1906
C HA P T E R
The Thyroid:
Pathophysiology and
Thyroid Function Testing
William E. Winter, M.D., Desmond Schatz, M.D.,
and Roger L. Bertholf, Ph.D.*
he thyroid is a butterfly-shaped gland located in the
front of the neck, just above the trachea. The fully
T developed thyroid gland in a human is composed of
two lobes connected by a thin band of tissue, the isthmus,
which gives the gland the appearance of a butterfly. The gland
is closely attached to the trachea in the anterior aspect of the
neck. Anatomically and embryologically, the thyroid gland
is two glands in one: the thyroid follicular cells produce
thyroid hormones and the parafollicular (or C) cells secrete
calcitonin.21
THYROID GLAND: STRUCTURAL AND
FUNCTIONAL ONTOGENY
Embryologically at day 24, the thyroid gland develops from
an anterior outpouching of the foregut. From this thyroid
diverticulum, the thyroid gland descends, and the process is
usually complete at 7 weeks. Failure of descent can produce
(1) a lingual thyroid gland (located at the base of the tongue),
(2) an ectopic gland, or (3) a hypoplastic or aplastic gland.
Coincident with thyroid development, the parafollicular (C
cells) cells (producing calcitonin) develop.
The fetus is dependent on the transplacental passage of
maternal thyroid hormone for the first half of gestation.26,29,65,225
Although only minute amounts of maternal thyroid hormone
cross the placenta, maternal hypothyroidism in the first 20
weeks of gestation may adversely impact fetal central nervous
system (CNS) development, leading to neuropsychologic
impairment in infants and children.178 Worldwide, the most
common cause of hypothyroidism during pregnancy is iodine
deficiency. Conversely, in developed countries, where iodine
*The authors gratefully acknowledge the previous contributions of L. M. Demers and C. Spencer, on which portions of this
chapter are based.
1905
55
fortification is widespread, the most common cause of
primary hypothyroidism is Hashimoto thyroiditis (chronic
autoimmune thyroiditis/inflammation of the thyroid gland).
Hypothalamic thyrotropin-releasing hormone (TRH)
stimulates thyrotropin [thyroid-stimulating hormone (TSH)],
which in turn stimulates thyroid hormone synthesis. By 10
weeks, fetal thyroid follicles and thyroxine synthesis are
demonstrable. By the mid second trimester, maturation of
the hypothalamic-pituitary-thyroid axis occurs, so that by 20
weeks, the fetus is becoming responsible for its own produc-
tion of thyroid hormone. Thyroxine-binding globulin (TBG)
and thyroxine are first detectable in fetal serum at 8 to 10
weeks gestation and increase thereafter until they plateau at
35 to 37 weeks (Figure 55-1). Thyroxine [tetraiodothyronine
(T4); see Table 55-1 for nomenclature and abbreviations) and
TSH rise until birth, with T4 near 10 g/dL and TSH reach-
ing a concentration of 7 to 10 mIU/L. Beginning at 30
weeks, T4 is converted to triiodothyronine (T3) by increased
activity of the type 1 deiodinase (D1), so that T3 concentra-
tions rise, while reverse T3 (rT3) concentrations decline. At 30
weeks, T3 is less than 15 ng/dL; at term, T3 is 50 ng/dL.
Within hours of birth, TSH, T4, and T3 rise rapidly (Figure
55-2).52 Cold stress is believed to be responsible for the
massive TSH surge to concentrations of 70 to 100 mIU/L. By
2 to 3 days, the TSH concentration falls in most cases to less
than 20 mIU/L. Total T4 peaks near 17 g/dL. T4 then falls
to adult concentrations by 1 to 2 months of age. The postbirth
rise in T3 results from increased thyroid gland release in
response to the rising TSH concentration and increased con-
version of T4 to T3 due to maturation of the type 1 deiodinase
enzyme (D1).
Section V Pathophysiology
Changes in Fetal Thyroid Function During Gestation
Fetal thyroid develops ~4 wks
Figure 55-1 Changes in fetal thyroid function during gestation. During the rst half
of gestation, the fetus is dependent on transplacental passage of thyroid hormone.
After mid gestation, the fetus produces its own thyroid hormone.
TSH
Relative concentration
0 1
Days after birth
Figure 55-2 Concentrations of thyroid hormones for 5
days after birth. After birth, an immediate TSH surge
peaks at 30 minutes. T3 and T4 rapidly rise, peaking at
24 hours after delivery. The greater initial rise in T3 as
compared with T4 in the rst 24 hours of life likely
represents acutely increased conversion of T4 to T3.
Following their peak concentrations, T4 and T3 decline
to stable concentrations.
THYROID GLAND ANATOMY
The adult thyroid gland weighs between 15 and 20 g. However,
in disease states, the gland can attain a weight of several
hundred grams. The thyroid gland is normally bilobed, with
the right lobe somewhat larger than the left lobe. In adults,
TRH
TSH
T4
rT3
TSH
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
T3
10 20 30 40
Weeks of Gestation
the lobes are 2.0 to 2.5 cm thick by 2.0 to 2.5 cm wide by
4 cm high. The lobes are connected by the isthmus, which
is 0.5 cm thick by 2.0 cm wide by 1 to 2 cm high.
Microscopically, the thyroid gland is composed of follicles
or acini (Figure 55-3, A). The outside of the follicular unit/
acinus is enveloped by a basement membrane. Along the
Total T4
inside of the basement membrane are squamous or cuboidal
epithelial follicular cells. The height of the follicular cells
reflects their biochemical activity: the greater their height, the
more thyroid hormone synthetic activity is occurring. The
Total T3 basal pole of the follicular cell is oriented toward the base-
ment membrane, and the apical pole is oriented toward the
center of the follicle (Figure 55-3, B). In the center of the fol-
licle, lined by the apical aspects of the follicular cells, is the
2 3 4 5 lacuna, which contains colloid composed predominantly of
thyroglobulin.
On average, follicles are 200 microns wide (compared
with a normal red blood cell diameter of 7 microns). The
parafollicular cells usually are located below the basement
membrane but are not adjacent to the lumen (lacuna) of the
follicle. Without immunohistochemical staining for calcito-
nin, the parafollicular cells are very difficult to identify.
Branches of the common carotid artery (the superior
thyroid arteries) and the subclavian arteries (the inferior
thyroid arteries) supply the thyroid. It is surprising to
note that on a per gram basis, the thyroid gland receives
more blood than the kidney: 4 to 6 mL/min/g of thyroid
tissue versus 3 mL/min/g of kidney. In cases of hyperthy-
roidism, blood flow to the entire thyroid gland can exceed
1000 mL/min, which is 20% of the normal cardiac output
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-
cell
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
space
and approximately 80% of T3 is formed by extrathyroidal
Total triiodothyronine T3
Colloid monodeiodination of T4 to T3.
(3,5,3-triiodothyronine)
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
hormone)
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.
space
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
Apical
(Thyroxine) thyronine thyronine coupling; this can lead to an 800-fold difference in the con-
pole
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
Cap

and Conditions basolateral poles of the follicular cells and tight junctions
mRNA
illary

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
Tg
lume
n

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
symporter
expression by the liver), (6) increase vitamin requirements, I
and (7) regulate calcium and phosphorous metabolism. Colloid
TPO
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
I I I I
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

H3N COO H3N COO H3 N COO
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
O O O
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
OH OH OH
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
H3N COO H3N COO H3 N COO
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
O O O

OH Monoiodination OH Diiodination OH I
OH I I
H H I H H H I I OH OH OH
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
Diiodotyrosine
(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
transcription. Thus, increased D1 activity promotes periph- free T3 (FT3)-to-T4 (FT4) ratio is affected by a genetic poly-
eral conversion of T4 to T3 in hyperthyroidism. Therapeuti- morphism in the D1 gene (DIO1; the single-nucleotide poly-
cally, D1 is inhibited by propylthiouracil, which is used to morphism rs2235544).143
treat hyperthyroidism. In contrast to the effect of thyroid Each of the deiodinase enzymes has a single transmem-
hormone on D1, D2 expression is suppressed by thyroid brane domain. The deiodinases are attached to the inner
hormone. The Km for the D1-T4 complex is approximately plasma membrane (D1 and D3) or endoplasmic reticulum
1000-fold greater than the corresponding Km for D2 or (D2), and the active site of the enzyme is on the cytoplasmic
D3. Regulation of the T4T3 and T4 rT3 conversions is domain. Therefore, thyroid hormone must enter the cyto-
dependent on the tissue distribution of D1, D2, and D3. The plasm of cells to be metabolized. In addition to thyroid
hormone metabolism via deiodination, small amounts of T4
are glucuronidated or sulfated and excreted in the bile. Intes-
tinal cleavage of glucuronidated T4 releases T4 that can then
Thyroid gland
be reabsorbed from the intestine back into the circulation.
Until recently it was believed that both T4 and T3 entered
cells by passive diffusion across the plasma membrane.
However, evidence has shown that thyroid hormones cross
~20%
plasma membranes using specific transporters.74 One impor-
tant thyroid hormone transporter is monocarboxylate
H3N COO H3N COO transporter 8 (MCT8).118 The solute carrier family of mono-
CH2 CH2 carboxylate transporters has 14 members (MCT1 through
MCT14); examples of other monocarboxylates transported
I I I I by these carriers are lactate and pyruvate.
O D1 O It has been shown that MCT10 (SLC16A10; chromosome
~80% 6q) transports iodothyronines and aromatic amino acids
I I I across membranes, and MCT10 is likely more preferential
OH OH
than MCT8 in transporting T3 over T4 across plasma
T4 T3 membranes. Organic anion transporting polypeptide 1C1
(OATP1C1) also has high affinity for thyroid hormone and
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.
(80%) comes from peripheral monodeiodination of T4 Transport of thyroid hormone into neurons is especially
to T3 [catalyzed by type 1 deiodinase (D1) and type 2 critical for normal CNS development and function (Figure
deiodinase (D2)] with only a minority of T3 (20%) 55-11). OATP1C1 may be responsible for thyroid hormone
coming directly from the thyroid gland. uptake and release by astrocytes, although this has not yet
CNS Vasculature
Overlapping endothelial cells
Astrocyte
processes Astrocyte Neuron
TR
T3 MCT8
T4 -- D2 T3
T4 T3
D3
T2
Possibly: OATP1C1
Capillary basement
membrane
Figure 55-11 Transport of thyroid hormones to neurons. Before thyroid hormones
are transported to neurons,T4 and T3 pass through endothelial cells and astrocytes.
Organic aniontransporting polypeptide 1C1 (OATP1C1) may allow astrocyte uptake
of T4 and T3.Within the astrocyte,T4 may be converted to T3 via type 2 deiodinase (D2).
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
receptor (TR) or can be converted to 3,3-T2 via type 3 deiodinase (D3).
1912 Section V
been definitively demonstrated. Within the astrocyte, T4 is
converted to T3 via D2 attached to the astrocyte membrane.
T3 may exit the astrocyte via OATP1C1 to be taken up by
neurons via MCT8. Within the neuron, T3 can interact with
the intranuclear thyroid hormone receptor (TR), or can be
converted to 3,3-T2 via membrane-attached D3.
Although T4 is converted to T3 outside of target organs
(the liver converts T4 to T3 and then releases T3 back into the
circulation), target organs can also deiodinate T4 to T3 so that
T3 can bind to TRs. The liver, kidney, brain, brown fat, ante-
rior pituitary, pineal, heart, skeletal muscle, and placenta all
express a 5-deiodinase capable of deriving T3 from T4.
T3 binds to specific intranuclear TRs to exert its hormonal
effects. In humans (and various animals), thyroid hormone
upregulates the expression of growth hormone,110 myelin
basic protein, the alpha-myosin heavy chain, and a sarcoplas-
mic reticulum calcium ATPase, while downregulating the
expression of the TSH beta chain and the beta-myosin heavy
chain. In part, suppression of TSH beta chain synthesis con-
tributes to negative feedback.
TRs are members of the steroid hormone supergene family
and serve as transcription factors that include domains for
DNA binding, T3 binding, and transactivation. T3 binds to the
TRs with 15-fold greater affinity than T4. The TRs form het-
erodimers with the retinoid X receptor (RXR) to interact with
the thyroid response elements (TREs) in DNA. Monomers
and homodimers of TRs can also bind to the TREs. TRs have
zinc-finger structures that form alpha helices for interaction
with the response elements.
Two types of TRs are known: TR and TR (Table 55-2).218
TR is encoded by the thyroid hormone receptor-alpha
(THRA) gene on chromosome 17q11.2. Of its two expressed
isoforms (TR1 and TR2), only TR1 binds thyroid
hormone. A TR3 isoform produced by alternative splicing
has also been reported. TR1 is expressed throughout the
body, but its mRNA is in highest concentrations in the heart,
brain, liver, and kidneys.
TR is encoded by 11 exons on the thyroid hormone
receptor-beta (THRB) gene on chromosome 3p24.3. Three
isoforms of TR exist: TR1, TR2, and TR3. The highest
concentrations of TR1 are found in heart, kidney, liver, and
brain. Expression of TR2 has been identified in the anterior
pituitary, hypothalamus, retina, cochlea of the inner ear, and
TABLE 55-2 Thyroid Hormone Receptor (TR) Sizes
Amino Acids kDa
TR1 410 47
TR2 490* 55
TR1 461 53
TR2 514 58
*Extended C-terminal region (does not bind T3).
Extended N-terminal region.
Pathophysiology
developing brain. Defects in TR1 can produce resistance to
thyroid hormone; defects in TR have not been reported.
Within most cells, 90% of T3 is cytosolic and 10% is
intranuclear. An exception is seen in the pituitary gland,
where T3 is distributed approximately equally between the
cytoplasm and the nucleus. A T3-binding protein (CTBP, or
C-T3BP) has been identified that may influence the intracel-
lular distribution of thyroid hormone; however, this protein
does not exhibit receptor activity.
It is important to consider the comparative biological
activity of circulating T4 and T3. On a molar basis, there is
100 times as much circulating T4 as T3. Typical reference
intervals for T4 and T3 are 60 to 135 nmol/Land 1 to 3.5,
nmol/L, respectively. The difference in T4 and T3 concentra-
tions narrows, however, to a 10-fold excess of free (active) T4,
compared with unbound T3, in the circulation. Because T3 is
4 to 15 times as biologically active as T4, the difference in
effectiveness narrows further. It is debated whether the effects
of T4 exceed those of T3, or whether they equally influence
the tissues. Within the cells of the anterior pituitary, large
intracellular amounts of T3 are derived from monodeiodin-
ation of T4, so circulating FT4 appears to be the major regula-
tor of pituitary TSH secretion. This is an important concept
that explains why TSH remains normal in the early stages of
the sick euthyroid syndrome, when FT4 is normal, preventing
a rise in TSH, yet the T3 is decreased.
In the pituitary, most T3 is derived from intrapituitary
conversion of T4 to T3. Within target tissues, if more T3 comes
from T4 (via target cell 5-deiodination) than from the circu-
lation itself, T4 has a major effect on tissues. Nevertheless
because most T3 is derived from T4, within the circulation or
within a target cell, T4 is often considered to be a prohormone
in the generation of T3.
Compared with the kidney, liver, and cerebral cortex,
much higher concentrations of TRs are found in the anterior
pituitary gland and brown adipose tissue. In both of the latter
tissues, two thirds to three quarters of the receptors are occu-
pied by T3, with slightly more than half of the T3 derived from
intracellular conversion of T4 to T3 via D2, versus T3 taken up
from the circulation (derived from T4 outside the target tissue
by D1 and D2). Most of the hepatic and renal T3 comes from
circulating hormone. On the other hand, the vast majority
of T3 in the cerebral cortex is derived intracellularly from T4
by monodeiodination of T4 to T3.
PHYSIOLOGY
Thyroid Hormone Negative Feedback Control
of TRH and TSH
Thyroid hormone synthesis and secretion are controlled by a
negative feedback system involving the hypothalamus, the
pituitary, and the thyroid follicular cells (Figure 55-12).30 In
contrast to other hypothalamic-anterior pituitary target organ
systems that utilize a negative feedback system, however, the
major site of thyroid hormone feedback is at the level of
the anterior pituitary thyrotrophsnot at the level of the
hypothalamus.
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1913
Hypothalamus
glutamate terminus is required for TRH bioactivity. The PVN
are located in the anterior hypothalamus, rostral to the optic
Minor site of
TRH negative feedback chiasm. TRH concentrations rise in thyroid hormone defi-
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 negative
feedback Anterior Pituitary Thyrotroph Physiology
Thyroid Thyrotrophs, the anterior pituitary cells that secrete thyroid-
stimulating hormone (TSH; thyrotropin), express TRH
receptors, which are G-proteincoupled receptors with seven
T4, T3 transmembrane domains. When TRH binds to its receptor,
TBP T4 T3 the thyrotroph depolarizes, triggering calcium influx. Conse-
quently, increased free cytosolic calcium activates the Ca2+-
phosphatidylinositol cascade, effecting TSH release, synthesis,
Target and glycosylation of alpha and beta TSH subunits. In relative
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
Figure 55-12 Metabolic control of thyroid hormones.
may lack potency as the result of insufficient glycosylation,
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
activity of the hormone when injury or disease results in a
thyrotropin) from anterior pituitary thyrotrophs. TSH
stimulates the release of T4 and T3 from the thyroid TRH deficiency.
TRH modifies the sensitivity of the thyrotroph to thyroid
gland, although most T3 comes from peripheral
monodeiodination of T4 to T3. More than 99% of T4 and T3 hormone negative feedback; increased TRH makes the thy-
is bound to various thyroid hormonebinding proteins. rotroph less sensitive to inhibition with decreasing TRH
T4 and T3 negatively feed back on the hypothalamus and, makes the thyrotroph more sensitive to negative feedback.
more powerfully, the pituitary. T4 and T3 enter into target The mechanism for TRH control involves reduced expression
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
thyroid hormone excess. An inverse logarithmic relationship
H
C N exists between TSH and FT4 concentrations (Figure 55-14); a
CH 50% decline in FT4 concentration results in a 100-fold increase
HN
O NH2 in TSH.
