormones, L-thyroxine (tetraiodothyronine, T₄) and L-triiodothyronine (T₃) (Figure 2-1, below). Iodine is an indispensable component of the thyroid hormones, comprising 65% of T₄'s weight, and 58% of T₃'s. The thyroid hormones are the only iodine-containing compounds with established physiologic significance in vertebrates.

The term "iodine" occasionally causes confusion because it may refer to the iodine atom itself but also to molecular iodine (I₂). In this chapter "iodine" refers to the element in general, and "molecular iodine" refers to I₂. "Iodide" refers specifically to the ion I^-.

Ingested iodine is absorbed through the small intestine and transported in the plasma to the thyroid, where it is concentrated, oxidized, and then incorporated into thyroglobulin (Tg) to form MIT and DIT and later T₄ and T₃ (Figure 2-2). After a variable period of storage in thyroid follicles, Tg is subjected to proteolysis and the released hormones are secreted into the circulation, where specific binding proteins carry them to target tissues. This chapter discusses these broad steps as: (a) iodine availability and absorption; (b) uptake of iodide by the thyroid; (c) oxidation of iodide, which involves the thyroperoxidase (TPO), H₂O₂, and H₂O₂ generation; (d) Tg, whose iodination leads to hormone formation; (e) storage of thyroid hormones in a Tg-bound form; (f) Tg breakdown and hormone release; (g) control of synthesis and secretion by iodine supply and TSH; and (h) effects of drugs and other external agents on the process.

Fig. 2-2: The iodide cycle. Ingested iodide is trapped in the thyroid, oxidized, and bound to tyrosine to form iodotyrosines in thyroglobulin (TG); coupling of iodotyrosyl residues forms T4 and T3. Hormone secreted by the gland is transported in serum. Some T4 is deiodinated to T3. The hormone exerts its metabolic effect on the cell and is ultimately deiodinated; the iodide is reused or excreted in the kidney. A second cycle goes on inside the thyroid gland, with deiodination of iodotyrosines generating iodide, some of which is reused without leaving the thyroid.

The production of thyroid hormones is based on the organization of thyroid epithelial cells in functional units, the thyroid follicles. A single layer of polarized cells (Fig. 2-4A) forms the enveloppe of a spherical structure with an internal compartment, the follicle lumen. Thyroid hormone synthesis is dependent on the cell polarity that conditions the targeting of specific membrane protein, either on the external side of the follicle (facing the blood capillaries) or on the internal side (at the cell-lumen boundary) and on the tightness of the follicle lumen that allows the gathering of substrates and the storage of products of the reactions.Thyroid hormone secretion relies on the existence of stores of pre-synthetized hormones in the follicle lumen and cell polarity-dependent transport and handling processes leading to the delivery of hormones into the blood stream.

IODINE AVAILABILITY AND TRANSPORT

The daily iodine intake of adult humans varies from less than 10 µg in areas of extreme deficiency to several hundred milligrams for some persons receiving medicinal iodine. Milk, meat, vitamin preparations, medicines, radiocontrast material, and skin antiseptics are important sources (Table 2-1) (1;2). In the United States, the average intake in 1960 was about 100-150 µg/day, then rose to 200-500 µg/day in the following decade. It is currently about 150 µg/day (3). The use of iodate as a bread conditioner in the baking industry greatly increased average iodine consumption; this additive has been replaced more recently by other conditioners that do not contain iodine. Iodophors as sterilizing agents in the milk industry also added much iodine to the food chain, but this source may also be diminishing. In the USA and elsewhere, most consumers are unaware of the amount of iodine they ingest. Commerce and manufacturing technology rather than health dictate the presence of iodine in most products. The amounts of iodine are usually unrevealed, and changes in them unannounced.
In the USA, where iodized salt use is optional, about 70% of the population consumes table salt containing approximately 76 ppm iodine (76 mg I/kg salt). Most prepared food in the USA and Europe uses uniodized salt (Switzerland and Macedonia are exceptions) and only about 15% of the daily salt intake is added at the table, so iodized salt in these areas makes only a modest contribution to daily iodine intake (4). The National Health and Nutrition Examination Surveys (NHANES) showed that the median national urinary iodine excretion in the USA in samples collected between 1988 and 1994 was 145 µg/L, a marked decrease from the 321 µg/L in a similar survey two decades before (5;6). Estimates from the NHANES (2001) are about 160 µg/L. The Total Diet Study of the U.S. Food and Drug Administration reported a parallel decrease in iodine consumption between 1970 and 1990 (7) . These fluctuations in iodine intake result from changes in societal and commercial practices that are largely unrecognized and unregulated. Canada mandates that all salt for human consumption contain KI at 100 ppm (76 ppm as iodine). Calculations of the representative Canadian diet in 1986 estimated slightly over 1 mg iodine/person/day, of which iodized salt contributed over half (8). Urinary iodine excretion in a group of men in Ottawa in 1990 was less than 50% of that in the Canadian national survey of 1975 (9), suggesting a decrease in dietary intake there as well as in the USA. Some countries have areas with very high iodine intake (10), from dietary custom (e.g., seaweeds in Japan) or from iodine-rich soil and water (e.g., a few places in China). But many countries have had some degree of iodine deficiency (11) in at least part of their territory. This has been corrected by the widespread programs of iodine prophylaxis promoted by ICCIDD (12).
Too much iodine increases the incidence of iodine-induced hyperthyroidism, autoimmune thyroid disease and perhaps thyroid cancer. Too little causes goiter, hypothyroidism and their consequences i.e.features of the so-called iodine deficiency disorders (5). The global push to eliminate iodine deficiency in the current decades has put both excess and deficiency of iodine in the spotlight. Some countries have already moved rapidly from severe iodine deficiency to iodine excess, while others are only now recognizing iodine deficiency as a problem (5;12). Their experience, as well as that in the USA and Canada, emphasizes the need for continued monitoring to assess trends in iodine intake.
Medicinal sources can provide iodine in amounts much larger than those consumed in an average diet (Table 2-1). For example, 200 mg of amiodarone contains 75 mg of iodine. Radiographic contrast materials typically contain grams of iodine in covalent linkage, and significant amounts (milligrams) may be liberated in the body. Skin disinfectants (e.g., povidone iodine) and iodine-based water purification systems can greatly augment iodine intake. At the other end, some individuals with little consumption of dairy products and of iodized salt have low iodine intakes.

 

Table 2-1. Some common sources of iodine in adults USA (1,2)
Dietary iodine	Daily intake (µg)
Dairy products	52
Grains	78
Meat	31
Mixed dishes	26
Vegetables	20
Desserts	20
Eggs	10
Iodized salt	380
Other iodine sources	(µg)
Vitamin/mineral prep (per tablet)	150
Amiodarone (per tablet)	75,000
Povidone iodine (per mL)	10,000
Ipodate (per capsule)	308,000

Most dietary iodine is reduced to iodide before absorption throughout the gut, principally in the small intestine. Absorption is virtually complete. Iodinated amino acids, including T₄ and T₃, are transported intact across the intestinal wall. Short-chain iodopeptides may also be absorbed without cleavage of peptide bonds (13). Iodinated dyes used in radiography are absorbed intact, but some deiodination occurs later. Except in the postabsorptive state, the concentration of iodide in the plasma is usually less than 10 µg/L. Absorbed iodide has a volume of distribution numerically equal to about 38% of body weight (in kilograms) (14), mostly extracellular, but small amounts are found in red cells and bones.

