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1. THYROID
HORMONES
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2.  Thyroid gland produces two principal
hormones … thyroxine & tri iodo
thyronine which regulate the metabolic rate of
the body.
 Iodine is essential for the synthesis of thyroid
hormones
 More than half of the body’s total content is
found in the thyroid gland

10. Hypothalamo pituitary axis
The hypothalamo-pituitary
axis is a classical
negative feedback
regulatory mechanism in
which secretion of TSH
is modulated by thyroid
hormones. Release of
TSH from the pituitary
gland is stimulated by
thyrotropin releasing
hormone (TRH) from the
hypothalamus.

11. Hypothalamo pituitary axis
 A small increase in T3
and T4 produces a
diminished TSH
response to TRH at the
pituitary level.
 T3 and T4 act at the
hypothalamic level by
inhibiting mRNA for TRH
synthesis.
 Only unbound fractions
of hormone are
metabolically active and
only this free hormone
has an inhibitory effect
on the secretory activity
of the thyroid.
 dopamine
physiologically inhibits
TSH secretion
 glucocorticoids have
been shown to dull the
response of the
pituitary to TRH
 oestrogens increase
the sensitivity of
thyrotrophs to TRH

12. Mechanism of thyroid hormone
receptor action

13. Actions of thyroid hormones
 Brain----growth&development of
nervous system
 Bone&tissue growth– linear growth &
maturation of bones
 CVS-- increased contractility,heart
rate &cardiac output
 GUT—increased absorption of
nutrients, increased motility
 Liver -increased
gluconeogenesis&glyco genolysis
 Adipose tissue –increased
lipolysis
 Muscle –increased protein
catabolism in skeletal muscle
 Kidney -increased erythropoietin
synthesis
 Respiration- increased central
stimulation of respiration
 Energy metabolism -increased
BMR,increased oxygen
consumption,increased heat
production stimulation of Na-K-
ATP ase

14. Wolff-chaikoff effect
 Iodine deficiency increases thyroid blood flow
& upregulates the NIS , stimulating more
efficient uptake.
 Excess iodide transiently inhibits thyroid iodide
organification ,a phenomenon known as the
wolff-chaikoff effect

15. The functional unit of thyroid is
thyroid follicle. Normal
follicle

16. Thyroid follicle with out TSH
Thyroid follicle with high TSH
stimulation

18. High T3 or T4 gives
 decreased TSH
subunit synthesis
 inactive
thyrotrophs may
lose the capacity
to respond to
reduced T3 or T4
levels

19.  inhibits TSH release
 potentiates the
effect of thyroid
hormones on
thyrotrophs, ie
thyroid hormone has
inhibitory effects on
TSH release
 derives from the
median eminence of
the hypothalamus
 thyrotropin
releasing
hormone, ie
stimulates TSH
release
somatostatin TRH

20.  Iodine deficiency
 Hasimoto’s thyroiditis
 Thyroidectomy
 Radiation therapy
 Drugs-lithium,antithyroid
drugs and PAS
 Absent or ectopic thyroid
gland
 Dyshormonogenesis
 TSH receptor mutation
Hypopituitarism
 Tumors,pituitary
surgery, irradiation/infi
ltration, sheehan’s
syndrome & isolated
TSH deficiency
Hypothalamic disease
 Trauma & infiltration
Primary hypo thyroidism Secondary hypotyroidism

21. cretinism
- congential absence of T3 and T4 or
chronic iodine deficiency during childhood
- retarded growth
- sluggish movements
- mental deficiencies

22. myxedema
- low rate of metabolism and lethargy
- decreased body temp
- decreased heart rate
- outer skin becomes scaley
- myxodema – swelling of sub-cu connective
tissues

23.  Grave’ disease
 Toxic multinodular
goitre
 Toxic adenoma
 Functioning metastatic
thyroid carcinoma
 TSH receptor mutation
 Struma ovarii
 Iodine excess
 TSH secreting
pituitary adenoma
 Thyroid hormone
resistance syndrome
 Chorionic
gonadotropin
secreting tumours
 Gestational
thyrotoxicosis
Primary hyperthyroidism Secondary hyper thyroidism

24. hyperthyroidism
- Grave’s Disease
- tall stature, hyperactivity
- high rate of metabolism
- high body temp
- high heart rate

27. Thyroid function in pregnancy
Four factors alter thyroid function in pregnancy
 Transient increase in hcG during first trimester
which stimulates TSH-R
 The estrogen induced rise in TBG during the
first trimester which is sustained during
pregnancy
 Alterations in the immune system ,leading to
onset, exacerbation ,or amelioration of an
underlying auto immune thyroid disease
 Increased urinary iodide excretion ,which can
cause impaired thyroid hormone production