C
C TSH is a 30 kDa heterodimeric glycoprotein that shares
O CH2 CH a subunit with luteinizing hormone (LH), follicular stimulat-
H CH2 ing hormone (FSH), and human chorionic gonadotropin
N C C
CH N H C N (hCG).174 All four hormones contain a 14.7 kDa alpha sub-
CH2
O C H C
CH2 O H2
unit (gene location: chromosome 6q21.1-q23). Each of these
C hormones has a unique beta subunit that is responsible for
H2
the specific activity of the hormone. The 15.6 kDa TSH
(pyro)Glu-His-Pro(NH2)
beta chain is encoded by a three-exon gene located on chro-
Figure 55-13 Chemical structure of thyrotropin-releasing mosome 1p. The alpha chain contains two oligosaccharides,
hormone (TRH). Note that TRH is a tripeptide and the TSH beta subunit contains one oligosaccharide
(L-pyroglutamyl-L-histidyl-L-prolinamide). modification.
Thyroid Follicular Cell Physiology
The effects of TSH on the thyroid follicular cell are mediated
Hypothalamic Physiology through TSH receptor (TSHR)G-proteinadenyl cyclase cells is stimulated by cAMP, phospholipase C, insulin-like
Thyrotropin-releasing hormone (TRH; thyrotropin-releasing coupled synthesis of intracellular cyclic adenosine mono-
factor, thyroliberin, or protirelin) is encoded on chromosome phosphate (cAMP).197 At supraphysiologic concentrations
3, and the modified tripeptide l-pyroglutamyl-l-histidyl-l- (100 normal), TSH will signal through the inositol-
prolinamide is secreted by the paraventricular nuclei (PVN) phosphate diacylglycerol cascade, activating phospholipase C
in the hypothalamus (Figure 55-13). Cyclization of the (PLC) and raising intracellular calcium concentrations, with
1914 Section V Pathophysiology
TSH - Free T4 Relationships in
Ambulatory Patients with Stable Thyroid Status
1000
100
TSH
mIU/L
10
4.0
TSH
Reference
Interval
0.4
100x
0.1
2x
0.01
Undetectable
Hypo- FT4 Reference
Hyperthyroid
Thyroid Interval
9 23 pmol/L
{ Free T4
0.7 1.8 ng/dL
Figure 55-14 The log-linear relationship between thyroid-stimulating hormone
(TSH) and FT4. A twofold change in TSH is associated with an approximate 100-fold
change in circulating FT4 concentrations. (Modied from Spencer CA, LoPresti JS, Patel A,
Guttler RB, Eigen A, Shen D, et al. Applications of a new chemiluminometric thyrotropin assay to
subnormal measurement. J Clin Endocrinol Metab 1990;70:453-60. Copyright 1990, The Endocrine
Society.)
subsequent stimulation of H2O2 generation and iodide efflux
into the follicular lumen. THYROID HORMONES IN THE CIRCULATION
The TSHR is a 743 amino acid, 84.5 kDa protein generated Both T4 and T3 are highly bound to plasma proteins in circu-
after cleavage of a 21 amino acid pre- (or leader) sequence. lation. Protein-bound T4 and T3 serve as thyroid hormone
After post-translational glycosylation, the TSHR is 100 kDa. reservoirs within plasma. In the thyroid gland, Tg serves as
The first nine exons encode the 398 amino acid N-terminal a reservoir. The unbound fraction of circulating thyroid
extracellular domain. The extracellular domain contains six hormone is the biologically active form, so FT4 concentra-
N-glycosylation sites that make up the TSH-binding domain. tions correlate more closely with the clinical status of the
The TSHR may be in either of two conformations: the on patient than do total T4 concentrations.15,142 However, eleva-
conformation, with active signaling, or the off conforma- tions in FT3 usually correlate with the clinical status of the
tion, where signaling does not occur. TSH binding to the patient, low FT3 concentrations do not always equate with
TSHR puts it in the on conformation, leading to stimulation clinical hypothyroidism, as evidenced in the sick euthyroid
of the thyroid gland. syndrome (see later). Similarly, elevated free concentrations
Within the TSH-stimulated thyroid follicular cell, the of T4 or T3 do not always correlate with hyperthyroidism
activity of the sodium-iodine symporter increases; increased because of the possibility of peripheral thyroid hormone
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
colloid with consequent degradation and release of T4 and binding proteins can profoundly affect the total concentrations
T3 from Tg are also noted. Replication of thyroid follicular of T4 and T3 without significant changes in the free hormone
concentrations.
growth factor-I (IGF-I), and a fibroblastic growth factor T4 binds to thyroxine-binding globulin (TBG; a 54 kDa
(FGF)-mediated kinase. TSH stimulation increases the size 1-globulin), thyroxine-binding prealbumin (TBPA, or trans-
and number of thyroid follicular cells. Likewise, follicular thyretin; 54 kDa), and albumin. T3 binds almost exclusively
cellular hyperplasia is seen in Graves disease, when an ago- to TBG and albumin; TBPA binds only negligible amounts of
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-
affinity)
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
methadone)

RAIU
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
hyperthyroxinemia)
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

Elevated
FT4
Elevated Normal Decreased
Clinically: Hyper-
Euthyroid thyroid
Thyroid TSH- Primary
hormone dependent hypothyroidism
resistance hyper-
thyroidism
Subclinical
hypothyroidism
Figure 55-16 Suggested algorithm for the laboratory evaluation of thyroid function.
If hypothalamic or pituitary disease is suspected, initial
testing requires measurement of both TSH and FT4 (with FT4
being more important). FT4 is used to establish the presence
of the biochemical euthyroid state, hypothyroidism, or hyper-
thyroidism. When FT4 is low and TSH is low or normal,
central hypothyroidism may be present. If FT4 is low with
only a mild elevation in TSH, central hypothyroidism is still
possible because of discordance in the ratio of immunoreac-
tivity to bioactivity, with a decrement in bioactivity from
abnormal TSH glycosylation from pituitary disease or TRH
deficiency. In the setting of clinical hyperthyroidism, central
hyperthyroidism is diagnosed with an elevated FT4 and
normal or elevated TSH.
HYPOTHYROIDISM
Hypothyroidism is defined as a deficiency in thyroid hormone
secretion and action that produces a variety of clinical signs
and symptoms of hypometabolism.73,84,198 This common dis-
order occurs in 2 to 15% of the population, more commonly
in women than in men. The risk of developing hypothyroid-
ism increases with age.39
Clinical symptoms suggesting hypothyroidism include
(1) mental dullness (including mental retardation in children
with untreated or undertreated congenital hypothyroidism),
(2) somnolence, (3) increased sleeping, (4) lethargy, (5) easy
fatigability, (6) hoarseness or deepening of the voice, (7) hair
loss, (8) weight gain, (9) cold intolerance, (10) menstrual
irregularities, (11) infertility, (12) growth failure, (13) delayed
puberty in adolescents, (14) constipation, (15) muscle weak-
ness or cramps, and (16) depressed affect or frank clinical
depression.171 Physical signs compatible with hypothyroidism
include (1) bradycardia, (2) decreased pulse pressure, (3) cool
Normal Decreased
No further FT4
testing
Elevated Normal Decreased
T3
Elevated Normal
Primary Central
hyper- hypothyroidism
thyroidism
T3 toxicosis Subclinical
hyperthyroidism
and/or dry skin, (4) puffy eyes, (5) loss of the outer lateral
eyebrows, (6) delayed relaxation phase of reflexes (hung-up
reflexes), (7) myopathy, (8) carotenemia, (9) occasional galac-
torrhea, and (10) radiologic evidence of delayed bone age in
children. In cases of severe hypothyroidism, congestive heart
failure or coma may develop.90 In children with untreated
congenital hypothyroidism, severe growth failure and mental
retardation ensue. The development of biological and bio-
chemical processes is delayed in cases of congenital hypothy-
roidism. For example, affected infants often have prolonged
jaundice as the result of immaturity of UDP-glucuronyl
transferase.
A rare (and controversial) manifestation of Hashimoto
thyroiditis is encephalopathy.216 However, no definitive clini-
cal test is available to diagnose encephalopathy resulting from
Hashimoto thyroiditis. The diagnosis is considered when
encephalopathy of unknown origin is associated with auto-
antibodies against the thyroid gland.
Laboratory evidence compatible with hypothyroidism
encompasses hyponatremia, a normocytic or macrocytic
anemia, elevated creatine kinase (from myopathy), and
hypercholesterolemia and/or hypertriglyceridemia [from
decreased lipoprotein-lipase activity and decreased low-
density lipoprotein (LDL) receptor expression]. Anatomi-
cally, stimulation of the TSH receptor can cause follicular cell
hyperplasia and goiter. Goiter can also be caused by glandular
infiltration (e.g., Hashimoto thyroiditis, in which the gland is
heavily infiltrated by lymphocytes).
Based on TSH and FT4, the causes of hypothyroidism are
classified as primary thyroid gland failure (low FT4 and
increased TSH; primary hypothyroidism) or central hypo-
thyroidism (low FT4 and usually a normal or low TSH con-
centration). Central hypothyroidism results from pituitary
BOX 55-2 Causes of Primary Hypothyroidism
Endogenous Disorders
Autoimmune thyroid disease
Hashimoto thyroiditis
Atrophic thyroiditis
Late-stage Graves disease
Postpartum thyroiditis
Inborn errors in thyroid hormone biosynthesis
(dyshormonogenesis)
Na+/iodide pump dysfunction
Inadequate organification/iodinationthyroperoxidase
dysfunction
Defective thyroglobulin
Deiodinase deficiency
Pendred syndromehypothyroidism and deafness
Developmental disorders involving the thyroid gland
Congenital hypothyroidism: aplasia, hypoplasia
Ectopic thyroid: lingual thyroid, thyroglossal duct cyst
Consumptive hypothyroidism
Exogenous Disorders
Iodine excess/deficiency
Drugs
Thionamides
Lithium
Nitroprusside
Amiodarone
Biologicals (e.g., interferon, interleukin-2)
Dietary goitrogens
Radiation-induced hypothyroidism
Surgical removal of the thyroid gland
Viral or bacterial thyroiditis
disease (secondary hypothyroidism from TSH deficiency) or
hypothalamic disease (tertiary hypothyroidism from TRH
deficiency).
Primary Hypothyroidism
The causes of primary hypothyroidism are classified as endog-
enous or exogenous (Box 55-2).38 Endogenous disorders are
conditions that develop within the patient such as autoim-
mune thyroid gland dysfunction, inborn errors, and develop-
mental abnormalities. Exogenous disorders are conditions
that originate outside the patient such as iodine deficiency or
excess, goitrogen or drug effects, and postsurgical hypothy-
roidism or hypothyroidism following radioactive iodine
treatment.
Autoimmune Hypothyroidism
Excluding the newborn period, autoimmune thyroid disease
(AITD) is the most common cause of thyroid disease
and primary hypothyroidism.111,148,203 Hashimoto thyroiditis
(chronic lymphocytic thyroiditis) leads to destruction of the
thyroid follicular cells through a cell-mediated autoimmune
process.102 Histologically, the gland is infiltrated with lym-
phocytes and plasma cells to the extent that secondary lym-
phoid follicles develop within the thyroid gland that are
nodes. Initially, the gland is usually enlarged. Over time, with
destruction of the gland, the gland can atrophy or become
firm. In the rare condition of Riedel thyroiditis (Riedel disease
or struma, ligneous struma, ligneous thyroiditis, chronic
fibrous thyroiditis), the thyroid gland becomes fibrotic with
possible attachment to adjacent structures that can produce,
for example, tracheal compression.144 Subacute (viral) thy-
roiditis may also cause Riedel thyroiditis.31 Not all cases of
Riedel thyroiditis are presaged by Hashimoto thyroiditis.
Although atrophy may occur in Hashimoto thyroiditis,
atrophic thyroiditis may also occur when autoantibodies
against the TSH receptor bind to the receptor and block the
action of endogenous TSH. TSH receptor blocking autoanti-
bodies can cross the placenta during pregnancy, causing tran-
sient hyperthyrotropinemia in infants (elevated TSH with a
normal T4) or even transient congenital hypothyroidism.
Therefore atrophic thyroiditis falls under the rubric of AITD.
The diagnosis of Hashimoto thyroiditis is supported by
recognition of autoantibodies against TPO or Tg.113 Ninety
percent of patients with chronic lymphocytic thyroiditis (the
histologic description of Hashimoto thyroiditis) have anti-
thyroperoxidase autoantibodies (TPOA), antithyroid micro-
somal autoantibodies (TMA), and/or antithyroglobulin
autoantibodies (TgA), making these autoantibodies excellent
markers for Hashimoto thyroiditis.182 TPOA testing was ini-
tially pursued by testing for TMA (see section on thyroid
autoantibody testing). TMA testing is being replaced by spe-
cific testing for TPOA using TPO as the antigen in the immu-
noassay. TPOA/TMA positivity is more common at the time
of diagnosis than TgA positivity with TgA usually appearing
later in the disease process. TgA overall are less common than
TMA or TPOA. Ultrasound has limited value and is not rou-
tinely used to detect Hashimoto thyroiditis.
In many families, AITD appears to be inherited in an
autosomal dominant pattern although some family members
develop Hashimoto thyroiditis and other family members
develop Graves disease. Several genetic loci have been associ-
ated with susceptibility to Hashimoto thyroiditis (or AITD):
HLA-DR, CTLA-4, CD40, FCRL3, Tg, the TSH receptor,
PTPN22, and the IL-2 receptor. However, no single Mende-
lian locus explains the apparent autosomal dominant pattern
of inheritance of AITD.66 It is most appropriate to describe
AITD as polygenic and multifactorial, indicating that both
environment and multiple genes provide susceptibility to
these very common disorders.
Nonendocrine and endocrine autoimmune disorders can
occur with increased frequency in people with AITD. Chronic
lymphocytic gastritis causing pernicious anemia may accom-
pany AITD, particularly in older patients. AITD also occurs
commonly in association with type 1 diabetes and pernicious
anemia.61,91,104 Another, less common, association is AITD
and Addison disease.82
AITD can occur as part of autoimmune polyglandular
syndrome type 2 (APS 2) and, more rarely, APS 1.161,175 APS
2 affects women more often than men with onset in child-
hood or early adulthood and is diagnosed when Addison
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
disease (or adrenal autoantibodies) occurs together with
AITD and/or type 1 diabetes. In contrast to the polygenic
nature of APS 2, mutations in AIRE (the autoimmune regula-
tor gene that is a transcription factor; gene location: chromo-
some 21q22.3) produce autoimmune polyglandular syndrome
type 1 (APS 1), which is inherited in an autosomal recessive
pattern, thus affecting males and females equally.18,93
Another variant of AITD is postpartum thyroiditis
(PPT).192 PPT develops presumably as a consequence of a
decline in the natural immunosuppression of pregnancy fol-
lowing delivery. PPT follows 5% of all pregnancies and
10% of pregnancies in women with type 1 diabetes. The
clinical phenotype of PPT is transient hypothyroidism,
hyperthyroidism, or both (one following the other) with a
return to the euthyroid state by approximately 1 year postpar-
tum. Women with thyroid autoantibodies are at highest risk
for PPT. Later in life, permanent clinical evidence of AITD
can develop in women afflicted with PPT. Thyroid autoim-
munity, even in the absence of hypothyroidism, may impair
fertility in women and increase the risk of spontaneous abor-
tion in those women who do become pregnant.154 Therefore
an indication for thyroid autoantibody testing in the absence
of hypothyroidism would be female infertility or recurrent
miscarriage. Convincing data suggest that T4 treatment of
pregnant women who are positive for thyroid autoantibodies
(especially TPOA) leads to a higher frequency of miscarriage
(13.8%) than is seen in pregnant women who lack TPOA
(2.4% rate of miscarriage), and that T4 treatment of the
TPOA-positive group reduces the rate of miscarriage to
approximately 3.5%.136
Inborn Errors in Thyroid Hormone Biosynthesis
Inborn errors in thyroid hormone biosynthesis (dyshor-
monogenesis) are rare causes of primary hypothyroidism.
These defects usually present early in life and can appear in
newborns as a goiter. In very rare cases, asphyxia from tra-
cheal compression has been reported.213 Biochemical defects
include iodine transport defects from loss-of-function muta-
tions in the NIS (no increase in the perchlorate discharge);
defects in thyroperoxidase, DUOX2,130 and pendrin (with
increased perchlorate discharge); thyroglobulin deficiency;
and iodotyrosine dehalogenase mutations (potentially causing
iodine deficiency through loss of MIT, DIT, and iodine in the
urine).129
Developmental Hypothyroidism Disorders
Developmental causes of primary hypothyroidism involve
aplasia or hypoplasia of the thyroid gland and ectopic and
lingual thyroid glands.202 These disorders account for 75%
of cases of congenital hypothyroidism. Other causes of con-
genital hypothyroidism include thyroid dyshormonogenesis
(10% of cases; see earlier), hypothalamic or pituitary abnor-
malities (5% of cases). Ten percent of cases of congenital
hypothyroidism are transient.