The thyroid and kidneys remove most iodide from the plasma. The renal clearance of iodide is 30-50 mL plasma/min (14-16) and appears largely independent of the load of iodide or other anions. In certain species, such as the rat, large chloride loads can depress iodide clearance. In humans, renal iodide clearance depends principally on glomerular filtration, without evidence of tubular secretion or of active transport with a transfer maximum (17). Reabsorption is partial, passive, and depressed by an extreme osmotic diuresis. Hypothyroidism may decrease and hyperthyroidism may increase renal iodide clearance, but the changes are not marked (14;18).

On iodine diets of about 150 µg/day, the thyroid clears iodide from 10-25 mL of serum (average, 17 mL) per minute (14). The total effective clearance rate in humans is thus 45-60 mL/min, corresponding to a decrease in plasma iodide of about 12%/hr. Thyroidal iodide clearance may reach over 100 mL/min in iodine deficiency, or as low as 3 or 4 mL/min after chronic iodine ingestion of 500-600 µg/day.

The salivary glands and the stomach also clear iodide and small but detectable amounts appear in sweat and in expired air. Breast milk contains large amounts of iodide, mainly during the first 24 hours after ingestion (19). Its content is directly proportional to dietary iodine. For example, in one part of the USA with community adult iodine intake of about 300 µg daily per person, breast milk contained about 18 µg iodine/dL, while in an area of Germany consuming 15 µg iodine per capita daily, the breast milk iodine concentration was only 1.2 µg/dL (20). Milk is the source of virtually all the newborn's iodine, so milk substitutes need to provide adequate amounts.

UPTAKE OF IODINE BY THE THYROID

Thyroid cells extract and concentrate iodide from plasma (21;22). As shown in Fig. 2-3, shortly after administration, radioiodide is taken up from the blood and accumulates within thyroid follicular cells. About 20% of the iodide perfusing the thyroid is removed at each passage through the gland (23). The normal thyroid maintains a concentration of free iodide 20 to 50 times higher than that of plasma, depending on the amount of available iodine and the activity of the gland (24). This concentration gradient may be more than 100:1 in the hyperactive thyroid of patients with Graves' disease. The thyroid can also concentrate other ions, including bromide, astatide, pertechnetate, rhenate, and chlorate, but not fluoride (25;26).

The protein responsible for iodide transport, the so-called sodium/iodide symporter or NIS, is located at the basolateral plasma membrane of thyrocytes (Fig. 2-4.). NIS-mediated I^- accumulation is a Na⁺-dependent active transport process that couples the energy released by the inward translocation of Na⁺ down to its electrochemical gradient to the simultaneous inward translocation of I^- against its electrochemical gradient. The maintenance of the Na⁺ gradient acting as the driving force is insured by Na⁺-K⁺-ATPase. NIS belongs to the sodium/glucose cotransport family as the SLC5A5 member. Iodide transport is energy-dependent and requires O₂. Ouabain, digitoxin, and other cardiac glycosides block transport in vitro (27;28). Iodide uptake by thyroid cells is dependent on membrane ATPase. During gland hyperplasia, iodide transport usually varies concordantly with plasma membrane Na⁺-K⁺-activated, ouabain-sensitive ATPase activity (29).

NIS cDNA was first cloned in rat FRTL-5 cells by Dai et al. (30). The rat NIS gene gives rise to a 3kb transcript with an open reading frame of 1,854 nucleotides encoding a polypeptide chain of 618 amino acids. The mature protein is a glycoprotein with an apparent molecular mass of 85kDa (31;32).It has 13 membrane spanning domains, with the carboxy terminus in the cytoplasm and the amino terminus located outside the cells (33). In the model of Levy et al. (34), a Na⁺ ion first binds to the transporter which, in the presence of iodide, forms a complex that then transfers iodide and two Na⁺ ions to the cell interior.

The human NIS gene located on chromosome 19 (35) codes for a protein of 643 amino acids that is 84% homologous with rat NIS (36). The mouse NIS polypeptide chain (37) has the same size (618 amino acids) as the rat NIS. At variance with other species, three different transcripts are generated from the porcine NIS gene by alternative splicing (38); the main form encodes a polypeptide of 643 amino acids as human NIS.

Functional studies clearly show that NIS is responsible for most of the events previously described for iodide concentration by the thyroid. TSH stimulates NIS expression (39;40) and iodide transport (31;32). TSH exerts its regulatory action at the level of transcription through a thyroid-specific far-upstream enhancer denominated NUE (NIS Upstream Enhancer) that contains binding sites for the transcription factor Pax8 and a cAMP response element-like sequence. This original demonstration made on the rat NIS gene (41) has now been extended to human (42) and mouse (43) NIS genes. It has been suggested that TSH could also regulate NIS expression at post-transcriptional level (44). Data from TSH receptor-null mice (44-46) clearly show that TSH is required for expression of NIS. Moderate doses of iodide in the TSH-stimulated dog thyroid inhibit expression of the mRNAs for NIS and TPO, while not affecting that for Tg and TSH receptor (40) . The decrease in thyroid iodide transport resulting from excess iodide administration (escape from the Wolff-Chaikoff effect, see further) is related to a decrease in NIS expression (40;47). Both NIS mRNA and NIS protein are suppressed by TGFb, which also inhibits iodide uptake (48;49). Reviews focus on NIS and its functional importance (50;51).

Several mutations in the NIS gene causing defective iodide transport have been reported in humans (52-59). The most commonly found mutation corresponds to a single base alteration T354P in the ninth putative transmembrane domain of NIS (55). Site directed mutagenesis of rat NIS cDNA to substitute for threonine at residue 354 and transfection into COS cells lead to loss of iodide transport activity (60). Other mutations lead to truncated NIS (58) or to alterations of membrane targeting of the NIS protein (59) . NIS expression is increased in Grave’s disease and hyperactive nodules (61-63) and decreased in adenomas and carcinomas (64;65) appearing as cold nodules at scintigraphy.In hypofuctioning benign or malignant tumors, the impairment of iodide transport would result from both transcriptional and post-transcriptional alterations of NIS expression (66).Other tissues that concentrate iodide also show NIS expression, including salivary glands (67) and mammary glands (68;69).

Iodide supply of follicular lumen involves a two-step transport process: the active transport across the basolateral plasma membrane of thyrocytes by NIS and a passive transport across the apical plasma membrane. The protein(s) insuring the second step is (are) not yet identified. A potential iodide transporter has been proposed: pendrin (70;71). Pendrin, encoded by the PDS gene (72) and composed of 780 amino acids, is expressed in different organs including kidney, inner ear and thyroid. In the thyroid, pendrin is a 110kDA membrane glycoprotein (73), selectively located at the apical plasma membrane (74). Its activity as transporter of anions including iodide has been demonstrated in different experimental systems (71;75-77). Pendrin belongs to the SLC family under the reference SLC26A4. However, the implication of pendrin in thyroid iodide transport remains uncertain for several reasons. First, there is still no direct demonstration of a pendrin-mediated efflux of iodide from thyrocytes to the follicular lumen. Second, the genetic alterations of the PDS gene found in patients with the Pendred syndrome, which lead to a loss of the anion transport activity of pendrin and to a constant and severe hearing loss, only have a moderate impact on the thyroid functioning, generally a euthyroid goiter (78). Third, PDS knock-out mice (79) do not show any thyroid dysfunction. In summary, contrary to NIS for which the anion selectivity (25) corresponds to what was expected, the ion selectivity of thyroid pendrin remains to be elucidated. In the thyroid as in the kidney, pendrin could act primarily as a chloride/bicarbonate anion exchanger. Rather than pendrin, anoctamin-1/TEM 16A, a calcium-activated chloride channel, seems to be responsible for most of the iodide efflux accross the apical membrane of the thyrocytes (80).

