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1906 Section V Pathophysiology

C HA P T E R Changes in Fetal Thyroid Function During Gestation


The Thyroid: TSH

Pathophysiology and
Thyroid Function Testing
William E. Winter, M.D., Desmond Schatz, M.D.,

Fetal thyroid develops ~4 wks

Colloid present in fetal thyroid ~11 wks
Fetal thyroid functional ~12 wks
Hypothalamic-portal system ~20 wks
Fetal TSH increases ~20 wks
Fetal thyroid hormone production ~18-20 wks

TRH in CNS ~5 wks

Thyroid descent to neck ~7 wks
TRH in hypothalamus: ~10-14 wks
TSH in pituitary and serum ~10 wks
and Roger L. Bertholf, Ph.D.*

10 20 30 40
Weeks of Gestation
he thyroid is a butterfly-shaped gland located in the fortification is widespread, the most common cause of Figure 55-1 Changes in fetal thyroid function during gestation. During the rst half
front of the neck, just above the trachea. The fully primary hypothyroidism is Hashimoto thyroiditis (chronic of gestation, the fetus is dependent on transplacental passage of thyroid hormone.
T developed thyroid gland in a human is composed of autoimmune thyroiditis/inflammation of the thyroid gland). After mid gestation, the fetus produces its own thyroid hormone.
two lobes connected by a thin band of tissue, the isthmus, Hypothalamic thyrotropin-releasing hormone (TRH)
which gives the gland the appearance of a butterfly. The gland stimulates thyrotropin [thyroid-stimulating hormone (TSH)],
is closely attached to the trachea in the anterior aspect of the which in turn stimulates thyroid hormone synthesis. By 10 the lobes are 2.0 to 2.5 cm thick by 2.0 to 2.5 cm wide by
neck. Anatomically and embryologically, the thyroid gland weeks, fetal thyroid follicles and thyroxine synthesis are 4 cm high. The lobes are connected by the isthmus, which
is two glands in one: the thyroid follicular cells produce demonstrable. By the mid second trimester, maturation of is 0.5 cm thick by 2.0 cm wide by 1 to 2 cm high.
thyroid hormones and the parafollicular (or C) cells secrete the hypothalamic-pituitary-thyroid axis occurs, so that by 20 Microscopically, the thyroid gland is composed of follicles
calcitonin.21 weeks, the fetus is becoming responsible for its own produc- or acini (Figure 55-3, A). The outside of the follicular unit/
tion of thyroid hormone. Thyroxine-binding globulin (TBG) acinus is enveloped by a basement membrane. Along the
and thyroxine are first detectable in fetal serum at 8 to 10 Total T4
inside of the basement membrane are squamous or cuboidal
THYROID GLAND: STRUCTURAL AND weeks gestation and increase thereafter until they plateau at epithelial follicular cells. The height of the follicular cells
FUNCTIONAL ONTOGENY 35 to 37 weeks (Figure 55-1). Thyroxine [tetraiodothyronine reflects their biochemical activity: the greater their height, the

Relative concentration
Embryologically at day 24, the thyroid gland develops from (T4); see Table 55-1 for nomenclature and abbreviations) and more thyroid hormone synthetic activity is occurring. The
an anterior outpouching of the foregut. From this thyroid TSH rise until birth, with T4 near 10 g/dL and TSH reach- Total T3 basal pole of the follicular cell is oriented toward the base-
diverticulum, the thyroid gland descends, and the process is ing a concentration of 7 to 10 mIU/L. Beginning at 30 ment membrane, and the apical pole is oriented toward the
usually complete at 7 weeks. Failure of descent can produce weeks, T4 is converted to triiodothyronine (T3) by increased center of the follicle (Figure 55-3, B). In the center of the fol-
(1) a lingual thyroid gland (located at the base of the tongue), activity of the type 1 deiodinase (D1), so that T3 concentra- licle, lined by the apical aspects of the follicular cells, is the
(2) an ectopic gland, or (3) a hypoplastic or aplastic gland. tions rise, while reverse T3 (rT3) concentrations decline. At 30 0 1 2 3 4 5 lacuna, which contains colloid composed predominantly of
Coincident with thyroid development, the parafollicular (C weeks, T3 is less than 15 ng/dL; at term, T3 is 50 ng/dL. Days after birth thyroglobulin.
cells) cells (producing calcitonin) develop. Within hours of birth, TSH, T4, and T3 rise rapidly (Figure On average, follicles are 200 microns wide (compared
Figure 55-2 Concentrations of thyroid hormones for 5
The fetus is dependent on the transplacental passage of 55-2).52 Cold stress is believed to be responsible for the days after birth. After birth, an immediate TSH surge
with a normal red blood cell diameter of 7 microns). The
maternal thyroid hormone for the first half of gestation.26,29,65,225 massive TSH surge to concentrations of 70 to 100 mIU/L. By peaks at 30 minutes. T3 and T4 rapidly rise, peaking at parafollicular cells usually are located below the basement
Although only minute amounts of maternal thyroid hormone 2 to 3 days, the TSH concentration falls in most cases to less 24 hours after delivery. The greater initial rise in T3 as membrane but are not adjacent to the lumen (lacuna) of the
cross the placenta, maternal hypothyroidism in the first 20 than 20 mIU/L. Total T4 peaks near 17 g/dL. T4 then falls compared with T4 in the rst 24 hours of life likely follicle. Without immunohistochemical staining for calcito-
weeks of gestation may adversely impact fetal central nervous to adult concentrations by 1 to 2 months of age. The postbirth represents acutely increased conversion of T4 to T3. nin, the parafollicular cells are very difficult to identify.
system (CNS) development, leading to neuropsychologic rise in T3 results from increased thyroid gland release in Following their peak concentrations, T4 and T3 decline Branches of the common carotid artery (the superior
impairment in infants and children.178 Worldwide, the most response to the rising TSH concentration and increased con- to stable concentrations. thyroid arteries) and the subclavian arteries (the inferior
common cause of hypothyroidism during pregnancy is iodine version of T4 to T3 due to maturation of the type 1 deiodinase thyroid arteries) supply the thyroid. It is surprising to
deficiency. Conversely, in developed countries, where iodine enzyme (D1). note that on a per gram basis, the thyroid gland receives
THYROID GLAND ANATOMY more blood than the kidney: 4 to 6 mL/min/g of thyroid
The adult thyroid gland weighs between 15 and 20 g. However, tissue versus 3 mL/min/g of kidney. In cases of hyperthy-
in disease states, the gland can attain a weight of several roidism, blood flow to the entire thyroid gland can exceed
*The authors gratefully acknowledge the previous contributions of L. M. Demers and C. Spencer, on which portions of this hundred grams. The thyroid gland is normally bilobed, with 1000 mL/min, which is 20% of the normal cardiac output
chapter are based. the right lobe somewhat larger than the left lobe. In adults, of an adult.

Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1907 1908 Section V Pathophysiology

Basal lamina the alpha (inner) ring, and the 3 and 5 positions are on the Iodination of tyrosine to ultimately produce T4 and T3
TABLE 55-1 Nomenclature and Abbreviations
beta (outer) ring. The biologically important thyroid hor- involves the tyrosine residues on thyroglobulin.86 An isomer
for Thyroid Tests Follicular mones are 3,5,3,5-tetraiodothyronine [thyroxine (T4)] and of T3 is reverse T3 (rT3), in which the alpha ring is monoiodin-
Name Abbreviation 3,5,3-triiodothyronine (T3). Debate continues over whether ated and the beta ring is di-iodinated, producing 3,3,5-l-tri-
T4 has any intrinsic biological activity, or whether it is a iodothyronine. Reverse T3 is not biologically active. Almost
Hormones prohormone. all rT3 is formed by the extrathyroidal conversion of T4 to rT3,
Total thyroxine T4 Interfollicular
and approximately 80% of T3 is formed by extrathyroidal
Total triiodothyronine T3
Colloid monodeiodination of T4 to T3.
Thyroid hormone biosynthesis begins with the active
Free thyroxine FT4 Parafollicular H3N COO H3N COO H3N COO
transport of iodide (I) into the thyroid gland via the Na+/I
Free triiodothyronine FT3 C cells CH2 CH2 CH2
Thyrotropin (thyroid-stimulating TSH (sodium-iodide) symporter (NIS, SLC5A; SLC = solute
A carrier; chromosome 19p12-13.2; Figure 55-5).20 Iodide
I I I I I transport is inhibited by lithium, which competes with
Reverse T3 (3,3,5-triiodothyronine) rT3 O O O
Interfollicular sodium, but the NIS transports other anions besides iodide.
Serum-Binding Proteins In the follicular cells, the iodine concentration is 30- to
I I I I I 40-fold greater than the circulating concentration. When the
Thyroxine-binding globulin TBG OH OH OH
Thyroxine-binding prealbumin TBPA Basal thyroid gland is stimulated by TSH, even larger iodide con-
lamina T4 T3 rT3
(transthyretin) Tetraiodo- 3,5,3 3,3,5 centration gradients can result. Antithyroid drugs such as
Thyroxine-binding proteins TBP thyronine Triiodo- Triiodo- propylthiouracil and methimazole inhibit iodination and
(Thyroxine) thyronine thyronine coupling; this can lead to an 800-fold difference in the con-
Tests for Autoimmune Thyroid Disease Figure 55-4 Chemical structures of T4, T3, and rT3. centration of iodide in the thyroid gland versus the plasma.
Thyroglobulin autoantibodies TGA
Thyroid microsomal autoantibodies TMA Tight
junction Basolateral
Thyroperoxidase autoantibodies TPOA pole
TSH receptor autoantibodies TRA Capillary basement membrane T4 , T 3
Thyroid-stimulating immunoglobulins TSI B Follicle basement membrane
Thyrotropin-binding inhibitory TBII Figure 55-3 A, Basic unit of the thyroid gland. The follicle
immunoglobulin is the basic unit of the thyroid gland. It is composed of T4, T3, MIT
Primary lysosome
thyroid follicular cells surrounding the colloid. Outside
Other Hormones, Thyroid-Related Proteins, the follicular cells is a basal lamina. B, Apical and DIT, AAs

and Conditions basolateral poles of the follicular cells and tight junctions

Thyrotropin-releasing hormone TRH between the follicular cells. Parafollicular (C cells) that Secondary
Thyroglobulin Tg secrete calcitonin can be found beneath or outside the lysosome Dehalogenase

Thyroid hormone receptor TR basal lamina. Not pictured between the follicles are
capillaries and broblasts. Pinocytotic uptake Golgi Tg
Thyroperoxidase TPO RER
TSH receptor autoantibodies TRA Tg-iodination I
Thyroid hormone receptor-alpha THRA and coupling Tg
Thyroid hormone receptor-beta THRB (4) stimulate protein synthesis and carbohydrate metabolism,
Autoimmune thyroid disease AITD (5) increase synthesis and degradation of cholesterol and tri- Tg
Calcitonin CT glycerides (e.g., regulation of low density lipoprotein receptor Na/I
expression by the liver), (6) increase vitamin requirements, I
and (7) regulate calcium and phosphorous metabolism. Colloid
Thyroid hormones maintains the basal metabolic rate and
BIOLOGICAL FUNCTION thus regulates the metabolism of endogenous and exogenous I
Thyroid hormones (T4 and T3) bind to intranuclear receptors substances. Hypothyroidism impairs the excretion of many
that function as transcription factors and thereby regulate drugs, with hyperthyroidism accelerating their clearance. Pendrin
gene expression. Thyroid hormones have ubiquitous effects Figure 55-5 Synthesis of thyroid hormones begins with absorption of iodide by the
on growth and development in the fetus, child, and adoles- thyroid follicular cell and the Na+/I symporter (NIS). From the cytoplasm, iodide
cent, and they regulate calorigenesis and metabolic rate BIOCHEMISTRY moves into the lacunae via pendrin. Within the lacunae, thyroperoxidase (TPO)
throughout life. At a molecular level, thyroid hormones Thyroid hormone is derived from the amino acid tyrosine. and the dual oxidases [DUOX (not depicted)] convert iodide to iodine, leading to
iodination of tyrosine residues on thyroglobulin (Tg). Tg is synthesized in the cell and
(1) increase oxygen consumption within tissues via increased Thyronine is produced by substitution of a second phenol
exported to the lacunae. TPO is responsible for the coupling of monoiodotyrosine
membrane transport (cycling of sodium/potassium ATPase moiety for the phenolic hydrogen on tyrosine, producing a
(MIT) and di-iodotyrosine (DIT) to form T3, and di-iodotyrosine and di-iodotyrosine
with increased synthesis and consumption of adenosine diphenyl ether having two phenol rings attached to one to form T4. Upon uptake of iodinated Tg (containing T4 and T3) and fusion of this
triphosphate), (2) enhance mitochondrial metabolism another through an ether linkage (Figure 55-4). phagosome-like vesicle with a primary lysosome, Tg is degraded in a secondary
(stimulation of mitochondrial respiration and oxidative There are four possible sites for iodine attachment to thy- lysosome, releasing T4 and T3 into the circulation and MIT and DIT undergo
phosphorylation), (3) increase sensitivity to catecholamines ronine at the meta positions on both phenyl rings, designated deiodination via a dehalogenase to recycle the iodine for new thyroid hormone
with increased heart rate and myocardial contractility, the 3, 5, 3, and 5 positions. The 3 and 5 positions are on synthesis.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1909 1910 Section V Pathophysiology

Once in the thyroid follicular cells, cytoplasmic iodide Hydrogen peroxide (H2O2) serves as the terminal electron OH Coupling OH
moves into the lacuna (colloid) via pendrin (SLC26A4 gene on acceptor, forming H2O2.185 Hydrogen peroxide is generated I H reaction I H
chromosome 7q21-34) (see Figure 55-5).88 Pendrin is a passive at the apical membrane by the action of DUOX1 and DUOX2 Thyroperoxidase
iodide-transporting glycoprotein identified during studies of (DUOX was previously known as THOX, or thyroid oxidase).
patients with Pendred syndrome. Pendred syndrome is an DUOX is a dual oxidase that has domains analogous to the OH O
autosomal recessive disorder of dyshormonogenesis that is domains found in nicotinamide adenine dinucleotide phos- Monoiodotyrosine
characterized by goiter and sensorineural deafness. phate (NADPH) oxidoreductases. Hypothyroidism due to (within Tg)
Thyroglobulin (Tg) is necessary for thyroid hormone syn- DUOX mutations has been reported.130
thesis. Similar to other proteins, Tg is synthesized in follicular TPO catalyzes the monoiodination of Tg tyrosines to
epithelial cells by ribosomes located on the endoplasmic monoiodotyrosine (MIT) (Figure 55-6). Di-iodination of Diiodotyrosine T3 (within Tg)
reticulum (see Figure 55-5). Tg may represent up to 50% of tyrosine forms di-iodotyrosine (DIT) (Figure 55-6). After Tg (within Tg)
protein synthesis in the thyroid gland and may account for is iodinated to form MIT and DIT, TPO catalyzes the cou- Figure 55-8 Chemical coupling of one molecule of monoiodotyrosine and one
75% of glandular total protein. pling reaction to produce T4 (Figure 55-7) and T3 (Figure molecule of di-iodotyrosine to produce one molecule of T3. The reaction is catalyzed
The 42-exon gene encoding Tg is located on chromosome 55-8). In the coupling reaction, T3 (bound to Tg) is formed by thyroperoxidase.
8q24 and spans 250 kilobases. Tg is a glycoprotein homodi- from one DIT and one MIT residue with the transfer of one
mer of 660 kDa. A total of 134 tyrosine residues are found in monoiodinated phenolic group to a DIT residue. T4 (bound

the homodimer, and 25 to 30 of these residues are iodinated. to Tg) is formed from two DIT residues with the transfer of
However, T4 is formed only at residues 5, 1290, and 2553, and one di-iodinated phenolic group to another DIT residue. CH2 CH2 CH2
T3 is formed only at residue 2746. The Tg sedimentation coef- Tg, along with its T3 and T4 residues, remains in the colloid,
ficient of 19S is similar to that of circulating pentameric IgM, providing a reservoir of thyroid hormone. With TSH stimula- I I D1,D2 I I
D3 I
reflecting the large size of Tg. TSH is the principal stimulator tion, the apical pole of the follicular cell releases colloid into
of Tg synthesis. Thyroid transcription factor 1 (TTF1) inter- a vesicle by pinocytosis (see Figure 55-5). The follicular cell
acts with the Tg promoter to stimulate Tg mRNA synthesis. digests the intravesicular colloid containing Tg after fusion of I I I I I
Although most Tg is secreted into the follicular lumen, a the phagosome body with a primary lysosome. Fusion of the T3 T4 rT3
small amount of Tg is released from the follicular cells without endocytosed Tg with the primary lysosome forms a secondary
D3 D1,D2
transport into the colloid, and this Tg is not iodinated. Of lysosome, in which digestion of Tg occurs, releasing T4, T3, D1,D2 D3
consequence in autoimmune thyroid disease, iodination MIT, DIT, and amino acids. By diffusion, the lipophilic T4 and
increases the immunogenicity of Tg. Increased dietary iodine T3 molecules exit the lysosome and cross the follicular cell
exposure is associated with the development or expression of plasma membrane to be captured in thyroid capillaries, where
Hashimoto thyroiditis.112 the vast majority of thyroid hormone is protein bound. Only CH2 CH2 CH2
Once in the lacuna, iodide is oxidized (organified) to 0.03% of total T4 is free (unbound and bioactive), and only
an iodine radical by the thyroperoxidase (TPO) enzyme. 0.3% of total T3 is unbound and bioactive. I I I