Rare causes of congenital hypothyroidism involve loss-of-
function mutations in the pituitary TRH receptor,23 the TSH
beta chain,128 and the TSH receptor.19,58 Mutations in anterior
1919 1920 Section V Pathophysiology
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
PIT1) can lead to TSH deficiency. Causes of transient con- the thyroid gland, more T4 is converted to T3 to maintain the
genital hypothyroidism include (1) severe maternal iodine euthyroid state overall. Endemic goiters can develop nodular- of Thyroid Function
deficiency, (2) acute iodine exposure, (3) transplacental ity (with or without hemorrhage into the nodule); in such Cause Drug Effect
passage of thionamides taken for treatment of maternal cases, neoplasia must be excluded. Large goiters can produce
hyperthyroidism, (4) transplacental transfer of TSH receptor dysphagia, obstruction of the trachea, or compression of the Inhibit TSH Dopamine T4; T3;
blocking antibodies, (5) hypothyroxinemia of prematurity, recurrent laryngeal nerves. A rare cause of iodine deficiency secretion l-dopa TSH
and (6) heterozygosity for a DUOX2 mutation. is nephrotic syndrome, with increased urinary loss of iodine Glucocorticoids
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 ;
thyroidism in North America reveals a general population A significant danger of iodine deficiency is maternal hypo-
hormone Lithium TSH
frequency of congenital hypothyroidism of 1 in 4000. Con- thyroidism leading to an insufficient supply of thyroid
synthesis or
genital hypothyroidism is more common in Hispanic infants, hormone to the fetus in the first half of gestation, when the
release
less common in Caucasian infants, and least common in fetus is entirely dependent on maternal thyroid hormone.
Inhibit Amiodarone T3; rT3;
African American infants. The male-to-female ratio is 1 : 2. A Thus maternal hypothyroidism can produce a reduction in
conversion of Glucocorticoids , ,
higher frequency of congenital hypothyroidism is seen in the IQ of the affected child. Maternal iodine deficiency will
T4 to T3 Propranolol T4 and
infants with Down syndrome (1 in 140). produce fetal iodine deficiency.
Propylthiouracil FT4; ,
Clinically, fewer than 1 in 20 hypothyroid babies are Although it is logical that iodine deficiency would produce
Radiographic TSH
recognized in the newborn nursery by physicians. This hypothyroidism because iodine is a necessary component in
contrast agents
emphasizes the critical need for newborn biochemical screen- the synthesis of thyroid hormone, it is ironic that excess
Inhibit binding of Salicylates T4; T3;
ing. Early intervention improves clinical outcomes in these iodine can interfere with normal thyroid gland function.106
T4/T3 to serum Phenytoin FT4E,
cases. However, some studies report that even with rapid However, excessive iodine does suppress thyroid gland func-
proteins Carbamazepine ,
treatment after birth, the IQ of affected children may be tion. High doses of iodine [super-saturated potassium iodide
Furosemide FT4; 
10 points below that of their unaffected siblings. An (SSKI) or Lugols solution] are routinely used before planned
Nonsteroidal TSH
increased frequency of neuropsychologic deficits has been surgical thyroidectomy for Graves disease, to inhibit further
anti-inflammatory
reported in children with appropriately treated congenital thyroid hormone release and decrease the vascularity of the
agents
hypothyroidism. gland to reduce surgical blood loss.
Heparin (in vitro
In North America, screening for congenital hypothyroid- Excess iodine reduces thyroid hormone release and inhib-
effect)
ism is carried out predominantly by measuring total T4 on its organification of iodine and iodination of Tg (the Wolff-
Stimulate Phenobarbital T4; FT4;
DBSs. A common practice is to measure TSH in babies who Chaikoff effect). The Wolff-Chaikoff effect can speculatively
metabolism of Phenytoin  TSH
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
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;
hypothyroidism is made, and the infant is transferred to sources of excess iodine include amiodarone (an antiarrhyth- absorption of hydroxide TSH
a referral center emergently to be evaluated and started on mic drug), povidone-iodine (used to disinfect the skin before ingested T4 Ferrous sulfate
thyroxine replacement therapy (e.g., 10 to 15 g/kg/d). If the surgery), and radiologic contrast agents that contain iodine. Cholestyramine
TSH is between 20 and 59 mIU/L, the infant is clinically Excess iodine does not usually cause permanent thyroid dys- Colestipol
evaluated and repeat thyroid function testing is performed function because the gland normally recovers (escapes) Iron sucralfate
before thyroid hormone replacement is started. If the TSH from suppression after approximately 10 days of high-dose Soybean
is less than 20 mIU/L, primary hypothyroidism is excluded. iodine administration. However, if the patient has underlying preparations
Screening with T4 only testing will lead to missed rare cases disease that may involve the thyroid (e.g., Hashimoto thy- Kayexalate
of central hypothyroidism (1/100,000) and cases of hypothy- roiditis, Graves disease), escape from the Wolff-Chaikoff Increase in Estrogen T4; T3;
roidism wherein TSH is elevated but T4 is not yet depressed. effect is less likely and permanent hypothyroidism can concentration Clofibrate  FT4;
In Europe, many screening programs use a TSH-first strategy. develop. of T4-binding Opiates (heroin,  TSH
Although TSH screening will detect subclinical hypothyroid- proteins methadone)
ism (see later), cases of central hypothyroidism will be missed. Drug-Induced Hypothyroidism
5-Fluorouracil
Transcription factor mutations involving TTF-1 and Pax8 Various drugs affect thyroid gland function (Table 55-4).63 Perphenazine
have been recognized in some children with congenital hypo- Collectively known as thionamides or thioureas, propylthio- Decrease in Androgens T4; T3;
thyroidism. It is not clear whether molecular testing will uracil (PTU), methimazole, and carbimazole inhibit the concentration Glucocorticoids  FT4;
prove useful in future screening programs for congenital oxidation of iodide and the subsequent binding of iodine of T4-binding  TSH
hypothyroidism. to tyrosine residues in Tg. Other drugs with thionamide- proteins
like activity include ethionamide, aminoglutethimide, phen-
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
an immunosuppressive effect of thionamides on thyroid
edition. Philadelphia, Pa: JB Lippincott, 1995:299-306; Stockigt JR. Thyroid
Worldwide, the most common cause of goiter is iodine defi- autoimmunity. hormone changes in critical illness: the sick euthyroid syndrome. Diagn
ciency. In iodine-deficient areas, especially mountainous PTU, methimazole, and carbimazole are commonly used Endo Metab 1997;15:39-46.
areas, iodine deficiency produces endemic goiter. Frank to treat hyperthyroidism. (Note: Carbimazole is not available , Reduced serum concentration; , increased serum concentration; ,
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
drug decreases peripheral conversion of T4 to T3 through
inhibition of the D1 deiodinase. Because of this effect (in
addition to the direct suppressive effect of PTU on the thyroid
gland), some endocrinologists argue that PTU should be
chosen over methimazole for the treatment of hyperthyroid-
ism. However, recognition of serious or even fatal liver disease
in a small (but significant) number of children treated with
PTU is increasing.100 Therefore, many pediatric endocrinolo-
gists discourage the routine use of PTU for the treatment of
hyperthyroidism in children. Reduced granulocyte counts
and subsequent infections are serious but uncommon side
effects of thionamides. Therefore the white blood cell count
and the differential count should be monitored during such
therapy.
A potential advantage of methimazole over PTU is its
longer half-life (PTU t1/2 = 1.5 hours and methimazole t1/2 =
6 hours). Both PTU and methimazole cross the placenta, can
interfere with fetal thyroid function, and can cause goiter.
Additionally, methimazole has been associated with aplasia
cutis and choanal atresia. Therefore during pregnancy, PTU
is the preferred drug in the treatment of maternal
hyperthyroidism.
A variety of other drugs have been known to cause hypo-
thyroidism. For example, lithium, which is used in the treat-
ment of bipolar disorder (manic-depressive illness), can
induce hypothyroidism.85 The action of lithium appears
similar to that of high-dose iodine, inhibiting thyroid
hormone release and organification of iodine. Prolonged use
of nitroprusside, a drug used to treat acute-onset, severe
hypertension or severe heart failure by inducing preload and
afterload reduction, may lead to hypothyroidism. Cyanide
(CN) released from nitroprusside is metabolically converted
to thiocyanate (SCN), which inhibits iodide uptake by the
thyroid gland. Fortunately, nitroprusside only rarely causes
hypothyroidism. Amiodarone is an antiarrhythmic drug that
contains two iodine atoms per molecule and can induce
hypothyroidism or hyperthyroidism.22,204 Various recom-
binant DNA-derived biologicals (e.g., interferon-alpha,
interleukin-2) used to treat chronic viral hepatitis or cancer
have been found to cause thyroid dysfunction (hypothyroid-
ism or hyperthyroidism).124
Surgical and Radiation-Induced Hypothyroidism
Surgical removal of the thyroid gland will produce hypothy-
roidism. External irradiation of the thyroid gland (e.g., treat-
ment of lymphoma or Hodgkin disease) or ingestion of
radioactive iodine can ablate the gland, causing hypothyroid-
ism.123 Administration of radioactive iodine to pregnant
women can lead to ablation of the fetal thyroid gland, causing
congenital hypothyroidism.
Viral or Bacterial Thyroiditis
Only rarely do viral or bacterial infections of the thyroid
gland occur. Even more rarely do these entities damage the
thyroid gland sufficiently to produce hypothyroidism. Viral
infection of the thyroid gland is termed subacute thyroiditis
(de Quervain, granulomatous, or giant cell thyroiditis) and
1921
can produce generalized thyroid gland tenderness. Bacterial
infection of the thyroid gland is termed acute thyroiditis and
can produce a thyroid abscess.3
Central Hypothyroidism
In a patient with clinical evidence of hypothyroidism, sup-
ported by laboratory findings of a low FT4, if TSH is not
elevated to the extent predicted (by FT4), or if TSH is within
or below the reference interval, central hypothyroidism
should be considered.92,229 Isolated pituitary TSH deficiency
is rare, as most patients with secondary hypothyroidism also
have other anterior pituitary hormone deficiencies (panhypo-
pituitarism). Causes of central hypothyroidism (hypotha-
lamic or pituitary disease) include (1) tumor, (2) hemorrhage,
(3) trauma, (4) malformation, (5) postinfectious damage, and
(6) postsurgical damage. Also, several rare autosomal reces-
sive or dominant hereditary disorders involving transcription
factor mutations can cause hypopituitarism and TSH defi-
ciency: examples include paired-like homeodomain tran-
scription factor-1 (Pit-1) and PROphet of Pit-1 (PROP1)
mutations.131
Subclinical Hypothyroidism
Subclinical hypothyroidism is defined by a persistent eleva-
tion in TSH (6 to 12 weeks or longer) in the setting of FT4
concentrations that are repeatedly found within the reference
interval.49 Other conditions wherein TSH is elevated but FT4
is normal encompass recent reinstitution of thyroid hormone
replacement therapy (FT4 returns to normal before TSH
declines), poor compliance with treatment in primary hypo-
thyroidism, recovery from nonthyroidal illness, positively
interfering heterophilic antibodies [e.g., human antimouse
antibodies (HAMA)] in double-antibody immunoassays, and
thyroid hormone resistance.
Subclinical hypothyroidism is very common, affecting 3 to
8% of the general population. By the sixth decade of life, 10%
of the population exhibits laboratory findings consistent with
subclinical hypothyroidism. Consequences of subclinical
hypothyroidism include a high risk of progression to clinical
hypothyroidism, dyslipidemia, vascular endothelial dysfunc-
tion, increased risk of cardiovascular disease and death, and
possible neurocognitive deficits. The general consensus is that
if TSH is repeatedly 10 mIU/L or greater, thyroid hormone
replacement therapy is warranted. It is controversial whether
thyroid hormone therapy is beneficial when TSH is less than
10 mIU/L, except during pregnancy, wherein any persistent
TSH elevation requires treatment.
Controversy is ongoing over the appropriate upper limit
of the reference interval for TSH. Basing the TSH reference
interval on the central 95% of the general, healthy population
results in an upper limit of normal between 4.0 and 5.0 mIU/L
for many TSH assays. If patients with thyroid autoantibodies
such as TPOA, a family history of thyroid disease, or an
abnormal thyroid gland ultrasound are excluded from the
reference population, the upper limit of the reference interval
declines to approximately 2.5 to 3.0 mIU/L. Therefore, an
upper limit of 2.5 to 3.0 mIU/L for TSH may be a truer
1922 Section V
definition of normal, because the reference population mostly
excludes individuals with endogenous thyroid disease or pro-
pensity to develop thyroid disease. However, it is unclear how
patients with TSH concentrations between 2.5/3.0 mIU/L and
4.0/5.0 mIU/L should be managed. It has been argued that
lowering the upper limit of the reference interval will provide
no clear patient benefit and inevitably will lead to confusion
and possible overtreatment of subclinical hypothyroidism.
Overtreatment carries the risk of predisposing the patient to
osteoporosis and atrial fibrillation. As noted previously, the
value of treatment when TSH is between 4.0/5.0 and 9.9
mIU/L is currently debated. Another component of the TSH
reference interval debate is that according to National Health
and Nutrition Examination Survey (NHANES) data,9,134A,209
TSH rises as populations age. For example, in healthy adults
age 80 and older, the upper limit of the reference interval for
TSH is 7.5 mIU/L.
Thyroid Hormone Monitoring During
Replacement Therapy
The thyroid hormone replacement of choice is Na+-l-
thyroxine (levothyroxine; T4). Several brands of T4 are avail-
able, including Synthroid (Abbott, North Chicago, Ill),
Levothroid (Forest, St Louis, Mo) and Levoxyl (King, Bristol,
Tenn). USP (United States Pharmacopeia) Thyroid or mix-
tures of T4 and T3 should not be used for thyroid hormone
replacement. USP Thyroid is a desiccated extract of whole
animal thyroid containing variable proportions of T3 and T4.
Because its exact formulation is highly variable and is not
standardized, USP Thyroid will not produce stable concentra-
tions of thyroid hormone over the long term, leading to
periods of excess or deficient thyroid hormone replacement.
Despite many trials, fixed mixtures of T3 plus T4examples
include Naturethroid (RLC, Tempe, Ariz) and Thyrolar
(Forest)have not been shown to be clinically superior to T4
replacement alone. Administering T4 alone allows the body
to regulate the production of T3. Recall that normally 80% of
T3 comes from peripheral monodeiodination of T4 to T3. The
sodium salt of T3 (liothyronine) is available in tablet (Cytomel,
King) and injectable (Triostat, King) forms. Considerable
controversy surrounds whether generic preparations of T4 are
as effective as brand name preparations because of variable
consistency and/or absorption from the gastrointestinal tract.
Anecdotal reports have suggested that brand name prepara-
tions are clinically superior to generics.
Thyroxine should be taken at the same time of day for
clinical consistency. The timing of FT4 measurements should
also be standardized because FT4 may rise abruptly within
hours of oral thyroxine ingestion. Thyroxine is best absorbed
without food. A 2009 study demonstrated that TSH was
lowest when thyroxine was taken in the fasting state (mean
TSH, 1.06 mIU/L) versus when taken with breakfast (2.93
mIU/L) or at bedtime (2.19 mIU/L).12 Many reasons have
been put forth for variations in TSH measurements over
time,52 including intraindividual variation of 15 to 35%.6 The
2003 National Academy of Clinical Biochemistry Laboratory
Pathophysiology
Practice Management Guideline on thyroid function testing
reported intraindividual coefficients of variation for TSH of
19.3% for 1 week, 20.6% for 6 weeks, and 22.4% for 1 year.13
A significant source of biological variation in TSH is the
pulsatile pattern of secretion and the fairly short half-life of
TSH (approximately 1 to 2 hours).80 In one study, TSH was
released in approximately 18 pulses over the course of a day,
and TSH half-life was 60 to 90 minutes. The half-life of TSH
may vary between individuals, depending on interindividual
differences in the degree of sialylation and sulfation of
TSH.47,149
FT4 and TSH are usually measured approximately 6 weeks
after the beginning of thyroxine treatment for hypothyroid-
ism. The dose of thyroxine should be titrated upward until
FT4 is in the upper half of the reference interval. TSH returns
to the reference interval more slowly than the rise of FT4 into
the reference interval occurs in response to therapy. Ideally
during treatment, TSH should be maintained within the ref-
erence interval. As the dose of thyroxine is increased during
the initial months of therapy, some endocrinologists will
allow TSH to be slightly suppressed (but >0.02 to 0.04 mIU/L)
if the patient feels substantially better. It can be argued that,
particularly in women, it is prudent to measure bone mineral
density by dual-energy x-ray absorptiometry (DEXA) scan at
least once a year to screen for bone loss that could result from
overtreatment with thyroxine. Overtreatment of hypothy-
roidism is not uncommon.7
HYPERTHYROIDISM
Hyperthyroidism, also known as thyrotoxicosis, is the clinical
syndrome that results from elevated concentrations of free
thyroid hormone in the plasma, associated with clinical
evidence of hypermetabolism.135,139,178A Symptoms of thy-
rotoxicosis include (1) nervousness, (2) erratic behavior,
(3) emotional lability, (4) restlessness, 5) sleeplessness,
(6) difficulty concentrating, (7) smooth and/or shiny hair
and/or skin, (8) weight loss, (9) excessive sweating, (10) heat
intolerance, (11) menstrual irregularities, and (12) diarrhea
or frequent bowel movements. Physical signs compatible
with hyperthyroidism include tachycardia, atrial arrhyth-
mias, systolic murmurs, increased pulse pressure, a bounding
pulse, warm and/or damp skin, softened texture of the
skin, tremor, increased reflexes, eyelid retraction (and
other signs of ophthalmopathy in Graves disease), and
hypocholesterolemia.
Based on TSH and FT4, the causes of hyperthyroidism may
be classified as primary (elevated FT4 and decreased TSH) or
central (elevated FT4 and normal to elevated TSH). Central
hyperthyroidism can be further parsed into pituitary (sec-
ondary hyperthyroidism from TSH excess) and hypothalamic
(tertiary hyperthyroidism from TRH excess) causes. In
primary hyperthyroidism, TSH is typically less than 0.05
mIU/L. With current assays, a TSH concentration within the
reference interval nearly always excludes the diagnosis of
primary hyperthyroidism.