Fig. 2-4: NIS-mediated transport of iodide. A, immunolocalization of the human NIS protein at the basolateral plasma membrane of thyrocytes in their typical follicle organization. B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses (33). C, transport of iodide from the extracellular fluid (or plasma) to the thyroid follicle lumen. The uptake of iodide at the basolateral plasma membrane of thyrocytes must be active; it operates against an electrical gradient (0 - 50 mV) and a concentration gradient, [ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle lumen should be a passive process, the electrical and concentration gradients being favorable.

Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and bound to tyrosyl residues in Tg. A significant proportion of intrathyroidal iodide is free for about 10-20 minutes after administration of a radioactive tracer (81), but in the steady state, iodide contributes less than 1% of the thyroid total iodine. A major fraction of the intrathyroidal free iodide pool comes from deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation. Some data suggest that iodide entering the gland by active transport segregates from that generated by deiodination of Tg within the gland (82;83). Once in the thyroid, iodide is organically bound at a rate of 50 to 100% of the pool each minute (24;84). The proportion of an iodide load that is bound varies little, despite wide shifts in daily intake. In contrast, NIS activity is sensitive to both iodine availability and TSH stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide available for hormonogenesis.

IODINE IN OTHER TISSUES

The thyroid is not the only organ to concentrate iodine; the others endowed with this capacity are salivary glands, gastric mucosa, mammary glands, and choroid plexus. Ductal cells of the salivary glands express NIS (67) . The plasma membrane of the mammary gland epithelium contains a NIS protein with a molecular mass different from that of thyroid NIS (~75 kDa vs ~90 kDa). In the mammary gland, NIS is processed differently after translation and subjected to regulation by lactogenic stimuli (68). It has been reported that over 80% of human breast cancer samples express this symporter. As it is absent in normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible target for radioiodine therapy (69). The thyroid, salivary glands, and gastric mucosa share a common embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is inhibited by thiocyanate, perchlorate, and cardiac glycosides. TSH stimulates transport only in the thyroid. An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk. Iodide concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit, but needs to be remembered for possible insights into pathways as yet undiscovered.

Iodine, particularly in the form of I₂, may enter additional metabolic pathways outside the thyroid. Rats administered I₂ orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide (85). In another comparison of I₂ versus iodide, administration of iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I₂. Additionally, I₂ decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased the former and had no effect on the latter (86).

THYROPEROXIDASE (TPO)

After concentrating iodide, the thyroid rapidly oxidizes it and binds it to tyrosyl residues in Tg, followed by coupling of iodotyrosines to form T₄ and T₃. The process requires the presence of iodide, a peroxidase (TPO), a supply of H₂O₂, and an iodine acceptor protein (Tg).

Thyroperoxidase oxidizes iodide in the presence of H₂O₂. In crude thyroid homogenates, enzyme activity is associated to cell membranes. It can be solubilized using detergents such as deoxycholate or digitonin. The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely related porphyrin (87;88). Chemical removal of the prosthetic group inactivates the enzyme, and recombination with the heme protein restores activity (89). The apoprotein from human thyroid is not always fully saturated with its prosthetic group (90). Some congenitally goitrous children have poor peroxidase function because the apoprotein has weak binding for the heme group (90).

Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients with autoimmune thyroid disease (AITD), led to identification of TPO. These antibodies were found to react with proteins of 101-107 kDa and to immunoprecipitate thyroid peroxidase (TPO), thus identifying microsomal antigen as TPO (91-95). A monoclonal antibody to purified microsomal antigen or antibodies directed againt thyroperoxidase were then used to clone human TPO (96-98). Different laboratories then cloned TPO from various species: pig (99), rat (100), and mouse (101). Kimura et al. (96) cloned two different cDNAs of humanTPO.TPO1 coded for a protein of 933 residues and TPO2 was identical to TPO1 except that it lacked exon 10 and was composed of 876 residues. Both forms occur in normal and abnormal human thyroid tissue. The C-terminal portion of the proteins exhibits a hydrophobic segment (residues 847-871), likely corresponding to a transmembrane domain; thus, TPO has a short intracellular domain and most of the polypeptide chain is extracellular (Fig. 2-5A). TPO1 is active, but TPO2 appears enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface in transfected cell lines (102). Different degradative pathways exist for the two forms (103). Several other TPO variants resulting from exon skipping have been identified; they appear either active or inactive (104). Pig TPO contains 926 amino acids (99) ; mannose-rich oligosaccharide units occupy four of its five glycosylation sites (105).

Human TPO, which has 46% nucleotide and 44% amino acid sequence homology with human myeloperoxidase, clearly belongs to the same protein family. The TPO gene resides on chromosome 2p13, spans over 150 kbp, and has 17 exons (106). As NIS, Tg, and the TSH receptor (TSHr), TPO expression is controlled by the TSH cAMP pathway (107) through thyroid-specific transcription factors. These include TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 (108;109). Tg and TPO genes have the same binding sites for TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 in their promoters, and the genes for both have TTF-1/NKx2.1 sites in enhancer regions.

Inactivating mutations in the TPO gene are responsible for a subtype of congenital hypothyroidism characterized by thyroid dyshormonogenesis due to iodide organification defect. More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism (110;111).

TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation. TPO is then transported to the Golgi where it is subjected to terminal glycosylation and packaged into transport vesicles along with Tg (112) (Fig. 2-6). These vesicles fuse with the apical plasma membrane in a process stimulated by TSH. TPO delivered at the apical pole of thyrocytes exposes its catalytic site with the attached heme in the thyroid follicular lumen (113). TPO activity is restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the perinuclear part of the endoplasmic reticulum (114;115). Most of this intracellular protein is incompletely or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO has complex carbohydrate units. Glycosylation is essential for enzymatic activity (115). Chronic TSH stimulation increases the amount of TPO and its targetting at the apical membrane (116).

H ₂ O ₂ GENERATING SYSTEM

By definition, a peroxidase requires H₂O₂ for its oxidative function. A large body of older work (reviewed in (117)) investigated possible sources using various in vitro models (117-120). It was already suggested in 1971 that H₂O₂ would be produced at the apical plasma membrane of the thyrocyte by an enzyme that requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway (121). Further biochemical studies showed that the enzymatic complex producing H₂O₂ for TPO is a membrane-bound NADPH-dependent flavoprotein (122-126). H₂O₂ produced by this NADPH-dependent protein is the limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is sufficient (127-129). In human thyroid, the H₂O₂ production and iodination process are stimulated by the calcium-phosphatidylinositol pathway (129). The quantity of H₂O₂ produced is important especially in stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes. While the activated leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr (130;131). Thus thyroid cells may be exposed to high doses of H₂O₂ and have to adapt to it by developing highly regulated generator and efficient protective systems.

More than twenty years passed between the initial biochemical studies and the cloning of Duox as the catalytic enzymatic core of the H₂O₂ thyroid generating system. By two independent molecular strategies Duox enzymes were uncovered from the thyroid. Starting from a purified fraction of pig thyroid membrane bound NADPH flavoprotein, the team of C. Dupuy isolated p138 ^Tox which turned out to be Duox2 lacking the first 338 residues (132). Simultaneously, De Deken et al cloned two cDNAs encoding NADPH oxidases using the strategy based on the functional similarities between H₂O₂ generation in the leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system would belong to the known gp91^phox gene family and display sequence similarities with gp91^phox, now called NOX2. Screenings of two cDNA libraries at low stringency with a NOX2 probe enabled the isolation of two sequences coding for two NADPH oxidases of 1551 and 1548 amino acids respectively initially named Thox1 and Thox2 (133). The encoded polypeptides display 83% sequence similarity and are clearly related to gp91^phox (53 and 47% similarity over 569 amino acids of the C-terminal end) . The whole protein is composed of : a N-terminus ecto-sequence of 500 amino acids showing a similarity of 43% with thyroperoxidase (hence named Dual oxidase-Duox in the present terminology); a first transmembrane segment preceding a large cytosolic domain which contains two calcium binding EF-hand motifs; the C-terminal portion componed of six transmembrane segments, harbouring the four His and two Arg characteristic of the Nox family protein heme binding site and the conserved FAD- and NADPH-binding sites at the extreme C-terminal cytolic portion (Fig. 2-5B). Duox proteins are localized like TPO at the apical plasma membrane of the thyrocyte as fully glycosylated forms (~190kDa) and in the endoplasmic reticulum as high mannose glycosylated forms (~180kDa) (Fig. 2-6C).

