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M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
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

More than half of the bodys total content is
found in the thyroid gland

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

Hypothalamo pituitary axis
A small increase in T

and T
produces a
diminished TSH
response to TRH at the
pituitary level.
and T
act at the
hypothalamic level by
inhibiting mRNA for TRH
Only unbound fractions
of hormone are
metabolically active and
only this free hormone
has an inhibitory effect
on the secretory activity
of the thyroid.

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

Mechanism of thyroid hormone
receptor action
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

GUTincreased absorption of
nutrients, increased motility

Liver -increased
gluconeogenesis&glyco genolysis
Adipose tissue increased

Muscle increased protein
catabolism in skeletal muscle

Kidney -increased erythropoietin

Respiration- increased central
stimulation of respiration

Energy metabolism -increased
BMR,increased oxygen
consumption,increased heat
production stimulation of Na-K-
ATP ase
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
The functional unit of thyroid is
thyroid follicle. Normal
Thyroid follicle with out TSH
Thyroid follicle with high TSH

High T3 or T4 gives

decreased TSH
subunit synthesis
thyrotrophs may
lose the capacity
to respond to
reduced T
or T

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
releasing hormone,
ie stimulates TSH
somatostatin TRH
Iodine deficiency
Hasimotos thyroiditis
Radiation therapy
drugs and PAS
Absent or ectopic thyroid
TSH receptor mutation
sheehans syndrome
& isolated TSH
Hypothalamic disease
Trauma & infiltration
Primary hypo thyroidism Secondary hypotyroidism
- congential absence of T3 and T4 or
chronic iodine deficiency during childhood

- retarded growth

- sluggish movements

- mental deficiencies

- low rate of metabolism and lethargy

- decreased body temp

- decreased heart rate

- outer skin becomes scaley

- myxodema swelling of sub-cu connective
Grave disease
Toxic multinodular
Toxic adenoma
Functioning metastatic
thyroid carcinoma
TSH receptor mutation
Struma ovarii
Iodine excess
TSH secreting
pituitary adenoma
Thyroid hormone
resistance syndrome
secreting tumours

Primary hyperthyroidism Secondary hyper thyroidism
- Graves Disease

- tall stature, hyperactivity

- high rate of metabolism

- high body temp

- high heart rate

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
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
Iodine supplementation is considered to be
important in women with precarious iodine
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 50g/day during pregnancy
Thyroid function tests
Estimation of thyroid
Total T4
Total T3
Estimation of free
hormone fraction
Free T4 fraction
Free T3 fraction

Estimates of free
FT4E (T4 X %FT4)
FT3E (T3 X % FT3)
T4: TBG ratio

Thyroid function tests
Serum binding
Thyroxine binding
Thyroxine binding
Tests for auto immune
thyroid disease
Anti thyroglobulin
Anti microsomal Abs
Anti TPO antibodies
TSH receptor anti
Other hormones &
thyroid related
Measurement of T4,T3 &rT3
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
Sandwich ELISA

Determination of free thyroid
Direct assays currently serve as reference

Indirect assays - more widely available for
general laboratory use
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
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
Indirect methods
More convenient & less expensive than direct
Automated immuno assay instuments
Two step immunoassay
One step immunoassay
These methods estimate free hormone conc.
by using antibody extraction techniques
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
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)
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

Residual free I-T3 then adsorbed by resin is
removed from the sample and then adsorbed/bound
I is measured
FT4 index

Unlike direct free T4 methods , index methods
measure both the serum total T4 & the free T4
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(g/dl) x % thyroid uptake/ 100

Plasma TSH
Method- Immunoassay
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

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

Determination of thyroid binding
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
Calculation of T4:TBG & T3:TBG
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
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 g/dl

Thyroglobulin is used primarily as tumor marker in
pts carrying a diagnosis of differentiated thyroid

Tg levels are elevated in
Thyroid follicular &papillary carcinoma
Certain non neoplastic conditions like..,
Thyroid adenoma
Subacute thyroiditis
hashimotos thyroiditis
Graves disease
Serum Tg conc. are
not increased in pts
with medullary thyroid
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

Determination of antithyroid
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)
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
individualsthey may have occult or
subclinical thyroid dysfunction
CHEMILUMINISCENCE based immunometry
Radioimmunometric technique
Reference value is 2U/ml(with sensitive
chemiluminiscence assay)
Detectable conc. Of TPO Ab are seen in
hashimotos thyroiditis,idiopathic myxedema,
graves disease, Type 1 IDDM

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

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
Graves disease
TSI measurement is also used for following the
course of therapy & predicting relapse & remission
Radio active iodine
Radioactive iodine uptake by thyroid gland and
thyroid scanning with Tc 99 are of diagnostic
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
Normal ranges
T3 :120-190 ng/dl
r T3 : 10-25 ng/dl
T4 : 5-12 g/dl
Thyroglobulin:3-5 g/dl
TRH :5-60 ng/L
TSH :0.5-5 U/ L
Thyroxine binding globulin :1-2 mg/dl