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1923 1924 Section V Pathophysiology

Causes of Hyperthyroidism
The causes of hyperthyroidism are listed in Box 55-3. Endog-
enous disorders causing hyperthyroidism include intrinsic
thyroid disease (primary hyperthyroidism), ectopic thyroid
tissue (struma ovarii),166 and disorders of the hypothalamus
or pituitary causing excess TSH secretion (secondary hyper-
thyroidism). Exogenous causes of hyperthyroidism (disease
related to external factors) encompass infectious origins
(thyroid gland inflammation and destruction), iodine-
induced hyperthyroidism, and thyroid hormone ingestion
(thyrotoxicosis factitia).
The first line of treatment for primary hyperthyroidism is
controversial. Administration of radioactive iodine is the first
choice in many adults and older children with Graves disease.
When medical therapy is chosen, thionamides are used to
suppress thyroid gland hyperactivity.195 In the immediate
period following the start of thionamide therapy, TSH mea-
surements may poorly reflect the patients thyroid status
because TSH can remain suppressed for months even after
the patient becomes clinically euthyroid. This is similar to the
treatment of hypothyroidism, where 6 weeks or more can
elapse before TSH returns to normal even with adequate thy-
roxine replacement.
After 1 to 2 years of thionamide therapy, if disease remis-
sion has not occurred, radioactive iodine is usually advised.71
An alternative therapy is thyroidectomy, particularly in spe-
cialized centers with extensive surgical experience. However,
with radioactive iodine ablation of the gland, there is no
surgical risk of inadvertent parathyroidectomy or recurrent
laryngeal nerve injury. As a consequence of radioactive iodine
treatment or thyroidectomy, primary hypothyroidism can
BOX 55-3 Causes of Hyperthyroidism
Endogenous Thyroid Disorders
Autoimmune thyroid disease
Graves disease
Hashitoxicosis
Postpartum thyroiditis
Gain-of-function mutation in the TSH receptor
Toxic nodule
Toxic multinodular goiter
Toxic adenoma
Familial
Struma ovarii
hCG-induced hyperthyroidism
Gestational transient thyrotoxicosis
TSH receptor sensitivity to hCG
hCG-secreting tumors
Secondary hyperthyroidism (e.g., central hyperthyroidism)
Exogenous Disorders
Thyroid destruction from viral or bacterial thyroiditis
Iodine-induced hyperthyroidism
Thyroid hormone ingestion (thyrotoxicosis factitia)
subsequently develop, necessitating surveillance for hypothy-
roidism with yearly TSH measurements.
T3 Toxicosis
T3 toxicosis is defined by the presence of clinical hyperthy-
roidism in a patient with suppressed TSH, normal FT4, and
elevated T3 (or FT3).50 FT4 may be normal in early hyperthy-
roidism when a very mild excess of thyroid hormone is
causing increased thyroidal and peripheral deiodination of T4
to T3, raising the latter but not the former. Recall that D1
conversion of T4 to T3 is enhanced in hyperthyroidism. T3
toxicosis is also possible when hyperthyroidism occurs in the
presence of mild iodine deficiency, when iodine is sufficient
to synthesize excessive amounts of T3 but not T4. Last, it is
known that stimulation of the TSHR enhances the produc-
tion of T3 more than T4. T3 toxicosis is not a unique or indi-
vidual disease, but rather can be considered a phase in the
natural progression of primary hyperthyroidism that may or
may not be recognized by the clinician.
Graves Disease
Graves disease affects approximately 0.4% of the U.S. popula-
tion and results from agonistic autoantibodies that bind to,
and activate, the TSH receptor, producing excess release of
thyroid hormone and subsequent clinical hyperthyroidism.24
Women are more frequently affected by Graves disease than
men, by a 5 : 1 ratio.
The classical clinical triad observed in patients with
Graves disease consists of (1) goiter and biochemical hyper-
thyroidism, (2) exophthalmos, and (3) nonpitting pretibial
myxedema.220 The presence of exophthalmos strongly sug-
gests Graves disease in a hyperthyroid patient. Exophthalmos
appears to have an autoimmune cause. Retro-orbital tissues
may express TSH receptors, and consequent to stimulatory
TSH receptor autoantibodies, retro-orbital tissue hyperplasia
occurs, producing exophthalmos.17 Myxedema is the accu-
mulation of mucopolysaccharides in subcutaneous tissue that
cause visible and palpable swelling. The potential finding of
myxedema in both Graves disease and Hashimoto thyroiditis
(with hypothyroidism) is not readily explained.
Similar to Hashimoto thyroiditis, genetic susceptibility to
Graves disease is polygenic.66 In practice, Hashimoto thy-
roiditis is often associated with HLA-DR5 while Graves
disease is associated with HLA-DR3.
TPOA (including TMA) and TgA are seen in both Hashi-
moto thyroiditis and Graves disease but usually are found in
higher concentrations in Hashimoto thyroiditis. However,
these autoantibodies do not distinguish between the two con-
ditions. Testing for TSH receptor autoantibodies should be
reserved for cases where the diagnosis of Graves disease is in
doubt. TSH receptor autoantibody (TRA) testing is available
for stimulatory autoantibodies [TSH receptor stimulatory
autoantibodies (TSI)] and receptor-binding autoantibodies
[thyrotropin-binding inhibitory immunoglobulins (TBII)].
Usually, both TSI and TBII tests are positive in patients with
Graves disease.
Hashitoxicosis and Postpartum Thyroiditis
During the clinical course of Hashimoto thyroiditis, if a
period of accelerated destruction of thyroid follicular cells
occurs, subsequent release of thyroid hormone can produce
a transient interval of hyperthyroidism, termed Hashitoxico-
sis.133 Hashitoxicosis should be differentiated from Graves
disease because the treatments for these two conditions are
different. Hashitoxicosis is self-limited, and if any treatment
is required, -blockers (such as propranolol) are used to sup-
press the effects of excess catecholamines, such as tachycar-
dia. TRA are usually positive in patients with Graves disease
and negative in Hashitoxicosis. In addition, the RAIU is ele-
vated in Graves disease but is not elevated in Hashitoxicosis.
Patients with postpartum thyroiditis can experience a period
of transient, usually self-limited, hyperthyroidism from accel-
erated breakdown of thyroid tissue. Subacute or acute thy-
roiditis can produce a period of transient hyperthyroidism.
Toxic Nodular or Multinodular Goiter
A subset of patients with nodular or multinodular goiter can
develop hyperthyroidism.35,155,181 When hyperthyroidism is
caused by these lesions, the term toxic has been used (toxic
multinodular goiter). In the older literature, toxic multinodu-
lar goiter is referred to as Plummer disease.
The cause of nodular or multinodular goiter is not well
characterized. One theory is that these disorders arise from
colloid goiters.37 A colloid goiter itself is a disorder of unknown
origin, in which the thyroid gland enlarges because of
increased size of the follicular lacunae.5 Microscopically, the
enlarged lacunae display wide variation in size. However,
colloid goiters do not display the follicular cell hyperplasia
typical of Graves disease. Patients with colloid goiter lack
thyroid autoantibodies, and no evidence suggests a defect in
thyroid hormone biosynthesis. Over many years, some
regions of colloid goiters undergo further hypertrophy, while
other areas atrophy, producing nodules that may become pal-
pable. TRA are negative in toxic multinodular goiter.214
In some cases of nodular or multinodular goiter, the
hyperactive tissue can display a somatic gain-of-function
mutation in the TSH receptor or Gs protein, so that the recep-
tor is perpetually in the on confirmation or functions as
though it is in the on position, in the absence of TSH
binding to the receptor.201 This mutation occurs in at least
60% of cases of nodular or multinodular goiter with hyper-
thyroidism. However, in the other 40% of cases, the cause of
the hyperthyroidism is unknown. Somatically acquired gain-
of-function mutations in the Gs alpha subunit produce many
of the symptoms of McClune-Albright syndrome, including
thyrotoxicosis.
Sometimes follicular adenomas are autonomously hyper-
active, producing hyperthyroidism (toxic adenomas).87 Other
than elevated FT4 and depressed TSH from hyperthyroidism,
no specific laboratory tests can diagnose nodular or multi-
nodular goiter, or a toxic adenoma. Toxic (hyperactive, or
hot) nodules are very rarely carcinomas. Cold nodules
(nodules that do not synthesize thyroid hormone and do not
take up radioactive iodine or technetium) may result from
TSH receptor loss-of-function mutations. Any nodule that is
not hot may be cancerous, and fine-needle aspiration biopsy
should be strongly considered.
Gain-of-Function Mutations in the TSH Receptor
A familial, autosomal dominant form of nonGraves disease
hyperthyroidism that is due to gain-of-function mutations in
the TSH receptor has been described.89 The gain-of-function
mutation places the TSH receptor in the on position in the
absence of ligand (TSH) binding. In infants homozygous for
such mutations, neonatal thyrotoxicosis, even requiring emer-
gency thyroidectomy, has been observed. Certain heterozy-
gous mutations have caused infantile hyperthyroidism.217
Central Hyperthyroidism
TSH-secreting anterior pituitary adenomas are very rare.103
This diagnosis is suggested by (1) clinical hyperthyroidism,
(2) elevated FT4, (3) normal to elevated TSH concentration,
and (4) evidence of a pituitary mass on computed tomogra-
phy (CT) scan or magnetic resonance imaging (MRI). Inci-
dentalomas of the pituitary are found in approximately 5% of
the general population; therefore a CT or MRI anomaly is not
pathognomonic for a pituitary adenoma. A thyrotropinoma
can produce a mass displacement effect, resulting in deficien-
cies of other anterior pituitary hormones caused by compres-
sion of pituitary tissue.96
Rarely, selective pituitary resistance to the effects of thyroid
hormone is present, yet peripheral sensitivity is normal.226
This condition produces clinical hyperthyroidism because
only elevated concentrations of thyroid hormones suppress
TSH. The subsequent rise in TSH promotes thyroid hormone
synthesis and release from the thyroid gland, producing clini-
cal hyperthyroidism because the peripheral tissues have
normal responsiveness to thyroid hormone. Selective pitu-
itary resistance to thyroid hormone feedback is suggested
when the pituitary CT scan or MRI is normal. Unique muta-
tions in TR causing selective pituitary resistance have been
described.169
Hyperthyroidism Due to Human
Chorionic Gonadotropin
Human chorionic gonadotropin (hCG)induced hyperthy-
roidism is observed in gestational transient thyrotoxicosis,
TSH receptor sensitivity to appropriate hCG concentrations
during pregnancy, and hCG-secreting tumors.94,97,163 Gesta-
tional transient thyrotoxicosis occurs in 2 to 3% of all preg-
nancies, and results from activation of TSH receptors by
hCG, which is greatly elevated during pregnancy. The degree
of hyperthyroidism is typically mild, and treatment is not
usually required. In contrast to Graves disease, thyroid auto-
antibodies are negative. If PTU or methimazole is used, fetal
monitoring for thyroid dysfunction is important, because
these drugs cross the placenta, potentially causing fetal hypo-
thyroidism and goiter.
Fetal thyroid gland suppression by thionamides is a
concern when any pregnant woman with hyperthyroidism is
treated. The size of the fetal thyroid gland can be monitored
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
by ultrasound. TSH and T4 can be measured in fetal blood
obtained by cordocentesis (a high-risk procedure) or in the
amniotic fluid.34 Besides the analytical challenge of measur-
ing the very low concentrations of these hormones in non-
plasma body fluids, finding an appropriate reference interval
can be difficult. Also, it is unlikely that many laboratories
have validated measurements of thyroid hormone or TSH in
amniotic fluid. Hyperemesis gravidarum (severe vomiting
during pregnancy) has been associated with gestational tran-
sient thyrotoxicosis and may be related to very high hCG
concentrations.147
Tumors that secrete hCG, such as choriocarcinoma, hyda-
tidiform mole, or metastatic embryonal carcinoma, have
caused hyperthyroidism through hCG stimulation of the
TSH receptor.212
Iodine-Induced Hyperthyroidism
In a patient with iodine deficiency and underlying Graves
disease or nodular/multinodular goiter, iodine administra-
tion may lead to hyperthyroidism.132 Before iodine replenish-
ment is provided, hyperthyroidism is likely to be kept in
check by iodine deficiency.193 The phenomenon of iodine-
induced hyperthyroidism is termed the Jod-Basedow syn-
drome (or phenomenon). Distinct from these two disorders
(iodine-induced hyperthyroidism in iodine-deficient Graves
disease and nodular/multinodular goiter) are rare patients
without underlying thyroid disease who paradoxically
develop hyperthyroidism with iodine treatment.10,101
Sources of iodine besides the diet include administered
iodinated radiocontrast dyes, Lugols solution, topical anti-
septics, iodine-containing expectorants, and amiodarone.152
The typical intake of iodine in the United States is 150 to
200 g/d, one 200 mg tablet of amiodarone contains 75 mg
of iodine, of which 6 mg is liberated (corresponding to at least
30 times the usual daily intake!).
Other Exogenous Causes of Hyperthyroidism
Thyroid gland destruction from any cause, including nonau-
toimmune thyroiditis from viral (subacute) or bacterial
(acute) causes, can release excessive amounts of thyroid
hormone, producing hyperthyroidism.179,180 Intentional or
unintentional ingestion of excess thyroid hormone can also
produce hyperthyroidism.76 In one interesting report, hyper-
thyroidism developed in people who ate hamburger meat that
included bovine thyroid glands added during the grinding
process.146
When thyroid hormone is ingested or released from an
inflamed or damaged thyroid gland, subsequent suppression
of TSH reduces thyroidal iodine uptake, which is reflected in
a reduction in the RAIU test. Likewise, glandular suppression
will lower circulating Tg concentrations. If T3 is ingested (e.g.,
Cytomel), hyperthyroidism can ensue, producing an unusual
set of biochemical abnormalities: suppressed TSH, sup-
pressed FT4 (and total T4, because of TSH suppression), and
elevated total T3 (and FT3).
Maternal Graves disease can cause fetal (and transient
neonatal) hyperthyroidism from the transplacental passage of
1925 1926 Section V Pathophysiology
89
TSI. In these cases, neonatal hyperthyroidism, although
H3N COO H3N COO H3 N COO
potentially serious and possibly fatal if not recognized and
CH2 CH2 CH2
appropriately treated, is usually self-limited.
Thyroid Storm and Apathic Hyperthyroidism I I D1,D2 I I
D3 I
O O O
Thyroid storm (also, thyrotoxic crisis) is an uncommon syn-
drome of severe and accelerating hyperthyroidism potentially
I I I I I
manifested in (1) tachycardia, (2) restlessness, (3) high- OH OH OH
output congestive heart failure, (4) fever greater than 41 C, T3 T4 rT3
(5) extreme irritability, (6) delirium or coma, (7) hypoten- D3 D1,D2
sion, and (8) vomiting or diarrhea.134 Although laboratory D1,D2 D3
tests reveal suppressed TSH and elevated FT4 and T3 concen-
trations, thyroid storm remains a clinical diagnosis. Thyroid
H3N COO H3N COO H3 N COO
storm cannot be diagnosed on the basis of the magnitude of
FT4 or T3 concentrations, or of changes observed in these CH2 CH2 CH2
hormones compared with previous measurements.
In older individuals, an unusual variant of hyperthyroid- I I I
O O O
ism is termed apathetic hyperthyroidism.127 In this disorder,
biochemical evidence of thyroid hormone excess is observed
in the absence of any clinical evidence of hyperthyroidism. I I I
OH OH OH
Alternatively, only a single organ system may be affected. 3,3T2
3,5 T2 3,5T2
Subclinical Hyperthyroidism Figure 55-17 Effects of altered deiodination. In states of nonthyroidal illness (e.g.,
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.
tions are normal identifies subclinical hyperthyroidism.55
Patients with subclinical hyperthyroidism have increased fre-
quencies of atrial fibrillation and osteoporosis. In the absence
of a clinically compatible cardiac arrhythmia or significantly TABLE 55-5 Nonthyroidal Illness
reduced bone mineral density, there is little justification for Initial Mid Course Prolonged Resolution
placing a patient with subclinical hyperthyroidism on thion-
amide therapy. The treatment of patients with asymptomatic TSH Nl Nl, Decr Decr Incr
hyperthyroidism is much more controversial than the treat- T4 Nl Variable Decr Decr
ment of those with asymptomatic hypothyroidism. T3 Decr () Decr ( ) Decr ( ) Decr ()
rT3 Incr (+) Incr (+ +) Incr (+) Incr (+)
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
illness, or chronic illness can result in thyroid function
changes characterized as nonthyroidal illness (NTI; sick pituitary, the pituitary is exposed to adequate concentrations contributing to the patients acute need for hospitalization
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).
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
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
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
(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
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
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
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-
corticoids and -blockers can also inhibit the conversion of concentration in NTI. lamic or pituitary disease has TSH less than 0.05 mIU/L,
T4 to T3. As mentioned previously, one effect of propylthio- With prolonged NTI, disordered hypothalamic-pituitary hyperthyroidism is a possibility. With hospitalized patents, a
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-
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
and is considered to be an adaptive and beneficial response above the reference interval and remain elevated until FT4 management of the patient.
to illness. The recognition that NTI does not represent and T3 return to normal, usually after 1 to 2 months. The
hypothyroidism is important because thyroid hormone sequential stages in NTI are illustrated in Table 55-5. Thyroid Hormone Resistance
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-
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
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
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
and T4 (and FT3 and FT4) are required to maintain the euthy-
roid state. Therefore, in the absence of clinical hyperthyroid-
ism, if the biochemical picture of elevated T4, FT4, T3, and FT3
is present with TSH in the upper reference interval or mildly
elevated, thyroid hormone resistance is likely. Because of TSH
stimulation of the thyroid, glandular enlargement may be
seen because there is no resistance to TSH. Some affected
patients may, in fact, have mild clinical findings of hypothy-
roidism. If patients with thyroid hormone resistance are
incorrectly treated with thyroid-suppressing drugs, clinical
hypothyroidism may be induced.