Duox1 and Duox2 genes are co-localized on chromosome 15q15.3, span 75kb, have opposite transcriptional orientations and are separated by a ~16kb region (Fig. 2-6A). Duox1 gene is more telomeric, spans 36 kb and is composed of 35 exons; two first of them are non-coding. Duox2 spans 21.5 kb and is composed of 34 exons; the first being non-coding (134).

In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia, in oocytes (135-137) and Duox2 in gastrointestinal mucosa and salivary glands (138;139). Multiple functions are attributed to Duox enzymes: airway fuid acidification (140), mucin secretion (141), wound healing (142;143) and innate hoste defense (144-147) .

Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the gut (144;145;148;149) . Beside these killing mechanisms, Duox and H₂O₂ are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e.g. in bronchi and intestine (146;150). In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte would increase the efficiency of H₂O₂ producer-consumer system (151-153).

 

Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker. The proteins involved in the synthesis of thyroid hormones are expressed just after the thyroid precursor cells have completed their migration from the primitive pharynx and reached their final location around the trachea (154;155). This final morphological maturation begins in mouse with the expression of Tg at embryonic day 14 followed one day later (E15) by the expression of TPO, NIS, TSH receptor and Duox concomitant with the apparition of iodinared Tg (46;156).

 

Until 2006, the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state. Several cell lines transfected with Duox1 and/or Duox2 showed Duox expression completely retained in the endoplasmic reticulum in their immature form without displaying any production of H₂O₂ (157). HEK293 cells transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent manner (158). The reconstitution of a Duox-based functional H₂O₂ generating system requires a maturation factor called DuoxA. The two human DuoxA paralogs were initially identified as thyroid specific expressed genes by in silico screenings of multiple parallel signature sequencing data bases (159). The two genes are located on chromosome 15 in the Duox1/Duox2 intergenic region in a tail to tail orientation, DuoxA1 facing Duox1 and DuoxA2 facing Duox2 (Fig. 2-6A.). DuoxA2 ORF spans 6 exons and encodes a 320 amino acid protein predicted to compose five transmembrane segments, a large external loop presenting N-glycosylation sites between the second and third transmembrane helices and a C-terminal cytoplamic region (Fig. 2-6B). DuoxA1 gene was initially annotated “homolog of Drosophila Numb-interacting protein: NIP” (160). Four alternatively spliced DuoxA1 variants have been identified (161). One of the most expressed transcript, DuoxA1α, is the closest homolog of DuoxA2 and encodes a 343 amino acid protein (58% identity of sequence with DuoxA2) adopting the same predicted structure.

In heterologous systems DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where they probably form complexes. Only the Duox1/DuoxA1 and Duox2/DuoxA2 pairs produce the highest levels of H₂O₂ as they undergo the glycosylation steps through the Golgi. Duox2/DuoxA1 pair does not produce H₂O₂ but rather superoxide anions and Duox1/DuoxA2 is unable to produce any ROS. In addition it has been shown that the type of Duox-dependent ROS poduction is dictated by defined sequences in DuoxA (162). This means that the Duox activators promote Duox maturation but also are parts of the H₂O₂ generating complex (163;164). Mice deficient in DuoxA maturation factors present a maturation defect of Duox, lacking the N-glycan processing, and a loss of H₂O₂ production. These mice develop severe goitrous congenital hypothyroidism with undetectable serum T4 and high serum TSH levels (165).

The reconstitution of this functional H₂O₂ producing system has been useful to measure and compare the intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma membrane under stimulation of the major signalling pathways active in the thyroid. It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs. However, the two oxidase enzymatic activities are differently regulated after activation of the two main signalling cascades in the thyroid. Duox1 but not Duox2 activity is stimulated by the cAMP dependent cascade triggered by forskolin (EC50=0.1µM) via protein kinase A-mediated phosphorylation on serine 955 of Duox1. In contrast, phorbol esters, at low concentrations, induce Duox2 phosphorylation via protein kinase C activation associated with high H₂O₂ generation (EC50= 0.8nM) (166). These results suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H₂O₂ to TPO to oxidize iodide and couple iodotyrosines.

 

From in vitro and in vivo data it has been concluded that Duox-DuoxA constitutes the major if not the unique component of the hormonogenic thyroid H₂O₂ generating system. The bidirectional promoter allows the coexpression of Duox and DuoxA in the same tissue but the mechanisms regulating their transcription are not well and definitely characterized (167;168). It has been recently shown that Th2 cytokines, IL4 and IL13, up-regulate Duox2 and DuoxA2 genes in human thyrocytes through an activation of Jak-Stat pathway opening new perspectives for a better understanding of the eventual role of Duox in autoimmune diseases (169).

 

Defects in Duox and/or DuoxA were rapidly recognized possible causes of congenital hypothyroidism (CH) due to thyroid dyshormonogenesis in patients born with a hyperplastic thyroid or developing a goiter postnatally when T4 treatment is delayed after birth.

The first screening of mutations in Duox genes in 2002 was performed on 9 patients who had idiopathic congenital hypothyroidism with positive ClO4^- discharge (>10%), one with permanent and 8 with transient hypothyroidism (170). They were identified in the Netherlands by neonatal screening and followed up to determine the evolution of CH with the time. One of the patients with total organification defect (TIOD) presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a partial organification defect (PIOD). Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations (Q686X, R701X, S965fsX994) meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH. It is noteworthy that this hypothyroid status was limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable in adulthood since adult heterozygotes in these families presented normal TSH serum levels. The only case with severe permanent CH was homozygous for a nonsense mutation (R434X= protein devoid of the catalytic core) leading to the conclusion at this time of a complete inability to synthesize thyroid hormone in absence of Duox2. No mutation was detected in Duox1.

With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to make the correlation between genotype and phenotype as initially described.

Indeed, subsequent studies have shown a link between biallelic Duox2 defects and PIOD. Patients with compound heterozygous missense (R376W) and a nonsense mutation (R842X), leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia. This suggests that Duox1 can compensate at least partially for the defect in Duox2 (171). Varela et al.. described also two cases of permanent CH with compound heterozygous missense and nonsense or splicing mutations (Q36H and S965fsX994; G418fsX482 and g.IVS19-2A>C conducting to inactive proteins) responsible for congenital goiter with a PIOD (172).

 

The phenotype-genotype correlation suggested by the work of Moreno et al. is no longer clear. Maruo et al. described a series of transient CH characterized by biallelic defects in Duox2: in one family, four siblings were compound heterozygous for early frameshift mutations (L479SfsX2 and K628RfsX10) resulting in a presumed complete loss of Duox2 activity (not tested at this time). Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement therapy ceased to be necessary by 9yr of age (173). A French-Canadian patient with a transient CH initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense mutation (G1518S) inherited from the father and a deletion removing the part of the gene coding for the catalytic core of Duox2 inherited from the mother. In vitro test proved that the missense mutant protein was totally inactive (174). This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles (175-177).