OH Monoiodination OH Diiodination OH I
Thyroperoxidase Thyroperoxidase 3,3T2
3,5 T2 3,5T2
Figure 55-9 Metabolic pathways of T4. Type 1 deiodinase (D1) converts T4 to T3 or
rT3. D1 then converts T3 to 3,3-T2, while D1 converts rT3 to 3,3-T2. Type 2
deiodinase (D2) converts T4 to T3 and rT3 to 3,3-T2. Type 3 deiodinase (D3) converts
Tyrosine Monoiodotyrosine Tyrosine Diiodotyrosine T4 to rT3 and T3 to 3,3-T2.
(within Tg) (within Tg) (within Tg) (within Tg)
Figure 55-6 Monoiodination and di-iodination of tyrosine.
Within the cytoplasm of the follicular cell, the released activity expressed in the CNS, anterior pituitary, brown fat,
MIT and DIT are stripped of iodine by dehalogenase (Dhal) placenta, heart, skeletal muscle, and thyroid; and type 3 (D3),
OH Coupling OH
to produce free iodide ions, which can be recycled immedi- with inner ring deiodinase activity identified in CNS, liver,
I I reaction I I ately for the synthesis of new thyroid hormone.64 Two deha- endometrium, and placenta. Approximately 40% of T4 is deio-
Thyroperoxidase logenase genes have been described: Dhal1 and Dhal1b. dinated to T3 by D1 or D2, and 45% is deiodinated to rT3
Normally, only negligible amounts of MIT and DIT are by D1 or D3. About 80% of circulating T3 comes from 5-
OH O released from the thyroid gland into the circulation. However, deiodination of T4; only 20% of T3 is released directly from
I I I I loss-of-function mutations in the dehalogenase enzyme the thyroid gland (Figure 55-9). Almost all circulating rT3
(within Tg) potentially lead to iodine loss in the urine and increased results from 5-deiodination of T4. By regulating the conver-
concentrations of DIT and MIT in the circulation. sion of T4 to T3, the body, in part, regulates the metabolic rate.
The peripheral metabolism of thyroid hormones is very D1 converts T4 to rT3 (via 5-deiodination) and T4 to T3
Diiodotyrosine T4 (within Tg) complex.191 Three homodimeric deiodinases are present: type (via 5-deiodination) (Figure 55-10). The preferred substrates
(within Tg) 1 (D1), with inner and outer ring deiodinase activities found for D1 are sulfated T3 (converted to 3,3-T2 via 5-deiodination)
Figure 55-7 Chemical coupling of two molecules of di-iodotyrosine to produce a in liver, kidney, thyroid, and possibly the anterior pituitary and rT3 (converted to 3,3-T2 via 5-deiodination). D1 activity
molecule of T4 (thyroxine). The reaction is catalyzed by thyroperoxidase. gland (Figure 55-9); type 2 (D2), with outer ring deiodinase is stimulated by thyroid hormone through increased gene
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1911 1912 Section V Pathophysiology

transcription. Thus, increased D1 activity promotes periph- free T3 (FT3)-to-T4 (FT4) ratio is affected by a genetic poly- been definitively demonstrated. Within the astrocyte, T4 is developing brain. Defects in TR1 can produce resistance to
eral conversion of T4 to T3 in hyperthyroidism. Therapeuti- morphism in the D1 gene (DIO1; the single-nucleotide poly- converted to T3 via D2 attached to the astrocyte membrane. thyroid hormone; defects in TR have not been reported.
cally, D1 is inhibited by propylthiouracil, which is used to morphism rs2235544).143 T3 may exit the astrocyte via OATP1C1 to be taken up by Within most cells, 90% of T3 is cytosolic and 10% is
treat hyperthyroidism. In contrast to the effect of thyroid Each of the deiodinase enzymes has a single transmem- neurons via MCT8. Within the neuron, T3 can interact with intranuclear. An exception is seen in the pituitary gland,
hormone on D1, D2 expression is suppressed by thyroid brane domain. The deiodinases are attached to the inner the intranuclear thyroid hormone receptor (TR), or can be where T3 is distributed approximately equally between the
hormone. The Km for the D1-T4 complex is approximately plasma membrane (D1 and D3) or endoplasmic reticulum converted to 3,3-T2 via membrane-attached D3. cytoplasm and the nucleus. A T3-binding protein (CTBP, or
1000-fold greater than the corresponding Km for D2 or (D2), and the active site of the enzyme is on the cytoplasmic Although T4 is converted to T3 outside of target organs C-T3BP) has been identified that may influence the intracel-
D3. Regulation of the T4T3 and T4 rT3 conversions is domain. Therefore, thyroid hormone must enter the cyto- (the liver converts T4 to T3 and then releases T3 back into the lular distribution of thyroid hormone; however, this protein
dependent on the tissue distribution of D1, D2, and D3. The plasm of cells to be metabolized. In addition to thyroid circulation), target organs can also deiodinate T4 to T3 so that does not exhibit receptor activity.
hormone metabolism via deiodination, small amounts of T4 T3 can bind to TRs. The liver, kidney, brain, brown fat, ante- It is important to consider the comparative biological
are glucuronidated or sulfated and excreted in the bile. Intes- rior pituitary, pineal, heart, skeletal muscle, and placenta all activity of circulating T4 and T3. On a molar basis, there is
tinal cleavage of glucuronidated T4 releases T4 that can then express a 5-deiodinase capable of deriving T3 from T4. 100 times as much circulating T4 as T3. Typical reference
Thyroid gland
be reabsorbed from the intestine back into the circulation. T3 binds to specific intranuclear TRs to exert its hormonal intervals for T4 and T3 are 60 to 135 nmol/Land 1 to 3.5,
Until recently it was believed that both T4 and T3 entered effects. In humans (and various animals), thyroid hormone nmol/L, respectively. The difference in T4 and T3 concentra-
cells by passive diffusion across the plasma membrane. upregulates the expression of growth hormone,110 myelin tions narrows, however, to a 10-fold excess of free (active) T4,
However, evidence has shown that thyroid hormones cross basic protein, the alpha-myosin heavy chain, and a sarcoplas- compared with unbound T3, in the circulation. Because T3 is
plasma membranes using specific transporters.74 One impor- mic reticulum calcium ATPase, while downregulating the 4 to 15 times as biologically active as T4, the difference in

tant thyroid hormone transporter is monocarboxylate expression of the TSH beta chain and the beta-myosin heavy effectiveness narrows further. It is debated whether the effects
H3N COO H3N COO transporter 8 (MCT8).118 The solute carrier family of mono- chain. In part, suppression of TSH beta chain synthesis con- of T4 exceed those of T3, or whether they equally influence
CH2 CH2 carboxylate transporters has 14 members (MCT1 through tributes to negative feedback. the tissues. Within the cells of the anterior pituitary, large
MCT14); examples of other monocarboxylates transported TRs are members of the steroid hormone supergene family intracellular amounts of T3 are derived from monodeiodin-
I I I I by these carriers are lactate and pyruvate. and serve as transcription factors that include domains for ation of T4, so circulating FT4 appears to be the major regula-
O D1 O It has been shown that MCT10 (SLC16A10; chromosome DNA binding, T3 binding, and transactivation. T3 binds to the tor of pituitary TSH secretion. This is an important concept
~80% 6q) transports iodothyronines and aromatic amino acids TRs with 15-fold greater affinity than T4. The TRs form het- that explains why TSH remains normal in the early stages of
I I I across membranes, and MCT10 is likely more preferential erodimers with the retinoid X receptor (RXR) to interact with the sick euthyroid syndrome, when FT4 is normal, preventing
than MCT8 in transporting T3 over T4 across plasma the thyroid response elements (TREs) in DNA. Monomers a rise in TSH, yet the T3 is decreased.
T4 T3 membranes. Organic anion transporting polypeptide 1C1 and homodimers of TRs can also bind to the TREs. TRs have In the pituitary, most T3 is derived from intrapituitary
(OATP1C1) also has high affinity for thyroid hormone and zinc-finger structures that form alpha helices for interaction conversion of T4 to T3. Within target tissues, if more T3 comes
Figure 55-10 Metabolic sources of T4 and T3. The only
source of T4 is the thyroid gland. However, most T3
may be an important component of the transport of T3 and T4. with the response elements. from T4 (via target cell 5-deiodination) than from the circu-
(80%) comes from peripheral monodeiodination of T4 Transport of thyroid hormone into neurons is especially Two types of TRs are known: TR and TR (Table 55-2).218 lation itself, T4 has a major effect on tissues. Nevertheless
to T3 [catalyzed by type 1 deiodinase (D1) and type 2 critical for normal CNS development and function (Figure TR is encoded by the thyroid hormone receptor-alpha because most T3 is derived from T4, within the circulation or
deiodinase (D2)] with only a minority of T3 (20%) 55-11). OATP1C1 may be responsible for thyroid hormone (THRA) gene on chromosome 17q11.2. Of its two expressed within a target cell, T4 is often considered to be a prohormone
coming directly from the thyroid gland. uptake and release by astrocytes, although this has not yet isoforms (TR1 and TR2), only TR1 binds thyroid in the generation of T3.
hormone. A TR3 isoform produced by alternative splicing Compared with the kidney, liver, and cerebral cortex,
has also been reported. TR1 is expressed throughout the much higher concentrations of TRs are found in the anterior
CNS Vasculature body, but its mRNA is in highest concentrations in the heart, pituitary gland and brown adipose tissue. In both of the latter
Overlapping endothelial cells brain, liver, and kidneys. tissues, two thirds to three quarters of the receptors are occu-
TR is encoded by 11 exons on the thyroid hormone pied by T3, with slightly more than half of the T3 derived from
receptor-beta (THRB) gene on chromosome 3p24.3. Three intracellular conversion of T4 to T3 via D2, versus T3 taken up
processes Astrocyte Neuron isoforms of TR exist: TR1, TR2, and TR3. The highest from the circulation (derived from T4 outside the target tissue
concentrations of TR1 are found in heart, kidney, liver, and by D1 and D2). Most of the hepatic and renal T3 comes from
brain. Expression of TR2 has been identified in the anterior circulating hormone. On the other hand, the vast majority
T3 MCT8 pituitary, hypothalamus, retina, cochlea of the inner ear, and of T3 in the cerebral cortex is derived intracellularly from T4
T4 -- D2 T3
by monodeiodination of T4 to T3.
T4 T3
Thyroid Hormone Negative Feedback Control
Possibly: OATP1C1 TABLE 55-2 Thyroid Hormone Receptor (TR) Sizes of TRH and TSH
Capillary basement
membrane Amino Acids kDa Thyroid hormone synthesis and secretion are controlled by a
negative feedback system involving the hypothalamus, the
Figure 55-11 Transport of thyroid hormones to neurons. Before thyroid hormones TR1 410 47 pituitary, and the thyroid follicular cells (Figure 55-12).30 In
are transported to neurons,T4 and T3 pass through endothelial cells and astrocytes. TR2 490* 55
Organic aniontransporting polypeptide 1C1 (OATP1C1) may allow astrocyte uptake contrast to other hypothalamic-anterior pituitary target organ
TR1 461 53 systems that utilize a negative feedback system, however, the
of T4 and T3.Within the astrocyte,T4 may be converted to T3 via type 2 deiodinase (D2).
TR2 514 58 major site of thyroid hormone feedback is at the level of
T3 then may leave the astrocyte via OATP1C1 to enter neurons via monocarboxylate
transporter 8 (MCT8).Within the neuron,T3 can interact with a thyroid hormone *Extended C-terminal region (does not bind T3). the anterior pituitary thyrotrophsnot at the level of the

receptor (TR) or can be converted to 3,3-T2 via type 3 deiodinase (D3). Extended N-terminal region. hypothalamus.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1913 1914 Section V Pathophysiology

glutamate terminus is required for TRH bioactivity. The PVN TSH - Free T4 Relationships in
are located in the anterior hypothalamus, rostral to the optic Ambulatory Patients with Stable Thyroid Status
Minor site of
TRH negative feedback chiasm. TRH concentrations rise in thyroid hormone defi- 1000
ciency with TRH declining when thyroid hormone is in
Pituitary excess. TRH is delivered to the anterior pituitary gland via
Major site of the hypothalamic-pituitary portal system. TSH
TSH negative mIU/L
feedback Anterior Pituitary Thyrotroph Physiology 10
Thyroid Thyrotrophs, the anterior pituitary cells that secrete thyroid- 4.0
stimulating hormone (TSH; thyrotropin), express TRH TSH
receptors, which are G-proteincoupled receptors with seven Reference
T4, T3 transmembrane domains. When TRH binds to its receptor, 0.4
TBP T4 T3 the thyrotroph depolarizes, triggering calcium influx. Conse- 100x
quently, increased free cytosolic calcium activates the Ca2+-
phosphatidylinositol cascade, effecting TSH release, synthesis, 2x
Target and glycosylation of alpha and beta TSH subunits. In relative 0.01
cell terms, TRH has a greater effect on TSH glycosylation than
T4 T3 TR
hormone release.165 However, glycosylation of TSH is neces-
sary for normal TSH bioactivity. When TRH is deficient, TSH Hypo- FT4 Reference
Figure 55-12 Metabolic control of thyroid hormones. Hyperthyroid
may lack potency as the result of insufficient glycosylation, Thyroid Interval
Thyrotropin-releasing hormone (TRH) from the
hypothalamus enters the hypothalamic-pituitary portal yet nonglycosylated TSH may retain much of its immunore-
system to release thyroid-stimulating hormone (TSH; activity. Therefore, immunoassays for TSH may not reflect the 9 23 pmol/L
activity of the hormone when injury or disease results in a { Free T4
thyrotropin) from anterior pituitary thyrotrophs. TSH 0.7 1.8 ng/dL
stimulates the release of T4 and T3 from the thyroid TRH deficiency.
TRH modifies the sensitivity of the thyrotroph to thyroid Figure 55-14 The log-linear relationship between thyroid-stimulating hormone
gland, although most T3 comes from peripheral
(TSH) and FT4. A twofold change in TSH is associated with an approximate 100-fold
monodeiodination of T4 to T3. More than 99% of T4 and T3 hormone negative feedback; increased TRH makes the thy-
change in circulating FT4 concentrations. (Modied from Spencer CA, LoPresti JS, Patel A,
is bound to various thyroid hormonebinding proteins. rotroph less sensitive to inhibition with decreasing TRH Guttler RB, Eigen A, Shen D, et al. Applications of a new chemiluminometric thyrotropin assay to
T4 and T3 negatively feed back on the hypothalamus and, makes the thyrotroph more sensitive to negative feedback. subnormal measurement. J Clin Endocrinol Metab 1990;70:453-60. Copyright 1990, The Endocrine
more powerfully, the pituitary. T4 and T3 enter into target The mechanism for TRH control involves reduced expression Society.)
tissues, where T4 is converted to T3 with T3 binding to the
of TRs in the thyrotroph following TRH stimulation. When
thyroid hormone receptor (TR).
the thyrotroph is less sensitive to negative feedback from
thyroid hormone, TSH release is potentiated. Therefore, TSH
secretion rises in thyroid hormone deficiency and declines in subsequent stimulation of H2O2 generation and iodide efflux
thyroid hormone excess. An inverse logarithmic relationship into the follicular lumen. THYROID HORMONES IN THE CIRCULATION
C N exists between TSH and FT4 concentrations (Figure 55-14); a The TSHR is a 743 amino acid, 84.5 kDa protein generated Both T4 and T3 are highly bound to plasma proteins in circu-
CH 50% decline in FT4 concentration results in a 100-fold increase after cleavage of a 21 amino acid pre- (or leader) sequence. lation. Protein-bound T4 and T3 serve as thyroid hormone
O NH2 in TSH. After post-translational glycosylation, the TSHR is 100 kDa. reservoirs within plasma. In the thyroid gland, Tg serves as
C TSH is a 30 kDa heterodimeric glycoprotein that shares The first nine exons encode the 398 amino acid N-terminal a reservoir. The unbound fraction of circulating thyroid
O CH2 CH a subunit with luteinizing hormone (LH), follicular stimulat- extracellular domain. The extracellular domain contains six hormone is the biologically active form, so FT4 concentra-
H CH2 ing hormone (FSH), and human chorionic gonadotropin N-glycosylation sites that make up the TSH-binding domain. tions correlate more closely with the clinical status of the
CH N H C N (hCG).174 All four hormones contain a 14.7 kDa alpha sub- The TSHR may be in either of two conformations: the on patient than do total T4 concentrations.15,142 However, eleva-
CH2 O H2
unit (gene location: chromosome 6q21.1-q23). Each of these conformation, with active signaling, or the off conforma- tions in FT3 usually correlate with the clinical status of the
C hormones has a unique beta subunit that is responsible for tion, where signaling does not occur. TSH binding to the patient, low FT3 concentrations do not always equate with
the specific activity of the hormone. The 15.6 kDa TSH TSHR puts it in the on conformation, leading to stimulation clinical hypothyroidism, as evidenced in the sick euthyroid
beta chain is encoded by a three-exon gene located on chro- of the thyroid gland. syndrome (see later). Similarly, elevated free concentrations
Figure 55-13 Chemical structure of thyrotropin-releasing mosome 1p. The alpha chain contains two oligosaccharides, Within the TSH-stimulated thyroid follicular cell, the of T4 or T3 do not always correlate with hyperthyroidism
hormone (TRH). Note that TRH is a tripeptide and the TSH beta subunit contains one oligosaccharide activity of the sodium-iodine symporter increases; increased because of the possibility of peripheral thyroid hormone
(L-pyroglutamyl-L-histidyl-L-prolinamide). modification. synthesis of thyroglobulin (Tg) and TPO, increased genera- resistance syndromes and rare MCT8 loss-of-function muta-
tion of hydrogen peroxide and NADPH, and pinocytosis of tions. Alterations in the concentrations of thyroid hormone
Thyroid Follicular Cell Physiology colloid with consequent degradation and release of T4 and binding proteins can profoundly affect the total concentrations
The effects of TSH on the thyroid follicular cell are mediated T3 from Tg are also noted. Replication of thyroid follicular of T4 and T3 without significant changes in the free hormone
Hypothalamic Physiology through TSH receptor (TSHR)G-proteinadenyl cyclase cells is stimulated by cAMP, phospholipase C, insulin-like concentrations.
Thyrotropin-releasing hormone (TRH; thyrotropin-releasing coupled synthesis of intracellular cyclic adenosine mono- growth factor-I (IGF-I), and a fibroblastic growth factor T4 binds to thyroxine-binding globulin (TBG; a 54 kDa
factor, thyroliberin, or protirelin) is encoded on chromosome phosphate (cAMP).197 At supraphysiologic concentrations (FGF)-mediated kinase. TSH stimulation increases the size 1-globulin), thyroxine-binding prealbumin (TBPA, or trans-
3, and the modified tripeptide l-pyroglutamyl-l-histidyl-l- (100 normal), TSH will signal through the inositol- and number of thyroid follicular cells. Likewise, follicular thyretin; 54 kDa), and albumin. T3 binds almost exclusively
prolinamide is secreted by the paraventricular nuclei (PVN) phosphate diacylglycerol cascade, activating phospholipase C cellular hyperplasia is seen in Graves disease, when an ago- to TBG and albumin; TBPA binds only negligible amounts of
in the hypothalamus (Figure 55-13). Cyclization of the (PLC) and raising intracellular calcium concentrations, with nistic autoantibody stimulates the TSH receptor. T3 (Table 55-3).176 On serum protein electrophoresis, TBPA
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1915 1916 Section V Pathophysiology