Another form of thyroid hormone resistance results from
defective conversion of T4 to T3. This condition results from
a mutation in the sequence-binding protein 2 (gene name:
SECISBP2), which influences the synthesis of cellular
deiodinases.
Euthyroid Hyperthyroxinemia
Euthyroid hyperthyroxinemia exists when total T4 is elevated
yet the patient is clinically euthyroid.158,205 Because FT4 mea-
surements have supplanted total T4 measurements, euthyroid
hyperthyroxinemia is not commonly encountered in modern
clinical practice.
Besides TBG elevations and the thyroid hormone resis-
tance syndromes, euthyroid hyperthyroxinemia can also
result from acute illness (through a mechanism that is
not well understood) or abnormalities in albumin and
transthyretin. Familial dysalbuminemic hyperthyroxinemia
is an autosomal dominant disorder characterized by an
albumin variant that has a greater binding affinity for T4
(but not T3).16,28 Although TSH, FT4, and total T3 (and
FT3) are all normal, total T4 is elevated. Mutations in trans-
thyretin can lead to familial euthyroid thyroxine excess, an
autosomal dominant disorder producing abnormal thyroid
function tests similar to what is observed in familial dysalbu-
minemic hyperthyroxinemia.14 These two disorders can be
TABLE 55-6 Patterns of Thyroid Dysfunction
TSH
Primary hypothyroidism Increased
Subclinical primary hypothyroidism Increased
Primary hyperthyroidism Decreased
T3 toxicosis Decreased
Subclinical primary hyperthyroidism Decreased
Thyroid hormone resistance due to Normal to increased
defects in the thyroid hormone
receptor
Thyroid hormone resistance due to Mildly increased
defects in thyroid hormone
metabolism
Thyroid hormone resistance due to Normal to increased
defects in thyroid hormone
transport into cells
1927
distinguished from conditions involving overproduction of
TBG by measuring TBG concentration.
MCT8 Mutations
Loss-of-function mutations in MCT8, causing a rare form of
X-linked mental retardation, have been reported.74,211 Because
MCT8 is important for the transport of thyroid hormone into
neurons, there are profound adverse effects on the develop-
ment of the nervous system in affected males. In addition to
mental retardation and hypotonia, an unusual pattern of
thyroid function abnormalities occurs, with increased T3,
normal to decreased T4 (and FT4), normal to elevated TSH,
and low rT3 concentrations.
Defective cellular uptake of T3 can, in part, explain ele-
vated T3 concentrations in the blood. However, evidence indi-
cates that not all tissues display equivalent defects in thyroid
hormone uptake, and this may explain the decline in T4 and
rT3. Increased T3 concentrations in the liver may also promote
T4 and rT3 clearance. If the pituitary or hypothalamus is less
sensitive to negative feedback from thyroid hormones
(because T3 does not normally enter thyrotrophs or the hypo-
thalamic PVN), theoretically, TSH concentrations would rise.
The elevation in TSH may further contribute to elevated T3
concentrations caused by increased T3 production by the
thyroid gland. The observation that MCT8 mutations exist
requires recognition of a new pattern of thyroid function
studies. Various patterns of thyroid function tests are shown
in Table 55-6.
SCREENING FOR THYROID DYSFUNCTION
Other than newborn screening for congenital hypothyroid-
ism, the value of thyroid function testing in asymptomatic
individuals is highly controversial.72,196,210 Excluding newborn
screening for congenital hypothyroidism, so far no compel-
ling data show that screening asymptomatic individuals,
FT4 Comments
Decreased stimulation via the TSH receptor, (2) the mass of thyroid
Normal tissue present in the individual, and (3) the presence of
Increased T3: increased
Normal T3: increased
Normal T3: normal
Increased T3: increased; rT3: increased
Increased T3: decreased; rT3: increased
Normal to decreased T3: increased; rT3: decreased
1928 Section V
including pregnant women or the elderly, improves the clini-
cal outcome of the patient.
DRUG EFFECTS ON THYROID FUNCTION
Various drugs alter thyroid function in a large number of
ways.60,170 For example, dopamine, l-dopa, glucocorticoids,
and somatostatin all depress TSH, potentially leading to a
fall in thyroid hormone concentrations. As noted previ-
ously, iodine (including amiodarone) and lithium impair
thyroid hormone synthesis or release, causing primary
hypothyroidism.85
Many drugs inhibit conversion of T4 to T3, including amio-
darone, glucocorticoids, -blockers, propylthiouracil, and
radiographic contrast agents. While T3 declines and rT3 rises,
FT4 may increase, decrease, or remain unchanged. Other
drugs can displace T4 and T3 from thyroid hormonebinding
proteins; salicylates, phenytoin, carbamazepine, furosemide,
nonsteroidal anti-inflammatory agents, and heparin (in vitro
effect) lower total T4 and T3 by occupying binding sites
on proteins, yet FT4 remains within the reference interval
or rises.
Increased metabolism of thyroid hormone lowers T4 and
T3. Phenobarbital, phenytoin, carbamazepine, and rifampicin
can lower T4 and T3. Because (1) aluminum hydroxide,
(2) ferrous sulfate, (3) cholestyramine, (4) colestipol, (5) iron
sucralfate, (6) soybean preparations, and (7) Kayexalate all
inhibit thyroid hormone absorption, their effects should be
considered in any patient being treated with thyroxine
replacement.
THYROGLOBULIN
The biology of Tg was extensively reviewed in the biochem-
istry section of this chapter. Some Tg normally enters the
circulation by direct exocytosis from thyroid follicular cells
without being iodinated. Tg is normally detected in the cir-
culation in concentrations between 3 and 40 ng/mL. The
highest Tg concentrations are in cord blood and are noted
during the first weeks of life. Between 2 and 6 weeks of age,
Tg concentrations are between 10 and 250 ng/mL,51 and they
decline until adulthood.141 Three factors influence the con-
centration of Tg in the plasma: (1) the degree of thyroid gland
thyroid disease or damage, which leads to increased release
of Tg into the circulation.
When TSH is within the reference interval, each gram of
thyroid tissue provides approximately 1 ng/mL of Tg to the
circulation. At TSH concentrations less than 0.1 mIU/L, each
gram of thyroid tissue releases only about 0.5 ng/dL of Tg
into the plasma. Trauma to the thyroid gland, inflammation
(such as subacute thyroiditis or amiodarone-induced thy-
roiditis), surgical removal, or irradiation produces elevated
Tg concentrations. Following fine-needle aspiration, Tg can
be elevated for up to 3 weeks. Tg is typically decreased in
acquired hypothyroidism, congenital hypothyroidism (from
Pathophysiology
thyroid hypoplasia), factitious hyperthyroidism (because of
TSH suppression), and following thyroidectomy.
Clinical Utility of Thyroglobulin Measurements
Thyroid cancer is classified according to the cell of origin
(follicular vs. parafollicular) and the degree of differentiation
(cancers of follicular origin, including differentiated papillary,
follicular, or Hrthle cell carcinoma, vs. anaplastic thyroid
cancer). Following surgery for differentiated thyroid carci-
noma, the completeness of tumor excision can be assessed by
measuring serum Tg concentrations.54,67 If thyroglobulin
autoantibodies are present (see later), Tg measurements are
unreliable.
Following thyroidectomy and/or radioactive iodine
therapy, plasma Tg should be undetectable unless residual
thyroid tissue or metastatic lesions are present. To ensure
that any remnant thyroid, or metastatic tumor, tissue is stim-
ulated to produce Tg, the patients TSH must not be sup-
pressed (postoperative thyroid hormone replacement must
be discontinued), or, alternatively, exogenous recombinant
DNA-produced TSH (thyrogen-alpha; Thyrogen, Genzyme,
Cambridge, Mass) may be given.140 Exogenous TSH admin-
istration normally stimulates at least a fivefold increase in Tg
if responsive tissue is present. With a local thyroid tumor
remnant (following incomplete thyroidectomy) or a well-
differentiated thyroid tumor, TSH can stimulate a tenfold
increase in Tg concentration.188 With poorly differentiated
tumors, Tg rises less than threefold after TSH. One caveat is
that the differentiated thyroid cancer should be shown to
produce Tg before surgery is performed, lest the patient be
judged to be in remission following surgery but his or her
cancer is a rare nonTg-secreting differentiated thyroid
carcinoma.
Tg measurements have other clinical uses. For example,
the absence of detectable Tg in the serum of an infant with
congenital hypothyroidism supports the diagnosis of thyroid
dysgenesis. In cases of congenital hypothyroidism where Tg
is undetectable, but thyroid gland tissue is identified by
thyroid scan, a Tg gene mutation should be considered. In
cases of suspected factitious hyperthyroidism (hyperthyroid-
ism caused by exogenous thyroid hormone ingestion), Tg will
be suppressed; in Graves disease, Tg is measurable.
ANALYTICAL METHODS
The approach to the laboratory assessment of thyroid func-
tion has changed considerably over the past 2 decades. This
change was driven by two technologic advances: (1) a
roughly 100-fold improvement in the lower detection limits
of analytical methods to measure TSH, and (2) widespread
availability of free (unbound) T4 assays on automated plat-
forms. An unfortunate consequence of this change, however,
was the emergence of a confusing array of terms used to
describe the analytical methods that measure thyroid hor-
mones. Terms such as direct, indirect, index, one-step,
and two-step are used in nonstandard and often inconsis-
tent ways in reference to FT4 assays.120 In addition, the term
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
analog (or analogue, in the UK) is often used to describe
FT4 immunoassays, as though these methods involve a tech-
nology that is unique from other competitive and noncom-
petitive binding immunoassays that make use of a chemically
modified antigen, when the approach basically is the same.
Finally, generational numbers have been applied to TSH
immunoassays to designate the analytical sensitivity. In other
analytes (such as troponin T), generational numbers mostly
refer to improvements in specificity, and more sensitive assays
for analytes such as C-reactive protein are simply designated
as high sensitivity. Except where helpful to establish histori-
cal relationships, use of these terms will be avoided in favor
of more standard immunoassay terminology.
Measurement of Thyrotropin (TSH)
Human thyrotropin [thyroid-stimulating hormone (TSH)] is
a 28 to 30 kDa dimeric glycoprotein consisting of a 92 amino
acid alpha subunit (identical to the alpha subunit of human
chorionic gonadotropin, follicle-stimulating hormone, and
luteinizing hormone) and a 112 to 118 amino acid beta
subunit. Functionally important domains are present on both
alpha and beta subunits; neither is active alone. Residues 113
to 118 of the beta subunit undergo post-translational proteo-
lytic cleavage without affecting hormonal activity, so the
C-terminal end of the beta subunit apparently is not neces-
sary for receptor binding.
Although bioassays are available for measurement of TSH
activity, and radioimmunoassay (RIA) has been used as well,
nearly all modern TSH methods are two-site sandwich het-
erogeneous immunoassays involving an enzyme or chemilu-
minescent label. Migration to more sensitive TSH methods
about 2 decades ago prompted a change in the strategy for
laboratory investigation of thyroid dysfunction, establishing
TSH as its foundation. Sensitivity was the essential catalyst of
this change because TSH concentrations may be decreased in
nonthyroidal illness, and the lower limit of detection was
necessary to distinguish between nonthyroidal causes of sup-
pressed TSH and thyrotoxicosis, which is associated with
TSH concentrations less than 0.01 mIU/L.
The generational classification divides TSH methods by
analytical sensitivities separated by roughly one log unit.
Thus, first-, second-, and third-generation methods have
functional sensitivities of 1.0, 0.1, and 0.01 mIU/L, respec-
tively. Some methods claim a detection limit of 0.001 mIU/L
and, following this convention, are called fourth-generation
methods. These definitions are not formal but were loosely
adopted by manufacturers of TSH assays and are used more
for marketing purposes than to express analytical perfor-
mance by standard criteria.
In 1991, the Nomenclature Committee of the American
Thyroid Association recommended that the functional detec-
tion limit of serum TSH assays should be determined on
the basis of low-end interassay precision characteristics.69
The Committee recommended that precision [coefficient
of variation (CV)] at the lower reporting limit should be
10 to 15%, but no worse than 20%. Currently, functional
sensitivity is defined as the lowest concentration of TSH at
1929
168,190
which an interassay CV of 20% or less can be achieved.
The American Thyroid Association determined that only
TSH assays with third-generation (0.01 mIU/L) functional
sensitivity are sufficient for use as screening tests for hyper-
thyroidism; their recommendation is consistent with the
National Academy of Clinical Biochemistry Laboratory
Medicine Practice Guideline13 for assessment of thyroid
function.
An additional complication in defining the analytical per-
formance of TSH assays is that the hormone is subject to
structural heterogeneity because of genetic polymorphisms
and post-translational modifications affecting the carbohy-
drate content, as well as proteolytic removal of the six
C-terminal beta subunit residues. There was poor agreement
between various TSH immunoassays until an International
Reference Preparation (IRP) isolated from pituitary tissue
was made available in the 1980s. In 1999, a recombinant
human TSH IRP was created,157 but significant differences
have been noted between recombinant TSH and isolates from
pituitary tissue, primarily as the result of modifications of
polysaccharide moieties on TSH, which are highly cell spe-
cific. Pituitary-derived TSH contains polysaccharides that are
mostly sulfated, however the recombinant hormone is pri-
marily sialylated.157 These modifications produce epitopic
diversity and change the antigenic profile of the protein.
Efforts have been made to chemically modify recombinant
TSH to more closely resemble the circulating form of the
hormone. This approach eventually may result in better
agreement between various TSH immunoassays.40
Principles of TSH Immunoassays
Modern automated TSH immunoassays typically involve a
double-antibody approach [for which the enzyme-linked
immunosorbent assay (ELISA) is the prototype], with the
capture antibody directed toward the alpha subunit and a
signal antibody that recognizes an epitope on the unique beta
subunit.
Heterogeneous immunoassays differ in the method used
to separate bound and free fractions before application of the
signal antibody. The various designs of automated heteroge-
neous immunoassays are discussed in Chapter 16.
As noted previously, TSH immunoassays are calibrated
with a reference preparation of TSH (World Health Organiza-
tion Second International Reference Preparation, 2nd IRP
80/558) and are expressed as milli-international units of bio-
logical activity per liter of serum (mIU/L), a value established
by bioassay. In contrast to the inverse dose-response curve
produced in radioimmunoassay, immunometric assays gen-
erate a proportional dose-response curve, with larger signals
corresponding to higher concentrations of analyte. Because
the physiologic interval of TSH concentrations is typically
three orders of magnitude, high-dose hook effects are rarely
encountered with TSH immunoassays. Automated heteroge-
neous immunoassays for TSH offer improved sensitivity and
rapid turnaround time, along with a wider linear measure-
ment range, when compared with traditional competitive
immunoassays such as RIA.
1930 Section V
A wide variety of TSH methods are now available.
Most TSH methods use a detection antibody labeled with a
chemiluminescent (or electrochemiluminescent) tracer.119,156
Some assays use peroxidase, alkaline phosphatase, photo-
metric, and fluorescent167 labels. Other TSH methods are
based on fluorescent labels using europium chelates,78 chemi-
luminescent compounds such as acridinium esters or ruthe-
nium, or bioluminescent molecules such as recombinant
aequorin.
Chemiluminescence-based immunoassays are available in
several configurations.109 For example, one assay involves a
polystyrene tube coated with a monoclonal capture antibody
and a polyclonal detection antibody conjugated to an acri-
dinium ester; the acridinium ester emits a photon when
reacted with hydrogen peroxide in alkaline solution.189
Another approach uses an antibody-coated tube, a polyclonal
antibody conjugated to alkaline phosphatase, and an adaman-
tyl dioxetane phosphate ester as substrate; on dephosphoryla-
tion, the substrate decomposes and emits a sustained glow of
light.25 Cross-reactivity of this TSH assay with luteinizing
hormone, follicle-stimulating hormone, human chorionic
gonadotropin, or the free beta subunit of glycoprotein hor-
mones is less than 0.001%.
Specimen Collection and Storage
Serum or plasma may be used for TSH measurements. TSH
is stable for 5 days at 2 to 8 C, and for at least 1 month when
stored frozen. For newborn screening, whole blood may be
collected by heel puncture 48 to 72 hours after birth.
Comments on TSH Measurements
Secretion of TSH is circadian; peak concentrations of TSH
occur between 0200 and 0400, and the nadir occurs between
1700 and 1800. Low-amplitude oscillations occur throughout
the day. The nocturnal increase in TSH is lost in critical
illness and after surgery. TSH surges immediately after birth,
reaching a peak of 25 to 160 mIU/L within 30 minutes,
and declining back to cord blood concentrations by postpar-
tum day 3. TSH concentrations stabilize to near adult con-
centrations within the first few weeks of life. In the first
trimester of pregnancy, TSH concentrations decline as hCG
stimulates the maternal thyroid gland to produce thyroid
hormone, sometimes leading to a TSH concentration that is
just below the lower limit of the reference interval. Reference
intervals for adult TSH concentrations are the same for men
and women, and no significant racial influence on concentra-
tions of this hormone has been noted. The distribution of
TSH concentrations in healthy populations is log-Gaussian
(or log-normal); reference intervals should be determined
nonparametrically.
Measurement of Total Thyroxine (T4)
Thyroxine [3,5,3,5-tetraiodothyronine (T4)] is the principal
hormone secreted by the thyroid gland (see Figure 55-4), and
its circulating concentration is under the influence of TSH.
The hormone is synthesized from tyrosine residues on thyro-
globulin in the colloid of the thyroid gland. Circulating T4 is
Pathophysiology
highly (>99.9%) protein bound, and the fraction that is bound
to protein is biologically inactive.