 

The first homozygous nonsense mutation in DuoxA2 (Y246X) that resulted in a non-functional protein tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a dyshormonogenic goiter (164;178). The mild phenotype can be explained by a partial maintenance of H₂O₂ production by Duox2/DuoxA1 as demonstrated in vitro. A high level of functional redundancy in Duox/DuoxA system could also explained the mild transient hypothyroidism in a patient with a novel biallelic DuoxA2 mutation and one allele of Duox2 and DuoxA1(179).

The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or by the activation of Duox1 or DuoxA1 in peculiar circumstances.

Fig 2-6: A, Localization of Duox and DuoxA genes on chromosomes 15q15.3. B, Schematic representation of the predicted structure of DuoxA (from (156). C, Immmunolocalization of human Duox and TPO at the apical membrane of the thyrocyte (upper: Duox immunostaining, middle:preimmune serum, lower:TPO immunostaining) (130).

THYROGLOBULIN (Tg)

Thyroglobulin is the most abundant protein in the thyroid gland; its concentration within the follicular lumen can reach 200-300 g/L. Its main function is to provide the polypeptide backbone for synthesis and storage of thyroid hormones (180). It also offers a convenient depot for iodine storage and retrieval when external iodine availability is scarce or uneven. Neosynthesised Tg polypeptide chains entering the lumen of the rough endoplasmic reticulum (RER) are subjected to core glycosylation, dimerise and are transferred to the Golgi where they undergo terminal glycosylation (Fig. 2-7). Iodination and hormone formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle colloid content.

The Tg peptide chain derives from a gene of more than 200 kbp located on chromosome 8 in humans. The human Tg gene consisting of 48 exons (181) gives rise to a 8.5kb transcript that translates a 2,749 residue peptide (in addition to a 19-residue signal peptide) (182;183). The primary structure deduced from cDNA is also known for bovine, rat, and mouse (184-186). The biochemical traits of human Tg have been reviewed in (187). The N-terminal part of Tg has regions of highly conserved internal homology (10 motives of about 60 amino acids) which appears in several other proteins and are referred to as ‘thyroglobulin type-1 domains’. Such domains have been found to be potent inhibitors of cysteine proteases (188). This finding might be of importance, because these proteases are active in Tg proteolysis (see below). It has been suggested that this region of the Tg molecule may modulate its own degradation and hormone release (189). In the Tg-type 1 repeats, cysteine and proline residues are found in constant position; they may have an important role in the tridimensional structure of the protein. The proximal region of the C-terminal half portion of Tg contains five repeats of another type of cysteine-rich motives. The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide bonds, probably gives rise to peculiar structural constraints. The C-terminal portion of Tg is homologous with acetylcholinesterases (190). Because binding to cell membranes is one feature of acetylcholinesterases, perhaps Tg C-terminus has a similar role. It was reported that the acetylcholinesterase-homology region of Tg could function as a dimerization domain (178;191-193).Furthermore, three highly conserved thioredoxin boxes have been identified in mammalian Tg between residues 1,440 and 1,474; these boxes might be involved in disulfide bond formation leading to intermolecular cross-linking of Tg molecules inside the follicle lumen (194) . Tg gene expression is controlled by the same main thyroid-specific transcription factors that regulate synthesis of TPO (108):TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 that bind at the same sites in Tg as they do in TPO. Hydrogen peroxide might be a regulatory factor of Tg expression, based on experimental work showing increased Tg promoter activity with reduced Pax-8 and TTF-1 (195-198). If substantiated, this proposal offers another point of integration between H₂O₂ generation and transcription of NIS, Tg and TPO genes, all of which being regulated by TSH.

Maturation of the Tg polypeptide chain begins while still on the RER. It undergoes core glycosylation and then monomers fold into stable dimers. Arvan and co-workers (199-204) have mapped this process and emphasize the role of molecular chaperones. The latter are essential for folding the new Tg molecules, and those that are folded improperly are not allowed to proceed further. The principal molecular chaperones are BiP, GRP 94, ERP 72, and calnexin. Only Tg molecules that pass this quality control system unscathed can proceed towards the secretory pathway. Glycosylation is a key event in Tg maturation. Carbohydrates comprise about 10% of Tg weight (205). Human Tg may contain four different types of carbohydrate units. The "polymannose" units consist only of mannose and N-acetylglucosamine. The "complex unit" has a core of three mannose residues with several chains of N-acetylglucosamine, galactose, and fucose or sialic acid extending from them. Both these types of unit are common in glycoproteins and are linked to peptide through an asparagine-N-acetylglucosamine bond. About three quarters of the potential N-glycosylation sites in human Tg are occupied, mostly with the complex unit (206). Two additional units have been found in human Tg; one contains galactosamine and is linked to the hydroxyl group of serine, the other is a chondroitin sulfate unit containing galactosamine and glucuronic acid (207) .

Failure in Tg folding can lead to disease as in the cog/cog mouse; these animals have a large thyroid with a distended ER and sparse Tg storage in follicles (208). Their Tg shows abnormal folding and decreased export from the ER in association with increased levels of several molecular chaperones. In the Tg cDNA of cog/cog mouse, Kim et al.(185) identified a single base substitution that changes leucine to proline at position 2,263. Correction of this defect by site-directed mutagenesis returned Tg export to normal in transfected cells. The cog/cog mouse is an example of endoplasmic reticulum storage disease (209). Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disorders (210). In each situation, the underlying defect appears to be a mutation in the coding sequence of exportable proteins. The ER retains the abnormal proteins, which cannot then proceed for further maturation. Several reports describe a similar pathogenesis for cases of congenital goiter and hypothyroidism in humans, although these are not as well characterized. Ohyama et al. (211) investigated a five-year-old euthyroid goitrous boy with high thyroidal radioiodine uptake, a positive perchlorate discharge test, apparently normal H₂O₂ generation and peroxidase activity in gland tissue, and low amounts of Tg in thyroid tissue overall, but large amounts in the RER. In another report, two hypothyroid goitrous sibs had a 138 bp segment missing between positions 5,590-5,727 in hTg mRNA, translating into a Tg polypeptide chain that lacked 46 residues (212). A third example described four subjects with congenital hypothyroid goiter from two unrelated families (213). Their thyroid tissue showed accumulation of Tg intracellularly with distension of the ER and large increases in activity of specific molecular chaperones, but with failure of Tg to reach the Golgi or the follicular lumen; this case was put forward as an ER storage disease similar to the cog/cog mouse (213).

Tg also contains sulphur and phosphorus. The former is present in the chondroitin sulfate and the complex carbohydrate units, although its form and role are not known (214). Several studies have reported phosphate in Tg, up to 12 mol. per mol Tg. Of this, about half is in the complex carbohydrate units, the remainder is present as phosphoserine and phosphotyrosine (215-217). This may relate to protein kinase A activity (218).

THYROGLOBULIN IODINATION AND HORMONE SYNTHESIS

The step preliminary to thyroid hormone formation is the attachment of iodine to tyrosyl residues in Tg to produce MIT and DIT. This process occurs at the apical plasma membrane-follicle lumen boundary and involves H₂O₂, iodide, TPO, and glycosylated Tg. All rendezvous at the apical membrane to achieve Tg iodination (Fig. 2-8).

First, iodide must be oxidized to an iodinating form. An extensive literature has sought to identify the iodinating species, but the issue is still not resolved (see (219) for a detailed review). One scheme proposes that oxidation produces free radicals of iodine and tyrosine, while both are bound to TPO to form MIT which then separates from the enzyme (Fig.2-5C). Further reaction between free radicals of iodine and MIT gives DIT. Experimental studies by Taurog (219) and others suggest that the TPO reduction occurs directly in a two electron reaction. A second proposal, based on studies of rapid spectral absorption changes (88;220;221), is that TPO-I⁺ is the iodination intermediate and that the preferred route is oxidation of TPO by H₂O₂ followed by two electron oxidation of I^- to I⁺, which then reacts within a tyrosine. As a third possibility, Taurog (219) proposed a reaction between oxidized TPO and I^- to produce hypoiodite (OI^-), which also involves a two electron reaction. Whatever the precise nature of the iodinating species, it is clear that iodide is oxidized by H₂O₂ and TPO, and transferred to the tyrosyl groups of Tg. All tyrosine residues of Tg are not equally accessible to iodination. The molecule has about 132 tyrosyl residues among its two identical chains; at most, only about 1/3 of the tyrosyls are iodinated. As isolated from the thyroid, Tg rarely contains more than 1% iodine or about 52 iodine atoms.