migrates in the prealbumin region (hence, its name). TBG TBG excess (an X-linked dominant condition), male hemi- In most endogenous hyperthyroid states, the RAIU is (goiter) is typically evaluated by measurement of TSH
(chromosome Xq11-23) is a 395 amino acid acidic glycopro- zygotes display threefold to fivefold elevations in TBG con- increased; in hypothyroid states, the RAIU is decreased. In and thyroid hormones, and on the basis of (1) history,
tein with one iodothyronine-binding site. TBG has 4 hetero- centration, while female heterozygotes typically have twofold thyrotoxicosis, measurement of the RAIU at 6 hours may be (2) physical examination, and (3) laboratory results; patients
saccharide sidechains with 5 to 9 sialic acid moieties. As the to threefold elevations in TBG. Abnormal forms of albumin helpful because a hyperactive thyroid gland takes up the may be classified as (1) euthyroid, (2) hypothyroid, or
degree of sialylation increases (an effect of estrogen), the half- (familial dysalbuminemic hyperthyroxinemia) and TBPA radioiodine at a very rapid rate and may have released some (3) hyperthyroid.95,125,153
life of TBG increases, thus raising TBG concentrations. (familial euthyroid thyroid excess) alter the concentrations of of the tracer by 24 hours. Otherwise, scanning at 24 hours Patients presenting with a thyroid mass are typically
Increased concentrations of TBG raise total T4, yet FT4 total T4, yet do not affect FT4, total T3, or FT3. These condi- alone might reveal a falsely lower measurement of the RAIU. euthyroid. Suspicious masses are typically followed by
remains within the euthyroid interval as the result of negative tions are discussed later, along with antithyroid hormone As iodine has become more plentiful in the diet, the reference ultrasound-guided fine-needle aspiration of the mass with
feedback (see Figure 55-12). Similarly, decreased TBG con- autoantibodies that raise total thyroid hormone concentra- interval for the RAIU has declined. cytologic examination. A thorough discussion of all forms of
centrations lower total T4 while maintaining normal FT4. tions. Isolated euthyroid hypertriiodothyroninemia (with all thyroid cancer is beyond the scope of this chapter, although
Causes of elevations and depressions in thyroid hormone other thyroid parameters normal) caused by a rare form of Thyroid Scans the role of Tg measurements will be examined as a tumor
binding protein (TBP) are shown in Box 55-1. In congenital dysalbuminemia has been reported. Administration of radioactive iodine or technetium pertech- marker for differentiated thyroid cancers.67
netate allows visualization of thyroid tissue in the neck and If clinical findings do not suggest an abnormality in the
throughout the body. In such studies, the scanner provides hypothalamic-pituitary thyroid axis, laboratory evaluation is
RADIOGRAPHIC THYROID TESTING an image of the thyroid gland that can reveal low uptake, high begun with measurement of TSH (Figure 55-16).4,95A If TSH
TABLE 55-3 Thyroid Hormone Transport in Plasma Thyroid gland location, anatomy, and function is also assessed uptake, and anatomic disorders such as (1) hemithyroid, a is within the reference interval, thyroid dysfunction generally
TBP TBG TTR Albumin radiographically. For example, thyroidal uptake of radioactive toxic (hyperactive) nodule; (2) a cold nodule (a nodule that can be excluded. If TSH is below the reference interval, mea-
iodine (I-123 or I-131) or technetium pertechnetate (99mTc- fails to take up the radioactive tracer); or (3) ectopic thyroid surement of FT4 is indicated. If TSH is depressed and FT4 is
Concentration 4-5.4 mg/dL 10-20 mg/dL 3.5-5 g/dL pertechnetate; TcO4) is assessed over time as an index of tissue. Thyroid scans have been used after thyroidectomy to elevated, the biochemical diagnosis of primary hyperthyroid-
Affinity for T4 High Modest Low thyroid function.116 detect residual thyroid tissue at the surgical site, as well as ism is established. Although total T3 can be measured at this
T4 capacity, 22 120 1000 The degree of uptake of an exogenously administered differentiated thyroid cancer metastases. point (and is expected to be greatly elevated), this value
mcg/dL radioactive tracer versus time reflects the activity of the should not change the diagnosis or the initial therapy. If TSH
thyroid gland (Figure 55-15). Radioactive iodine or techne- Perchlorate Discharge is depressed and FT4 is within the reference interval, T3
Distribution tium pertechnetate can be used. The radioactive iodine Normally, iodide is rapidly transported into the thyroid gland should be measured in search of T3 toxicosis. The authors
T4 67% 20% 13% uptake (RAIU) is typically expressed as the counts measured via the NIS. Within minutes of entry into the thyroid gland, favor total T3 measurements over FT3 measurements because
T3 53% 1% 46% (via scanning of the thyroid gland) divided by the total counts intracellular iodide is transported into the lacunae via pendrin total T3 measurements are both more accurate and less expen-
administered. The reference interval for the RAIU is usually and undergoes oxidation, leading to iodination of tyrosine sive than FT3 measurements. If TSH is depressed and both
5 to 25%, which means that 5 to 25% of administered radioac- residues on thyroglobulin. DUOX and thyroperoxidase are FT4 and T3 are normal on more than one occasion, subclinical
tive tracers are present in the thyroid gland at the time of the responsible for the formation of organified iodine, and thy- hyperthyroidism is diagnosed. This assumes that causes of
BOX 55-1 Alterations in the Concentration or scan (usually at 24 hours). roperoxidase is responsible for the iodination of tyrosine. TSH suppression such as high-dose glucocorticoids and
Afnity of Thyroid HormoneBinding Proteins TPO couples MIT and DIT to produce T3, and DIT and DIT dopamine have been excluded. If TSH is depressed, FT4 is
to produce T4. The NIS also concentrates other anions within normal, and T3 is elevated, the diagnosis of T3 toxicosis is
Increases in
the thyroid gland, such as thiocyanate (SCN), pertechnetate established.50
A. Thyroxine-binding globulin (TBG) concentration (or
(TcO4), and perchlorate (ClO4). If TSH is above the reference interval, FT4 should be mea-
1. Genetic (inherited) causes 50% The perchlorate discharge test is used to detect defects in sured. If FT4 is depressed, the biochemical diagnosis of
2. Nonthyroidal illness (HIV infection, infectious and thyroid gland iodide oxidation or iodination of Tg. Radioac- primary hypothyroidism is established. Measurement of T3
chronic active hepatitis, estrogen-producing tumors, Hyperthyroid tive iodine is administered prior to perchlorate, and any (or FT3) provides no essential or additional information when
acute intermittent porphyria) radioiodine still in the follicular cells that has not yet been hypothyroidism is a clinical consideration. Furthermore, in
3. Normal physiology (pregnancy, newborn) incorporated into colloidal Tg is released.227 Perchlorate primary hypothyroidism, T3 declines later than T4 because
4. Drug use (oral contraceptives, estrogens, tamoxifen, does not block iodination of Tg. If the NIS has transported elevated TSH concentrations in primary hypothyroidism

radioiodide into the thyroid gland, but the iodide is not yet stimulate T3 production more than T4 production. Whether
B. Prealbumin binding (familial euthyroid thyroxine excess) Normal incorporated into Tg after perchlorate administration, a this is due to limited availability of iodine during an attempt
C. Albumin binding (familial dysalbuminemic Hyperthyroid with
rapid uptake supranormal amount of radioiodine is released from the at increased thyroid hormone production or the increased
thyroid gland (an increase in the perchlorate discharge of speed of synthesis (T3 synthesis requires only one iodin-
D. T4 binding by antibodies (autoimmune thyroid disease,
radioiodine occurs). ation versus two for T4 synthesis) is unknown.
hepatocellular carcinoma) Hypothyroid
Causes of increased radioiodide discharge after perchlo- If TSH is elevated and FT4 is within the reference interval
Decreases in rate administration include defects in pendrin, thyroperoxi- on more than one occasion in an asymptomatic patient, sub-
A. TBG concentration 0 6 12 24 48 hours dase, and DUOX. Alternatively, if an inborn error is present clinical hypothyroidism is diagnosed (assuming that hetero-
1. Genetic (inherited) determination in the NIS (e.g., a loss-of-function mutation), release of radio- philic antibodies have been excluded as a cause of TSH
2. Nonthyroidal illness (major illness or surgical stress, iodide is not increased after perchlorate because radioiodide elevation).8,145 Elevated TSH and FT4 raise the possibility of
chronic liver disease, protein-losing enteropathy, Figure 55-15 Radioactive iodine uptake (RAIU) as a
was not initially taken up by the thyroid gland. central hyperthyroidism or thyroid hormone resistance.
nephrotic syndrome) function of time. RAIU is dependent on the activity of
These disorders are differentiated clinically: patients with
3. Drug use (androgens, anabolic steroids, large doses of the thyroid gland. In hyperthyroidism, excessive uptake
of radioactive iodine occurs. In hypothyroidism, uptake thyroid hormone resistance usually are euthyroid or, at worst,
glucocorticoids) CLINICAL CONDITIONS mildly hypothyroid, however, those with true hyperthyroid-
B. TBG-binding capacity (drugs bound to TBG such as of radioactive iodine is decient. In cases of severe
hyperthyroidism, RAIU may peak before 24 hours and a Because the signs and symptoms of thyroid dysfunction ism clinically manifest thyrotoxicosis (see later). Because of
salicylates and phenytoin)
6 hour measurement is necessary to detect an elevated are extremely variable, thyroid function studies are often the log-linear relationship of TSH to FT4, FT4 usually is
C. Prealbumin concentration
uptake. measured in clinical practice.36,41 An enlarged thyroid gland normal as long as TSH is between 0.5 and 10 mcIU/mL.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1917 1918 Section V Pathophysiology

TSH similar to the secondary follicles observed in normal lymph

BOX 55-2 Causes of Primary Hypothyroidism nodes. Initially, the gland is usually enlarged. Over time, with
destruction of the gland, the gland can atrophy or become
Elevated Normal Decreased Endogenous Disorders
Autoimmune thyroid disease
firm. In the rare condition of Riedel thyroiditis (Riedel disease
Hashimoto thyroiditis or struma, ligneous struma, ligneous thyroiditis, chronic
FT4 No further FT4 Atrophic thyroiditis fibrous thyroiditis), the thyroid gland becomes fibrotic with
testing Late-stage Graves disease possible attachment to adjacent structures that can produce,
Postpartum thyroiditis for example, tracheal compression.144 Subacute (viral) thy-
Elevated Normal Decreased Elevated Normal Decreased
Inborn errors in thyroid hormone biosynthesis roiditis may also cause Riedel thyroiditis.31 Not all cases of
(dyshormonogenesis) Riedel thyroiditis are presaged by Hashimoto thyroiditis.
T3 Na+/iodide pump dysfunction Although atrophy may occur in Hashimoto thyroiditis,
Inadequate organification/iodinationthyroperoxidase atrophic thyroiditis may also occur when autoantibodies
Clinically: Hyper- Elevated Normal dysfunction
Euthyroid thyroid against the TSH receptor bind to the receptor and block the
Defective thyroglobulin
action of endogenous TSH. TSH receptor blocking autoanti-
Deiodinase deficiency
Pendred syndromehypothyroidism and deafness
bodies can cross the placenta during pregnancy, causing tran-
Thyroid TSH- Primary Primary Central sient hyperthyrotropinemia in infants (elevated TSH with a
hormone dependent hypothyroidism hyper- hypothyroidism Developmental disorders involving the thyroid gland
resistance hyper- thyroidism Congenital hypothyroidism: aplasia, hypoplasia normal T4) or even transient congenital hypothyroidism.
thyroidism Ectopic thyroid: lingual thyroid, thyroglossal duct cyst Therefore atrophic thyroiditis falls under the rubric of AITD.
Consumptive hypothyroidism The diagnosis of Hashimoto thyroiditis is supported by
Subclinical T3 toxicosis Subclinical recognition of autoantibodies against TPO or Tg.113 Ninety
hypothyroidism hyperthyroidism
Exogenous Disorders percent of patients with chronic lymphocytic thyroiditis (the
Figure 55-16 Suggested algorithm for the laboratory evaluation of thyroid function. Iodine excess/deficiency histologic description of Hashimoto thyroiditis) have anti-
Drugs thyroperoxidase autoantibodies (TPOA), antithyroid micro-
Thionamides somal autoantibodies (TMA), and/or antithyroglobulin
If hypothalamic or pituitary disease is suspected, initial and/or dry skin, (4) puffy eyes, (5) loss of the outer lateral autoantibodies (TgA), making these autoantibodies excellent
testing requires measurement of both TSH and FT4 (with FT4 eyebrows, (6) delayed relaxation phase of reflexes (hung-up Amiodarone
markers for Hashimoto thyroiditis.182 TPOA testing was ini-
being more important). FT4 is used to establish the presence reflexes), (7) myopathy, (8) carotenemia, (9) occasional galac- Biologicals (e.g., interferon, interleukin-2) tially pursued by testing for TMA (see section on thyroid
of the biochemical euthyroid state, hypothyroidism, or hyper- torrhea, and (10) radiologic evidence of delayed bone age in Dietary goitrogens autoantibody testing). TMA testing is being replaced by spe-
thyroidism. When FT4 is low and TSH is low or normal, children. In cases of severe hypothyroidism, congestive heart Radiation-induced hypothyroidism cific testing for TPOA using TPO as the antigen in the immu-
central hypothyroidism may be present. If FT4 is low with failure or coma may develop.90 In children with untreated Surgical removal of the thyroid gland noassay. TPOA/TMA positivity is more common at the time
only a mild elevation in TSH, central hypothyroidism is still congenital hypothyroidism, severe growth failure and mental Viral or bacterial thyroiditis of diagnosis than TgA positivity with TgA usually appearing
possible because of discordance in the ratio of immunoreac- retardation ensue. The development of biological and bio- later in the disease process. TgA overall are less common than
tivity to bioactivity, with a decrement in bioactivity from chemical processes is delayed in cases of congenital hypothy- TMA or TPOA. Ultrasound has limited value and is not rou-
abnormal TSH glycosylation from pituitary disease or TRH roidism. For example, affected infants often have prolonged disease (secondary hypothyroidism from TSH deficiency) or tinely used to detect Hashimoto thyroiditis.
deficiency. In the setting of clinical hyperthyroidism, central jaundice as the result of immaturity of UDP-glucuronyl hypothalamic disease (tertiary hypothyroidism from TRH In many families, AITD appears to be inherited in an
hyperthyroidism is diagnosed with an elevated FT4 and transferase. deficiency). autosomal dominant pattern although some family members
normal or elevated TSH. A rare (and controversial) manifestation of Hashimoto develop Hashimoto thyroiditis and other family members
thyroiditis is encephalopathy.216 However, no definitive clini- Primary Hypothyroidism develop Graves disease. Several genetic loci have been associ-
cal test is available to diagnose encephalopathy resulting from The causes of primary hypothyroidism are classified as endog- ated with susceptibility to Hashimoto thyroiditis (or AITD):
HYPOTHYROIDISM Hashimoto thyroiditis. The diagnosis is considered when enous or exogenous (Box 55-2).38 Endogenous disorders are HLA-DR, CTLA-4, CD40, FCRL3, Tg, the TSH receptor,
Hypothyroidism is defined as a deficiency in thyroid hormone encephalopathy of unknown origin is associated with auto- conditions that develop within the patient such as autoim- PTPN22, and the IL-2 receptor. However, no single Mende-
secretion and action that produces a variety of clinical signs antibodies against the thyroid gland. mune thyroid gland dysfunction, inborn errors, and develop- lian locus explains the apparent autosomal dominant pattern
and symptoms of hypometabolism.73,84,198 This common dis- Laboratory evidence compatible with hypothyroidism mental abnormalities. Exogenous disorders are conditions of inheritance of AITD.66 It is most appropriate to describe
order occurs in 2 to 15% of the population, more commonly encompasses hyponatremia, a normocytic or macrocytic that originate outside the patient such as iodine deficiency or AITD as polygenic and multifactorial, indicating that both
in women than in men. The risk of developing hypothyroid- anemia, elevated creatine kinase (from myopathy), and excess, goitrogen or drug effects, and postsurgical hypothy- environment and multiple genes provide susceptibility to
ism increases with age.39 hypercholesterolemia and/or hypertriglyceridemia [from roidism or hypothyroidism following radioactive iodine these very common disorders.
Clinical symptoms suggesting hypothyroidism include decreased lipoprotein-lipase activity and decreased low- treatment. Nonendocrine and endocrine autoimmune disorders can
(1) mental dullness (including mental retardation in children density lipoprotein (LDL) receptor expression]. Anatomi- occur with increased frequency in people with AITD. Chronic
with untreated or undertreated congenital hypothyroidism), cally, stimulation of the TSH receptor can cause follicular cell Autoimmune Hypothyroidism lymphocytic gastritis causing pernicious anemia may accom-
(2) somnolence, (3) increased sleeping, (4) lethargy, (5) easy hyperplasia and goiter. Goiter can also be caused by glandular Excluding the newborn period, autoimmune thyroid disease pany AITD, particularly in older patients. AITD also occurs
fatigability, (6) hoarseness or deepening of the voice, (7) hair infiltration (e.g., Hashimoto thyroiditis, in which the gland is (AITD) is the most common cause of thyroid disease commonly in association with type 1 diabetes and pernicious
loss, (8) weight gain, (9) cold intolerance, (10) menstrual heavily infiltrated by lymphocytes). and primary hypothyroidism.111,148,203 Hashimoto thyroiditis anemia.61,91,104 Another, less common, association is AITD
irregularities, (11) infertility, (12) growth failure, (13) delayed Based on TSH and FT4, the causes of hypothyroidism are (chronic lymphocytic thyroiditis) leads to destruction of the and Addison disease.82
puberty in adolescents, (14) constipation, (15) muscle weak- classified as primary thyroid gland failure (low FT4 and thyroid follicular cells through a cell-mediated autoimmune AITD can occur as part of autoimmune polyglandular
ness or cramps, and (16) depressed affect or frank clinical increased TSH; primary hypothyroidism) or central hypo- process.102 Histologically, the gland is infiltrated with lym- syndrome type 2 (APS 2) and, more rarely, APS 1.161,175 APS
depression.171 Physical signs compatible with hypothyroidism thyroidism (low FT4 and usually a normal or low TSH con- phocytes and plasma cells to the extent that secondary lym- 2 affects women more often than men with onset in child-
include (1) bradycardia, (2) decreased pulse pressure, (3) cool centration). Central hypothyroidism results from pituitary phoid follicles develop within the thyroid gland that are hood or early adulthood and is diagnosed when Addison
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1919 1920 Section V Pathophysiology