Instrument-Based Methods
Electron capture gas chromatography,150 high-performance
liquid chromatography,68 and isotope dilution tandem mass
spectrometry126,183,184A have been used in measuring T4 in
human serum. The latter method has been suggested as a
reference method for assay of T4. With this technique, tritium-
labeled T4 is added as an internal standard before extraction,
derivatization, and quantitation of T4 by combined gas
chromatographymass spectrometry.
Immunoassays
Most clinical laboratories measure total T4 by using an auto-
mated competitive immunoassay. Many T4 immunoassays
use high-affinity polyclonal antibodies produced against an
albumin-T4 conjugate. These polyclonal antibodies are highly
specific and display minimal cross-reactivity with T3. Thyro-
globulin sometimes has been used as the immunogen because
it contains iodinated tyrosine residues that are the precursors
of T4 and T3. Monoclonal antibodies against T4 have also been
developed.
Immunoassays of total T4 measure both free and protein-
bound hormone. Accurate measurement of total T4 therefore
requires dissociation of the hormone from serum proteins
(thyroxine-binding globulin, albumin, and transthyretin)
that bind more than 99.9% of circulating T4. Dissociation of
T4 from albumin is not a concern because the association
constant of T4 for anti-T4 antibodies (usually 109 L/mol)
is several orders of magnitude higher than the association
constant for albumin-bound T4 (approximately 1.6 106 L/
mol). However, association constants for T4 binding to
thyroxine-binding globulin (TBG) and transthyretin are
higher: 2 1010 L/mol and 2 108 L/mol, respectively. T4
can be dissociated from transthyretin by the addition of
barbital, which competitively inhibits T4 binding to this
protein. Various blocking agents have been used to dissociate
T4 from TBG. The most common of these blocking agents is
8-anilino-1-naphthalene-sulfonic acid (ANS), although salic-
ylate, thimerosal, and phenytoin have been used to displace
T4 from TBG. In some assays, proportional bias has been
attributed to incomplete release of T4 from serum-binding
proteins.138
Considerable effort has been directed toward the develop-
ment of immunoassays that do not require the use and
measurement of radioactivity, and nonisotopic assays for T4
are widely available for use on various automated immuno-
assay systems, or adaptable to automated chemistry plat-
forms. In recent College of American Pathologists Ligand
Assay Surveys, the number of participants using RIA to
measure total T4 was insufficient to report as a peer group. A
variety of different labels have been used in the design of
nonisotopic immunoassays. Enzymes such as horseradish
peroxidase, alkaline phosphatase, and alpha-D-galactosidase
are popular, in addition to fluorescent and chemiluminescent
labels.
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing 1931 1932 Section V Pathophysiology

Heterogeneous enzyme immunoassays for T4 often involve
solid-phase separation systems. Some methods immobilize
the anti-T4 antibodies on a plastic surface (e.g., polystyrene
beads, test tubes, microwells) so that bound and free analyte
may be separated by decanting and washing. For example,
one automated assay uses test tubes that contain polystyrene
beads coated with a monoclonal anti-T4 capture antibody.11
T4 in the specimen is liberated from binding proteins by ANS
and competes with a T4alkaline phosphatase conjugate for
binding sites on antibodies attached to the polystyrene beads.
After the unbound label is rinsed away, the bound enzyme is
measured using a chemiluminescent substrate. This combina-
tion of an enzyme label with a chemiluminescent substrate
has been applied to other automated T4 procedures as well.
An antibody-coated tube enzyme immunoassay has been
devised for measurement of T4 and includes a peroxidase-T4
conjugate with a photometric substrate.
Several enzyme immunoassays attach paramagnetic par-
ticles to the anti-T4 antibodies so that free and bound T4 can
be separated magnetically. In one automated scheme, plastic
cups contain magnetic beads coated with a T4 antibody.
Endogenous T4, displaced from its binding proteins by ANS,
competes with alkaline phosphataselabeled T4 for a limited
number of binding sites on the immobilized antibody. The
magnetized beads are washed to remove unbound enzyme-
labeled T4 and then are incubated with a fluorogenic sub-
strate. As with all competitive immunoassays, the amount of
enzyme-labeled T4 captured on the magnetic beads is inversely
proportional to the T4 concentration in the specimen.
Homogeneous enzyme immunoassays have been devel-
oped for measuring serum T4 concentration. Chapter 16 dis-
cusses several approaches to homogeneous immunoassays. In
the enzyme-multiplied immunoassay technique (EMIT) for
T4, glucose-6-phosphate dehydrogenase (G-6-PDH) is cova-
lently linked to T4 as the enzyme label. When T4-specific
antibodies bind to the enzyme-labeled T4, enzyme activity is
inhibited as the result of steric obstruction of the substrate-
binding site. The cloned enzyme donor immunoassay
(CEDIA) technique for measuring T4 is based on the use of
cloned fragments of alpha-galactosidase, a tetrameric hydro-
lase enzyme that converts alpha-galactosides into galactose.
The CEDIA approach involves two fragments of the mono-
meric subunit of alpha-galactosidase, the larger of which is
called the acceptor, and the smaller of which is called the
donor. In solution, the two fragments spontaneously associate
to form a monomeric subunit, which subsequently associates
with three other monomers to form the active enzyme. In the
CEDIA method, T4 is labeled with the donor fragment of
alpha-galactosidase monomer, and antibody binding to the
labeled T4 prevents association with the acceptor fragment;
hence, enzyme activity is lost.
Fluorescent probes have been used to measure T4, and
both heterogeneous and homogeneous immunoassays based
on fluorescent labels are available. An example of the latter
is the fluorescence polarization immunoassay (FPIA). In
the FPIA, fluorescein-labeled T4 competes with unlabeled
hormone in the patient specimen for binding sites on anti-T4
antibodies. Polarized excitation light (485 nm) induces fluo-
rescence of the fluorescein tracer. Polarization of the emitted
light (525 to 550 nm) requires that the orientation of the
tracer remain fixed for the time interval between excitation
and fluorescence; this interval is typically nanoseconds. The
unbound T4-fluorescein conjugate has a rotational frequency
of femtoseconds1, so the orientation of the uncomplexed
tracer is randomized between excitation and fluorescence,
and polarization is lost. However, when fluorescein-labeled
T4 is bound to antibody, the rotational frequency is several
orders of magnitude slower, so excitation and fluorescence
occur in the same plane, and polarization is maintained.
Heterogeneous fluorescent immunoassays for T4 based on
lanthanide rare earth elements and time-resolved fluores-
cence are also available. The use of europium chelates as fluo-
rescent probes is particularly attractive because of their wide
Stokes shifts and long fluorescence decay times, which
improve the specificity of the measurement by limiting inter-
ference from scattered excitation light and endogenous fluo-
rophores. In a typical competitive immunoassay design, T4
in the specimen competes with europium-labeled T4 for a
limited number of binding sites on monoclonal T4 antibodies.
Antibody-bound and free antigen fractions are separated by
adsorption of T4 antibodies to the surface of a microwell
coated with antimouse immunoglobulin (Ig)G. After incuba-
tion and washing, the europium ions are dissociated from the
bound phase, converted into a highly fluorescent chelate, and
measured in a fluorometer with time resolution capability. A
variation on this technique involves attachment of biotin to
a monoclonal T4 antibody and streptavidin coupled to the
europium chelate.
Most contemporary automated immunoassays for mea-
suring total T4 use chemiluminescent labels. In one design,
endogenous T4 competes with T4 coupled to paramagnetic
particles for binding sites on a mouse monoclonal antibody
labeled with acridinium ester. After incubation, the labeled
T4 is captured on a magnetized surface that is subsequently
washed to remove the unbound label. The antibody-bound
labeled T4 is quantified in a luminometer after hydrogen per-
oxide is added to initiate the chemiluminescence reaction.
Electrochemiluminescence has also been applied to
total T4 measurement. In this technology, a ruthenium
tris(bipyridyl) [Ru(bpy)32+] complex is used as the antibody
label. In the presence of tripropylamine and an applied elec-
trical potential, the ruthenium complex undergoes a chemi-
luminescent reaction.
Particle-enhanced immunoassays involving turbidimetric
measurements have been developed for measuring T4 in
serum. These homogeneous immunoassays are based on
competition between T4 in the specimen and a T4-ficoll con-
jugate for binding sites on a monoclonal antibody coupled to
microparticles. The rate of formation of cross-linked com-
plexes is inhibited by competing T4 and is measured turbidi-
metrically by the change in absorbance at 600 nm.
Dry-chemistry immunoassay systems, which do not
require liquid reagents or sample pretreatment, have also
been developed for T4. One such system uses a thin-film
multilayer test module consisting of three distinct layers
mounted on a plastic support. The serum specimen is
applied to the top layer of film, which contains phenoxynaph-
thalene sulfonic acid, to displace T4 from binding proteins.
The released T4 passes through an iron oxide screen into
the detection layer, where the hormone competes with
rhodamine-labeled T4 for binding sites on immobilized T4
antibodies. Unbound fluorescent-labeled T4 diffuses into the
opaque layers above the screen, where it cannot be detected.
The fluorescent signal of the remaining antibody-bound
labeled T4 fraction is measured fluorometrically. This signal
is inversely proportional to the concentration of T4 in the
specimen.
Specimen Collection and Storage
Serum is the preferred specimen for the measurement of T4,
but plasma with EDTA or heparin as anticoagulant has also
been used. Plasma may form fibrin clots after freezing and
thawing, however, and may produce spurious results in
methods that are susceptible to changes in specimen viscosity.
Gel barrier collection devices do not have any apparent
adverse effect on T4 methods. T4 is a stable analyte with no
appreciable change in concentration for up to 7 days at room
temperature, or 30 days when frozen. As with most analytes,
repeated freezing and thawing of specimens should be
avoided because it can produce concentration gradients that
require vigorous mixing to alleviate. Mild to moderate hemo-
lysis and lipemia do not significantly affect most T4 immuno-
assays; however, grossly hemolyzed specimens should be
avoided because of dilutional effects. Intracellular T4 concen-
trations are very low because thyroxine-binding proteins are
mostly extracellular. T4 autoantibodies interfere with some
immunoassays and may produce erroneously low or high
results, depending on the method.215
Heelstick capillary specimens have been used for T4 mea-
surement, as have dried blood specimens collected on filter
paper. Dried blood specimens are stable and easily trans-
ported and are widely used in the United States to screen
neonates for congenital hypothyroidism.115 When this collec-
tion technique is used, a one eighth inch dot is punched out
from the blood-saturated filter paper and T4 is extracted into
a buffer before the assay is performed. The filter paper should
not be exposed to extreme heat or light.
Comments on Total T4 Measurements
Total T4 concentration alone provides limited clinical infor-
mation, because it reflects mostly inactive (protein-bound)
hormone. In fact, it is reasonable to question whether isolated
measurements of total T4 currently have any value in the
assessment of thyroid disease. In conjunction with free
hormone measurements, total T4 may reveal protein-binding
abnormalities that influence the ratio of bound to free
hormone, and total T4 should inversely correlate with TSH
activity in the absence of protein-binding abnormalities. In
patients with normal serum thyroxine-binding capacity
(normal albumin, TBG, and transthyretin), total T4 is propor-
tional to the active free hormone concentration. Methods for
measuring free T4 are widely available and largely supplant
the need for total T4 measurements. As discussed later, free
(unbound) T4 measurements are more useful clinically, except
in unusual circumstances.
Cord blood T4 concentrations are lower in preterm than
in full-term neonates, and they correlate positively with birth
weight in full-term infants. At birth, serum total T4 concen-
trations are higher in neonates because of the maternal
estrogen-induced increase in serum TBG; free T4 concentra-
tions are near adult concentrations. Total T4 rises abruptly in
the first few hours after birth and declines gradually until
adolescence. In males, T4 production declines as they mature
sexually, but this phenomenon is not observed in females.
Analytical performance goals have been recommended for
thyroid hormone assays.13 When total T4 is used to diagnose
thyroid disease, the suggested goals for maximum bias and
imprecision (coefficient of variation) are 2.9% and 5.7%,
respectively. When the T4 assay is used to monitor changes in
an individual over time, bias and imprecision goals are 1.3%
and 2.6%, respectively.
Measurement of Triiodothyronine (T3)
Triiodothyronine [3,5,3-triiodothyronine (T3)] is the princi-
pal active thyroid hormone, and although a small amount of
T3 is secreted directly by the thyroid gland, most of this
hormone is produced by deiodination of T4 in the peripheral
tissues, primarily in the liver. Similar to T4, T3 is highly bound
to protein, with less than 1% of the total concentration as free
active hormone. Total T3 measurements have limited clinical
use in that the T3 concentration usually reflects its progenitor,
T4. However, clinical conditions are known that affect periph-
eral conversion of T4 to T3 (discussed earlier), resulting in a
clinical hypothyroid state in the presence of normal or ele-
vated T4, with normal (or even elevated) TSH. Compared
with T4, T3 is less tightly bound to serum proteins by about
an order of magnitude, so displacement of bound T3 from
proteins is essentially complete in the presence of conven-
tional blocking agents such as ANS. T3 does not ordinarily
displace T4 from thyroid hormonebinding proteins.
Radioimmunoassays
Numerous commercial RIA kits have been introduced for
measuring total T3 concentrations in serum. These methods
are similar to the RIAs previously described for T4, except that
a 125I-T3 tracer and T3-specific antibody are used. Most ana-
lytical designs incorporate antibodies bound to a solid phase,
such as the wall of a cuvette or paramagnetic particles. As
with T4 methods, ANS can be used to release the hormone
from proteins without disturbing T3 binding to antibody.
Nonisotopic Immunoassays
Nonisotopic immunoassays for T3 are similar to total T4
methods. Many of the T3 methods have been developed for
use on automated immunoassay systems, and some are com-
patible with chemistry platforms. Many commercial methods
use enzyme labels, such as peroxidase or alkaline phospha-
tase, conjugated to T3 or anti-T3 antibodies. Enzyme activity
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
99
is determined by using a variety of sensitive photometric,
fluorescent, or chemiluminescent substrates. Immunoassays
for T3 that use fluorescent and chemiluminescent labels are
also available, and as with T4 assays, chemiluminescence-
based methods on automated platforms dominate the present
clinical laboratory market. Both heterogeneous and homoge-
neous immunoassays for T3 have been described.81
Specimen Collection and Storage
Serum is the preferred specimen, but plasma with ethylene-
diaminetetraacetic acid (EDTA) or heparin as anticoagulant
may be used. Serum specimens should be tested within 24
hours of collection, or stored at 2 to 8 C if tested beyond 24
hours. Frozen specimens are stable for at least 30 days.
Repeated freezing and thawing of the specimens should be
avoided for reasons described earlier. Turbid samples may
require centrifugation before testing.
Comments on Total T3 Measurements
Significant discrepancies have been observed when the results
of different T3 immunoassays were compared using reference
sera. Interlaboratory quality assurance (proficiency testing)
results also demonstrate higher analytical variance for T3
compared with T4 methods. Among the factors that have
been suggested to account for the poorer analytical perfor-
mance of T3 immunoassays are the lower concentration of T3
in serum, the greater antibody cross-reactivity, protein inter-
ferences, and the different assay limits of detection. Perfor-
mance goals for bias and precision have been suggested for
T3.13 When a total T3 assay is used for diagnosis, the suggested
goals for maximum bias and imprecision are 5.7% and 11.5%
(CV), respectively. For therapeutic guidance, bias and impre-
cision goals are 2.6% and 5.2%, respectively. Total T3 mea-
surements are useful in the diagnosis and monitoring of
hyperthyroid patients with suppressed TSH and normal free
T4 concentrations (T3-thyrotoxicosis); T3 measurements
have only limited value in euthyroid and hypothyroid
patients.83 Thyroid hormone replacement for hypothyroid
patients is based on TSH and FT4 measurements.
Measurement of Reverse Triiodothyronine (rT3)
Reverse T3 is produced by monodeiodination of the alpha
ring of T4 to 3,3,5-triiodothyronine and is biologically inert.
Several radioimmunoassay methods for measuring rT3 have
been developed. Antibodies to rT3 generally are obtained by
immunizing rabbits with rT3-human serum albumin (HSA)
[or bovine serum albumin (BSA)] conjugates.32 Cross-
reactivity of anti-rT3 antibodies with T4 varies between 0.01%
and 0.15%, and insignificant cross-reactivity of these antibod-
ies with monoiodinated and di-iodinated thyronines is
noted.117 Similar to T3 and T4, rT3 is highly bound to TBG,
transthyretin, and albumin in serum but is displaced by
ethanol extraction or ANS.
Commercial assays for measurement of rT3 are available,
but none has been adapted to an automated platform because
rT3 measurement has limited diagnostic value.
1933
Specimen Collection and Storage
Specimen requirements for rT3 are essentially the same as
those previously described for T3.
Comments on rT3 Measurements
Reverse T3 in serum is produced almost exclusively from
peripheral deiodination of T4 by 5-deiodinase (D1 or D3)
enzymes. Although deiodination of T4 in the peripheral
tissues produces roughly equal amounts of T3 and rT3, the
serum concentration of rT3 is typically lower than that of T3
because it is metabolized and cleared more rapidly. Serum rT3
concentrations are elevated at birth, but decrease to stable
concentrations by about the fifth day of life. Reverse T3 in
amniotic fluid decreases with increasing gestational age. The
use of rT3 to diagnose sick euthyroid syndrome has been
questioned because of its poor sensitivity. Renal failure is
associated with low rT3 concentrations.
Measurement of Free Thyroid Hormones
No practical methods are available for measuring the exact
concentration of unbound, active thyroid hormones. Bioas-
says can measure the activity of T3 and T4 but are imprecise
and are not easily adapted to automated chemistry analyzers.
Direct potentiometry measures the activity of various elec-
trolytes, independent of the inactive bound fraction, but elec-
trodes that respond to thyroid hormones have not been
developed. Methods that remove unbound T4 inevitably
disrupt the equilibrium between bound and free hormone;
therefore at best, FT4 methods only approximate the true
active concentration of the hormone.
