Thyroid hormone structure and function

1. THYROID HORMONE
DR.K.RAVI BABU B.Sc.,MBBS., MD BIOCHEMISTRY

2. INTRODUCTION
300 million people in the world are suffering
from thyroid disorders and among them about
42 million people reside in India.
 Thyroid disorders are more common in
women than in men, may be associated with
estrogen and progesterone.
 overt thyroid dysfunction is associated with
significant morbidity and mortality.

3. INTRODUCTION
 Hypothyroidism itself contributes to morbidity
from osteoporosis, hyperlipidemia,
hypercholesterolemia, cardiovascular and
neuropsychiatry disease in the population.
 the prevalence and pattern of thyroid disorders
depends on gender, age, ethnic and geographical
factors and especially on iodine intake.
 Still thyroid disorders especially hypothyroidism,
both subclinical and overt, contributes
significantly to burden of thyroid disorders in
India.

16. EXTRATHYROIDAL FACTORS THAT AFFECT
THYROID FUNCTION continue……
 Concentrations of thyrotropin, thyroid hormones, thyroid
antibodies, and thyroid-binding proteins are to varying
degrees determined genetically.
 Epidemiologic factors such as
 age,
 gender,ethnicity,
 body mass index,
 smoking, pregnancy,
 nutritional iodine,
 season,
 nonthyroidal disease,
 radiation,
 and medication.

17. FACTORS THAT AFFECT
THYROID FUNCTION continue……
In children, concentrations of plasma tT3, fT3,
tT4, free T4 (fT4), and TSH decrease with age,
but tT4 seem to increase again in girls during
puberty.
TBG concentration increases until 5 years and
then decreases with age and is also gender
dependent
Seasonal variations in TSH occur, leading to an
increased false-positive rate of congenital
hypothyroidism in screening programs in the
winter (0.9%) compared with the summer
(0.6%).

18. FACTORS THAT AFFECT
THYROID FUNCTION continue……
In adults, TSH increase with age and are
higher in whites than in blacks or Hispanics.
Antithyroid antibody concentrations increase
with age, are higher in women than men, and
higher in whites than in blacks.

19. FACTORS THAT AFFECT
THYROID FUNCTION
Radiation exposure, nuclear or medical, or
environmental contaminants, increase the risk
of autoimmune thyroid disease.
T4 secretion and degradation decrease with
age,but plasma fT4 concentrations remain
largely unchanged with increasing age.
In older adults (age older than ~60 years),
plasma TSH concentrations increase; the
upper limit especially increases markedly

20. FACTORS THAT AFFECT
THYROID FUNCTION.
acute or chronic nonthyroidal illness leading to
decreased plasma fT3, low or normal TSH, high rT3,
and relatively normal fT4 concentrations.
 As a result, the diagnoses of both subclinical and
overt hypothyroidism and hyperthyroidism may
pose challenges.

21. Higher TSH concentrations with age are associated
with increased longevity.
147. Atzmon G, Barzilai N, Hollowell JG, et al. Extreme
longevity is associated with increased serum
thyrotropin. J Clin Endocrinol Metab 2009;94:1251–4.
148. Atzmon G, Barzilai N, Surks MI, et al. Genetic
predisposition to elevated serum thyrotropin is
associated with exceptionallongevity. J Clin Endocrinol
Metab 2009;94:4768–75.

22. Age, Gender, and Circadian Rhythm.
The TSH surge during the neonatal period .
 TSH can reach in excess of 80 mIU/L but
typically drop to below 20 U/L in the first day
of life .
falling into the adult reference limits during
the first month.

23. Age, Gender, and Circadian Rhythm.
TSH has a circadian rhythm with peak levels at
around midnight and nadir in the morning
through noon.
 Sleep deprivation causes a rise in TSH.
The circadian rhythm of TSH is lost during
illness.