disease (or adrenal autoantibodies) occurs together with pituitary transcription factors (HESX1, LHX3, LHX4, PROP1, iodine deficiency, more T3 is synthesized than T4, and within
TABLE 55-4 Effects of Some Drugs on Tests
AITD and/or type 1 diabetes. In contrast to the polygenic PIT1) can lead to TSH deficiency. Causes of transient con- the thyroid gland, more T4 is converted to T3 to maintain the
nature of APS 2, mutations in AIRE (the autoimmune regula- genital hypothyroidism include (1) severe maternal iodine euthyroid state overall. Endemic goiters can develop nodular- of Thyroid Function
tor gene that is a transcription factor; gene location: chromo- deficiency, (2) acute iodine exposure, (3) transplacental ity (with or without hemorrhage into the nodule); in such Cause Drug Effect
some 21q22.3) produce autoimmune polyglandular syndrome passage of thionamides taken for treatment of maternal cases, neoplasia must be excluded. Large goiters can produce
type 1 (APS 1), which is inherited in an autosomal recessive hyperthyroidism, (4) transplacental transfer of TSH receptor dysphagia, obstruction of the trachea, or compression of the Inhibit TSH Dopamine T4; T3;
pattern, thus affecting males and females equally.18,93 blocking antibodies, (5) hypothyroxinemia of prematurity, recurrent laryngeal nerves. A rare cause of iodine deficiency secretion l-dopa TSH
Another variant of AITD is postpartum thyroiditis and (6) heterozygosity for a DUOX2 mutation. is nephrotic syndrome, with increased urinary loss of iodine Glucocorticoids
(PPT).192 PPT develops presumably as a consequence of a Dried blood spot (DBS) screening for congenital hypo- often in the form of thyroid hormone.53,114 Somatostatin
Inhibit thyroid Iodine T 4; T 3 ;
decline in the natural immunosuppression of pregnancy fol- thyroidism in North America reveals a general population A significant danger of iodine deficiency is maternal hypo-
hormone Lithium TSH
lowing delivery. PPT follows 5% of all pregnancies and frequency of congenital hypothyroidism of 1 in 4000. Con- thyroidism leading to an insufficient supply of thyroid
synthesis or
10% of pregnancies in women with type 1 diabetes. The genital hypothyroidism is more common in Hispanic infants, hormone to the fetus in the first half of gestation, when the
clinical phenotype of PPT is transient hypothyroidism, less common in Caucasian infants, and least common in fetus is entirely dependent on maternal thyroid hormone.
Inhibit Amiodarone T3; rT3;
hyperthyroidism, or both (one following the other) with a African American infants. The male-to-female ratio is 1 : 2. A Thus maternal hypothyroidism can produce a reduction in
conversion of Glucocorticoids , ,
return to the euthyroid state by approximately 1 year postpar- higher frequency of congenital hypothyroidism is seen in the IQ of the affected child. Maternal iodine deficiency will
T4 to T3 Propranolol T4 and
tum. Women with thyroid autoantibodies are at highest risk infants with Down syndrome (1 in 140). produce fetal iodine deficiency.
Propylthiouracil FT4; ,
for PPT. Later in life, permanent clinical evidence of AITD Clinically, fewer than 1 in 20 hypothyroid babies are Although it is logical that iodine deficiency would produce
Radiographic TSH
can develop in women afflicted with PPT. Thyroid autoim- recognized in the newborn nursery by physicians. This hypothyroidism because iodine is a necessary component in
contrast agents
munity, even in the absence of hypothyroidism, may impair emphasizes the critical need for newborn biochemical screen- the synthesis of thyroid hormone, it is ironic that excess
Inhibit binding of Salicylates T4; T3;
fertility in women and increase the risk of spontaneous abor- ing. Early intervention improves clinical outcomes in these iodine can interfere with normal thyroid gland function.106
T4/T3 to serum Phenytoin FT4E,
tion in those women who do become pregnant.154 Therefore cases. However, some studies report that even with rapid However, excessive iodine does suppress thyroid gland func-
proteins Carbamazepine ,
an indication for thyroid autoantibody testing in the absence treatment after birth, the IQ of affected children may be tion. High doses of iodine [super-saturated potassium iodide
Furosemide FT4; 
of hypothyroidism would be female infertility or recurrent 10 points below that of their unaffected siblings. An (SSKI) or Lugols solution] are routinely used before planned
Nonsteroidal TSH
miscarriage. Convincing data suggest that T4 treatment of increased frequency of neuropsychologic deficits has been surgical thyroidectomy for Graves disease, to inhibit further
pregnant women who are positive for thyroid autoantibodies reported in children with appropriately treated congenital thyroid hormone release and decrease the vascularity of the
(especially TPOA) leads to a higher frequency of miscarriage hypothyroidism. gland to reduce surgical blood loss.
Heparin (in vitro
(13.8%) than is seen in pregnant women who lack TPOA In North America, screening for congenital hypothyroid- Excess iodine reduces thyroid hormone release and inhib-
(2.4% rate of miscarriage), and that T4 treatment of the ism is carried out predominantly by measuring total T4 on its organification of iodine and iodination of Tg (the Wolff-
Stimulate Phenobarbital T4; FT4;
TPOA-positive group reduces the rate of miscarriage to DBSs. A common practice is to measure TSH in babies who Chaikoff effect). The Wolff-Chaikoff effect can speculatively
metabolism of Phenytoin  TSH
approximately 3.5%.136 have the lowest 10% of T4s measured on the day of testing. be interpreted as an autoregulatory response of the thyroid
iodothyronines Carbamazepine
Usually if the TSH is 60 mcIU/mL or greater (after the first gland to avoid excessive production of thyroid hormone
Inborn Errors in Thyroid Hormone Biosynthesis Rifampicin
24 to 48 hours of life), a presumptive diagnosis of primary when exposed to high doses of exogenous iodine. Nondietary Inhibit Aluminum T4; FT4;
Inborn errors in thyroid hormone biosynthesis (dyshor- hypothyroidism is made, and the infant is transferred to sources of excess iodine include amiodarone (an antiarrhyth- absorption of hydroxide TSH
monogenesis) are rare causes of primary hypothyroidism. a referral center emergently to be evaluated and started on mic drug), povidone-iodine (used to disinfect the skin before ingested T4 Ferrous sulfate
These defects usually present early in life and can appear in thyroxine replacement therapy (e.g., 10 to 15 g/kg/d). If the surgery), and radiologic contrast agents that contain iodine. Cholestyramine
newborns as a goiter. In very rare cases, asphyxia from tra- TSH is between 20 and 59 mIU/L, the infant is clinically Excess iodine does not usually cause permanent thyroid dys- Colestipol
cheal compression has been reported.213 Biochemical defects evaluated and repeat thyroid function testing is performed function because the gland normally recovers (escapes) Iron sucralfate
include iodine transport defects from loss-of-function muta- before thyroid hormone replacement is started. If the TSH from suppression after approximately 10 days of high-dose Soybean
tions in the NIS (no increase in the perchlorate discharge); is less than 20 mIU/L, primary hypothyroidism is excluded. iodine administration. However, if the patient has underlying preparations
defects in thyroperoxidase, DUOX2,130 and pendrin (with Screening with T4 only testing will lead to missed rare cases disease that may involve the thyroid (e.g., Hashimoto thy- Kayexalate
increased perchlorate discharge); thyroglobulin deficiency; of central hypothyroidism (1/100,000) and cases of hypothy- roiditis, Graves disease), escape from the Wolff-Chaikoff Increase in Estrogen T4; T3;
and iodotyrosine dehalogenase mutations (potentially causing roidism wherein TSH is elevated but T4 is not yet depressed. effect is less likely and permanent hypothyroidism can concentration Clofibrate  FT4;
iodine deficiency through loss of MIT, DIT, and iodine in the In Europe, many screening programs use a TSH-first strategy. develop. of T4-binding Opiates (heroin,  TSH
urine).129 Although TSH screening will detect subclinical hypothyroid- proteins methadone)
ism (see later), cases of central hypothyroidism will be missed. Drug-Induced Hypothyroidism
Developmental Hypothyroidism Disorders Transcription factor mutations involving TTF-1 and Pax8 Various drugs affect thyroid gland function (Table 55-4).63 Perphenazine
Developmental causes of primary hypothyroidism involve have been recognized in some children with congenital hypo- Collectively known as thionamides or thioureas, propylthio- Decrease in Androgens T4; T3;
aplasia or hypoplasia of the thyroid gland and ectopic and thyroidism. It is not clear whether molecular testing will uracil (PTU), methimazole, and carbimazole inhibit the concentration Glucocorticoids  FT4;
lingual thyroid glands.202 These disorders account for 75% prove useful in future screening programs for congenital oxidation of iodide and the subsequent binding of iodine of T4-binding  TSH
of cases of congenital hypothyroidism. Other causes of con- hypothyroidism. to tyrosine residues in Tg. Other drugs with thionamide- proteins
genital hypothyroidism include thyroid dyshormonogenesis like activity include ethionamide, aminoglutethimide, phen-
(10% of cases; see earlier), hypothalamic or pituitary abnor- Hypothyroidism Caused by Iodine Deficiency ylbutazone, and para-aminosalicylic acid. Evidence suggests Data from Smallridge RD. Chapter 33. Thyroid function tests. In: Becker
or Excess KL, ed. Principles and practice of endocrinology and metabolism, 7th
malities (5% of cases). Ten percent of cases of congenital an immunosuppressive effect of thionamides on thyroid
edition. Philadelphia, Pa: JB Lippincott, 1995:299-306; Stockigt JR. Thyroid
hypothyroidism are transient. Worldwide, the most common cause of goiter is iodine defi- autoimmunity. hormone changes in critical illness: the sick euthyroid syndrome. Diagn
Rare causes of congenital hypothyroidism involve loss-of- ciency. In iodine-deficient areas, especially mountainous PTU, methimazole, and carbimazole are commonly used Endo Metab 1997;15:39-46.
function mutations in the pituitary TRH receptor,23 the TSH areas, iodine deficiency produces endemic goiter. Frank to treat hyperthyroidism. (Note: Carbimazole is not available , Reduced serum concentration; , increased serum concentration; ,
beta chain,128 and the TSH receptor.19,58 Mutations in anterior hypothyroidism, however, is far less common because with in the United States.) When large doses of PTU are used, the no change.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1921 1922 Section V Pathophysiology

drug decreases peripheral conversion of T4 to T3 through can produce generalized thyroid gland tenderness. Bacterial definition of normal, because the reference population mostly Practice Management Guideline on thyroid function testing
inhibition of the D1 deiodinase. Because of this effect (in infection of the thyroid gland is termed acute thyroiditis and excludes individuals with endogenous thyroid disease or pro- reported intraindividual coefficients of variation for TSH of
addition to the direct suppressive effect of PTU on the thyroid can produce a thyroid abscess.3 pensity to develop thyroid disease. However, it is unclear how 19.3% for 1 week, 20.6% for 6 weeks, and 22.4% for 1 year.13
gland), some endocrinologists argue that PTU should be patients with TSH concentrations between 2.5/3.0 mIU/L and A significant source of biological variation in TSH is the
chosen over methimazole for the treatment of hyperthyroid- Central Hypothyroidism 4.0/5.0 mIU/L should be managed. It has been argued that pulsatile pattern of secretion and the fairly short half-life of
ism. However, recognition of serious or even fatal liver disease In a patient with clinical evidence of hypothyroidism, sup- lowering the upper limit of the reference interval will provide TSH (approximately 1 to 2 hours).80 In one study, TSH was
in a small (but significant) number of children treated with ported by laboratory findings of a low FT4, if TSH is not no clear patient benefit and inevitably will lead to confusion released in approximately 18 pulses over the course of a day,
PTU is increasing.100 Therefore, many pediatric endocrinolo- elevated to the extent predicted (by FT4), or if TSH is within and possible overtreatment of subclinical hypothyroidism. and TSH half-life was 60 to 90 minutes. The half-life of TSH
gists discourage the routine use of PTU for the treatment of or below the reference interval, central hypothyroidism Overtreatment carries the risk of predisposing the patient to may vary between individuals, depending on interindividual
hyperthyroidism in children. Reduced granulocyte counts should be considered.92,229 Isolated pituitary TSH deficiency osteoporosis and atrial fibrillation. As noted previously, the differences in the degree of sialylation and sulfation of
and subsequent infections are serious but uncommon side is rare, as most patients with secondary hypothyroidism also value of treatment when TSH is between 4.0/5.0 and 9.9 TSH.47,149
effects of thionamides. Therefore the white blood cell count have other anterior pituitary hormone deficiencies (panhypo- mIU/L is currently debated. Another component of the TSH FT4 and TSH are usually measured approximately 6 weeks
and the differential count should be monitored during such pituitarism). Causes of central hypothyroidism (hypotha- reference interval debate is that according to National Health after the beginning of thyroxine treatment for hypothyroid-
therapy. lamic or pituitary disease) include (1) tumor, (2) hemorrhage, and Nutrition Examination Survey (NHANES) data,9,134A,209 ism. The dose of thyroxine should be titrated upward until
A potential advantage of methimazole over PTU is its (3) trauma, (4) malformation, (5) postinfectious damage, and TSH rises as populations age. For example, in healthy adults FT4 is in the upper half of the reference interval. TSH returns
longer half-life (PTU t1/2 = 1.5 hours and methimazole t1/2 = (6) postsurgical damage. Also, several rare autosomal reces- age 80 and older, the upper limit of the reference interval for to the reference interval more slowly than the rise of FT4 into
6 hours). Both PTU and methimazole cross the placenta, can sive or dominant hereditary disorders involving transcription TSH is 7.5 mIU/L. the reference interval occurs in response to therapy. Ideally
interfere with fetal thyroid function, and can cause goiter. factor mutations can cause hypopituitarism and TSH defi- during treatment, TSH should be maintained within the ref-
Additionally, methimazole has been associated with aplasia ciency: examples include paired-like homeodomain tran- Thyroid Hormone Monitoring During erence interval. As the dose of thyroxine is increased during
cutis and choanal atresia. Therefore during pregnancy, PTU scription factor-1 (Pit-1) and PROphet of Pit-1 (PROP1) Replacement Therapy the initial months of therapy, some endocrinologists will
is the preferred drug in the treatment of maternal mutations.131 The thyroid hormone replacement of choice is Na+-l- allow TSH to be slightly suppressed (but >0.02 to 0.04 mIU/L)
hyperthyroidism. thyroxine (levothyroxine; T4). Several brands of T4 are avail- if the patient feels substantially better. It can be argued that,
A variety of other drugs have been known to cause hypo- Subclinical Hypothyroidism able, including Synthroid (Abbott, North Chicago, Ill), particularly in women, it is prudent to measure bone mineral
thyroidism. For example, lithium, which is used in the treat- Subclinical hypothyroidism is defined by a persistent eleva- Levothroid (Forest, St Louis, Mo) and Levoxyl (King, Bristol, density by dual-energy x-ray absorptiometry (DEXA) scan at
ment of bipolar disorder (manic-depressive illness), can tion in TSH (6 to 12 weeks or longer) in the setting of FT4 Tenn). USP (United States Pharmacopeia) Thyroid or mix- least once a year to screen for bone loss that could result from
induce hypothyroidism.85 The action of lithium appears concentrations that are repeatedly found within the reference tures of T4 and T3 should not be used for thyroid hormone overtreatment with thyroxine. Overtreatment of hypothy-
similar to that of high-dose iodine, inhibiting thyroid interval.49 Other conditions wherein TSH is elevated but FT4 replacement. USP Thyroid is a desiccated extract of whole roidism is not uncommon.7
hormone release and organification of iodine. Prolonged use is normal encompass recent reinstitution of thyroid hormone animal thyroid containing variable proportions of T3 and T4.
of nitroprusside, a drug used to treat acute-onset, severe replacement therapy (FT4 returns to normal before TSH Because its exact formulation is highly variable and is not
hypertension or severe heart failure by inducing preload and declines), poor compliance with treatment in primary hypo- standardized, USP Thyroid will not produce stable concentra- HYPERTHYROIDISM
afterload reduction, may lead to hypothyroidism. Cyanide thyroidism, recovery from nonthyroidal illness, positively tions of thyroid hormone over the long term, leading to Hyperthyroidism, also known as thyrotoxicosis, is the clinical
(CN) released from nitroprusside is metabolically converted interfering heterophilic antibodies [e.g., human antimouse periods of excess or deficient thyroid hormone replacement. syndrome that results from elevated concentrations of free
to thiocyanate (SCN), which inhibits iodide uptake by the antibodies (HAMA)] in double-antibody immunoassays, and Despite many trials, fixed mixtures of T3 plus T4examples thyroid hormone in the plasma, associated with clinical
thyroid gland. Fortunately, nitroprusside only rarely causes thyroid hormone resistance. include Naturethroid (RLC, Tempe, Ariz) and Thyrolar evidence of hypermetabolism.135,139,178A Symptoms of thy-
hypothyroidism. Amiodarone is an antiarrhythmic drug that Subclinical hypothyroidism is very common, affecting 3 to (Forest)have not been shown to be clinically superior to T4 rotoxicosis include (1) nervousness, (2) erratic behavior,
contains two iodine atoms per molecule and can induce 8% of the general population. By the sixth decade of life, 10% replacement alone. Administering T4 alone allows the body (3) emotional lability, (4) restlessness, 5) sleeplessness,
hypothyroidism or hyperthyroidism.22,204 Various recom- of the population exhibits laboratory findings consistent with to regulate the production of T3. Recall that normally 80% of (6) difficulty concentrating, (7) smooth and/or shiny hair
binant DNA-derived biologicals (e.g., interferon-alpha, subclinical hypothyroidism. Consequences of subclinical T3 comes from peripheral monodeiodination of T4 to T3. The and/or skin, (8) weight loss, (9) excessive sweating, (10) heat
interleukin-2) used to treat chronic viral hepatitis or cancer hypothyroidism include a high risk of progression to clinical sodium salt of T3 (liothyronine) is available in tablet (Cytomel, intolerance, (11) menstrual irregularities, and (12) diarrhea
have been found to cause thyroid dysfunction (hypothyroid- hypothyroidism, dyslipidemia, vascular endothelial dysfunc- King) and injectable (Triostat, King) forms. Considerable or frequent bowel movements. Physical signs compatible
ism or hyperthyroidism).124 tion, increased risk of cardiovascular disease and death, and controversy surrounds whether generic preparations of T4 are with hyperthyroidism include tachycardia, atrial arrhyth-
possible neurocognitive deficits. The general consensus is that as effective as brand name preparations because of variable mias, systolic murmurs, increased pulse pressure, a bounding
Surgical and Radiation-Induced Hypothyroidism if TSH is repeatedly 10 mIU/L or greater, thyroid hormone consistency and/or absorption from the gastrointestinal tract. pulse, warm and/or damp skin, softened texture of the
Surgical removal of the thyroid gland will produce hypothy- replacement therapy is warranted. It is controversial whether Anecdotal reports have suggested that brand name prepara- skin, tremor, increased reflexes, eyelid retraction (and
roidism. External irradiation of the thyroid gland (e.g., treat- thyroid hormone therapy is beneficial when TSH is less than tions are clinically superior to generics. other signs of ophthalmopathy in Graves disease), and
ment of lymphoma or Hodgkin disease) or ingestion of 10 mIU/L, except during pregnancy, wherein any persistent Thyroxine should be taken at the same time of day for hypocholesterolemia.
radioactive iodine can ablate the gland, causing hypothyroid- TSH elevation requires treatment. clinical consistency. The timing of FT4 measurements should Based on TSH and FT4, the causes of hyperthyroidism may
ism.123 Administration of radioactive iodine to pregnant Controversy is ongoing over the appropriate upper limit also be standardized because FT4 may rise abruptly within be classified as primary (elevated FT4 and decreased TSH) or
women can lead to ablation of the fetal thyroid gland, causing of the reference interval for TSH. Basing the TSH reference hours of oral thyroxine ingestion. Thyroxine is best absorbed central (elevated FT4 and normal to elevated TSH). Central
congenital hypothyroidism. interval on the central 95% of the general, healthy population without food. A 2009 study demonstrated that TSH was hyperthyroidism can be further parsed into pituitary (sec-
results in an upper limit of normal between 4.0 and 5.0 mIU/L lowest when thyroxine was taken in the fasting state (mean ondary hyperthyroidism from TSH excess) and hypothalamic
Viral or Bacterial Thyroiditis for many TSH assays. If patients with thyroid autoantibodies TSH, 1.06 mIU/L) versus when taken with breakfast (2.93 (tertiary hyperthyroidism from TRH excess) causes. In
Only rarely do viral or bacterial infections of the thyroid such as TPOA, a family history of thyroid disease, or an mIU/L) or at bedtime (2.19 mIU/L).12 Many reasons have primary hyperthyroidism, TSH is typically less than 0.05
gland occur. Even more rarely do these entities damage the abnormal thyroid gland ultrasound are excluded from the been put forth for variations in TSH measurements over mIU/L. With current assays, a TSH concentration within the
thyroid gland sufficiently to produce hypothyroidism. Viral reference population, the upper limit of the reference interval time,52 including intraindividual variation of 15 to 35%.6 The reference interval nearly always excludes the diagnosis of
infection of the thyroid gland is termed subacute thyroiditis declines to approximately 2.5 to 3.0 mIU/L. Therefore, an 2003 National Academy of Clinical Biochemistry Laboratory primary hyperthyroidism.
(de Quervain, granulomatous, or giant cell thyroiditis) and upper limit of 2.5 to 3.0 mIU/L for TSH may be a truer
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1923 1924 Section V Pathophysiology