In physiologic systems, proteins often act in much the
same way as the conjugate acid or base in a pH buffered solu-
tion, providing a large reservoir of bound, inactive ligand that
protects against precipitous changes in the total concentra-
tion of a hormone resulting from variations in glandular
output. In principle, the free, active concentration of T4 or T3
could be mathematically derived by measuring the total T4
concentration in a manner analogous to the way blood gas
analyzers use the Henderson-Hasselbalch equation to calcu-
late the bicarbonate concentration from measured pH and
PCO2 (from which the undissociated acid is estimated). The
cumulative dissociation constant for T4 from its binding pro-
teins is approximately 105, which is similar to the Ka of a
weak acid, producing a free T4 concentration that is approxi-
mately 0.03% of the total T4 concentration. However, this
approach is not satisfactory for two reasons: (1) T4 binds to
at least three different proteins, the concentrations of which
change in a variety of clinical conditions, and only one
(albumin) is routinely measured in clinical laboratories; and
(2) the affinities of T4-binding proteins for the hormone can
be altered by competing ligands, as well as by genetic poly-
morphisms that alter the protein structure and thyroid
hormonebinding affinities.
Clinical assessment of thyroid function in symptomatic
patients with normal TSH, or asymptomatic patients with
low or high TSH, has focused on measuring FT4, because
this can reveal whether TSH activity is consistent with the
1934 Section V
concentration of active hormone, regardless of abnormalities
in binding protein concentrations or affinities.
Analytical strategies for measuring FT4 have followed one
of two basic paths: (1) separate the bound and free fractions
and measure the concentration of T4 (immunometrically or
gravimetrically) in the free fraction; or (2) assay the free frac-
tion using a competitive T4 analog that does not bind to
albumin, TBG, or transthyretin. The former category includes
equilibrium dialysis and ultrafiltration (coupled with a variety
of immunochemical or mass spectrometric methods for mea-
suring T4 and T3 in the free fraction), and the latter comprises
an assortment of competitive and noncompetitive immuno-
assays available on automated systems. The term direct method
has been used to describe FT4 methods that measure the
concentration of free hormone (i.e., equilibrium dialysis
and ultrafiltration). Alternatively, the indirect method often
refers to an immunoassay that measures quantities of both
bound and free fractions, relying on calibration against speci-
mens with FT4 concentrations verified by direct methods.
Neither of these terms is consistently used, however, and both
approaches, to a greater or lesser degree, only approximate
the FT4 concentration.
The analytical validity and clinical utility of FT4 methods
have been debated for longer than 2 decades.43-45,121,122,222-224
Special reports from the Nomenclature Committee of the
American Thyroid Association of the National Academy of
Clinical Biochemistry69 the Clinical and Laboratory Stan-
dards Institute (CLSI) [formerly National Committee for
Clinical Laboratory Standards (NCCLS)] review some of
the issues and concerns regarding free thyroid hormone
measurements.
Direct (Reference) Methods
Direct measurement of FT4 (and FT3) in serum is technically
challenging because the amount of free hormone in normal
serum is exceedingly smalltypically less than 100 pmol/L
(or ng/L). The most reliable methods for measuring FT4
and FT3 in serum involve separation of free and bound
hormone fractions by equilibrium dialysis or ultrafiltration,
and subsequent measurement of the free concentration by a
sensitive analytical method such as immunoassay or mass
spectrometry. In equilibrium dialysis, the dialysis buffer
dilutes the specimen, so there is some effect on the equilib-
rium between bound and free hormones, but the effect is
minimal. Similarly, ultrafiltration changes the concentration
of T4-binding proteins in the retentate, changing the bound/
free equilibrium.
Equilibrium Dialysis
In a equilibrium dialysis method for FT4 and FT3 described
in 2008,230 200 L aliquots of serum were dialyzed against
200 L of a HEPES buffer containing physiologic concentra-
tions of sodium, chloride, potassium, magnesium, sulfate,
phosphate, calcium, and urea, with sodium azide added as a
preservative using a cellulose membrane (5 kDa molecular
weight cutoff). Specimens were dialyzed at 37 C for approxi-
mately 20 hours. After the addition of 13C-labeled internal
Pathophysiology
standards, T3 and T4 were extracted from the dialysate
onto a C5 guard column, and then were eluted into a polar
LC column [solid-phase extraction-liquid chromatography
(SPE-LC)] before analysis by tandem mass spectrometry. The
upper and lower limits of quantitation, using the criterion
of 15% total bias, were 400 and 1.0 ng/L, respectively. FT4
results using this method correlated well with equilibrium
dialysis/RIA. FT3 results by the SPE-LC-MS/MS method were
compared with equilibrium tracer dialysis (see later), and
a bias of 38 to 53% was observed. Others have reported
similar equilibrium dialysis methods using LC-MS/MS to
quantify FT4.208
Ultrafiltration
A second method for separating protein bound and free frac-
tions of T4 is ultrafiltration, which is significantly less time-
consuming than dialysis.200 In an ultrafiltration method
described by Soldin and coworkers,184 the serum specimen
was filtered through a 30 K MW cutoff filter by centrifugation
for 1 hour at 25 C and 2900 rpm in a fixed-angle rotor. T4
was measured in the ultrafiltrate by isotope dilution LC/
MS-MS. Results using this ultrafiltration method correlated
closely with equilibrium dialysis and immunoassay. In a 2009
report,77 performance of this method was assessed in four
different patient populations: (1) pediatric, (2) euthyroid
adults with thyroid disease (both before and after thyroidec-
tomy), (3) healthy nonpregnant women, and (4) pregnant
women. In all of these groups, FT4 values measured by ultra-
filtration and LC-MS/MS correlated inversely with log-
transformed TSH concentrations.
Equilibrium Tracer Dialysis
Equilibrium tracer dialysis is a variation of equilibrium dialy-
sis, except that isotopically labeled T4 or T3 tracer is added
to the specimen before dialysis is performed. When equilib-
rium is established, concentrations of the tracer on both sides
of the dialysis membrane are used to calculate the ratio of
bound to free hormone, so the FT4 (or FT3) concentration can
be calculated based on the total T4 (or T3) concentration. If
the amount of tracer added to the specimen is small in com-
parison with the endogenous hormone concentration,
minimal disruption of free/bound hormone is noted. This
method is thought to provide the best estimate of FT4 con-
centration, because radioassay methods for measuring T4
have very low limits of detection, and displacement of
hormone from binding proteins in the retentate is not
required. However, equilibrium tracer dialysis involves the
use of radioactive isotopes, a lengthy dialysis step, and instru-
mentation for measuring radioactivity. These considerations
limit its use for routine measurement of FT4 and FT3.
Comments on Free Hormone Measurements Using
Separation Techniques
Ultrafiltration provides a faster (an hour or less) means for
separating protein-bound and free hormone fractions com-
pared with equilibrium dialysis, which ordinarily requires
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-
tein concentration increases in the retentate during filtration.
Some ultrafiltration methods are performed at physiologic
temperature because of concerns about temperature-related
changes in the equilibrium concentration of free hormone,
but it is interesting to note that Soldin and associates found
that ultrafiltration at 25 C produced FT4 results that corre-
lated better with equilibrium dialysis (at 37 C) than with
ultrafiltration at 37 C.184
The reliability of ultrafiltration as a method for quantita-
tively separating free T3 and T4 fractions in serum has been
questioned. In a 2007 study, Fritz and colleagues56 demon-
strated poor recovery of isotopically labeled T4 and T3 when
reference materials prepared in protein-free serum filtrates
were passed through four commercially available ultrafiltra-
tion devices. Tritiated water was used as the internal standard
and was evenly distributed between ultrafiltrate and retentate
in all cases. However, large discrepancies between labeled
hormone concentrations in the filtrate and retentate were
observed. The authors of the study conclude that ultrafiltra-
tion is complex, poorly characterized, and incompletely
understood. Whether such anomalies also affect the results
of equilibrium dialysis, which is similarly based on the pre-
sumption that free hormone equilibrates across a semiperme-
able membrane, has not been studied.
Free Thyroid Hormone Immunoassays
Many free thyroid hormone assays are commercially available
for use on automated chemistry and immunoassay platforms.
Before the advent of automated methods for measuring free
T4, strategies emerged to approximate free hormone by mea-
suring the total T4 concentration and using an indirect
method to assess protein-binding capacity. These methods
are no longer useful because immunoassays to measure the
FT4 concentration are widely available. Even though FT4
immunoassays (both one- and two-step) are susceptible to
bias when serum proteins that bind the hormone deviate
significantly from normal concentrations, or when binding
affinity is altered by genetic polymorphisms or competing
ligands, in the vast number of patients, these assays provide
reliable estimates of FT4 concentration. Therefore, index
methods that estimate free hormone concentrations based on
protein-binding capacity are mostly obsolete. For more infor-
mation on index methods, see the previous edition of this
textbook.
General Considerations
Immunochemical methods for measuring FT4 follow two
basic strategies: (1) noncompetitive assays involve adsorption
of free hormone on immobilized anti-T4 antibodies, removal
of the supernatant, and quantitation of the unoccupied solid-
phase binding sites through addition of labeled T4; the cap-
tured amount of labeled T4 is inversely proportional to the
FT4 concentration; (2) alternatively, competitive immuno-
assays involve a labeled T4 derivative (often called analog,
the distinguishing feature of which is significantly reduced
affinity for T4-binding proteins) that is added to serum along
with anti-T4 antibodies, and after isolation of the antibody
fraction, the amount of labeled T4 analog captured by the
antibody will be inversely proportional to the endogenous
FT4 concentration. This approach is sometimes called one-
step, because only one separation of bound and free ligand
is involved, in contrast to two-step methods, which remove
the soluble protein fraction before the addition of the labeled
ligand. Another approach is to immobilize T4 analogs on a
solid support and measure the amount of labeled antibody
adsorbed on the solid support; in this design, captured label
will be inversely proportional to the concentration of FT4,
which competes for binding sites on the labeled antibody.
Analog methods measure the distribution of labeled T4
between endogenous binding proteins (TBP, transthyretin,
and albumin) and an anti-T4 antibody. Therefore, these
methods produce only an estimate of the FT4 concentration,
because the introduction of an exogenous binding protein
the T4 antibodydisrupts the equilibrium between free and
bound hormones in the specimen. If the assay is calibrated
with calibrators that have similar T4 proteinbinding capacity
to the specimen being assayed, and if the calibrators have
been validated using a reference method (such as equilibrium
dialysis), then the results provide a good estimate of FT4
concentration. However, biases will be introduced when the
concentrations, or affinities, of T4-binding proteins deviate
significantly from expected values, because calibration of
these methods assumes a normal serum T4-binding capacity.
The serum protein-binding capacity for thyroid hormones
can be affected by physiological variations in protein synthe-
sis (as in pregnancy and infancy), proteinopathies resulting
from liver dysfunction (such as hypoalbuminemia), nonthy-
roidal disease, genetic factors that produce proteins with
diminished or enhanced T4 binding, competing ligands, and
the presence of T4 autoantibodies. With the exceptions of
pregnancy and infancymost notably, prematuritymost of
these conditions are relatively rare.
Another source of bias in FT4 immunoassays that involve
a T4 analog is the binding of analog to serum proteins, which
has been demonstrated to occur.137 Because analog methods
are based on the theoretical principle of competition between
free analog and free endogenous T4 for binding sites on the
capture antibody, significant binding of the hormone analog
to serum proteins invalidates the theoretical model. The
extent to which these limitations affect the FT4 estimates
obtained by one-step analog methods has been debated (see
earlier).
Two-Step FT4 Immunoassays
The key feature of the two-step method is that the labeled
hormone is added only after serum-binding proteins have
been removed, ensuring that no competition exists between
antibody and serum proteins for binding of tracer. This does
not ensure that the assay is not susceptible to changes in
serum protein-binding capacity, because the initial step
involves equilibration of labeled FT4 between the capture
antibody and serum hormone-binding proteins. However,
results using this approach correlate well with reference
(equilibrium or ultrafiltration) methods across a wide variety
of clinical disorders.
The first commercially available two-step immunoassay
for FT4 was introduced in 1979. Subsequently, a number of
manual and automated procedures were developed for FT4
(and FT3). The earliest methods used radioactive labels and
antibody-coated tubes or microbeads, but these have been
largely replaced by automated methods involving nonisotopic
labels and a variety of solid-phase formats. Automated one-
step homogeneous FT4 immunoassays have largely sup-
planted the use of these methods.
One-Step FT4 Immunoassays
One-step FT4 immunoassay is a slightly confusing misno-
mer, because homogeneous immunoassays are typically one-
step, in that no separation is required. However, all one-step
approaches to immunochemical measurement of FT4 and FT3
are competitive, heterogeneous immunoassays, requiring
separation of bound and free ligand prior to measurement of
signal. These FT4 methods are, for the most part, variations
on the prototypical ELISA described in Chapter 16. Two
common approaches consist of one involving labeled ligand,
and the other using a labeled antibody.
In the labeled ligand scheme, anti-T4 antibody may be
immobilized to a solid support or bound to a paramagnetic
particle, which can be used to capture the antibody-ligand
complex. Free T4 in the specimen and the labeled T4 analog
compete for binding sites on the anti-T4 antibodies, and the
amount of label adsorbed by the antibody is inversely propor-
tional to the endogenous T4 concentration. Of course, capture
of endogenous T4 will disrupt the equilibrium between
protein-bound and free T4, so the assay really measures the
competition between thyroid hormonebinding proteins and
the anti-T4 antibody. Typical assays of this type remove 1 to
2% of the protein-bound T4 fractiona quantity that exceeds
the true FT4 concentration by at least two orders of magni-
tude. Theoretical arguments have been made that removal of
such a small amount of the total T4 causes minimal disruption
of the bound/free ratio; therefore a properly calibrated
method should produce a close estimate of the actual FT4
concentration.33,121,122
Another analytical strategy for measuring FT4 is to immo-
bilize a modified T4 analog to a solid support (or paramag-
netic particle), and allow a labeled anti-T4 antibody to
equilibrate between endogenous FT4 and the immobilized
analog. Evidence suggests that immobilized T4 analog has less
affinity for T4-binding proteins than the labeled free analog
described earlier,231 which makes the labeled antibody
methods less susceptible to bias resulting from binding of the
T4 analog to endogenous proteins. In the labeled antibody
method, after the unbound fraction is removed, the signal is
measured in the solid (or immobilized) phase and will be
inversely proportional to the FT4 concentration. As with
labeled ligand methods, substantially more endogenous T4 is
bound to antibody than is contained in the original free frac-
tion as the result of re-equilibration after free T4 binds to
antibody, but the fraction removed from binding proteins is
small (1 to 2%) compared with the total T4 concentration, so
the equilibrium bound/free ratio is not significantly affected.
These methods are calibrated with reference materials con-
taining normal quantities of T4-binding proteins, and in
which FT4 (or FT3) has been measured by a reference method
such as equilibrium dialysis or ultrafiltration.
Many commercially available FT4 and FT3 assays follow
one or the other of the labeled ligand or labeled antibody
schemes, differing only in (1) the label, (2) the analog, and
(3) the chemical method for immobilizing antibody or analog.
Radioactive isotopes, enzymes, fluorophores, and chemilu-
minescent and electrochemiluminescent labels have been
used in FT4 immunoassays, but most modern methods use
one of the latter two. The structure of the analog, which is a
modified form of T4 designed to have limited affinity for
thyroxine-binding protein, albumin, and transthyretin, is
often proprietary, although T4 analogs involving protein con-
jugates have been described in the literature.231 In these
experiments, T4 was conjugated to rabbit gamma globulin,
thyroglobulin, transferrin-1 (and -2), and ferritin. The anti-
body or analog are immobilized by biotin-avidin (or strepta-
vidin) or paramagnetic particles.
Selection of Tests for Measuring Free Thyroid Hormones
Immunoassays that estimate FT4 and FT3 using the approaches
just described generally produce reliable results in healthy
subjects, hyperthyroid and hypothyroid patients, and patients
with mild protein-binding abnormalities. Hence, for most
clinical situations, selection of a specific FT4 or FT3 method
can be based on factors such as (1) technical convenience,
(2) turnaround time, (3) commercial availability, and (4) cost.
In certain clinical conditions, however, free hormone esti-
mates may produce results with significant bias compared
with reference methods. Table 54-4 summarizes the types of
bias that have been observed between reference and estimate
methods for FT4 in patients with thyroid hormonebinding
protein abnormalities. One study172 examined the bias
between nine commercially available FT4 immunoassays and
equilibrium dialysis in patient specimens with low, normal,
and high concentrations of T4-binding proteins. Relative to
specimens with normal concentrations of binding proteins,
the low group had a mean TBG concentration 21% lower, and
the high group had a mean TBG concentration 167% higher.
Although little bias was observed for high-concentration
TBG specimens, significant negative bias was observed in
low-TBG concentration specimens. A 2009 study98 compared
FT4 measured by two immunoassays in nonpregnant women
and pregnant women subdivided by trimester. Although
results were not compared with FT4 measured by a reference
method, a large number (5 to 67%) of pregnant subjects had
FT4 concentrations below the assay manufacturers lower ref-
erence limit, although all specimens had normal total T4
concentrations.
In addition to pregnancy, other clinical conditions may
produce unreliable FT4 results. In patients with familial
dysalbuminemic hyperthyroxinemia (estimated incidence
among Caucasians: 1 : 10,000), a normally minor albumin
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
variant with a high affinity for T4 is overexpressed, resulting
in a larger fraction of albumin-bound T4 and elevated total
T4. The affinity of the albumin variant for T3 is not signifi-
cantly different from normal albumin. Patients with familial
dysalbuminemic hyperthyroxinemia are clinically euthyroid,
and free hormone concentrations are normal as measured by
reference methods and by most two-step immunoassays.