24. Measurement of TSH.
first-generation assays were sensitive enough only
to discriminate normal from hypothyroid subjects;
second-generation assays could detect TSH below
the reference interval but not well enough to
reliably discriminate primary hyperthyroidism from
other causes of low TSH.
“third generation,” that is, they should have a
coefficient of variation (CV) of less than 20%
(functional sensitivity) at a concentration of 0.01
mIU/L

25. the pituitary can produce immunoreactive but
biologically inactive TSH.
 This is often quoted as the reason that TSH
alone cannot be used as a front-line screening
strategy for thyroid disease.
Both serum and plasma are acceptable
substrates for TSH immunoassay.
 TSH is stable in serum for at least five days at
4°C, and at least 29 years at −25°C.

26. Immuno assay

27. “Hook effect,”
the assay response drops off at high analyte
concentrations (eg, tumor markers).
 One way of minimizing or eliminating the hook
effect is by ensuring that the concentrations of
capture and labeled antibody are sufficiently high
to cover analyte concentrations over the entire
analytical range of the assay.
with modern automated immunoassays, hook-
effects may occur silently and be undetectable.

28. Free Thyroid Hormones (fT4)
the measurement of non–protein-bound T4 in
circulation (fT4) is a more accurate reflection of
thyroid status than the total amount of T4 (free plus
bound T4 or tT4).
tT4 concentrations are clearly influenced by
changes in thyroid-binding proteins as well as HPT
axis regulation.

29. Free Thyroid Hormones (fT4)
Direct methods (equilibrium dialysis or
ultrafiltration )
 Indirect methods (immunoassay methods ) that
estimate fT4 in the presence of T4 binding proteins.
Immunoassays are further divided into one and
two-step methods depending on whether a wash
step is included to remove serum constituents
before the addition of the T4 immunoassay tracer.
Modern immunoassay methods are also “analog”
because chemically modified T4 probes are used
rather than historic radiolabeled hormones.

30.  Direct methods :fT4 is separated from protein-bound T4 using
a physical method such as dialysis or ultrafiltration.
 Competitive immunoassay methods were originally used to
measure T4 in the protein depleted fraction, but these
methods are now being replaced with mass spectrometric
methods.
 NEFA are the best described displacing agent.

31. Indirect immunoassay methods
fT4:tT4 equilibrium is maintained during
immunoassay to an extent sufficient to return a
clinically relevant estimation of fT4.
 One-step methods incubate the assay antibody and tracer in the presence of all
serum constituents.
 Two-step or “back-titration” methods allow T4 to equilibrate with the assay
antibody in the presence of all serum components but wash away un captured
components before back titrating with tracer.
 Both methods sequester a significant amount of T4 from the serum pool during
assay, the fT4:tT4 equilibrium is maintained sufficiently to provide a reliable
estimate of fT4 under most conditions.
 familial dysalbuminemic hyperthyroxinemia and TBG or TTE deficiency or excess.

32. Total Thyroxine and Triiodothyronine
 Competitive immunoassay use a displacing agent such as 8-
anilino-1-napthalene-sulfonic acid to release thyroid hormone
from high-affinity serum binding sites;

33. Thyroglobulin
 Active thyroid tissue.
 Tg concentrations are determined by thyroid mass; TSH stimulation;
and thyroid manipulation, including surgery, fine-needle aspiration
(FNA), or thyroid injury, DTC, congenital hypothyroidism.
 Anti-Tg or antireagent antibodies that interfere with the
immunoassay.
 Competitive immunoassay:(~5 ng/mL).
 Immunometric assay:(≤0.1 ng/mL)
 Peptide mass spectrometric assay:0.4 ng/mL
 Reference interval :3-42 ng/mL (euthyroid adult)

34. Antithyroid Peroxidase
Anti-TPO assay is a very sensitive marker for
Hashimoto thyroiditis,being affected by age, gender,
ethnicity, and iodine status.

35. Anti–Thyroid-Stimulating Hormone
Receptor Antibodies
The TSHR is a target for both blocking (anti-TSHRB) and
stimulating autoantibodies (anti-TSHRS).
Graves disease, which is the most common cause of
hyperthyroidism in adults, is caused by anti-TSHRS.
use a cloned TSHR with either labeled TSH or a labeled
monoclonal anti-TSHR as the competitive ligand.
 TRAb -pregnant women with a history of Graves
disease who are at risk of passing anti-TSHRB or anti-
TSHRS transplacentally.
a genetically modified TSHR -specific for anti-TSHRS.