28.  Iodine supplementation is considered to be
important in women with precarious iodine
intake
 Maternal hypothyroidism occurs in 2 to 3% of
women of child bearing age & is associated
with increased risk of developmental delay in
the offspring
 Thyroid hormone requirements are increased
by 25 to 50µg/day during pregnancy

29. THYROID FUNCTION
TESTS

30. Thyroid function tests
Estimation of thyroid
hormones
 Total T4
 Total T3
Estimation of free
hormone fraction
 Free T4 fraction
%FT4
 Free T3 fraction
%FT3
 THBR
Estimates of free
hormone
concentration
 FT4E (T4 X %FT4)
 FT3E (T3 X % FT3)
 FT4I (T4 X THBR)
 FT3I (T3 X THBR)
 T4: TBG ratio

31. Thyroid function tests
Serum binding
proteins
 Thyroxine binding
globulin
 Thyroxine binding
prealbumin
Tests for auto immune
thyroid disease
 Anti thyroglobulin
Abs
 Anti TPO antibodies
 TSH receptor anti
bodies
Other hormones &
thyroid related
proteins
 TRH
 Thyroglobulin
 calcitonin

32. Measurement of T4,T3 &rT3
 METHOD
 Immunoassay
 Chemiluminiscence
 The major clinical role for T3 measurements are in
the diagnosis & monitoring of hyperthyroid pts
with suppressed TSH &normal FT4
 r T3 test is not always elevated with illness.It is
seldom used in pts with euthyroid sick syndrome
 Specifially,renal failure is associated with low r T3
conc.

33. Sandwich ELISA

34. Radioimmunoassay

35. Determination of free thyroid
hormones
 Direct assays – currently serve as reference
methods
 Indirect assays - more widely available for
general laboratory use

36. Direct methods
 Direct measurement of FT4&FT3 is a technical
challenge as free hormone conc. are low in serum
healthy individuals
 Assays for free thyroid hormones must be capable of
measuring sub picomole amounts
 Only minimal dilution of serum specimens is allowed
as dilution alters the binding of drugs, FFAs and
other substances to serum proteins

37. Methods
 Equilibrium dialysis
 Ultra filtration techniques
these techniques physically separate free hormone
from protein bound hormone (before direct
measurement of the free fraction with a sensitive T4
or T3 immunoassay)
These methods are unaffected by variations in SBPs
or thyroid hormone auto antibodies

38. Indirect methods
 More convenient & less expensive than direct
methods
 Automated immuno assay instuments
 Two step immunoassay
 One step immunoassay
 These methods estimate free hormone conc.
by using antibody extraction techniques

39.  FT4 is 0.03% of total serum T4
 FT3 is 0.3% of total serum T3
 Because T3 is less firmly bound by TBG than is
T4 the dialyzable fraction of T3 is appreciably
greater (by almost 10 times) than that of T4

40. Free hormone estimates
 FT4E = total T4 X %FT4
 The free hormone fraction as measured
dialysis or ultra filtration of diluted serum
containing tracer T4 or t3 is multiplied by the
respective total hormone concentration to
obtain indirect estimates
 THBR = %uptake(patient serum)/% uptake
(reference serum)

41. Invitro I –T3resin uptake by Resin
 A known amount of I-T3 is added to a standard
volume of serum from a patient
 The amount of I-T3 which binds to the serum
proteins varies inversely with the endogenous
thyroid hormones already bound to serum
proteins(TBG)
 Residual free I-T3 then adsorbed by resin is
removed from the sample and then adsorbed/bound
I is measured

42. FT4 index
 Unlike direct free T4 methods , index methods
measure both the serum total T4 & the free T4
fraction
 They have an advtantage that they can define
whether an abnormal FT4 estimate is due to
abnormal hormone production or due to abnormal
protein binding
 An FT4 index is sometimes directly calculated
using the percentage T-uptake
 FT4I =total T4(µg/dl) x % thyroid uptake/ 100

44. Plasma TSH
Method- Immunoassay
-chemiluminiscence
Secretion of TSH occurs in a circadian fashion
Primary Hypothyroidism-TSH increased
Secondary hypothyroidism-TSH ,T3 ,T4 are low
Primary hyper thyroidism –TSH decreased
Secondary hyperthyroidism-TSH,T3,T4 high

45. TSH stimulation test
Measurement of serum T4 after TSH injection
 No response - primary
 Increase of T4- secondary
 Useful for distinguishing primary from
secondary hypothyroidism

46. TRH response test
 TRH administration will stimulate the
production of TSH
 Useful for differentiating hypothalamic from a
pituitary hypotyroidism
 There is increase of TSH after TRH in
hypothalamic disorder