Causes of Hyperthyroidism subsequently develop, necessitating surveillance for hypothy- Hashitoxicosis and Postpartum Thyroiditis TSH receptor loss-of-function mutations. Any nodule that is
The causes of hyperthyroidism are listed in Box 55-3. Endog- roidism with yearly TSH measurements. During the clinical course of Hashimoto thyroiditis, if a not hot may be cancerous, and fine-needle aspiration biopsy
enous disorders causing hyperthyroidism include intrinsic period of accelerated destruction of thyroid follicular cells should be strongly considered.
thyroid disease (primary hyperthyroidism), ectopic thyroid T3 Toxicosis occurs, subsequent release of thyroid hormone can produce
tissue (struma ovarii),166 and disorders of the hypothalamus T3 toxicosis is defined by the presence of clinical hyperthy- a transient interval of hyperthyroidism, termed Hashitoxico- Gain-of-Function Mutations in the TSH Receptor
or pituitary causing excess TSH secretion (secondary hyper- roidism in a patient with suppressed TSH, normal FT4, and sis.133 Hashitoxicosis should be differentiated from Graves A familial, autosomal dominant form of nonGraves disease
thyroidism). Exogenous causes of hyperthyroidism (disease elevated T3 (or FT3).50 FT4 may be normal in early hyperthy- disease because the treatments for these two conditions are hyperthyroidism that is due to gain-of-function mutations in
related to external factors) encompass infectious origins roidism when a very mild excess of thyroid hormone is different. Hashitoxicosis is self-limited, and if any treatment the TSH receptor has been described.89 The gain-of-function
(thyroid gland inflammation and destruction), iodine- causing increased thyroidal and peripheral deiodination of T4 is required, -blockers (such as propranolol) are used to sup- mutation places the TSH receptor in the on position in the
induced hyperthyroidism, and thyroid hormone ingestion to T3, raising the latter but not the former. Recall that D1 press the effects of excess catecholamines, such as tachycar- absence of ligand (TSH) binding. In infants homozygous for
(thyrotoxicosis factitia). conversion of T4 to T3 is enhanced in hyperthyroidism. T3 dia. TRA are usually positive in patients with Graves disease such mutations, neonatal thyrotoxicosis, even requiring emer-
The first line of treatment for primary hyperthyroidism is toxicosis is also possible when hyperthyroidism occurs in the and negative in Hashitoxicosis. In addition, the RAIU is ele- gency thyroidectomy, has been observed. Certain heterozy-
controversial. Administration of radioactive iodine is the first presence of mild iodine deficiency, when iodine is sufficient vated in Graves disease but is not elevated in Hashitoxicosis. gous mutations have caused infantile hyperthyroidism.217
choice in many adults and older children with Graves disease. to synthesize excessive amounts of T3 but not T4. Last, it is Patients with postpartum thyroiditis can experience a period
When medical therapy is chosen, thionamides are used to known that stimulation of the TSHR enhances the produc- of transient, usually self-limited, hyperthyroidism from accel- Central Hyperthyroidism
suppress thyroid gland hyperactivity.195 In the immediate tion of T3 more than T4. T3 toxicosis is not a unique or indi- erated breakdown of thyroid tissue. Subacute or acute thy- TSH-secreting anterior pituitary adenomas are very rare.103
period following the start of thionamide therapy, TSH mea- vidual disease, but rather can be considered a phase in the roiditis can produce a period of transient hyperthyroidism. This diagnosis is suggested by (1) clinical hyperthyroidism,
surements may poorly reflect the patients thyroid status natural progression of primary hyperthyroidism that may or (2) elevated FT4, (3) normal to elevated TSH concentration,
because TSH can remain suppressed for months even after may not be recognized by the clinician. Toxic Nodular or Multinodular Goiter and (4) evidence of a pituitary mass on computed tomogra-
the patient becomes clinically euthyroid. This is similar to the A subset of patients with nodular or multinodular goiter can phy (CT) scan or magnetic resonance imaging (MRI). Inci-
treatment of hypothyroidism, where 6 weeks or more can Graves Disease develop hyperthyroidism.35,155,181 When hyperthyroidism is dentalomas of the pituitary are found in approximately 5% of
elapse before TSH returns to normal even with adequate thy- Graves disease affects approximately 0.4% of the U.S. popula- caused by these lesions, the term toxic has been used (toxic the general population; therefore a CT or MRI anomaly is not
roxine replacement. tion and results from agonistic autoantibodies that bind to, multinodular goiter). In the older literature, toxic multinodu- pathognomonic for a pituitary adenoma. A thyrotropinoma
After 1 to 2 years of thionamide therapy, if disease remis- and activate, the TSH receptor, producing excess release of lar goiter is referred to as Plummer disease. can produce a mass displacement effect, resulting in deficien-
sion has not occurred, radioactive iodine is usually advised.71 thyroid hormone and subsequent clinical hyperthyroidism.24 The cause of nodular or multinodular goiter is not well cies of other anterior pituitary hormones caused by compres-
An alternative therapy is thyroidectomy, particularly in spe- Women are more frequently affected by Graves disease than characterized. One theory is that these disorders arise from sion of pituitary tissue.96
cialized centers with extensive surgical experience. However, men, by a 5 : 1 ratio. colloid goiters.37 A colloid goiter itself is a disorder of unknown Rarely, selective pituitary resistance to the effects of thyroid
with radioactive iodine ablation of the gland, there is no The classical clinical triad observed in patients with origin, in which the thyroid gland enlarges because of hormone is present, yet peripheral sensitivity is normal.226
surgical risk of inadvertent parathyroidectomy or recurrent Graves disease consists of (1) goiter and biochemical hyper- increased size of the follicular lacunae.5 Microscopically, the This condition produces clinical hyperthyroidism because
laryngeal nerve injury. As a consequence of radioactive iodine thyroidism, (2) exophthalmos, and (3) nonpitting pretibial enlarged lacunae display wide variation in size. However, only elevated concentrations of thyroid hormones suppress
treatment or thyroidectomy, primary hypothyroidism can myxedema.220 The presence of exophthalmos strongly sug- colloid goiters do not display the follicular cell hyperplasia TSH. The subsequent rise in TSH promotes thyroid hormone
gests Graves disease in a hyperthyroid patient. Exophthalmos typical of Graves disease. Patients with colloid goiter lack synthesis and release from the thyroid gland, producing clini-
appears to have an autoimmune cause. Retro-orbital tissues thyroid autoantibodies, and no evidence suggests a defect in cal hyperthyroidism because the peripheral tissues have
may express TSH receptors, and consequent to stimulatory thyroid hormone biosynthesis. Over many years, some normal responsiveness to thyroid hormone. Selective pitu-
BOX 55-3 Causes of Hyperthyroidism TSH receptor autoantibodies, retro-orbital tissue hyperplasia regions of colloid goiters undergo further hypertrophy, while itary resistance to thyroid hormone feedback is suggested
occurs, producing exophthalmos.17 Myxedema is the accu- other areas atrophy, producing nodules that may become pal- when the pituitary CT scan or MRI is normal. Unique muta-
Endogenous Thyroid Disorders mulation of mucopolysaccharides in subcutaneous tissue that pable. TRA are negative in toxic multinodular goiter.214 tions in TR causing selective pituitary resistance have been
Autoimmune thyroid disease cause visible and palpable swelling. The potential finding of In some cases of nodular or multinodular goiter, the described.169
Graves disease myxedema in both Graves disease and Hashimoto thyroiditis hyperactive tissue can display a somatic gain-of-function
Hashitoxicosis (with hypothyroidism) is not readily explained. mutation in the TSH receptor or Gs protein, so that the recep- Hyperthyroidism Due to Human
Postpartum thyroiditis Similar to Hashimoto thyroiditis, genetic susceptibility to tor is perpetually in the on confirmation or functions as Chorionic Gonadotropin
Gain-of-function mutation in the TSH receptor Graves disease is polygenic.66 In practice, Hashimoto thy- though it is in the on position, in the absence of TSH Human chorionic gonadotropin (hCG)induced hyperthy-
Toxic nodule
roiditis is often associated with HLA-DR5 while Graves binding to the receptor.201 This mutation occurs in at least roidism is observed in gestational transient thyrotoxicosis,
Toxic multinodular goiter
disease is associated with HLA-DR3. 60% of cases of nodular or multinodular goiter with hyper- TSH receptor sensitivity to appropriate hCG concentrations
Toxic adenoma
Familial TPOA (including TMA) and TgA are seen in both Hashi- thyroidism. However, in the other 40% of cases, the cause of during pregnancy, and hCG-secreting tumors.94,97,163 Gesta-
Struma ovarii moto thyroiditis and Graves disease but usually are found in the hyperthyroidism is unknown. Somatically acquired gain- tional transient thyrotoxicosis occurs in 2 to 3% of all preg-
hCG-induced hyperthyroidism higher concentrations in Hashimoto thyroiditis. However, of-function mutations in the Gs alpha subunit produce many nancies, and results from activation of TSH receptors by
Gestational transient thyrotoxicosis these autoantibodies do not distinguish between the two con- of the symptoms of McClune-Albright syndrome, including hCG, which is greatly elevated during pregnancy. The degree
TSH receptor sensitivity to hCG ditions. Testing for TSH receptor autoantibodies should be thyrotoxicosis. of hyperthyroidism is typically mild, and treatment is not
hCG-secreting tumors reserved for cases where the diagnosis of Graves disease is in Sometimes follicular adenomas are autonomously hyper- usually required. In contrast to Graves disease, thyroid auto-
Secondary hyperthyroidism (e.g., central hyperthyroidism) doubt. TSH receptor autoantibody (TRA) testing is available active, producing hyperthyroidism (toxic adenomas).87 Other antibodies are negative. If PTU or methimazole is used, fetal
for stimulatory autoantibodies [TSH receptor stimulatory than elevated FT4 and depressed TSH from hyperthyroidism, monitoring for thyroid dysfunction is important, because
Exogenous Disorders autoantibodies (TSI)] and receptor-binding autoantibodies no specific laboratory tests can diagnose nodular or multi- these drugs cross the placenta, potentially causing fetal hypo-
Thyroid destruction from viral or bacterial thyroiditis
[thyrotropin-binding inhibitory immunoglobulins (TBII)]. nodular goiter, or a toxic adenoma. Toxic (hyperactive, or thyroidism and goiter.
Iodine-induced hyperthyroidism
Usually, both TSI and TBII tests are positive in patients with hot) nodules are very rarely carcinomas. Cold nodules Fetal thyroid gland suppression by thionamides is a
Thyroid hormone ingestion (thyrotoxicosis factitia)
Graves disease. (nodules that do not synthesize thyroid hormone and do not concern when any pregnant woman with hyperthyroidism is
take up radioactive iodine or technetium) may result from treated. The size of the fetal thyroid gland can be monitored
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1925 1926 Section V Pathophysiology

by ultrasound. TSH and T4 can be measured in fetal blood TSI. In these cases, neonatal hyperthyroidism, although
obtained by cordocentesis (a high-risk procedure) or in the potentially serious and possibly fatal if not recognized and
amniotic fluid.34 Besides the analytical challenge of measur- appropriately treated, is usually self-limited.
ing the very low concentrations of these hormones in non-
plasma body fluids, finding an appropriate reference interval Thyroid Storm and Apathic Hyperthyroidism I I D1,D2 I I
D3 I
can be difficult. Also, it is unlikely that many laboratories Thyroid storm (also, thyrotoxic crisis) is an uncommon syn-
have validated measurements of thyroid hormone or TSH in drome of severe and accelerating hyperthyroidism potentially
amniotic fluid. Hyperemesis gravidarum (severe vomiting manifested in (1) tachycardia, (2) restlessness, (3) high- OH OH OH
during pregnancy) has been associated with gestational tran- output congestive heart failure, (4) fever greater than 41 C, T3 T4 rT3
sient thyrotoxicosis and may be related to very high hCG (5) extreme irritability, (6) delirium or coma, (7) hypoten- D3 D1,D2
concentrations.147 sion, and (8) vomiting or diarrhea.134 Although laboratory D1,D2 D3
Tumors that secrete hCG, such as choriocarcinoma, hyda- tests reveal suppressed TSH and elevated FT4 and T3 concen-
tidiform mole, or metastatic embryonal carcinoma, have trations, thyroid storm remains a clinical diagnosis. Thyroid
caused hyperthyroidism through hCG stimulation of the storm cannot be diagnosed on the basis of the magnitude of
TSH receptor.212 FT4 or T3 concentrations, or of changes observed in these CH2 CH2 CH2
hormones compared with previous measurements.
Iodine-Induced Hyperthyroidism In older individuals, an unusual variant of hyperthyroid- I I I
In a patient with iodine deficiency and underlying Graves ism is termed apathetic hyperthyroidism.127 In this disorder,
disease or nodular/multinodular goiter, iodine administra- biochemical evidence of thyroid hormone excess is observed
tion may lead to hyperthyroidism.132 Before iodine replenish- in the absence of any clinical evidence of hyperthyroidism. I I I
ment is provided, hyperthyroidism is likely to be kept in Alternatively, only a single organ system may be affected. 3,3T2
3,5 T2 3,5T2
check by iodine deficiency.193 The phenomenon of iodine-
induced hyperthyroidism is termed the Jod-Basedow syn- Subclinical Hyperthyroidism Figure 55-17 Effects of altered deiodination. In states of nonthyroidal illness (e.g.,
drome (or phenomenon). Distinct from these two disorders A persistent depression in TSH when FT4 and T3 concentra- the sick euthyroid syndrome), thyroid hormone deiodination patterns are altered,
with reduced T3 concentrations and elevated concentrations of rT3.
(iodine-induced hyperthyroidism in iodine-deficient Graves tions are normal identifies subclinical hyperthyroidism.55
disease and nodular/multinodular goiter) are rare patients Patients with subclinical hyperthyroidism have increased fre-
without underlying thyroid disease who paradoxically quencies of atrial fibrillation and osteoporosis. In the absence
develop hyperthyroidism with iodine treatment.10,101 of a clinically compatible cardiac arrhythmia or significantly TABLE 55-5 Nonthyroidal Illness
Sources of iodine besides the diet include administered reduced bone mineral density, there is little justification for Initial Mid Course Prolonged Resolution
iodinated radiocontrast dyes, Lugols solution, topical anti- placing a patient with subclinical hyperthyroidism on thion-
septics, iodine-containing expectorants, and amiodarone.152 amide therapy. The treatment of patients with asymptomatic TSH Nl Nl, Decr Decr Incr
The typical intake of iodine in the United States is 150 to hyperthyroidism is much more controversial than the treat- T4 Nl Variable Decr Decr
200 g/d, one 200 mg tablet of amiodarone contains 75 mg ment of those with asymptomatic hypothyroidism. T3 Decr () Decr ( ) Decr ( ) Decr ()
of iodine, of which 6 mg is liberated (corresponding to at least rT3 Incr (+) Incr (+ +) Incr (+) Incr (+)
30 times the usual daily intake!). Nonthyroidal Illness (Sick Euthyroid Syndrome)
Note: The severity of decreased T3 is proportional to the number of () signs. The degree of increase in rT3 is proportional to the number of (+) signs.
Any type of significant nutritional deprivation, acute severe
Other Exogenous Causes of Hyperthyroidism illness, or chronic illness can result in thyroid function
Thyroid gland destruction from any cause, including nonau- changes characterized as nonthyroidal illness (NTI; sick pituitary, the pituitary is exposed to adequate concentrations contributing to the patients acute need for hospitalization
toimmune thyroiditis from viral (subacute) or bacterial euthyroid syndrome; low T3 syndrome; Table 55-5).1 Sup- of T3 from intrapituitary conversion of T4 to T3. Therefore, (such as cardiovascular disease, heart failure, or coma).
(acute) causes, can release excessive amounts of thyroid pressed peripheral conversion of T4 to T3 by D1 and D2 leads although plasma T3 may decline in NTI, TSH concentrations In a hospitalized patient, thyroid replacement therapy
hormone, producing hyperthyroidism.179,180 Intentional or to decreased circulating T3 (and FT3), while diminished deg- do not rise (at least not before the recovery stage of NTI). The should be considered only if the TSH is significantly elevated
unintentional ingestion of excess thyroid hormone can also radation of rT3 by D1 raises circulating rT3 concentrations pulsatility of TSH is attenuated, yet TSH typically remains (20 mIU/L). When modest elevations in TSH are observed
produce hyperthyroidism.76 In one interesting report, hyper- (Figure 55-17). In experimental systems, interleukin (IL)-1 within the reference interval. Occasionally, paradoxical (5 to 19 mIU/L), measurement of rT3 may be helpful; rT3 is
thyroidism developed in people who ate hamburger meat that and IL-6 have been shown to impair D1 activity. T3 can decline increases in FT4 result from T4 displacement from thyroxine- low in hypothyroidism, but elevated in NTI. However, rT3
included bovine thyroid glands added during the grinding by as much as 50% with a twofold to threefold increase in rT3. binding proteins caused by elevated free fatty acid concentra- measurements usually are not essential to the diagnosis of
process.146 Poorly controlled diabetes mellitus and drugs such as gluco- tions, as well as other uncharacterized substances that rise in NTI. If a long-term hospitalized patient without hypotha-
When thyroid hormone is ingested or released from an corticoids and -blockers can also inhibit the conversion of concentration in NTI. lamic or pituitary disease has TSH less than 0.05 mIU/L,
inflamed or damaged thyroid gland, subsequent suppression T4 to T3. As mentioned previously, one effect of propylthio- With prolonged NTI, disordered hypothalamic-pituitary hyperthyroidism is a possibility. With hospitalized patents, a
of TSH reduces thyroidal iodine uptake, which is reflected in uracil is to block D1, inhibiting the conversion of T4 to T3. function causes a decline in TSH concentration, which results cooperative dialogue between the clinician and the laborato-
a reduction in the RAIU test. Likewise, glandular suppression Decreased T3 reduces oxygen and nutritional demands in a concurrent decline in FT4. During recovery, TSH can rise rian can be helpful in deciding the proper diagnosis and
will lower circulating Tg concentrations. If T3 is ingested (e.g., and is considered to be an adaptive and beneficial response above the reference interval and remain elevated until FT4 management of the patient.
Cytomel), hyperthyroidism can ensue, producing an unusual to illness. The recognition that NTI does not represent and T3 return to normal, usually after 1 to 2 months. The
set of biochemical abnormalities: suppressed TSH, sup- hypothyroidism is important because thyroid hormone sequential stages in NTI are illustrated in Table 55-5. Thyroid Hormone Resistance
pressed FT4 (and total T4, because of TSH suppression), and replacement therapy of patients affected with NTI does not Because of NTI, it can be very difficult to interpret thyroid Loss-of-function mutations in the TR chain (the intranu-
elevated total T3 (and FT3). improve clinical outcome and may be detrimental.48,79,207 function abnormalities in hospitalized patients. Thus, thyroid clear thyroid hormone receptor, beta chain) lead to the rare
Maternal Graves disease can cause fetal (and transient Because FT4 concentrations are usually normal in NTI (at function testing in inpatients should be carried out only when syndrome of thyroid hormone resistance.2 In this autosomal
neonatal) hyperthyroidism from the transplacental passage of least in the early stages), and D2 activity is sustained in the there is the legitimate suspicion that thyroid dysfunction is dominant condition, supraphysiologic concentrations of T3
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1927 1928 Section V Pathophysiology