36. Assay Interference
Autoantibodies particularly interfere with T4, fT4,
T3, and fT3.
Heterophilic antibodies interfere particularly with
TSH in immunometric assays and fT4 in competitive
assays.
• assay interference -0.5%.

37. Thyroid-Stimulating Hormone Assay
Interference.
Heterophile or antianimal antibodies can bind to
reagent antibodies, causing blocking (negative)
or crosslinking (positive) interference.
“macro-TSH” complexes, are likely to be
immunoreactive but biologically inactive.

38. Free Thyroxine Assay Interference.
 heterophile or anti-animal interference that blocks
T4 binding to the assay reagents (given a false-
positive result owing to the competitive assay
design)
 or by antianalyte antibodies (endogenous anti- T4
antibodies) sequestering the T4 tracer, again giving
a false-positive result.
inherited or acquired changes in binding proteins,
such as familial (FDH), or the presence of
 The effect on (NEFA) generation of heparin and
drugs such as phenytoin, which can displace T4
from albumin.

39. Fig. An algorithm to guide interpretation of thyroid function tests. Different patterns of thyroid
function tests and their causes.

41. Some general principles are outlined:
1. A TSH will not return an accurate assessment of the HPT
axis in the following.
 Recent treatment for thyrotoxicosis (TSH may remain
suppressed even when thyroid hormone concentrations
have normalized.)
 Nonthyroidal illness.
 Medications such as glucocorticoids will transiently depress
TSH.
 Central hypothyroidism (eg, hypothalamic and pituitary
disorders).
 TSH-secreting pituitary adenoma (TSHoma).
 Resistance to thyroid hormone.
 Disorders of thyroid hormone transport or metabolism.

42. Some general principles are outlined: continue……..
2.wide interindividual variation of fT4,
comparison with previous results is likely to be
more valuable than the use of generic
reference intervals.
TRAb : Graves disease.
3.In subclinical disease, consider the use of
antithyroid antibody measurement if TSH is
elevated.
fT3:borderline elevated fT4 concentrations and
T3 toxicosis.

43. Familial dysalbuminemic
hyperthyroxinemia (FDH)
assay artefact, to which most current fT4 assays are
susceptible to a variable degree.
 In the most common form of FDH (R218H), fT3 is
typically within the reference interval.
(Two other forms, L66P and R218P, are associated
with elevated T3 and both elevated T3 and T4,
respectively.)

44. Measurement of Urinary Iodine
Concentration
iodine intake and iodine deficiency .
Secondary measurements for estimating iodine
deficiency are thyroid size, TSH concentration, and
thyroid hormones.
The gold standard is 24-hour urine collection.
The inductively coupled plasma mass spectrometry
method has a lower limit of detection

45. Perchlorate Discharge Test
hypothyroidism or goiter results from enzyme defects
responsible for the incorporation of iodine into thyroid
hormone.
Iodine will be trapped within the thyrocytes but not
organified. The perchlorate discharge test detects
defects in iodine oxidation or the iodination of Tg.
.After a dose of labeled perchlorate, if there is an
enzyme defect, a supranormal amount of radioiodine is
released from the thyroid gland, and the perchlorate
discharge of radioiodine is increased.
If there is a defect in the NIS (eg, a loss of function
mutation), the release of radioiodine is not increased
after perchlorate because radioiodine was not initially
taken up by the thyroid gland.

46. Hypothyroidism
Hypothyroidism can be classified
according to:
 Age of onset (congenital or acquired)
 HPT level (primary (defect in the thyroid),or
secondary (defect in the hypothalamus or pituitary
gland, also called central hypothyroidism)
 Severity (overt [clinical], mild [subclinical])
 And duration (permanent or transient).

51. Subclinical Hypothyroidism
Subclinical hypothyroidism can be classified as
mild (TSH between 4 and 9.9 mIU/L) or severe
(TSH ≥10 mIU/L).
trimester and age-dependent cut-offs differ and
should be applied as appropriate.
The elevation in TSH should have persisted for 6
to 12 weeks
The prevalence increases with age and is higher
in women than in men.