The final step in hormone synthesis is the coupling of two neighbouring iodotyrosyl residues to form iodothyronine (Fig. 2-9). Two DIT form T4; one DIT and one MIT form T3. Coupling takes place while both acceptor and donor iodotyrosyl are in peptide linkage within the Tg molecule.The reaction is catalyzed by TPO, requires H₂O₂ (222-225) and is stringently dependent on Tg structure (226).The generation of the iodothyronine residue involves the formation of an ether bond between the iodophenol part of a donor tyrosyl and the hydroxyl group of the acceptor tyrosyl (Fig 2-10). After the cleavage reaction that gives the iodophenol, the alanine side chain of the donor tyrosyl remains in the Tg polypeptide chain as dehydroalanine (227-229). Observations both in vivo and in vitro show an appreciable delay in coupling after initial formation of iodotyrosines. A typical distribution for a Tg containing 0.5% iodine (a normal amount for iodine-sufficient individuals) is 5 residues MIT, 5 of DIT, 2.5 of T4 and 0.7 of T3 (180). More iodine increases the ratios of DIT/MIT and T4/T3, while iodine deficiency decreases them.

The distribution of hormone among several sites in the Tg molecule has been studied in a number of species (180;230-233). The most important is at tyrosyl 5, quite close to Tg N-terminus. It usually contains about 40% of Tg total T4. The second most important site is at tyrosyl 2554, which may contain for 20-25% of total T4. A third important site is at tyrosyl 2747, which appears favored for T3 synthesis in some species. Tyrosyl 1291 is prominent in T4 formation in guinea pigs and rabbits and very responsive to TSH stimulation. Incremental iodination of low iodine hTg in vitro, with lactoperoxidase as surrogate for TPO, led to the identification of the favored sites for iodination (234). Small increments of iodine go first to tyrosyl residues 2554, 130, 685, 847, 1448, and 5, in that order. Further addition increases the degree of iodination at these sites, iodinates some new tyrosyls, and results in thyroid hormone formation at residues 5, 2554, 2747, and 685, with a trace found at 1291, in that quantitative order. These data identified the most important hormonogenic sites in hTg, and also the favored sites for early iodination. The same work recognized three consensus sequences associated with iodination and hormone formation: i) Asp/Glu-Tyr at three of the four most important sites for hormone synthesis, ii) Ser/Thr-Tyr-Ser associated with hormone formation, including the C-terminal hormonogenic site that favors T3 in some species and iii)Glu-X-Tyr favoring early iodination, although usually not with hormone formation (Fig. 2-11).

Identifying the donor tyrosyls has attracted considerable investigational interest over the past several decades. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of T4 makes them potential donor candidates (234). On the basis of in vitro iodination of an N-terminal cyanogen bromide Tg peptide, Marriq et al. (235) concluded that residue 130 was a donor tyrosine for the major hormonogenic site at Tyr5. This conclusion was challenged by Xiao et al. (236) in a similar in vitro system. A baculovirus system expressing the 1-198 fragment of Tg, either normal or mutated on tyrosyl residues, showed that iodination of a fragment containing tyrosyls only at residue 5, 107 and 130 formed T4 as did the intact normal peptide, but this fragment could also form T4 with substitutions at residue 5 or 130 (237). Dunn et al.(238) who incorporated 14C-Tyr into beef thyroid slices followed by in vitro iodination and trypsin digestion of the N-terminal portion of Tg localized pyruvate (as a derivative of dehydroalanine) to residue 130 by mass spectrometry. They proposed that Tyr130 was the donor tyrosine for the most important hormonogenic site at Tyr5. Gentile et al. (239) used mass spectrometry to identify a peptide containing dehydroalanine at tyrosine 1375 of bTg and proposed this tyrosine as the donor for the hormonogenic site at residue 1291. Donors for the other major hormonogenic sites have not yet been identified.

In addition to its role as component of the iodoamino acids, iodine is associated with cleavage of peptide bonds of Tg, at least in vitro (180). This has been attributed to generation of free radicals during oxidation (240). Exposure of Tg to reducing agents yields an N-terminal peptide of about 20-26kDa, depending on the animal species, that contains the major hormonogenic site of Tg (241). This peptide appears in parallel with iodination or may slightly precede it (242). Further addition of iodine cleaves the 26kDa further, to produce an 18kDa (on human Tg), an event that also occurs with TSH stimulation (242). Thus, iodination-associated cleavage appears to be part of the maturation of the Tg molecule. These discrete N-terminal peptides have been found in all vertebrate Tg examined so far (231).

The amount of iodine has important effects on thyroid hormone production (243). The initial reaction between TPO and H₂O₂ produces the so-called "compound I," which oxidizes iodide and iodinates Tg. Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid hormones. However, if excessive iodine is present, conversion to compound II does not take place, and hormone synthesis is impaired. (Fig. 2-12) Other iodinated compounds occasionally inhibit the thyroid. Thyroalbumin excited considerable interest several decades ago. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid (244). Occasionally, large amounts are found in certain thyroid diseases, including Hashimoto's thyroiditis (245), congenital metabolic defects (246), thyrotoxicosis (247) and thyroid carcinoma (248). In all these cases, there are abnormalities in thyroid structure which might explain the access of serum albumin to intrathyroidal iodination sites. However, in physiological conditions, serum albumin can reach thyroid follicle lumina by transcytosis i.e. basolateral endocytosis and vesicular transport to the apical plasma membrane (249). The thyroid also iodinates lipids and many different iodolipids have been described after high doses of iodide in vitro (250;251). Of particular interest is 2-iodohexadecanal (252;253). It occurs in the thyroid of several species following administration of KI, and its amount increases linearly with additional iodine, in contrast to iodination of Tg which eventually is inhibited by excess iodide. This compound inhibits the action of NADPH oxidase, which is responsible for H₂O₂ production (254;255). These findings suggested that iodination of lipids impairs H₂O₂ production and, therefore, decreases further Tg iodination. This is the most probable mechanism for the Wolff-Chaikoff effect (128).

HORMONE STORAGE

Tg molecules vectorially delivered to the follicule lumen by exocytosis accumulates to reach uncommun concentrations i.e. 0.3-0.5 mM.The mechanism operating such a “packaging” is unknown. Water and ion extraction from the follicle lumen might represent an active process leading toTg concentration. As the follicle lumen is a site of Ca⁺⁺ accumulation (256;257), the high degree of compaction of lumenal Tg might depend on electrostatic interactions between Ca⁺⁺ and anionic residues of Tg, which is an acidic protein. Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma membrane-lumen boundary (257-259), where TPO and H₂O₂ generating system reside. The mature Tg molecules, now containing MIT, DIT, T4 and T3, remains extracellular in the lumen of thyroid follicles. Turnover of intrafollicular material or so-called colloid varies greatly with gland activity. For normal humans, the organic iodine pool (largely in intrafollicular material), turns over at a rate of about 1% per day (14). When the turnover increases, less Tg is stored, and with extreme hyperplasia, none is evident and the entire organic iodine content may be renewed daily (14). In this situation, secretion of Tg and resorption of Tg (see below) probably occur at similar rates and only tiny amounts of intrafollicular material are present at any time.