47. If the hypothalamo pituitary axis is normal .the
T3 and T4 secretions will be increased
An abnormal response is seen in
Hyperthyroidism – T4 elevated
 Hypopituitarism- T4 Levels subnormal
 Primary hypothyroidism-exaggerated response

50. Determination of thyroid binding
globulin
 TBG is the thyroid binding globulin with the
greatest affinity for T4
 TBG is very important for regulating the conc. And
availability of the FT4 hormone.
 Method - immunoassay
- commercial kit methods available
- chemiluminiscence
 Estrogen induced TBG excess and congenital
TBG deficiency are important abnormalities that
affect the test results

51. Calculation of T4:TBG & T3:TBG
ratios
 These ratios correlate with FT4 or FT3 conc.
And are particularly useful in sera with altered
TBG conc.
 failures:They may fail however to compensate
for TBG variants with reduced T4 affinity & for
abnormal albumin binding
 Ref . Interval is 3.8 to 4.5

52. Determination of thyroglobulin
 Method –immunometric assay method
 These assays are based on the use of two or
more monoclonal antibodies directed to
different portions of the Tg molecule
 Difficulty: interference with anti-Tg antibodies
as seen in pts with thyroid cancer
Heterophilic antibody interference(HAMA)
 Ref interval is 3 to 42 μg/dl

53.  Thyroglobulin is used primarily as tumor marker in
pts carrying a diagnosis of differentiated thyroid
carcinoma
Tg levels are elevated in
Thyroid follicular &papillary carcinoma
Certain non neoplastic conditions like..,
 Thyroid adenoma
 Subacute thyroiditis
 hashimoto’s thyroiditis
 Grave’s disease

54.  Serum Tg conc. are
not increased in pts
with medullary thyroid
carcinoma
 Serial measurements
of Tg is most useful in
detecting recurrence
of diff. thyroid
carcinoma following
surgical resection
 Tg determination is
used as an adjunct to
ultrasound and radio
iodine scanning
 Assessment of serum
Tg also aids in
management of
infants with congenital
hypo thyroidism
 In hyperthyroidism-Tg
Low conc.-
thyrotoxicosis factita

55. Determination of antithyroid
antibodies
Anti thyroid antiodies are found in autoimmune
diseases and certain malignancies
These autoantibodies are directed against
several thyroid and thyroid hormone antigens
 Tg (Tg Ab)
 Thyroid peroxidase(TPO Ab)
 Thyroid receptor(TR Ab)
 TSH,T4,T3

56.  The presence of TPO antibodies is a risk
factor for autoimmune thyroid dysfunction
 However there is a high prevalence of anti-
TPO antibodies in the elderly
 With sensitive assays,low conc of TPO
antibodies may be detected in some healthy
individuals—they may have occult or
subclinical thyroid dysfunction

57. Method
 RIA
 CHEMILUMINISCENCE based immunometry
 Radioimmunometric technique
Reference value is ≤2U/ml(with sensitive
chemiluminiscence assay)
Detectable conc. Of TPO Ab are seen in
hashimoto’s thyroiditis,idiopathic myxedema,
grave’s disease, Type 1 IDDM

58. Determination of thyrotropin
receptor antibodies
Thyrotropin receptor antibodies are a group of related
immunoglobulins that bind to TSH receptors
Seen in pts with Graves disease & other auto immune
thyroid disorders
These Ab s demonstrate substantial heterogeneity
Some cause thyroid stimulation , where as others
have no effect or decrease thyroid secretion by
blocking the action of TSH

59.  Invitro bioassays assess the capacity of
immunoglobulins to stimulate functional activity of
thyroid gland such as..,
1.adenylatecyclase stimulation
2. c AMP formation
3.colloid mobilization
4.iodothyronine release
TSI s are present in 95% of pts with untreated
Grave’s disease
TSI measurement is also used for following the
course of therapy & predicting relapse & remission

60. Radio active iodine
uptake(RAIU)
 Radioactive iodine uptake by thyroid gland and
thyroid scanning with Tc 99 are of diagnostic
value.

61. calcitonin
 Calcitonin is secreted by the para follicular or
C cells ,which arise from the neural crest & are
distributed through out the thyroid gland
 A marker for medullary thyroid carcinoma
(tumor of C cells)
 Ref range ≤ 25pg/m L in men and ≤20 pg/m L

62. Normal ranges
 T3 :120-190 ng/dl
 r T3 : 10-25 ng/dl
 T4 : 5-12 µg/dl
 Thyroglobulin:3-5 µg/dl
 TRH :5-60 ng/L
 TSH :0.5-5 µU/ L
 Thyroxine binding globulin :1-2 mg/dl

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