52. Subclinical Hypothyroidism continue…….
individuals with TSH between 3 and 5 mIU/L
more often are positive for TPO antibodies
and have a higher rate of progression to overt
hypothyroidism.
SCH is associated with risk of heart failure
with TSH of 10 mIU/L or greater.

53. Laboratory Diagnosis of Hypothyroidism
 Rpt TPO antibodies preferably after a 2- to 3-month
interval.
The presence of antibodies indicates -progression to
overt hypothyroidism.
TSH is elevated but fT4 is normal encompass recent
institution of Thyroid hormone replacement therapy
(fT4 returns to normal before TSH declines).
 Poor compliance with treatment in primary
hypothyroidism, recovery from nonthyroidal illness,
positively interfering heterophilic antibodies, (eg,
HAMA) in double-antibody immunoassays, and thyroid
hormone resistance.
In central hypothyroidism, the laboratory diagnosis is
based on low, normal, or slightly elevated TSH
combined with low tT4 or fT4 concentrations.

54. Central Hypothyroidism.
Persani L et al : In the Netherlands, the
neonatal screening program using a combined
TBG, TSH, and T4 strategy – incidence 1 in
16,000.

55. Thyrotoxicosis
European studies showed that the prevalence of
undiagnosed hyperthyroidism was 1.72%, the
the incidence rate of hyperthyroidism was about 51
per 100,000 per year.
The risk increases with age and is higher in
women,in iodine deficient areas, and in whites.
prevalence of subclinical hyperthyroidism is 0.6% to
2% depending on the cut-off used for TSH
concentration.
the prevalence is higher in women and increases
with age, with prevalence reported to be 2% in
people older than 65 years of age.

56. Thyrotoxicosis
Graves disease, the commonest cause of
hyperthyroidism,
women than in men (5:1). Graves disease
frequently is associated with other autoimmune
disorders.
There is a genetic susceptibility to Graves disease
envi ronmental with smoking.
Thyroid-stimulating hormone–secreting anterior
pituitary adenomas - 1%
TSH-secreting tumors may occur at any age and
occur with equal frequency in men and women.

58. Diagnosis and Differential Diagnosis of
Thyrotoxicosis
suppressed or low TSH and increased fT4 or
increased fT3 or total T3.
TSH is the most sensitive biomarker.
2% to 4% of patients with hyperthyroidism have
increased concentration of fT3 or tT3 but normal
concentration of fT4 (T3 thyrotoxicosis).
 Subclinical hyperthyroidism is defined as low TSH
with normal circulating concentrations of T4 and T3.

59. Thyrotoxicosis-etiology ?
iodine uptake of the thyroid gland is low in
thyroiditis and high in Graves disease and
autonomous nodules (single or multiple).
Technetium scintigraphy uses pertechnetate that is
trapped by the thyroid and not organified, resulting
in a low range of normal uptake and high
background .
USG is only indicated in the differential diagnosis of
thyrotoxicosis when radioactive iodine is
contraindicated (eg, pregnancy or breastfeeding.

60. Stimulating TSH receptor autoantibodies immunoassay:
analytical evaluation and clinical performance in Graves'
disease.
Autilio C1, Morelli R1, Locantore P2, Pontecorvi A2, Zuppi
C1, Carrozza C1.
• Among all TRAbs, only the stimulating ones (S-
TRAbs) are considered as the pathogenetic
marker of Graves' disease (GD).

61. Toxic Adenomas and Toxic
Multinodular Goiter
thyrocytes function and produce thyroid hormones
independently of thyrotropin (TSH)
it accounts for up to 60% of cases of thyrotoxicosis.
However, thyroid autonomy is rare in regions with
sufficient iodine supply (3%–10% of cases with
thyrotoxicosis).
Confirmation by
 thyroid function tests
 Presence of palpable or sonographically demonstrable
nodule(s)
 Increased radionuclide (eg, 99mTc) uptake in the
nodule(s) combined with a decreased uptake in the
surroundin extranodular thyroid tissue

62. Gain-of-Function Mutations of the Thyroid-Stimulating
Hormone Receptor
A familial AD form of hyperthyroidism -by gain-of-
function mutations in the TSH receptor .
The gain-of-function mutation places the TSHR in
the “on” position in the absence of ligand (TSH)
binding.
In infants homozygous for such mutations, neonatal
thyrotoxicosis, heterozygous mutations -infantile
hyperthyroidism.