Thyroglobulin as usually isolated from the thyroid is chiefly the 19S 660kDa dimer that has been glycosylated and iodinated. Iodination and hormone formation of Tg is more complex than generally thought because of the slow diffusion of molecules that are in a colloidal state in the follicle lumen. It has been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules (260;261). The diffusion coefficient of Tg which is about 26mm² / sec in water would only be in the order of 10-100mm² / hour in the thyroid follicle lumen. There is evidence for the presence of insoluble Tg in the form of globules of 20-120 microns, at a protein concentration of almost 600 mg/mL, in the lumen of thyroid follicles of different animal species (262). In human, about 34% of the gland Tg would be in this form (263). In pig, insoluble Tg contains more iodine than did the 660kDa Tg, and had virtually no thyroid hormone (264). Insoluble Tg has many internal crosslinks through disulfide bonds, dityrosine, and glutamyl-lysine bonds, the latter generated by transglutaminase (265). The formation of Tg multimers that probably results from oxidative processes might be limited by the presence of molecular chaperones such as the protein disulfide isomerase (PDI) and BiP in the follicle lumen (266).

THYROGLOBULIN ENDOCYTOSIS

To be useful, thyroid hormones must be released from Tg and delivered to the circulation for action at their distant target tissues. Depending on numerous factors including - the supply of iodide as substrate, the activity of enzymes catalyzing hormone formation, the concentration and physico-chemical state of Tg - the hormone content of lumenal Tg molecules varies to a rather large extent. Tg molecules newly arrived in the follicle lumen with no or a low hormone content would co-exist with “older” Tg exhibiting up to 6-8 hormone residues. The downstream processes responsible for the production of free thyroid hormones from these prohormonal molecules must therefore adequately manage the use of these lumenal heterogeneous Tg stores to provide appropriate amounts of hormones for peripheral utilization. One would expect to find i) control systems preventing excess hormone production that would result from the processing of excessive amounts of prohormonal Tg molecules and ii) checking systems avoiding the use of Tg molecules with no or a low hormone content.

The way the thyroid follicle proceeds to generate free hormones from stored hormone containing Tg molecules has been known for a long time. Tg molecules are first taken up by polarized thyrocytes (Fig. 2-13) and then conveyed to lysosomal compartments for proteolytic cleavage that release T4 and T3 from their peptide linkages. The first step represents the limiting point in the thyroid hormone secretory pathway. Over the last decade, there has been substantial improvement in the knowledge of the cellular and molecular mechanisms governing the internalization or endocytosis and intracellular transport of the prohormone, Tg. The evolution has first been to consider that it could proceed via a mechanism different from phagocytosis, also named macropinocytosis, evidenced in rats under acute TSH stimulation (reviewed in (267)). Results obtained in rats and dogs have been for a long time extrapolated to the different animal species including human. There is now a number of experimental data indicating that in the thyroid of different species under physiological circumstances, basal internalization of Tg, mainly if not exclusively, occurs via vesicle-mediated endocytosis or micropinocytosis (reviewed in (268)), while macropinocytosis results from acute stimulation (Fig. 2-14) (269;270).

The internalization process starts with the organization of microdomains at the apical plasma membrane of thyrocytes; these microdomains or pits, resulting from the recruitment and assembly of proteins (clathrin, adaptins…) on the cytoplasmic side of the membrane, invaginate to finally generate coated vesicles after membrane fission. Lumenal Tg molecules, either free or associated to membrane proteins acting as Tg receptors, enter the pits and are then sequestrated into the newly-formed vesicles (267-269). Tg internalization via vesicle-mediated endocytosis is regulated by TSH (268). The vesicles lose their coat and, through a complex fusion process, deliver their content into a first type of endocytic compartments, the early apical endosomes (270) (Fig 2-15). In these compartments, Tg molecules probably undergo sorting on the basis of recognition of different physico-chemical parameters either linked or independent such as the hormone content, exposed carbohydrates, conformation of peptide domains… A step of sorting appears as a prerequisite for subsequent differential cellular handling of Tg molecules. It has been shown that internalized Tg molecules can follow different intracellular pathways. Part of Tg molecules are conveyed via a vesicle transport system to the second type of endocytic compartments, late endosomes or prelysosomes. This route ending to lysosomes corresponds to the Tg degradation pathway for the generation of free thyroid hormones. It is reasonable to think that Tg molecules following this route are the more mature molecules (with a high hormone content) but, this has not been firmly demonstrated. The other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport towards the apical plasma membrane (271) or via a two-step vesicular transport to the Golgi apparatus and then to the apical plasma membrane (272). Alternately, Tg molecules are transported and released at the basolateral membrane domain of thyrocytes via transcytotic vesicles (262;273); a process accounting for the presence of Tg in plasma. The orientation of Tg molecules towards one or the other of these three routes requires the presence of receptors. However, one route could simply convey Tg molecules that are not selected for entering the other pathways.

Receptors involved in Tg endocytosis may operate at the apical plasma membrane for Tg internalization and downstream in apical early endosomes for Tg sorting. The requirement and/or the involvement of apical cell surface receptors has long been debated. Most investigators now recognize that receptors are not needed for internalization since Tg is present at a high concentration at the site of vesicle formation. So, Tg molecules are most likely internalized by fluid-phase endocytosis and not by receptor-mediated endocytosis. On the contrary, if apical membrane Tg receptors exist, their function would be to prevent the internalization of sub-classes of Tg molecules (274;275). As it is not conceivable that internalized Tg molecules could enter the different intracellular routes, described above, at random, Tg receptors must exist in early apical endosomes. A detailed review on potential Tg receptors has been made by Marino and Mc Cluskey (276).

The first candidate receptor, initially described by Consiglio et al.(277;278) was later identified as the asialoglycoprotein receptor composed of three subunits (RLH1,2 and 3). This receptor binds Tg at acidic pH and recognizes both sugar moities and peptide determinants on Tg (279). As low-iodinated Tg molecules are known to have a low sialic acid content, this receptor could be involved in sorting immature Tg molecules for recycling to the follicle lumen. A second receptor, still not identified, named N-acetylglucosamine receptor (280;281), presumably located in sub-apical compartments, interacts with Tg at acidic pH; it could also act as a receptor for recycling immature Tg molecules back to the follicle lumen. A third receptor; megalin, has more recently been discovered in the thyroid and has been the subject of extensive studies yielding convincing data (276;282-285). Megalin is an ubiquitous membrane protein belonging to the LDL receptor family. It is located in the apical region of thyrocytes and its expression is regulated by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity.In vitro and in vivo data indicate that Megalin is involved in the transcellular transport or transcytosis of Tg molecules, possibly with a low hormone content (286).

From the properties and subcellular location of these receptors, one can propose an integrated view of the sorting processes that would operate in early apical endosomes. The asialoglycoprotein receptor and/or the less defined N-acetylglucosamine receptor would recognize immature Tg for recycling and megalin would interact with Tg subjected to apical to basolateral transcytosis. The remaining Tg molecules would enter, without sorting, the functionally important pathway i.e. the prelysosome-lysosome route.

Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg internalization. Pseudopods representing extensions of the apical plasma membrane project into the follicle lumen and pinch off to form a resorption vacuole known as colloid droplet (287) .The colloid droplets then deliver their content to lysosomes. Pseudopod formation is one of the earliest effects of TSH on the gland, evident within several minutes after administration (288;289). In most species but perhaps not in rat, TSH stimulates macropinocytosis through the activation of the cyclic AMP cascade (290;291).