and T4 (and FT3 and FT4) are required to maintain the euthy- distinguished from conditions involving overproduction of including pregnant women or the elderly, improves the clini- thyroid hypoplasia), factitious hyperthyroidism (because of
roid state. Therefore, in the absence of clinical hyperthyroid- TBG by measuring TBG concentration. cal outcome of the patient. TSH suppression), and following thyroidectomy.
ism, if the biochemical picture of elevated T4, FT4, T3, and FT3
is present with TSH in the upper reference interval or mildly MCT8 Mutations Clinical Utility of Thyroglobulin Measurements
elevated, thyroid hormone resistance is likely. Because of TSH Loss-of-function mutations in MCT8, causing a rare form of DRUG EFFECTS ON THYROID FUNCTION Thyroid cancer is classified according to the cell of origin
stimulation of the thyroid, glandular enlargement may be X-linked mental retardation, have been reported.74,211 Because Various drugs alter thyroid function in a large number of (follicular vs. parafollicular) and the degree of differentiation
seen because there is no resistance to TSH. Some affected MCT8 is important for the transport of thyroid hormone into ways.60,170 For example, dopamine, l-dopa, glucocorticoids, (cancers of follicular origin, including differentiated papillary,
patients may, in fact, have mild clinical findings of hypothy- neurons, there are profound adverse effects on the develop- and somatostatin all depress TSH, potentially leading to a follicular, or Hrthle cell carcinoma, vs. anaplastic thyroid
roidism. If patients with thyroid hormone resistance are ment of the nervous system in affected males. In addition to fall in thyroid hormone concentrations. As noted previ- cancer). Following surgery for differentiated thyroid carci-
incorrectly treated with thyroid-suppressing drugs, clinical mental retardation and hypotonia, an unusual pattern of ously, iodine (including amiodarone) and lithium impair noma, the completeness of tumor excision can be assessed by
hypothyroidism may be induced. thyroid function abnormalities occurs, with increased T3, thyroid hormone synthesis or release, causing primary measuring serum Tg concentrations.54,67 If thyroglobulin
Another form of thyroid hormone resistance results from normal to decreased T4 (and FT4), normal to elevated TSH, hypothyroidism.85 autoantibodies are present (see later), Tg measurements are
defective conversion of T4 to T3. This condition results from and low rT3 concentrations. Many drugs inhibit conversion of T4 to T3, including amio- unreliable.
a mutation in the sequence-binding protein 2 (gene name: Defective cellular uptake of T3 can, in part, explain ele- darone, glucocorticoids, -blockers, propylthiouracil, and Following thyroidectomy and/or radioactive iodine
SECISBP2), which influences the synthesis of cellular vated T3 concentrations in the blood. However, evidence indi- radiographic contrast agents. While T3 declines and rT3 rises, therapy, plasma Tg should be undetectable unless residual
deiodinases. cates that not all tissues display equivalent defects in thyroid FT4 may increase, decrease, or remain unchanged. Other thyroid tissue or metastatic lesions are present. To ensure
hormone uptake, and this may explain the decline in T4 and drugs can displace T4 and T3 from thyroid hormonebinding that any remnant thyroid, or metastatic tumor, tissue is stim-
Euthyroid Hyperthyroxinemia rT3. Increased T3 concentrations in the liver may also promote proteins; salicylates, phenytoin, carbamazepine, furosemide, ulated to produce Tg, the patients TSH must not be sup-
Euthyroid hyperthyroxinemia exists when total T4 is elevated T4 and rT3 clearance. If the pituitary or hypothalamus is less nonsteroidal anti-inflammatory agents, and heparin (in vitro pressed (postoperative thyroid hormone replacement must
yet the patient is clinically euthyroid.158,205 Because FT4 mea- sensitive to negative feedback from thyroid hormones effect) lower total T4 and T3 by occupying binding sites be discontinued), or, alternatively, exogenous recombinant
surements have supplanted total T4 measurements, euthyroid (because T3 does not normally enter thyrotrophs or the hypo- on proteins, yet FT4 remains within the reference interval DNA-produced TSH (thyrogen-alpha; Thyrogen, Genzyme,
hyperthyroxinemia is not commonly encountered in modern thalamic PVN), theoretically, TSH concentrations would rise. or rises. Cambridge, Mass) may be given.140 Exogenous TSH admin-
clinical practice. The elevation in TSH may further contribute to elevated T3 Increased metabolism of thyroid hormone lowers T4 and istration normally stimulates at least a fivefold increase in Tg
Besides TBG elevations and the thyroid hormone resis- concentrations caused by increased T3 production by the T3. Phenobarbital, phenytoin, carbamazepine, and rifampicin if responsive tissue is present. With a local thyroid tumor
tance syndromes, euthyroid hyperthyroxinemia can also thyroid gland. The observation that MCT8 mutations exist can lower T4 and T3. Because (1) aluminum hydroxide, remnant (following incomplete thyroidectomy) or a well-
result from acute illness (through a mechanism that is requires recognition of a new pattern of thyroid function (2) ferrous sulfate, (3) cholestyramine, (4) colestipol, (5) iron differentiated thyroid tumor, TSH can stimulate a tenfold
not well understood) or abnormalities in albumin and studies. Various patterns of thyroid function tests are shown sucralfate, (6) soybean preparations, and (7) Kayexalate all increase in Tg concentration.188 With poorly differentiated
transthyretin. Familial dysalbuminemic hyperthyroxinemia in Table 55-6. inhibit thyroid hormone absorption, their effects should be tumors, Tg rises less than threefold after TSH. One caveat is
is an autosomal dominant disorder characterized by an considered in any patient being treated with thyroxine that the differentiated thyroid cancer should be shown to
albumin variant that has a greater binding affinity for T4 replacement. produce Tg before surgery is performed, lest the patient be
(but not T3).16,28 Although TSH, FT4, and total T3 (and SCREENING FOR THYROID DYSFUNCTION judged to be in remission following surgery but his or her
FT3) are all normal, total T4 is elevated. Mutations in trans- Other than newborn screening for congenital hypothyroid- cancer is a rare nonTg-secreting differentiated thyroid
thyretin can lead to familial euthyroid thyroxine excess, an ism, the value of thyroid function testing in asymptomatic THYROGLOBULIN carcinoma.
autosomal dominant disorder producing abnormal thyroid individuals is highly controversial.72,196,210 Excluding newborn The biology of Tg was extensively reviewed in the biochem- Tg measurements have other clinical uses. For example,
function tests similar to what is observed in familial dysalbu- screening for congenital hypothyroidism, so far no compel- istry section of this chapter. Some Tg normally enters the the absence of detectable Tg in the serum of an infant with
minemic hyperthyroxinemia.14 These two disorders can be ling data show that screening asymptomatic individuals, circulation by direct exocytosis from thyroid follicular cells congenital hypothyroidism supports the diagnosis of thyroid
without being iodinated. Tg is normally detected in the cir- dysgenesis. In cases of congenital hypothyroidism where Tg
culation in concentrations between 3 and 40 ng/mL. The is undetectable, but thyroid gland tissue is identified by
highest Tg concentrations are in cord blood and are noted thyroid scan, a Tg gene mutation should be considered. In
during the first weeks of life. Between 2 and 6 weeks of age, cases of suspected factitious hyperthyroidism (hyperthyroid-
TABLE 55-6 Patterns of Thyroid Dysfunction Tg concentrations are between 10 and 250 ng/mL,51 and they ism caused by exogenous thyroid hormone ingestion), Tg will
TSH FT4 Comments decline until adulthood.141 Three factors influence the con- be suppressed; in Graves disease, Tg is measurable.
centration of Tg in the plasma: (1) the degree of thyroid gland
Primary hypothyroidism Increased Decreased stimulation via the TSH receptor, (2) the mass of thyroid
Subclinical primary hypothyroidism Increased Normal tissue present in the individual, and (3) the presence of ANALYTICAL METHODS
Primary hyperthyroidism Decreased Increased T3: increased thyroid disease or damage, which leads to increased release The approach to the laboratory assessment of thyroid func-
T3 toxicosis Decreased Normal T3: increased of Tg into the circulation. tion has changed considerably over the past 2 decades. This
Subclinical primary hyperthyroidism Decreased Normal T3: normal When TSH is within the reference interval, each gram of change was driven by two technologic advances: (1) a
Thyroid hormone resistance due to Normal to increased Increased T3: increased; rT3: increased thyroid tissue provides approximately 1 ng/mL of Tg to the roughly 100-fold improvement in the lower detection limits
defects in the thyroid hormone circulation. At TSH concentrations less than 0.1 mIU/L, each of analytical methods to measure TSH, and (2) widespread
receptor gram of thyroid tissue releases only about 0.5 ng/dL of Tg availability of free (unbound) T4 assays on automated plat-
Thyroid hormone resistance due to Mildly increased Increased T3: decreased; rT3: increased
into the plasma. Trauma to the thyroid gland, inflammation forms. An unfortunate consequence of this change, however,
defects in thyroid hormone
(such as subacute thyroiditis or amiodarone-induced thy- was the emergence of a confusing array of terms used to
roiditis), surgical removal, or irradiation produces elevated describe the analytical methods that measure thyroid hor-
Thyroid hormone resistance due to Normal to increased Normal to decreased T3: increased; rT3: decreased
Tg concentrations. Following fine-needle aspiration, Tg can mones. Terms such as direct, indirect, index, one-step,
defects in thyroid hormone
be elevated for up to 3 weeks. Tg is typically decreased in and two-step are used in nonstandard and often inconsis-
transport into cells
acquired hypothyroidism, congenital hypothyroidism (from tent ways in reference to FT4 assays.120 In addition, the term
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1929 1930 Section V Pathophysiology

analog (or analogue, in the UK) is often used to describe which an interassay CV of 20% or less can be achieved. A wide variety of TSH methods are now available. highly (>99.9%) protein bound, and the fraction that is bound
FT4 immunoassays, as though these methods involve a tech- The American Thyroid Association determined that only Most TSH methods use a detection antibody labeled with a to protein is biologically inactive.
nology that is unique from other competitive and noncom- TSH assays with third-generation (0.01 mIU/L) functional chemiluminescent (or electrochemiluminescent) tracer.119,156
petitive binding immunoassays that make use of a chemically sensitivity are sufficient for use as screening tests for hyper- Some assays use peroxidase, alkaline phosphatase, photo- Instrument-Based Methods
modified antigen, when the approach basically is the same. thyroidism; their recommendation is consistent with the metric, and fluorescent167 labels. Other TSH methods are Electron capture gas chromatography,150 high-performance
Finally, generational numbers have been applied to TSH National Academy of Clinical Biochemistry Laboratory based on fluorescent labels using europium chelates,78 chemi- liquid chromatography,68 and isotope dilution tandem mass
immunoassays to designate the analytical sensitivity. In other Medicine Practice Guideline13 for assessment of thyroid luminescent compounds such as acridinium esters or ruthe- spectrometry126,183,184A have been used in measuring T4 in
analytes (such as troponin T), generational numbers mostly function. nium, or bioluminescent molecules such as recombinant human serum. The latter method has been suggested as a
refer to improvements in specificity, and more sensitive assays An additional complication in defining the analytical per- aequorin. reference method for assay of T4. With this technique, tritium-
for analytes such as C-reactive protein are simply designated formance of TSH assays is that the hormone is subject to Chemiluminescence-based immunoassays are available in labeled T4 is added as an internal standard before extraction,
as high sensitivity. Except where helpful to establish histori- structural heterogeneity because of genetic polymorphisms several configurations.109 For example, one assay involves a derivatization, and quantitation of T4 by combined gas
cal relationships, use of these terms will be avoided in favor and post-translational modifications affecting the carbohy- polystyrene tube coated with a monoclonal capture antibody chromatographymass spectrometry.
of more standard immunoassay terminology. drate content, as well as proteolytic removal of the six and a polyclonal detection antibody conjugated to an acri-
C-terminal beta subunit residues. There was poor agreement dinium ester; the acridinium ester emits a photon when Immunoassays
Measurement of Thyrotropin (TSH) between various TSH immunoassays until an International reacted with hydrogen peroxide in alkaline solution.189 Most clinical laboratories measure total T4 by using an auto-
Human thyrotropin [thyroid-stimulating hormone (TSH)] is Reference Preparation (IRP) isolated from pituitary tissue Another approach uses an antibody-coated tube, a polyclonal mated competitive immunoassay. Many T4 immunoassays
a 28 to 30 kDa dimeric glycoprotein consisting of a 92 amino was made available in the 1980s. In 1999, a recombinant antibody conjugated to alkaline phosphatase, and an adaman- use high-affinity polyclonal antibodies produced against an
acid alpha subunit (identical to the alpha subunit of human human TSH IRP was created,157 but significant differences tyl dioxetane phosphate ester as substrate; on dephosphoryla- albumin-T4 conjugate. These polyclonal antibodies are highly
chorionic gonadotropin, follicle-stimulating hormone, and have been noted between recombinant TSH and isolates from tion, the substrate decomposes and emits a sustained glow of specific and display minimal cross-reactivity with T3. Thyro-
luteinizing hormone) and a 112 to 118 amino acid beta pituitary tissue, primarily as the result of modifications of light.25 Cross-reactivity of this TSH assay with luteinizing globulin sometimes has been used as the immunogen because
subunit. Functionally important domains are present on both polysaccharide moieties on TSH, which are highly cell spe- hormone, follicle-stimulating hormone, human chorionic it contains iodinated tyrosine residues that are the precursors
alpha and beta subunits; neither is active alone. Residues 113 cific. Pituitary-derived TSH contains polysaccharides that are gonadotropin, or the free beta subunit of glycoprotein hor- of T4 and T3. Monoclonal antibodies against T4 have also been
to 118 of the beta subunit undergo post-translational proteo- mostly sulfated, however the recombinant hormone is pri- mones is less than 0.001%. developed.
lytic cleavage without affecting hormonal activity, so the marily sialylated.157 These modifications produce epitopic Immunoassays of total T4 measure both free and protein-
C-terminal end of the beta subunit apparently is not neces- diversity and change the antigenic profile of the protein. Specimen Collection and Storage bound hormone. Accurate measurement of total T4 therefore
sary for receptor binding. Efforts have been made to chemically modify recombinant Serum or plasma may be used for TSH measurements. TSH requires dissociation of the hormone from serum proteins
Although bioassays are available for measurement of TSH TSH to more closely resemble the circulating form of the is stable for 5 days at 2 to 8 C, and for at least 1 month when (thyroxine-binding globulin, albumin, and transthyretin)
activity, and radioimmunoassay (RIA) has been used as well, hormone. This approach eventually may result in better stored frozen. For newborn screening, whole blood may be that bind more than 99.9% of circulating T4. Dissociation of
nearly all modern TSH methods are two-site sandwich het- agreement between various TSH immunoassays.40 collected by heel puncture 48 to 72 hours after birth. T4 from albumin is not a concern because the association
erogeneous immunoassays involving an enzyme or chemilu- constant of T4 for anti-T4 antibodies (usually 109 L/mol)
minescent label. Migration to more sensitive TSH methods Principles of TSH Immunoassays Comments on TSH Measurements is several orders of magnitude higher than the association
about 2 decades ago prompted a change in the strategy for Modern automated TSH immunoassays typically involve a Secretion of TSH is circadian; peak concentrations of TSH constant for albumin-bound T4 (approximately 1.6 106 L/
laboratory investigation of thyroid dysfunction, establishing double-antibody approach [for which the enzyme-linked occur between 0200 and 0400, and the nadir occurs between mol). However, association constants for T4 binding to
TSH as its foundation. Sensitivity was the essential catalyst of immunosorbent assay (ELISA) is the prototype], with the 1700 and 1800. Low-amplitude oscillations occur throughout thyroxine-binding globulin (TBG) and transthyretin are
this change because TSH concentrations may be decreased in capture antibody directed toward the alpha subunit and a the day. The nocturnal increase in TSH is lost in critical higher: 2 1010 L/mol and 2 108 L/mol, respectively. T4
nonthyroidal illness, and the lower limit of detection was signal antibody that recognizes an epitope on the unique beta illness and after surgery. TSH surges immediately after birth, can be dissociated from transthyretin by the addition of
necessary to distinguish between nonthyroidal causes of sup- subunit. reaching a peak of 25 to 160 mIU/L within 30 minutes, barbital, which competitively inhibits T4 binding to this
pressed TSH and thyrotoxicosis, which is associated with Heterogeneous immunoassays differ in the method used and declining back to cord blood concentrations by postpar- protein. Various blocking agents have been used to dissociate
TSH concentrations less than 0.01 mIU/L. to separate bound and free fractions before application of the tum day 3. TSH concentrations stabilize to near adult con- T4 from TBG. The most common of these blocking agents is
The generational classification divides TSH methods by signal antibody. The various designs of automated heteroge- centrations within the first few weeks of life. In the first 8-anilino-1-naphthalene-sulfonic acid (ANS), although salic-
analytical sensitivities separated by roughly one log unit. neous immunoassays are discussed in Chapter 16. trimester of pregnancy, TSH concentrations decline as hCG ylate, thimerosal, and phenytoin have been used to displace
Thus, first-, second-, and third-generation methods have As noted previously, TSH immunoassays are calibrated stimulates the maternal thyroid gland to produce thyroid T4 from TBG. In some assays, proportional bias has been
functional sensitivities of 1.0, 0.1, and 0.01 mIU/L, respec- with a reference preparation of TSH (World Health Organiza- hormone, sometimes leading to a TSH concentration that is attributed to incomplete release of T4 from serum-binding
tively. Some methods claim a detection limit of 0.001 mIU/L tion Second International Reference Preparation, 2nd IRP just below the lower limit of the reference interval. Reference proteins.138
and, following this convention, are called fourth-generation 80/558) and are expressed as milli-international units of bio- intervals for adult TSH concentrations are the same for men Considerable effort has been directed toward the develop-
methods. These definitions are not formal but were loosely logical activity per liter of serum (mIU/L), a value established and women, and no significant racial influence on concentra- ment of immunoassays that do not require the use and
adopted by manufacturers of TSH assays and are used more by bioassay. In contrast to the inverse dose-response curve tions of this hormone has been noted. The distribution of measurement of radioactivity, and nonisotopic assays for T4
for marketing purposes than to express analytical perfor- produced in radioimmunoassay, immunometric assays gen- TSH concentrations in healthy populations is log-Gaussian are widely available for use on various automated immuno-
mance by standard criteria. erate a proportional dose-response curve, with larger signals (or log-normal); reference intervals should be determined assay systems, or adaptable to automated chemistry plat-
In 1991, the Nomenclature Committee of the American corresponding to higher concentrations of analyte. Because nonparametrically. forms. In recent College of American Pathologists Ligand
Thyroid Association recommended that the functional detec- the physiologic interval of TSH concentrations is typically Assay Surveys, the number of participants using RIA to
tion limit of serum TSH assays should be determined on three orders of magnitude, high-dose hook effects are rarely Measurement of Total Thyroxine (T4) measure total T4 was insufficient to report as a peer group. A
the basis of low-end interassay precision characteristics.69 encountered with TSH immunoassays. Automated heteroge- Thyroxine [3,5,3,5-tetraiodothyronine (T4)] is the principal variety of different labels have been used in the design of
The Committee recommended that precision [coefficient neous immunoassays for TSH offer improved sensitivity and hormone secreted by the thyroid gland (see Figure 55-4), and nonisotopic immunoassays. Enzymes such as horseradish
of variation (CV)] at the lower reporting limit should be rapid turnaround time, along with a wider linear measure- its circulating concentration is under the influence of TSH. peroxidase, alkaline phosphatase, and alpha-D-galactosidase
10 to 15%, but no worse than 20%. Currently, functional ment range, when compared with traditional competitive The hormone is synthesized from tyrosine residues on thyro- are popular, in addition to fluorescent and chemiluminescent
sensitivity is defined as the lowest concentration of TSH at immunoassays such as RIA. globulin in the colloid of the thyroid gland. Circulating T4 is labels.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1931 1932 Section V Pathophysiology