63. Central Hyperthyroidism
nonsuppressed
TSH in the presence of high levels of (fT3 and fT4).
pituitary mass on CT or MRI.
Artefacts -TSH-secreting tumors ( thyroid hormone
resistance, binding protein abnormalities), falsely
high fT4 results, and falsely high (given the high T4
TSH concentrations.
The molar ratio of α-subunit to TSH:DD
TSH-secreting pituitary adenoma, in which the ratio
of α-subunit to TSH is larger than 1 ng/mL.
 from thyroid hormone resistance syndrome, less
than or equal to 1 ng/mL.

64. FLOW CHART :DD betwwen TSHoma and TRH

65. Resistance to Thyroid Hormone
In TSH-secreting pituitary adenoma, α-subunit:TSH
serum concentrations is larger than 1 ng/mL;
in thyroid hormone resistance- less than or equal to
1 ng/mL.Further diagnosis is based on imaging and
genetic testing with THR beta gene sequencing*.
*Ortiga-Carvalho TM et al
TRH test or T3 suppression tests.

66. Hyperthyroidism Caused by Human
Chorionic Gonadotropin
Observed in gestational transient thyrotoxicosis,
resulting from TSHR sensitivity to (appropriately)
high hCG concentration during pregnancy and the
hyperemesis gravidarum, and to hCG-secreting
tumours.
Tumors that secrete hCG, such as choriocarcinoma,
hydatidiform mole, and metastatic embryonal
carcinoma, can cause hyperthyroidism through hCG
stimulation of the TSH receptor.

67. Subclinical Hyperthyroidism continue…
Mild if TSH is in the range 0.1 to 0.4 mIU/L.
Persistent subclinical hyperthyroidism : exogenous
iatrogenic overdose of levothyroxine or by endogenous
:primary hyperthyroidism such as Graves disease, toxic
multinodular goiter, or solitary autonomou nodule.
Transitory subclinical hyperthyroidism : treatment with
radioiodine or antithyroid drugs in patients previously
with overt hyperthyroidism or as part of thyroiditis.
Plasma TSH concentrations are lower in blacks, in older
adults, in cigarette smokers, during the first trimester of
pregnancy, in nonthyroidal illness, and in patients
treated with certain drugs (glucocorticoids, dopamine).
Apropriate reference intervals should be used
(pregnancy, age, and gender related

68. Subclinical Hyperthyroidism
Current ATA and AACE guidelines state that when
a TSH is persistently below 0.10 mIU/L.
Treatment* of subclinical thyrotoxicosis -age of
65 years, in postmenopausal women who are not
taking estrogens or bisphosphonates, and in
patients with osteoporosis or cardiac risk factors.
Bahn RS et l*.

69. Thyroid Disorders in Pregnancy and Postpartum
 4% ,
 Plasma tT3 and tT4 increase in -TBG concentration.
 This increase is caused by enhanced hepatic synthesis and
reduced metabolism (a result of increased estrogen levels)
early in pregnancy, 1.5-fold increase in TBG by 6 to 8 weeks
.
 TBG remains elevated throughout pregnancy.
 Placental hCG shares the same α subunit with TSH has a
unique β subunit and acts in early pregnancy as a TSH
agonist .
 hCG stimulation of the thyroid in early pregnancy leads to
 a physiological rise in T4 and T3, which, by the HPT axis -
causing TSH to fall.
 TSH is higher in singleton pregnancies than in twin
pregnancies.

72. Hypothyroidism in Pregnancy
• 2% to 3% of all iodine-sufficint SCH,Overt
hypothyroidism is 0.5% .
• the most common cause is endemic
• iodine deficiency, iodine-sufficient populations is
chronic autoimmune thyroiditis.
• 2% isolated hypothyroxinemia (eg, elevated thyroid
hormone without plasma TSH elevation and
without the presence of autoantibodies).
• About 10% to 20% of women in the childbearing
years have detectable autoantibodies (TPO or Tg
autoantibodies).