 

PROTEOLYTIC CLEAVAGE OF THYROGLOBULIN

Internalized Tg molecules that are conveyed to lysosome compartments are subjected to diverse hydrolytic reactions leading to the generation of free thyroid hormones and to complete degradation of the protein. Given its composition, Tg is likely the substrate for the different lysosomal enzymes: proteases, glycohydrolases, phosphatases, sulfatases.... Efforts have been made to identify proteases involved in the release of hormonal residues from their peptide linkage in Tg. Endopeptidases such as cathepsin D, H and L (292-299) are capable of cleaving Tg.

Initial cleavage would bring into play endopeptidases and resulting products would be further processed by exopeptidases. Dunn et al. (295) showed that cathepsin B has exopeptidase activity as well as an endopeptidase action (295;297). These investigators tested the activities of human enzyme preparations against the 20kDa N-terminal peptide from rabbit Tg, which contains the dominant T4 site at residue 5. Extended cathepsin B incubation produced the dipeptide T4-Gln, corresponding to residues 5 and 6 of Tg. The combination of cathepsin B with the exopeptidase dipeptidase I released T4 from this dipeptide, although lysosomal dipeptidase I alone was not effective. Thus, the combination of cathepsin B and lysosomal dipeptidase I was sufficient to release free thyroid hormone from its major site at residue 5. The exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg other than residue 5 (297). Thus, Tg probably undergoes selective cleavage reactions at its N- and C- terminal ends to release iodothyronines that are located nearby (297;300). Starting from highly purified preparations of thyroid lysosomes, Rousset et al. (301-303) have identified intralysosomal Tg molecules with very limited structural alterations but devoid of hormone residue. One may think that proteolysis of Tg occurs in two sequential steps; i) early and selective cleavages to release T3 and T4 residues and ii) delayed and complete proteolysis. The reduction of the very high number of disulfide bonds might be the limiting reaction between the two steps. The nature and the origin of the reducing compounds acting on Tg are not known. Noteworthy, the possibility of proteolytic cleavage of Tg inside the follicle lumen, before internalization, has been proposed (304-307) but not yet confirmed by other groups.

After Tg digestion, T4 and T3 must go from the lysosomal compartments to the cytoplasm and from the cytoplasm out of the cell to enter the circulation. It has been postulated for decades that thyroid hormones are released from thyrocytes by simple diffusion. There are many objections to this view (308). One of these comes from the chemical nature of iodothyronines; T4 and T3, which are generally considered as lipophylic compounds possess charges on both their proximal (amino acid side chain) and distal (phenolate) parts. As now known for the entry of thyroid hormones in peripheral target cells, the exit of thyroid hormones from thyrocytes probably involves membrane transporter(s). Details of hormone transport across the lysosomal membrane and then across the basolateral plasma membrane are unknown, including whether it is an active or passive process. At present, only a lysosomal membrane transporter for iodotyrosines has been reported (309;310). Nevertheless the role of newly cloned peripheral tissue thyroid transporters (311;312) in this process remains to be defined.

The type I and type II iodothyronine 5'-deiodinase is present in the thyroid (63;313;314) and deiodinate about 10% of T4 to T3. The extent of this intrathyroidal deiodination is increased when the thyroid is stimulated by TSH (315;316). Estimates of average normal secretion for euthyroid humans are 94-110 µg T4 and 10-22 µg T3 daily (317). The thyroid may also convert some T4 to 3,3'5'-T3 (reverse T3) within the thyroid. About 70% of the Tg iodine content is in the form of DIT and MIT, so this represents an important part of the intrathyroid iodine pool. Rather than lose it to the circulation, the thyroid deiodinates MIT and DIT and returns most of iodide to the intrathyroidal iodide pool. The responsible enzyme i.e. the iodotyrosine deiodinase is an NADPH-dependent flavoprotein with a estimated molecular weight of about 42kDa (318) and recently identified as DEHAL1(319-322). About 3-5 times more iodide is formed inside the gland each day by this deiodinase than enters the cell from the serum (14). The importance of the internal recycling of iodide is demontrated by congenitally goitrous subjects who harbour mutations in DEHAL gene (322) and cannot deiodinate iodotyrosines. These patients are successfully treated with large amounts of iodide (323). Some iodine is lost from the gland through inefficiency of its recycling by the iodotyrosine deiodinase (14;87;317;324). This leak may increase as the thyroid adapts to a high daily iodine intake (325), possibly as an autoregulatory process to prevent excessive Tg iodination. Much more iodide can be lost from diseased glands. Ohtaki et al. (87) found that some iodide leaks from all glands, including normal ones, but that the amount increases markedly with gland iodine content, presumably reflecting a dependence on dietary iodine intake. Fisher et al. (317) reported that about 38 µg iodide was released when the mean T4 secretion was 53 µg/day.

Among other products which are released or leak out from the thyroid, there is Tg (326;327). The secretion of Tg is clinically important. Its presence in serum can be detected by a routine assay and provides a sensitive (although not always specific) marker for increased thyroid activity. Attempts have been made to determine the biochemical characteristics of circulating Tg molecules in terms of iodine content (328), structural integrity (329) and hormone content (330). Serum levels are elevated in patients with hyperplastic thyroid or thyroid nodules including differentiated thyroid cancer. Tg measurement can identify congenital hyperplastic goiter, endemic goiter, and many benign multinodular goiters, but its greatest application is in the follow-up of differentiated thyroid cancer (331). Most papillary and follicular cancers retain some of the metabolic functions of the normal thyrocyte, including the ability to synthesize and secrete Tg. Subjects who have differentiated thyroid cancer treated by surgery and radioiodine should not have normal thyroid tissue left, and therefore, should not secrete Tg. If Tg is found in their serum, it reflects the continuing presence either of normal tissue, unlikely after its previous ablation, or of thyroid cancer. The depolarized cancer cells presumably secrete Tg directly in intercellular space. Tracking serum Tg levels is probably the most sensitive and practical means for the follow-up of such patients. It is more sensitive when the subject is stimulated by TSH. Until recently, this could only be done by withdrawal of thyroid hormone and consequent symptomatic hypothyroidism, but now recombinant human TSH can be administered to enhance the sensitivity of the serum Tg and thyroid scan (332-337).

CONTROL OF HORMONE SYNTHESIS

The most important controlling factors are iodine availability and TSH. Inadequate amounts of iodine lead to inadequate thyroid hormone production, increased TSH secretion and thyroid stimulation, and goiter in an attempt to compensate. Excess iodide acutely inhibits thyroid hormone synthesis, the Wolff-Chaikoff effect (243), apparently by inhibiting H₂O₂ generation, and therefore, blocking Tg iodination (127). A proposed mechanism is that the excess iodide leads to the formation of 2-iodohexadecanal (255), which is endowed with an inhibitory action on H₂O₂ generation.

TSH influences virtually every step in thyroid hormone synthesis and release. In humans the effects on secretion appear to be mediated through the cAMP cascade (see chapter 1) while the effects on synthesis are mediated by the Gq/phospholipase C cascade (338). Elsewhere in this chapter, we have mentioned instances of TSH regulation. To summarize, TSH stimulates the expression of NIS, TPO, Tg and the generation of H₂O₂ , increases formation of T3 relative to T4, alters the priority of iodination and hormonogenesis among tyrosyls and promotes the rapid internalization of Tg by thyrocytes. These several steps are interrelated and have the net effects of increasing the amount of iodine available to the cells and of making and releasing a larger amount and a more effective type of thyroid hormone (T3).

Anti-thyroid drugs are external compounds influencing thyroid hormone synthesis. The major inhibitory drugs are the thionamides: propylthiouracil and methimazole. In the thyroid, they appear to act by competing with tyrosyl residues of Tg for oxidized iodine, at least in the rat (219). Iodotyrosyl coupling is also inhibited by these drugs and appears more sensitive to their effects than does tyrosyl iodination.

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