Heterogeneous enzyme immunoassays for T4 often involve antibodies. Polarized excitation light (485 nm) induces fluo- multilayer test module consisting of three distinct layers measuring free T4 are widely available and largely supplant
solid-phase separation systems. Some methods immobilize rescence of the fluorescein tracer. Polarization of the emitted mounted on a plastic support. The serum specimen is the need for total T4 measurements. As discussed later, free
the anti-T4 antibodies on a plastic surface (e.g., polystyrene light (525 to 550 nm) requires that the orientation of the applied to the top layer of film, which contains phenoxynaph- (unbound) T4 measurements are more useful clinically, except
beads, test tubes, microwells) so that bound and free analyte tracer remain fixed for the time interval between excitation thalene sulfonic acid, to displace T4 from binding proteins. in unusual circumstances.
may be separated by decanting and washing. For example, and fluorescence; this interval is typically nanoseconds. The The released T4 passes through an iron oxide screen into Cord blood T4 concentrations are lower in preterm than
one automated assay uses test tubes that contain polystyrene unbound T4-fluorescein conjugate has a rotational frequency the detection layer, where the hormone competes with in full-term neonates, and they correlate positively with birth
beads coated with a monoclonal anti-T4 capture antibody.11 of femtoseconds1, so the orientation of the uncomplexed rhodamine-labeled T4 for binding sites on immobilized T4 weight in full-term infants. At birth, serum total T4 concen-
T4 in the specimen is liberated from binding proteins by ANS tracer is randomized between excitation and fluorescence, antibodies. Unbound fluorescent-labeled T4 diffuses into the trations are higher in neonates because of the maternal
and competes with a T4alkaline phosphatase conjugate for and polarization is lost. However, when fluorescein-labeled opaque layers above the screen, where it cannot be detected. estrogen-induced increase in serum TBG; free T4 concentra-
binding sites on antibodies attached to the polystyrene beads. T4 is bound to antibody, the rotational frequency is several The fluorescent signal of the remaining antibody-bound tions are near adult concentrations. Total T4 rises abruptly in
After the unbound label is rinsed away, the bound enzyme is orders of magnitude slower, so excitation and fluorescence labeled T4 fraction is measured fluorometrically. This signal the first few hours after birth and declines gradually until
measured using a chemiluminescent substrate. This combina- occur in the same plane, and polarization is maintained. is inversely proportional to the concentration of T4 in the adolescence. In males, T4 production declines as they mature
tion of an enzyme label with a chemiluminescent substrate Heterogeneous fluorescent immunoassays for T4 based on specimen. sexually, but this phenomenon is not observed in females.
has been applied to other automated T4 procedures as well. lanthanide rare earth elements and time-resolved fluores- Analytical performance goals have been recommended for
An antibody-coated tube enzyme immunoassay has been cence are also available. The use of europium chelates as fluo- Specimen Collection and Storage thyroid hormone assays.13 When total T4 is used to diagnose
devised for measurement of T4 and includes a peroxidase-T4 rescent probes is particularly attractive because of their wide Serum is the preferred specimen for the measurement of T4, thyroid disease, the suggested goals for maximum bias and
conjugate with a photometric substrate. Stokes shifts and long fluorescence decay times, which but plasma with EDTA or heparin as anticoagulant has also imprecision (coefficient of variation) are 2.9% and 5.7%,
Several enzyme immunoassays attach paramagnetic par- improve the specificity of the measurement by limiting inter- been used. Plasma may form fibrin clots after freezing and respectively. When the T4 assay is used to monitor changes in
ticles to the anti-T4 antibodies so that free and bound T4 can ference from scattered excitation light and endogenous fluo- thawing, however, and may produce spurious results in an individual over time, bias and imprecision goals are 1.3%
be separated magnetically. In one automated scheme, plastic rophores. In a typical competitive immunoassay design, T4 methods that are susceptible to changes in specimen viscosity. and 2.6%, respectively.
cups contain magnetic beads coated with a T4 antibody. in the specimen competes with europium-labeled T4 for a Gel barrier collection devices do not have any apparent
Endogenous T4, displaced from its binding proteins by ANS, limited number of binding sites on monoclonal T4 antibodies. adverse effect on T4 methods. T4 is a stable analyte with no Measurement of Triiodothyronine (T3)
competes with alkaline phosphataselabeled T4 for a limited Antibody-bound and free antigen fractions are separated by appreciable change in concentration for up to 7 days at room Triiodothyronine [3,5,3-triiodothyronine (T3)] is the princi-
number of binding sites on the immobilized antibody. The adsorption of T4 antibodies to the surface of a microwell temperature, or 30 days when frozen. As with most analytes, pal active thyroid hormone, and although a small amount of
magnetized beads are washed to remove unbound enzyme- coated with antimouse immunoglobulin (Ig)G. After incuba- repeated freezing and thawing of specimens should be T3 is secreted directly by the thyroid gland, most of this
labeled T4 and then are incubated with a fluorogenic sub- tion and washing, the europium ions are dissociated from the avoided because it can produce concentration gradients that hormone is produced by deiodination of T4 in the peripheral
strate. As with all competitive immunoassays, the amount of bound phase, converted into a highly fluorescent chelate, and require vigorous mixing to alleviate. Mild to moderate hemo- tissues, primarily in the liver. Similar to T4, T3 is highly bound
enzyme-labeled T4 captured on the magnetic beads is inversely measured in a fluorometer with time resolution capability. A lysis and lipemia do not significantly affect most T4 immuno- to protein, with less than 1% of the total concentration as free
proportional to the T4 concentration in the specimen. variation on this technique involves attachment of biotin to assays; however, grossly hemolyzed specimens should be active hormone. Total T3 measurements have limited clinical
Homogeneous enzyme immunoassays have been devel- a monoclonal T4 antibody and streptavidin coupled to the avoided because of dilutional effects. Intracellular T4 concen- use in that the T3 concentration usually reflects its progenitor,
oped for measuring serum T4 concentration. Chapter 16 dis- europium chelate. trations are very low because thyroxine-binding proteins are T4. However, clinical conditions are known that affect periph-
cusses several approaches to homogeneous immunoassays. In Most contemporary automated immunoassays for mea- mostly extracellular. T4 autoantibodies interfere with some eral conversion of T4 to T3 (discussed earlier), resulting in a
the enzyme-multiplied immunoassay technique (EMIT) for suring total T4 use chemiluminescent labels. In one design, immunoassays and may produce erroneously low or high clinical hypothyroid state in the presence of normal or ele-
T4, glucose-6-phosphate dehydrogenase (G-6-PDH) is cova- endogenous T4 competes with T4 coupled to paramagnetic results, depending on the method.215 vated T4, with normal (or even elevated) TSH. Compared
lently linked to T4 as the enzyme label. When T4-specific particles for binding sites on a mouse monoclonal antibody Heelstick capillary specimens have been used for T4 mea- with T4, T3 is less tightly bound to serum proteins by about
antibodies bind to the enzyme-labeled T4, enzyme activity is labeled with acridinium ester. After incubation, the labeled surement, as have dried blood specimens collected on filter an order of magnitude, so displacement of bound T3 from
inhibited as the result of steric obstruction of the substrate- T4 is captured on a magnetized surface that is subsequently paper. Dried blood specimens are stable and easily trans- proteins is essentially complete in the presence of conven-
binding site. The cloned enzyme donor immunoassay washed to remove the unbound label. The antibody-bound ported and are widely used in the United States to screen tional blocking agents such as ANS. T3 does not ordinarily
(CEDIA) technique for measuring T4 is based on the use of labeled T4 is quantified in a luminometer after hydrogen per- neonates for congenital hypothyroidism.115 When this collec- displace T4 from thyroid hormonebinding proteins.
cloned fragments of alpha-galactosidase, a tetrameric hydro- oxide is added to initiate the chemiluminescence reaction. tion technique is used, a one eighth inch dot is punched out
lase enzyme that converts alpha-galactosides into galactose. Electrochemiluminescence has also been applied to from the blood-saturated filter paper and T4 is extracted into Radioimmunoassays
The CEDIA approach involves two fragments of the mono- total T4 measurement. In this technology, a ruthenium a buffer before the assay is performed. The filter paper should Numerous commercial RIA kits have been introduced for
meric subunit of alpha-galactosidase, the larger of which is tris(bipyridyl) [Ru(bpy)32+] complex is used as the antibody not be exposed to extreme heat or light. measuring total T3 concentrations in serum. These methods
called the acceptor, and the smaller of which is called the label. In the presence of tripropylamine and an applied elec- are similar to the RIAs previously described for T4, except that
donor. In solution, the two fragments spontaneously associate trical potential, the ruthenium complex undergoes a chemi- Comments on Total T4 Measurements a 125I-T3 tracer and T3-specific antibody are used. Most ana-
to form a monomeric subunit, which subsequently associates luminescent reaction. Total T4 concentration alone provides limited clinical infor- lytical designs incorporate antibodies bound to a solid phase,
with three other monomers to form the active enzyme. In the Particle-enhanced immunoassays involving turbidimetric mation, because it reflects mostly inactive (protein-bound) such as the wall of a cuvette or paramagnetic particles. As
CEDIA method, T4 is labeled with the donor fragment of measurements have been developed for measuring T4 in hormone. In fact, it is reasonable to question whether isolated with T4 methods, ANS can be used to release the hormone
alpha-galactosidase monomer, and antibody binding to the serum. These homogeneous immunoassays are based on measurements of total T4 currently have any value in the from proteins without disturbing T3 binding to antibody.
labeled T4 prevents association with the acceptor fragment; competition between T4 in the specimen and a T4-ficoll con- assessment of thyroid disease. In conjunction with free
hence, enzyme activity is lost. jugate for binding sites on a monoclonal antibody coupled to hormone measurements, total T4 may reveal protein-binding Nonisotopic Immunoassays
Fluorescent probes have been used to measure T4, and microparticles. The rate of formation of cross-linked com- abnormalities that influence the ratio of bound to free Nonisotopic immunoassays for T3 are similar to total T4
both heterogeneous and homogeneous immunoassays based plexes is inhibited by competing T4 and is measured turbidi- hormone, and total T4 should inversely correlate with TSH methods. Many of the T3 methods have been developed for
on fluorescent labels are available. An example of the latter metrically by the change in absorbance at 600 nm. activity in the absence of protein-binding abnormalities. In use on automated immunoassay systems, and some are com-
is the fluorescence polarization immunoassay (FPIA). In Dry-chemistry immunoassay systems, which do not patients with normal serum thyroxine-binding capacity patible with chemistry platforms. Many commercial methods
the FPIA, fluorescein-labeled T4 competes with unlabeled require liquid reagents or sample pretreatment, have also (normal albumin, TBG, and transthyretin), total T4 is propor- use enzyme labels, such as peroxidase or alkaline phospha-
hormone in the patient specimen for binding sites on anti-T4 been developed for T4. One such system uses a thin-film tional to the active free hormone concentration. Methods for tase, conjugated to T3 or anti-T3 antibodies. Enzyme activity
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1933 1934 Section V Pathophysiology

99 Specimen Collection and Storage

is determined by using a variety of sensitive photometric, concentration of active hormone, regardless of abnormalities standards, T3 and T4 were extracted from the dialysate
fluorescent, or chemiluminescent substrates. Immunoassays Specimen requirements for rT3 are essentially the same as in binding protein concentrations or affinities. onto a C5 guard column, and then were eluted into a polar
for T3 that use fluorescent and chemiluminescent labels are those previously described for T3. Analytical strategies for measuring FT4 have followed one LC column [solid-phase extraction-liquid chromatography
also available, and as with T4 assays, chemiluminescence- of two basic paths: (1) separate the bound and free fractions (SPE-LC)] before analysis by tandem mass spectrometry. The
based methods on automated platforms dominate the present Comments on rT3 Measurements and measure the concentration of T4 (immunometrically or upper and lower limits of quantitation, using the criterion
clinical laboratory market. Both heterogeneous and homoge- Reverse T3 in serum is produced almost exclusively from gravimetrically) in the free fraction; or (2) assay the free frac- of 15% total bias, were 400 and 1.0 ng/L, respectively. FT4
neous immunoassays for T3 have been described.81 peripheral deiodination of T4 by 5-deiodinase (D1 or D3) tion using a competitive T4 analog that does not bind to results using this method correlated well with equilibrium
enzymes. Although deiodination of T4 in the peripheral albumin, TBG, or transthyretin. The former category includes dialysis/RIA. FT3 results by the SPE-LC-MS/MS method were
Specimen Collection and Storage tissues produces roughly equal amounts of T3 and rT3, the equilibrium dialysis and ultrafiltration (coupled with a variety compared with equilibrium tracer dialysis (see later), and
Serum is the preferred specimen, but plasma with ethylene- serum concentration of rT3 is typically lower than that of T3 of immunochemical or mass spectrometric methods for mea- a bias of 38 to 53% was observed. Others have reported
diaminetetraacetic acid (EDTA) or heparin as anticoagulant because it is metabolized and cleared more rapidly. Serum rT3 suring T4 and T3 in the free fraction), and the latter comprises similar equilibrium dialysis methods using LC-MS/MS to
may be used. Serum specimens should be tested within 24 concentrations are elevated at birth, but decrease to stable an assortment of competitive and noncompetitive immuno- quantify FT4.208
hours of collection, or stored at 2 to 8 C if tested beyond 24 concentrations by about the fifth day of life. Reverse T3 in assays available on automated systems. The term direct method
hours. Frozen specimens are stable for at least 30 days. amniotic fluid decreases with increasing gestational age. The has been used to describe FT4 methods that measure the Ultrafiltration
Repeated freezing and thawing of the specimens should be use of rT3 to diagnose sick euthyroid syndrome has been concentration of free hormone (i.e., equilibrium dialysis A second method for separating protein bound and free frac-
avoided for reasons described earlier. Turbid samples may questioned because of its poor sensitivity. Renal failure is and ultrafiltration). Alternatively, the indirect method often tions of T4 is ultrafiltration, which is significantly less time-
require centrifugation before testing. associated with low rT3 concentrations. refers to an immunoassay that measures quantities of both consuming than dialysis.200 In an ultrafiltration method
bound and free fractions, relying on calibration against speci- described by Soldin and coworkers,184 the serum specimen
Comments on Total T3 Measurements Measurement of Free Thyroid Hormones mens with FT4 concentrations verified by direct methods. was filtered through a 30 K MW cutoff filter by centrifugation
Significant discrepancies have been observed when the results No practical methods are available for measuring the exact Neither of these terms is consistently used, however, and both for 1 hour at 25 C and 2900 rpm in a fixed-angle rotor. T4
of different T3 immunoassays were compared using reference concentration of unbound, active thyroid hormones. Bioas- approaches, to a greater or lesser degree, only approximate was measured in the ultrafiltrate by isotope dilution LC/
sera. Interlaboratory quality assurance (proficiency testing) says can measure the activity of T3 and T4 but are imprecise the FT4 concentration. MS-MS. Results using this ultrafiltration method correlated
results also demonstrate higher analytical variance for T3 and are not easily adapted to automated chemistry analyzers. The analytical validity and clinical utility of FT4 methods closely with equilibrium dialysis and immunoassay. In a 2009
compared with T4 methods. Among the factors that have Direct potentiometry measures the activity of various elec- have been debated for longer than 2 decades.43-45,121,122,222-224 report,77 performance of this method was assessed in four
been suggested to account for the poorer analytical perfor- trolytes, independent of the inactive bound fraction, but elec- Special reports from the Nomenclature Committee of the different patient populations: (1) pediatric, (2) euthyroid
mance of T3 immunoassays are the lower concentration of T3 trodes that respond to thyroid hormones have not been American Thyroid Association of the National Academy of adults with thyroid disease (both before and after thyroidec-
in serum, the greater antibody cross-reactivity, protein inter- developed. Methods that remove unbound T4 inevitably Clinical Biochemistry69 the Clinical and Laboratory Stan- tomy), (3) healthy nonpregnant women, and (4) pregnant
ferences, and the different assay limits of detection. Perfor- disrupt the equilibrium between bound and free hormone; dards Institute (CLSI) [formerly National Committee for women. In all of these groups, FT4 values measured by ultra-
mance goals for bias and precision have been suggested for therefore at best, FT4 methods only approximate the true Clinical Laboratory Standards (NCCLS)] review some of filtration and LC-MS/MS correlated inversely with log-
T3.13 When a total T3 assay is used for diagnosis, the suggested active concentration of the hormone. the issues and concerns regarding free thyroid hormone transformed TSH concentrations.
goals for maximum bias and imprecision are 5.7% and 11.5% In physiologic systems, proteins often act in much the measurements.
(CV), respectively. For therapeutic guidance, bias and impre- same way as the conjugate acid or base in a pH buffered solu- Equilibrium Tracer Dialysis
cision goals are 2.6% and 5.2%, respectively. Total T3 mea- tion, providing a large reservoir of bound, inactive ligand that Direct (Reference) Methods Equilibrium tracer dialysis is a variation of equilibrium dialy-
surements are useful in the diagnosis and monitoring of protects against precipitous changes in the total concentra- Direct measurement of FT4 (and FT3) in serum is technically sis, except that isotopically labeled T4 or T3 tracer is added
hyperthyroid patients with suppressed TSH and normal free tion of a hormone resulting from variations in glandular challenging because the amount of free hormone in normal to the specimen before dialysis is performed. When equilib-
T4 concentrations (T3-thyrotoxicosis); T3 measurements output. In principle, the free, active concentration of T4 or T3 serum is exceedingly smalltypically less than 100 pmol/L rium is established, concentrations of the tracer on both sides
have only limited value in euthyroid and hypothyroid could be mathematically derived by measuring the total T4 (or ng/L). The most reliable methods for measuring FT4 of the dialysis membrane are used to calculate the ratio of
patients.83 Thyroid hormone replacement for hypothyroid concentration in a manner analogous to the way blood gas and FT3 in serum involve separation of free and bound bound to free hormone, so the FT4 (or FT3) concentration can
patients is based on TSH and FT4 measurements. analyzers use the Henderson-Hasselbalch equation to calcu- hormone fractions by equilibrium dialysis or ultrafiltration, be calculated based on the total T4 (or T3) concentration. If
late the bicarbonate concentration from measured pH and and subsequent measurement of the free concentration by a the amount of tracer added to the specimen is small in com-
PCO2 (from which the undissociated acid is estimated). The sensitive analytical method such as immunoassay or mass parison with the endogenous hormone concentration,
Measurement of Reverse Triiodothyronine (rT3) cumulative dissociation constant for T4 from its binding pro- spectrometry. In equilibrium dialysis, the dialysis buffer minimal disruption of free/bound hormone is noted. This
Reverse T3 is produced by monodeiodination of the alpha teins is approximately 105, which is similar to the Ka of a dilutes the specimen, so there is some effect on the equilib- method is thought to provide the best estimate of FT4 con-
ring of T4 to 3,3,5-triiodothyronine and is biologically inert. weak acid, producing a free T4 concentration that is approxi- rium between bound and free hormones, but the effect is centration, because radioassay methods for measuring T4
Several radioimmunoassay methods for measuring rT3 have mately 0.03% of the total T4 concentration. However, this minimal. Similarly, ultrafiltration changes the concentration have very low limits of detection, and displacement of
been developed. Antibodies to rT3 generally are obtained by approach is not satisfactory for two reasons: (1) T4 binds to of T4-binding proteins in the retentate, changing the bound/ hormone from binding proteins in the retentate is not
immunizing rabbits with rT3-human serum albumin (HSA) at least three different proteins, the concentrations of which free equilibrium. required. However, equilibrium tracer dialysis involves the
[or bovine serum albumin (BSA)] conjugates.32 Cross- change in a variety of clinical conditions, and only one use of radioactive isotopes, a lengthy dialysis step, and instru-
reactivity of anti-rT3 antibodies with T4 varies between 0.01% (albumin) is routinely measured in clinical laboratories; and Equilibrium Dialysis mentation for measuring radioactivity. These considerations
and 0.15%, and insignificant cross-reactivity of these antibod- (2) the affinities of T4-binding proteins for the hormone can In a equilibrium dialysis method for FT4 and FT3 described limit its use for routine measurement of FT4 and FT3.
ies with monoiodinated and di-iodinated thyronines is be altered by competing ligands, as well as by genetic poly- in 2008,230 200 L aliquots of serum were dialyzed against
noted.117 Similar to T3 and T4, rT3 is highly bound to TBG, morphisms that alter the protein structure and thyroid 200 L of a HEPES buffer containing physiologic concentra- Comments on Free Hormone Measurements Using
transthyretin, and albumin in serum but is displaced by hormonebinding affinities. tions of sodium, chloride, potassium, magnesium, sulfate, Separation Techniques
ethanol extraction or ANS. Clinical assessment of thyroid function in symptomatic phosphate, calcium, and urea, with sodium azide added as a Ultrafiltration provides a faster (an hour or less) means for
Commercial assays for measurement of rT3 are available, patients with normal TSH, or asymptomatic patients with preservative using a cellulose membrane (5 kDa molecular separating protein-bound and free hormone fractions com-
but none has been adapted to an automated platform because low or high TSH, has focused on measuring FT4, because weight cutoff). Specimens were dialyzed at 37 C for approxi- pared with equilibrium dialysis, which ordinarily requires
rT3 measurement has limited diagnostic value. this can reveal whether TSH activity is consistent with the mately 20 hours. After the addition of 13C-labeled internal 24 hours. Mass action, however, suggests that ultrafiltration
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1935 1936 Section V Pathophysiology