However, one-step immunoassays are likely to produce erro-
neously high FT4 estimates as the result of binding of the T4
analog to the variant albumin. Patients with circulating T4
autoantibodies, or with increased transthyretin, will produce
similarly elevated FT4 results by these immunoassays, because
both conditions result in enhanced T4-binding capacity in
the serum.
FT4 and FT3 methods may not be reliable in patients
with congenital TBG excess or deficiency, because the
immunoassay methods are calibrated with reference mate-
rials containing normal amounts of TBG, and most of
the circulating hormone is bound to this protein. Patients
with these conditions are clinically euthyroid, and their FT4
and FT3 are usually within their reference interval when
measured by reference methods (and sometimes by two-step
immunoassays).
Estimates of FT4 and FT3 may be affected by medications
that displace hormones from binding sites on serum proteins.
The free hormone concentration will rise in the presence of
such drugs, and in patients with normal thyroid function,
TSH will be suppressed and production of T4 will decrease,
resulting in lower total T4 with (after delay) normalized FT4.
With methods that use undiluted serum (ultrafiltration and
equilibrium dialysis), measurement of FT4 and FT3 in the
presence of competing drugs should be reliable. However,
methods that dilute the specimen diminish the effects
of inhibitors, and free hormone concentration may be
underestimated.
Measurement of Thyroxine-Binding
Globulin (TBG) and Other Thyroid
HormoneBinding Proteins
Thyroxine-binding globulin (TBG) is a 54 kDa protein that
binds T4 and T3 with high affinity (Ka 1010). Although TBG
has the lowest concentration of any of the three proteins that
bind T4, and its hormone binding sites are normally only 25%
saturated, about 70% of the total T4 and 80% of the total T3
is bound to TBG. Therefore, TBG concentration is the
primary regulator of total and free T4 and T3 concentrations.
Estrogen-induced TBG excess and congenital TBG deficiency
are the most significant TBG abnormalities that affect the
interpretation of thyroid function test results (Box 55-4).
Methods for Measuring TBG
Historical methods measured TBG indirectly by competition
between unlabeled and radiolabeled T4 for binding sites on
TBG, while blocking T4-binding sites on transthyretin and
albumin with a barbital buffer. After removal of the unbound
hormone, radioactivity retained in the protein fraction was
proportional to the TBG concentration. The presence of
1937
BOX 55-4 Alterations in the Concentration or
Afnity of Thyroid HormoneBinding Proteins
Increases in
A. Thyroxine-binding globulin (TBG) concentration (or
affinity)
1. Genetic (inherited) causes
2. Nonthyroidal illness (HIV infection, infectious and
chronic active hepatitis, estrogen-producing tumors,
acute intermittent porphyria)
3. Normal physiology (pregnancy, newborn)
4. Drug use (oral contraceptives, estrogens, tamoxifen,
methadone)
B. Prealbumin concentration
C. Albumin binding (familial dysalbuminemic
hyperthyroxinemia)
D. T4 binding by antibodies (autoimmune thyroid disease,
hepatocellular carcinoma)
Decreases in
A. TBG concentration
1. Genetic (inherited) determination
2. Nonthyroidal illness (major illness or surgical stress,
nephrotic syndrome)
3. Drug use (androgens, anabolic steroids, large doses of
glucocorticoids)
B. TBG-binding capacity (drugs bound to TBG such as
salicylates and phenytoin)
C. Prealbumin concentration
various drugs that inhibit T4 binding to TBG, or abnormal
transthyretin and albumin concentration or affinity, can
interfere with these assays and produce artificially low TBG
measurements.
Modern TBG methods measure the protein concentration
directly using a variety of immunochemical approaches. One
competitive, heterogeneous method measures the competi-
tion between endogenous TBG and labeled TBG for binding
to an immobilized anti-TBG antibody. After incubation,
bound and free fractions are separated by a variety of stan-
dard methods, and the concentration of label in the bound
fraction is inversely proportional to endogenous TBG
concentration. A competitive chemiluminescence enzyme
immunoassay for TBG uses peroxidase-labeled TBG and
anti-TBG antibody that is captured by a solid-phase second
antibody; bound conjugate is measured by chemilumines-
cence after the addition of luminol and hydrogen peroxide.
Another immunoassay for TBG is based on enhanced
microparticle turbidimetry, in which the presence of endo-
genous antigen inhibits the cross-linking of antigen-
microparticle complexes by anti-TBG antibody, reducing the
turbidity of the reaction mixture.
Specimen Collection and Storage
Serum is the preferred specimen; plasma with EDTA or
heparin as anticoagulant may also be used. Serum specimens
are best stored at 2 to 8 C if they will not be tested within 24
hours. If longer periods of storage are necessary, freezing the
1938 Section V
specimens is recommended. Frozen specimens are stable for
at least 30 days. Repeated freezing and thawing of the speci-
mens should be avoided. Turbid samples should be centri-
fuged before testing.
Thyroglobulin Measurement
Both competitive and noncompetitive immunoassays have
been applied to Tg measurement.190,228 Present-day competi-
tive immunoassays typically incubate the serum with anti-Tg
antibody; this is followed by the addition of 125I-labeled Tg.
The Tganti-Tg complex is precipitated by a second antibody,
separating antibody-bound from free 125I-labeled Tg. Longer
incubation generally enhances the sensitivity of the assay.
Noncompetitive (sandwich) immunoassays for Tg have
been developed using a variety of labels (radioactive isotope,
enzyme, chemiluminescent, and electrochemiluminescent).
The capture antibody and the labeled (or signal) antibody
recognize different epitopes on Tg that do not sterically inter-
fere with one another. The capture antibody can be attached
to a polystyrene bead or a paramagnetic bead to separate
bound and free fractions of antigen. Another variation is to
attach an antibody to biotin and separate the biotin-antibody-
Tg complex using avidin bound to a solid phase.
For most Tg double-antibody assays, the lower limit of
detection is near 0.5 to 1 ng/mL, with a coefficient of varia-
tion of 20%. Day-to-day coefficients of variation are usually
less than 6% near the center of the reportable interval but can
rise to 15% at very low or very high Tg concentrations.
A Tg reference preparation developed by the European
Community Bureau of Reference is used in many present-day
immunometric assays. With this reference preparation, inter-
assay variability is 30%, which exceeds intraindividual bio-
logical variability by a factor of three. Because of such
variability between different Tg assays, Tg monitoring
requires that the same assay be used longitudinally. Alterna-
tively, if a new Tg assay is introduced, the baseline Tg con-
centration for the patient should be re-established with the
new assay.
Technical challenges in Tg measurement include poor
interassay agreement, hook effects, interassay calibration
variability, and the need to reduce the lower limits of detec-
tion of Tg immunoassays for improved detection of residual
thyroid cancer, metastasis, or recurrence.75 A new generation
of Tg assays with enhanced lower limits of detection may
eliminate the need to withdraw thyroid hormone replace-
ment or administer rDNA TSH before Tg testing is
performed.62
Thyroglobulin Assay Interferences
Interference from human antimouse antibodies (HAMA),
other heterophile antibodies, or rheumatoid factor can affect
almost any immunoassay. Thyroglobulin autoantibodies
(TGA) in the patients blood also interfere with accurate mea-
surement of Tg.187 Unfortunately, anti-Tg autoantibodies are
much more common in thyroid cancer patients than in the
general population. TGA are reported in 15 to 35% of thyroid
cancer patients.
Pathophysiology
It is standard practice to search for TGA whenever Tg
is measured. The TGA assay should be sensitive to low
concentrations of TGA. TGA tend to decrease Tg measure-
ments in noncompetitive assays. Although some laboratories
might use polyethylene glycol to precipitate TGA-bound Tg
to measure the nonbound Tg fraction, practically speaking,
if TGA are detected, the Tg determination is not reliable.
Therefore, Tg measurements should not be used as tumor
markers in the clinical management of patients with demon-
strated TGA.
The interference of TGA with various two-site sandwich
immunoassays is unpredictable. TGA increase Tg con-
centrations in some assays. In other assays, TGA may
result in negative bias. Recovery studies generally are not
useful for determining whether TGA influences the Tg
concentration.107,186
When TGA are detected, their concentration may be
useful as a secondary marker of the mass of thyroid tissue
present in the patient. If TGA concentrations reflect the
degree of antigenic stimulation of B cells, higher TGA con-
centrations should correlate with greater amounts of thyroid
tissue in the patient. Therefore, in differentiated thyroid
cancers where TGA are positive, higher TGA concentrations
suggest more residual thyroid tissue and/or local or distal
spread of the tumor. Because of the uncertainty that TGA
positivity introduces into the interpretation of any Tg assay,
however, clinicians should rely principally on thyroid scans
to detect residual thyroid tissue in cases of differentiated
thyroid cancer when TGA are present.
Specimen Collection and Storage
The preferred specimen for Tg measurement is serum, but
EDTA or heparinized plasma may also be used. If not tested
within 24 hours, serum specimens are best stored at 2 to
8 C. If testing is delayed beyond a few days, the specimen
should be frozen until it is analyzed. Frozen specimens are
stable for at least 30 days. Repeated freeze-thaw cycles should
be avoided. Turbid samples should be centrifuged before
testing.
Reference Interval
The reference interval for Tg in euthyroid individuals varies
from a lower limit of 0.5 to 2 ng/mL to an upper limit of 20
to 42 ng/mL, depending on the method. One major reference
laboratory provides the following reference intervals: 0 to 11
months: 0.6 to 5.5 ng/mL; 1 to 11 years: 0.6 to 40.0 ng/mL;
and 12 years and older: 1.3 to 31.8 ng/mL. For patients
lacking a thyroid gland who are not receiving T4 replacement
therapy, Tg should not be detectable regardless of the patients
TSH concentration.
Novel Markers of Thyroid Tissue
in the Management of Differentiated
Thyroid Carcinoma
Another approach to the detection of residual thyroid cancer
is the measurement of Tg mRNA in the circulation.164 This
may be helpful when Tg cannot be measured because of TGA.
 Chapter 55 The Thyroid: Pathophysiology and Thyroid Function Testing
This procedure involves reverse-transcriptase polymerase
chain reaction amplification of mRNA in serum.159 This
nucleic acid approach may be valuable as an early marker of
relapse.160 However, the value of Tg mRNA detection is con-
troversial because of problems with both sensitivity and spec-
ificity.46,57 Thyroperoxidase and TSH receptor mRNAs are
alternative markers that are under investigation.162 In research
studies, the detection of minimal residual disease in thyroid
cancer has also included polymerase chain reaction detection
of cytokeratin 20.219
Thyroid Autoantibodies
Multiple autoantigens are targeted in AITD, including thyro-
peroxidase, thyroglobulin, the TSH receptor, a thyroid col-
loidal antigen, the NIS, and megalin.105,177 Autoantibodies that
appear to promote thyroid growth have also been described.206
These antibodies stimulate tritiated thymidine uptake, a
measure of cellular proliferation, without increasing cAMP
generationa measure of TSH-like stimulation. Autoanti-
bodies against T4 and T3 have also been described; and these
may interfere with measurement of these hormones but do
not appear to cause clinical disease.151,173
Thyroid microsomal autoantibodies (TMAs), thyroperoxi-
dase autoantibodies (TPOAs), and thyroglobulin autoanti-
bodies (TGAs) can be detected by a variety of methods,
including (1) indirect immunofluorescence, (2) the agar gel
diffusion precipitin technique, (3) agglutination (hemagglu-
tination or latex particle agglutination), (4) radioimmuno-
assay, (5) complement fixation, (6) ELISA techniques, and
(7) chemiluminescence-based immunometric assays.27,221
Modern assays have reduced the lower limits of detection of
these autoantibodies. With the older agglutination tech-
niques, red blood cells denatured with tannic acid treatment
were coated with microsomal antigen isolated from human
hyperplastic thyroid glands. TMA-positive sera would agglu-
tinate the tanned RBCs. Serial dilutions were performed
until agglutination was not detected. The last dilution before
the disappearance of agglutination represented the titer.
Newer immunoassays are more sensitive and can detect lower
concentrations of these autoantibodies. For example, some
assays attach TPOs to a solid support, and, with the addition
of patient serum, TPOA bind to the immobilized TPOs. A
labeled antihuman immunoglobulin antibody is added to
detect the TPOAs. Similar assays for TGAs are available.
Because of the interference of TGAs with Tg measurements,
TGAs assays with excellent low-end sensitivity are very
important.
It was initially believed that TGAs developed because thy-
roglobulin was released into the circulation following thyroid
follicular cell damage, thus exposing the immune system to a
previously sequestered antigen. However, as discussed previ-
ously, sensitive radioimmunoassays demonstrated that Tg
was present in normal serum; thus Tg does not appear to be
a sequestered antigen.
Two assay formats are commonly used for measuring
thyroid receptor autoantibodies (TRAs): measurement
of (1) thyroid-stimulating immunoglobulins (TSIs), and
1939
(2) thyrotropin-binding inhibitory immunoglobulins (TBIIs).
TRAs are detected using bioassays for TSIs and radioreceptor
assays for TBII. A report published in 2009 describes an auto-
mated assay for TBIIs.59 In most patients with Graves disease,
TSIs can be detected.194
TSIs are measured by adding patient serum to thyroid
follicular cells and assessing the release of T4 or cAMP (the
secondary messenger generated by stimulation of the TSH
receptor), or by observing colloid mobilization.199 A variety
of sources of thyroid follicular cells have been used, such as
thyroid slices or cultured human thyroid cell monolayers
(FRTL-5).
TBIIs are measured in a radioreceptor assay format.70
Patient serum is added to a solution containing TSH recep-
tors. Competition then occurs between 125I-labeled TSH and
TRAs in the patient serum for binding to TSH receptors. As
the concentration of TRAs rise in a patients serum, less
125
I-labeled TSH will bind to TSH receptors present in the
assay. The TBIIs assay does not distinguish agonistic from
antagonistic TRAs. Therefore, TBIIs typically are positive in
both Graves disease and atrophic thyroiditis, and negative in
Hashimoto thyroiditis. However, TSIs are negative in atrophic
thyroiditis. TSIs and TBIIs should be measured in pregnant
women with Graves disease (past or present) to assess the
risk of fetal or neonatal thyrotoxicosis from maternal IgG that
has crossed the placenta: if TSIs are present in high concen-
tration, the risk for the development of fetal or neonatal thy-
rotoxicosis is increased. TSIs are detected in 95% of patients
with untreated Graves disease. Several studies have shown
that higher TSIs concentrations predict relapse and lower
rates of remission. In atrophic thyroiditis in pregnant women,
TBIIs can cross the placenta, and this may result in transient
congenital hypothyroidism or transient hyperthyrotropin-
emia.108 On the other hand, TPOAs/TMAs and TGAs are not
associated with congenital hypothyroidism, indicating that
these autoantibodies are not pathogenic.42
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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-
mones of the hypothalamic-pituitary-gonadal axis,
R as well as the adrenal glands (see Chapter 54). These
hormones are crucial for reproductive function and include
gonadotropin-releasing hormone (GnRH), luteinizing
hormone (LH), follicle-stimulating hormone (FSH), and a
multitude of sex steroids. The sex steroids are synthesized by
the ovaries, testes, and adrenal glands and are responsible for
the manifestation of primary and secondary sex characteris-
tics. This chapter is divided into four sections: Male Repro-
ductive Biology, Female Reproductive Biology, Infertility, and
Methods.
MALE REPRODUCTIVE BIOLOGY
The mature testes synthesize both sperm and androgens. The
testes contain a structured network of tightly packed seminif-
erous tubules. The lumina of the seminiferous tubules are
lined by maturing germ cells and Sertoli cells. Sertoli cells
play a crucial role in sperm maturation and secrete inhibin, a
glycoprotein that inhibits the pituitary secretion of FSH. Sur-
rounding the seminiferous tubules are the interstitial Leydig
cells, the primary site of androgen production. The principal
androgen in man is testosterone, which serves a central role
in reproductive physiology. Testosterone is required for
sexual differentiation, spermatogenesis, and promotion and
maintenance of sexual maturity at puberty. At the cellular
level, these effects are mediated by binding of testosterone or
its more potent metabolite dihydrotestosterone (DHT) to the
androgen receptor or via aromatization to estradiol and sub-
sequent binding to the estrogen receptor. Testicular function
is under the control of the hypothalamic-pituitary-gonadal
axis.
*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.
1945
C HA P T E R
56
Reproductive
Hypothalamic-Pituitary-Gonadal Axis
Gonadotropin-releasing hormone (GnRH) is a decapeptide
synthesized in the hypothalamus and transported to the ante-
rior pituitary gland, where it stimulates the release of both
FSH and LH (see also Chapter 53).
In adult men, GnRH and thus LH and FSH are secreted in
pulsatile patterns. A circadian rhythm is present, with higher
concentrations found in the early morning hours and lower
concentrations in the late evening.302 LH acts on Leydig cells
to stimulate the conversion of cholesterol to pregnenolone.
FSH acts on Sertoli cells and spermatocytes and is central to
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-
back control of LH and FSH secretion, respectively. LH secre-
tion is inhibited by testosterone and by its metabolites,
estradiol and DHT. FSH may be elevated in disorders in
which Sertoli cell numbers (and hence inhibin concentra-
tions) are reduced. Likewise, a reduction in the number of
Leydig cells (and hence testosterone secretion) leads to
increased LH concentrations.
Androgens
Androgens are a group of C-19 steroids (Figure 56-1) res-
ponsible for masculinization of the genital tract and develop-
ment and maintenance of male secondary sex characteristics.
Testosterone is the principle androgen secreted in men.
Biosynthesis of Testosterone
Testosterone is synthesized primarily by the Leydig cells of
the testes (95%) and, to a lesser extent (5%) via peripheral
conversion from the precursors dehydroepiandrosterone
(DHEA) and androstenedione (which are synthesized in the