73. Diagnosis of hypothyroidism in pregnancy
• An elevated serum TSH concentration with
low concentrations of fT4, using trimester-
specific reference intervals.

74. Treatment and Monitoring.
ATA--decreased fT4, TSH >10.0 mIU/L irrespective of
fT4 concentration or with TPO autoantibodies.
SCH:TSH and fT4 every 4 weeks or so until 16 to 20
weeks of gestation and at least once between 26
and 32 weeks of gestation.
In euthyroid TPO autoantibody positive, TSH should
be evaluated every 4 weeks during the first half of
pregnancy and at least once between 26 and 32
weeks.

75. Thyrotoxicosis in Pregnancy
 Graves disease occurs in 0.1% to 1% of all pregnancies, TSH
and thyroid hormones should be measured every 4 to 6
weeks during pregnancy.
 , TRAb-at 24 to 28 weeks gestation because these
antibodies can cross the placenta, starting in late second
trimester.

76. Transient Gestational Hyperthyroidism and
Hyperemesis Gravidarum
• 2% to 3% of all pregnancies and results from
activation of TSH receptors by hCG.

77. Thyroid Autoantibodies in Euthyroid
Women
TPO antibodies and Tg antibodies can be
detected in 10% to 20% of pregnant women.

78. Postpartum Thyroiditis
 the presence of goiter, ophthalmopathy, and
thyroid receptor (TRAb) antibodies in Graves
disease with high iodine or technetium
uptake.
 these are usually not present in thyroiditis.
Approximately 4% to 9% of unselected
postpartum women develop postpartum
thyroiditis.

79. Thyroid Function Testing in Pregnancy
TSH:
 First trimester : 0.1 to 2.5 mIU/L
 • Second trimester : 0.2 to 3.0 mIU/L
 • Third trimester : 0.3 to 3.0 mIU/L
 LC–tandem MS combined with equilibrium dialysis or
ultrafiltration methods are more reliable for both
total and free hormone concentrations during
pregnancy

81. THYROID NEOPLASIA
TSH -thyroid nodule.
Suppressed TSH is in keeping with an autonomous
nodule
Differentiated Thyroid Cancer
Newer Tg assays have a functional sensitivity of 0.1
ng/mL.
Routine measurement of serum TSH, fT4, and fT3 is
required for patient follow-up.
• Immunometric assays-The hook effect.
• Serially measured Tg and TgAbs should be done on the
same analyzer using the same assay.

83. Medullary Thyroid Cancer (MTC)
Serum calcitonin can be used as a screening test in
patients with a family history of MTC.
CEA is useful for evaluating disease progression in
patients with clinically evident MTC and for
monitoring patients after thyroidectomy.
sporadic MTC can have RET gene mutations.
serum TSH and serum calcium should be measured
postoperatively.
automated noncompetitive immunoassays (eg,
immunochemiluminometric assays [ICMA])

84. THYROID DISEASE IN CHILDREN
• Neonatal hyperthyroidism may be caused by
transplacental passage of thyroid-stimulating
maternal Ig (due to active maternal Graves’ disease)
or by activating TSH receptor mutations .
• In older children and adolescents, the most common
cause of hyperthyroidism is Graves disease, and the
most common cause of hypothyroidism in iodine-
replete areas is Hashimoto thyroiditis.

85. Laboratory Diagnosis of Congenital
Hypothyroidism
 The initial screening occurs on the second to fifth day of
life.
 second specimen at 2 to 6 weeks of age.
 Some programs use cord blood at birth.
 heel prick on filter paper after birth.
 Thyroid hormone levels and TSH are higher in the first days
of life but have usually fall within the 2 to 4 weeks.

86. GENETICS
 65% TSH and thyroid hormone concentrations are genetically determined.
 20% genetic variation at the population level.
Genetics in Autoimmune Thyroid Disease
 (Tg and TSHR) and immune-regulatory genes, which are shared with
other autoimmune diseases.
 AITD immune-regulatory genes : FOXP3 and CD25 involved in
establishment of immune self-tolerance, and CD40, CTLA-4.
 HLADR3 carries the highest risk.
 AITD is higher among monozygotic twins than among dizygotic twins.