disrupts free/bound equilibrium concentrations, as the pro- with anti-T4 antibodies, and after isolation of the antibody (equilibrium or ultrafiltration) methods across a wide variety small (1 to 2%) compared with the total T4 concentration, so
tein concentration increases in the retentate during filtration. fraction, the amount of labeled T4 analog captured by the of clinical disorders. the equilibrium bound/free ratio is not significantly affected.
Some ultrafiltration methods are performed at physiologic antibody will be inversely proportional to the endogenous The first commercially available two-step immunoassay These methods are calibrated with reference materials con-
temperature because of concerns about temperature-related FT4 concentration. This approach is sometimes called one- for FT4 was introduced in 1979. Subsequently, a number of taining normal quantities of T4-binding proteins, and in
changes in the equilibrium concentration of free hormone, step, because only one separation of bound and free ligand manual and automated procedures were developed for FT4 which FT4 (or FT3) has been measured by a reference method
but it is interesting to note that Soldin and associates found is involved, in contrast to two-step methods, which remove (and FT3). The earliest methods used radioactive labels and such as equilibrium dialysis or ultrafiltration.
that ultrafiltration at 25 C produced FT4 results that corre- the soluble protein fraction before the addition of the labeled antibody-coated tubes or microbeads, but these have been Many commercially available FT4 and FT3 assays follow
lated better with equilibrium dialysis (at 37 C) than with ligand. Another approach is to immobilize T4 analogs on a largely replaced by automated methods involving nonisotopic one or the other of the labeled ligand or labeled antibody
ultrafiltration at 37 C.184 solid support and measure the amount of labeled antibody labels and a variety of solid-phase formats. Automated one- schemes, differing only in (1) the label, (2) the analog, and
The reliability of ultrafiltration as a method for quantita- adsorbed on the solid support; in this design, captured label step homogeneous FT4 immunoassays have largely sup- (3) the chemical method for immobilizing antibody or analog.
tively separating free T3 and T4 fractions in serum has been will be inversely proportional to the concentration of FT4, planted the use of these methods. Radioactive isotopes, enzymes, fluorophores, and chemilu-
questioned. In a 2007 study, Fritz and colleagues56 demon- which competes for binding sites on the labeled antibody. minescent and electrochemiluminescent labels have been
strated poor recovery of isotopically labeled T4 and T3 when Analog methods measure the distribution of labeled T4 One-Step FT4 Immunoassays used in FT4 immunoassays, but most modern methods use
reference materials prepared in protein-free serum filtrates between endogenous binding proteins (TBP, transthyretin, One-step FT4 immunoassay is a slightly confusing misno- one of the latter two. The structure of the analog, which is a
were passed through four commercially available ultrafiltra- and albumin) and an anti-T4 antibody. Therefore, these mer, because homogeneous immunoassays are typically one- modified form of T4 designed to have limited affinity for
tion devices. Tritiated water was used as the internal standard methods produce only an estimate of the FT4 concentration, step, in that no separation is required. However, all one-step thyroxine-binding protein, albumin, and transthyretin, is
and was evenly distributed between ultrafiltrate and retentate because the introduction of an exogenous binding protein approaches to immunochemical measurement of FT4 and FT3 often proprietary, although T4 analogs involving protein con-
in all cases. However, large discrepancies between labeled the T4 antibodydisrupts the equilibrium between free and are competitive, heterogeneous immunoassays, requiring jugates have been described in the literature.231 In these
hormone concentrations in the filtrate and retentate were bound hormones in the specimen. If the assay is calibrated separation of bound and free ligand prior to measurement of experiments, T4 was conjugated to rabbit gamma globulin,
observed. The authors of the study conclude that ultrafiltra- with calibrators that have similar T4 proteinbinding capacity signal. These FT4 methods are, for the most part, variations thyroglobulin, transferrin-1 (and -2), and ferritin. The anti-
tion is complex, poorly characterized, and incompletely to the specimen being assayed, and if the calibrators have on the prototypical ELISA described in Chapter 16. Two body or analog are immobilized by biotin-avidin (or strepta-
understood. Whether such anomalies also affect the results been validated using a reference method (such as equilibrium common approaches consist of one involving labeled ligand, vidin) or paramagnetic particles.
of equilibrium dialysis, which is similarly based on the pre- dialysis), then the results provide a good estimate of FT4 and the other using a labeled antibody.
sumption that free hormone equilibrates across a semiperme- concentration. However, biases will be introduced when the In the labeled ligand scheme, anti-T4 antibody may be Selection of Tests for Measuring Free Thyroid Hormones
able membrane, has not been studied. concentrations, or affinities, of T4-binding proteins deviate immobilized to a solid support or bound to a paramagnetic Immunoassays that estimate FT4 and FT3 using the approaches
significantly from expected values, because calibration of particle, which can be used to capture the antibody-ligand just described generally produce reliable results in healthy
Free Thyroid Hormone Immunoassays these methods assumes a normal serum T4-binding capacity. complex. Free T4 in the specimen and the labeled T4 analog subjects, hyperthyroid and hypothyroid patients, and patients
Many free thyroid hormone assays are commercially available The serum protein-binding capacity for thyroid hormones compete for binding sites on the anti-T4 antibodies, and the with mild protein-binding abnormalities. Hence, for most
for use on automated chemistry and immunoassay platforms. can be affected by physiological variations in protein synthe- amount of label adsorbed by the antibody is inversely propor- clinical situations, selection of a specific FT4 or FT3 method
Before the advent of automated methods for measuring free sis (as in pregnancy and infancy), proteinopathies resulting tional to the endogenous T4 concentration. Of course, capture can be based on factors such as (1) technical convenience,
T4, strategies emerged to approximate free hormone by mea- from liver dysfunction (such as hypoalbuminemia), nonthy- of endogenous T4 will disrupt the equilibrium between (2) turnaround time, (3) commercial availability, and (4) cost.
suring the total T4 concentration and using an indirect roidal disease, genetic factors that produce proteins with protein-bound and free T4, so the assay really measures the In certain clinical conditions, however, free hormone esti-
method to assess protein-binding capacity. These methods diminished or enhanced T4 binding, competing ligands, and competition between thyroid hormonebinding proteins and mates may produce results with significant bias compared
are no longer useful because immunoassays to measure the the presence of T4 autoantibodies. With the exceptions of the anti-T4 antibody. Typical assays of this type remove 1 to with reference methods. Table 54-4 summarizes the types of
FT4 concentration are widely available. Even though FT4 pregnancy and infancymost notably, prematuritymost of 2% of the protein-bound T4 fractiona quantity that exceeds bias that have been observed between reference and estimate
immunoassays (both one- and two-step) are susceptible to these conditions are relatively rare. the true FT4 concentration by at least two orders of magni- methods for FT4 in patients with thyroid hormonebinding
bias when serum proteins that bind the hormone deviate Another source of bias in FT4 immunoassays that involve tude. Theoretical arguments have been made that removal of protein abnormalities. One study172 examined the bias
significantly from normal concentrations, or when binding a T4 analog is the binding of analog to serum proteins, which such a small amount of the total T4 causes minimal disruption between nine commercially available FT4 immunoassays and
affinity is altered by genetic polymorphisms or competing has been demonstrated to occur.137 Because analog methods of the bound/free ratio; therefore a properly calibrated equilibrium dialysis in patient specimens with low, normal,
ligands, in the vast number of patients, these assays provide are based on the theoretical principle of competition between method should produce a close estimate of the actual FT4 and high concentrations of T4-binding proteins. Relative to
reliable estimates of FT4 concentration. Therefore, index free analog and free endogenous T4 for binding sites on the concentration.33,121,122 specimens with normal concentrations of binding proteins,
methods that estimate free hormone concentrations based on capture antibody, significant binding of the hormone analog Another analytical strategy for measuring FT4 is to immo- the low group had a mean TBG concentration 21% lower, and
protein-binding capacity are mostly obsolete. For more infor- to serum proteins invalidates the theoretical model. The bilize a modified T4 analog to a solid support (or paramag- the high group had a mean TBG concentration 167% higher.
mation on index methods, see the previous edition of this extent to which these limitations affect the FT4 estimates netic particle), and allow a labeled anti-T4 antibody to Although little bias was observed for high-concentration
textbook. obtained by one-step analog methods has been debated (see equilibrate between endogenous FT4 and the immobilized TBG specimens, significant negative bias was observed in
earlier). analog. Evidence suggests that immobilized T4 analog has less low-TBG concentration specimens. A 2009 study98 compared
General Considerations affinity for T4-binding proteins than the labeled free analog FT4 measured by two immunoassays in nonpregnant women
Immunochemical methods for measuring FT4 follow two Two-Step FT4 Immunoassays described earlier,231 which makes the labeled antibody and pregnant women subdivided by trimester. Although
basic strategies: (1) noncompetitive assays involve adsorption The key feature of the two-step method is that the labeled methods less susceptible to bias resulting from binding of the results were not compared with FT4 measured by a reference
of free hormone on immobilized anti-T4 antibodies, removal hormone is added only after serum-binding proteins have T4 analog to endogenous proteins. In the labeled antibody method, a large number (5 to 67%) of pregnant subjects had
of the supernatant, and quantitation of the unoccupied solid- been removed, ensuring that no competition exists between method, after the unbound fraction is removed, the signal is FT4 concentrations below the assay manufacturers lower ref-
phase binding sites through addition of labeled T4; the cap- antibody and serum proteins for binding of tracer. This does measured in the solid (or immobilized) phase and will be erence limit, although all specimens had normal total T4
tured amount of labeled T4 is inversely proportional to the not ensure that the assay is not susceptible to changes in inversely proportional to the FT4 concentration. As with concentrations.
FT4 concentration; (2) alternatively, competitive immuno- serum protein-binding capacity, because the initial step labeled ligand methods, substantially more endogenous T4 is In addition to pregnancy, other clinical conditions may
assays involve a labeled T4 derivative (often called analog, involves equilibration of labeled FT4 between the capture bound to antibody than is contained in the original free frac- produce unreliable FT4 results. In patients with familial
the distinguishing feature of which is significantly reduced antibody and serum hormone-binding proteins. However, tion as the result of re-equilibration after free T4 binds to dysalbuminemic hyperthyroxinemia (estimated incidence
affinity for T4-binding proteins) that is added to serum along results using this approach correlate well with reference antibody, but the fraction removed from binding proteins is among Caucasians: 1 : 10,000), a normally minor albumin
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1937 1938 Section V Pathophysiology

variant with a high affinity for T4 is overexpressed, resulting specimens is recommended. Frozen specimens are stable for It is standard practice to search for TGA whenever Tg
in a larger fraction of albumin-bound T4 and elevated total BOX 55-4 Alterations in the Concentration or at least 30 days. Repeated freezing and thawing of the speci- is measured. The TGA assay should be sensitive to low
T4. The affinity of the albumin variant for T3 is not signifi- Afnity of Thyroid HormoneBinding Proteins mens should be avoided. Turbid samples should be centri- concentrations of TGA. TGA tend to decrease Tg measure-
cantly different from normal albumin. Patients with familial fuged before testing. ments in noncompetitive assays. Although some laboratories
Increases in
dysalbuminemic hyperthyroxinemia are clinically euthyroid, A. Thyroxine-binding globulin (TBG) concentration (or
might use polyethylene glycol to precipitate TGA-bound Tg
and free hormone concentrations are normal as measured by Thyroglobulin Measurement to measure the nonbound Tg fraction, practically speaking,
reference methods and by most two-step immunoassays. 1. Genetic (inherited) causes Both competitive and noncompetitive immunoassays have if TGA are detected, the Tg determination is not reliable.
However, one-step immunoassays are likely to produce erro- 2. Nonthyroidal illness (HIV infection, infectious and been applied to Tg measurement.190,228 Present-day competi- Therefore, Tg measurements should not be used as tumor
neously high FT4 estimates as the result of binding of the T4 chronic active hepatitis, estrogen-producing tumors, tive immunoassays typically incubate the serum with anti-Tg markers in the clinical management of patients with demon-
analog to the variant albumin. Patients with circulating T4 acute intermittent porphyria) antibody; this is followed by the addition of 125I-labeled Tg. strated TGA.
autoantibodies, or with increased transthyretin, will produce 3. Normal physiology (pregnancy, newborn) The Tganti-Tg complex is precipitated by a second antibody, The interference of TGA with various two-site sandwich
similarly elevated FT4 results by these immunoassays, because 4. Drug use (oral contraceptives, estrogens, tamoxifen, separating antibody-bound from free 125I-labeled Tg. Longer immunoassays is unpredictable. TGA increase Tg con-
both conditions result in enhanced T4-binding capacity in methadone) incubation generally enhances the sensitivity of the assay. centrations in some assays. In other assays, TGA may
the serum. B. Prealbumin concentration Noncompetitive (sandwich) immunoassays for Tg have result in negative bias. Recovery studies generally are not
C. Albumin binding (familial dysalbuminemic
FT4 and FT3 methods may not be reliable in patients been developed using a variety of labels (radioactive isotope, useful for determining whether TGA influences the Tg
with congenital TBG excess or deficiency, because the D. T4 binding by antibodies (autoimmune thyroid disease,
enzyme, chemiluminescent, and electrochemiluminescent). concentration.107,186
immunoassay methods are calibrated with reference mate- hepatocellular carcinoma) The capture antibody and the labeled (or signal) antibody When TGA are detected, their concentration may be
rials containing normal amounts of TBG, and most of Decreases in recognize different epitopes on Tg that do not sterically inter- useful as a secondary marker of the mass of thyroid tissue
the circulating hormone is bound to this protein. Patients A. TBG concentration fere with one another. The capture antibody can be attached present in the patient. If TGA concentrations reflect the
with these conditions are clinically euthyroid, and their FT4 1. Genetic (inherited) determination to a polystyrene bead or a paramagnetic bead to separate degree of antigenic stimulation of B cells, higher TGA con-
and FT3 are usually within their reference interval when 2. Nonthyroidal illness (major illness or surgical stress, bound and free fractions of antigen. Another variation is to centrations should correlate with greater amounts of thyroid
measured by reference methods (and sometimes by two-step nephrotic syndrome) attach an antibody to biotin and separate the biotin-antibody- tissue in the patient. Therefore, in differentiated thyroid
immunoassays). 3. Drug use (androgens, anabolic steroids, large doses of Tg complex using avidin bound to a solid phase. cancers where TGA are positive, higher TGA concentrations
Estimates of FT4 and FT3 may be affected by medications glucocorticoids) For most Tg double-antibody assays, the lower limit of suggest more residual thyroid tissue and/or local or distal
that displace hormones from binding sites on serum proteins. B. TBG-binding capacity (drugs bound to TBG such as detection is near 0.5 to 1 ng/mL, with a coefficient of varia- spread of the tumor. Because of the uncertainty that TGA
salicylates and phenytoin)
The free hormone concentration will rise in the presence of tion of 20%. Day-to-day coefficients of variation are usually positivity introduces into the interpretation of any Tg assay,
C. Prealbumin concentration
such drugs, and in patients with normal thyroid function, less than 6% near the center of the reportable interval but can however, clinicians should rely principally on thyroid scans
TSH will be suppressed and production of T4 will decrease, rise to 15% at very low or very high Tg concentrations. to detect residual thyroid tissue in cases of differentiated
resulting in lower total T4 with (after delay) normalized FT4. A Tg reference preparation developed by the European thyroid cancer when TGA are present.
With methods that use undiluted serum (ultrafiltration and various drugs that inhibit T4 binding to TBG, or abnormal Community Bureau of Reference is used in many present-day
equilibrium dialysis), measurement of FT4 and FT3 in the transthyretin and albumin concentration or affinity, can immunometric assays. With this reference preparation, inter- Specimen Collection and Storage
presence of competing drugs should be reliable. However, interfere with these assays and produce artificially low TBG assay variability is 30%, which exceeds intraindividual bio- The preferred specimen for Tg measurement is serum, but
methods that dilute the specimen diminish the effects measurements. logical variability by a factor of three. Because of such EDTA or heparinized plasma may also be used. If not tested
of inhibitors, and free hormone concentration may be Modern TBG methods measure the protein concentration variability between different Tg assays, Tg monitoring within 24 hours, serum specimens are best stored at 2 to
underestimated. directly using a variety of immunochemical approaches. One requires that the same assay be used longitudinally. Alterna- 8 C. If testing is delayed beyond a few days, the specimen
competitive, heterogeneous method measures the competi- tively, if a new Tg assay is introduced, the baseline Tg con- should be frozen until it is analyzed. Frozen specimens are
Measurement of Thyroxine-Binding tion between endogenous TBG and labeled TBG for binding centration for the patient should be re-established with the stable for at least 30 days. Repeated freeze-thaw cycles should
Globulin (TBG) and Other Thyroid to an immobilized anti-TBG antibody. After incubation, new assay. be avoided. Turbid samples should be centrifuged before
HormoneBinding Proteins bound and free fractions are separated by a variety of stan- Technical challenges in Tg measurement include poor testing.
Thyroxine-binding globulin (TBG) is a 54 kDa protein that dard methods, and the concentration of label in the bound interassay agreement, hook effects, interassay calibration
binds T4 and T3 with high affinity (Ka 1010). Although TBG fraction is inversely proportional to endogenous TBG variability, and the need to reduce the lower limits of detec- Reference Interval
has the lowest concentration of any of the three proteins that concentration. A competitive chemiluminescence enzyme tion of Tg immunoassays for improved detection of residual The reference interval for Tg in euthyroid individuals varies
bind T4, and its hormone binding sites are normally only 25% immunoassay for TBG uses peroxidase-labeled TBG and thyroid cancer, metastasis, or recurrence.75 A new generation from a lower limit of 0.5 to 2 ng/mL to an upper limit of 20
saturated, about 70% of the total T4 and 80% of the total T3 anti-TBG antibody that is captured by a solid-phase second of Tg assays with enhanced lower limits of detection may to 42 ng/mL, depending on the method. One major reference
is bound to TBG. Therefore, TBG concentration is the antibody; bound conjugate is measured by chemilumines- eliminate the need to withdraw thyroid hormone replace- laboratory provides the following reference intervals: 0 to 11
primary regulator of total and free T4 and T3 concentrations. cence after the addition of luminol and hydrogen peroxide. ment or administer rDNA TSH before Tg testing is months: 0.6 to 5.5 ng/mL; 1 to 11 years: 0.6 to 40.0 ng/mL;
Estrogen-induced TBG excess and congenital TBG deficiency Another immunoassay for TBG is based on enhanced performed.62 and 12 years and older: 1.3 to 31.8 ng/mL. For patients
are the most significant TBG abnormalities that affect the microparticle turbidimetry, in which the presence of endo- lacking a thyroid gland who are not receiving T4 replacement
interpretation of thyroid function test results (Box 55-4). genous antigen inhibits the cross-linking of antigen- Thyroglobulin Assay Interferences therapy, Tg should not be detectable regardless of the patients
microparticle complexes by anti-TBG antibody, reducing the Interference from human antimouse antibodies (HAMA), TSH concentration.
Methods for Measuring TBG turbidity of the reaction mixture. other heterophile antibodies, or rheumatoid factor can affect
Historical methods measured TBG indirectly by competition almost any immunoassay. Thyroglobulin autoantibodies Novel Markers of Thyroid Tissue
between unlabeled and radiolabeled T4 for binding sites on Specimen Collection and Storage (TGA) in the patients blood also interfere with accurate mea- in the Management of Differentiated
TBG, while blocking T4-binding sites on transthyretin and Serum is the preferred specimen; plasma with EDTA or surement of Tg.187 Unfortunately, anti-Tg autoantibodies are Thyroid Carcinoma
albumin with a barbital buffer. After removal of the unbound heparin as anticoagulant may also be used. Serum specimens much more common in thyroid cancer patients than in the Another approach to the detection of residual thyroid cancer
hormone, radioactivity retained in the protein fraction was are best stored at 2 to 8 C if they will not be tested within 24 general population. TGA are reported in 15 to 35% of thyroid is the measurement of Tg mRNA in the circulation.164 This
proportional to the TBG concentration. The presence of hours. If longer periods of storage are necessary, freezing the cancer patients. may be helpful when Tg cannot be measured because of TGA.
Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1939 1940 Section V Pathophysiology

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Endocrinology and
Related Disorders
T. Scott Isbell, Ph.D., Emily Jungheim, M.D.,
and Ann M. Gronowski, Ph.D.*

eproductive endocrinology encompasses the hor- Hypothalamic-Pituitary-Gonadal Axis

mones of the hypothalamic-pituitary-gonadal axis, Gonadotropin-releasing hormone (GnRH) is a decapeptide
R as well as the adrenal glands (see Chapter 54). These synthesized in the hypothalamus and transported to the ante-
hormones are crucial for reproductive function and include rior pituitary gland, where it stimulates the release of both
gonadotropin-releasing hormone (GnRH), luteinizing FSH and LH (see also Chapter 53).
hormone (LH), follicle-stimulating hormone (FSH), and a In adult men, GnRH and thus LH and FSH are secreted in
multitude of sex steroids. The sex steroids are synthesized by pulsatile patterns. A circadian rhythm is present, with higher
the ovaries, testes, and adrenal glands and are responsible for concentrations found in the early morning hours and lower
the manifestation of primary and secondary sex characteris- concentrations in the late evening.302 LH acts on Leydig cells
tics. This chapter is divided into four sections: Male Repro- to stimulate the conversion of cholesterol to pregnenolone.
ductive Biology, Female Reproductive Biology, Infertility, and FSH acts on Sertoli cells and spermatocytes and is central to
Methods. the initiation (in puberty) and maintenance (in adulthood)
of spermatogenesis.297 Sex steroids and inhibin (a 32 kDa
protein secreted by the Sertoli cells) provide negative feed-
MALE REPRODUCTIVE BIOLOGY back control of LH and FSH secretion, respectively. LH secre-
The mature testes synthesize both sperm and androgens. The tion is inhibited by testosterone and by its metabolites,
testes contain a structured network of tightly packed seminif- estradiol and DHT. FSH may be elevated in disorders in
erous tubules. The lumina of the seminiferous tubules are which Sertoli cell numbers (and hence inhibin concentra-
lined by maturing germ cells and Sertoli cells. Sertoli cells tions) are reduced. Likewise, a reduction in the number of
play a crucial role in sperm maturation and secrete inhibin, a Leydig cells (and hence testosterone secretion) leads to
glycoprotein that inhibits the pituitary secretion of FSH. Sur- increased LH concentrations.
rounding the seminiferous tubules are the interstitial Leydig
cells, the primary site of androgen production. The principal Androgens
androgen in man is testosterone, which serves a central role Androgens are a group of C-19 steroids (Figure 56-1) res-
in reproductive physiology. Testosterone is required for ponsible for masculinization of the genital tract and develop-
sexual differentiation, spermatogenesis, and promotion and ment and maintenance of male secondary sex characteristics.
maintenance of sexual maturity at puberty. At the cellular Testosterone is the principle androgen secreted in men.
level, these effects are mediated by binding of testosterone or
its more potent metabolite dihydrotestosterone (DHT) to the Biosynthesis of Testosterone
androgen receptor or via aromatization to estradiol and sub- Testosterone is synthesized primarily by the Leydig cells of
sequent binding to the estrogen receptor. Testicular function the testes (95%) and, to a lesser extent (5%) via peripheral
is under the control of the hypothalamic-pituitary-gonadal conversion from the precursors dehydroepiandrosterone
axis. (DHEA) and androstenedione (which are synthesized in the

*The authors gratefully acknowledge the original contribution of R. J. Whitley, A. W. Meikle, and N. B. Watts, on which
portions of this chapter are based.