Thyroid Hormones :Clinical and Biochemical Insight

Dr. Abhishek Roy

JR-II, Dept. of Biochemistry,

Grant Govt. Medical College &

Sir J.J. Group of Hospitals, Mumbai

Email: mail@abhishek.ro

Date: 15/07/2014

Dealing with the topic

• Functions

• Biochemistry

• Physiology

• Radiographic Thyroid Testing

• Analytical Methods of the various thyroid hormones

• Clinical Correlations

Functions of Thyroid Hormones

• Increase O2 consumption within tissues via increased membrane transport.(More ATP consumed and more Na+-K+ ATPase function)

• Enhanced mitochondrial metabolism

• Increased sensitivity to catecholamines

• Stimulate protein synthesis and carbohydrate metabolism

• Increased synthesis and degradation of cholesterol and triglycerides.

• Increased Vitamin requirements

• Regulate Calcium and Phosphorus metabolism.

BIOCHEMISTRY OF THE HORMONES

• rT3- 3,3’,5’-L-triiodothyronine

• T3- 3,5,3’-L-triiodothyronine

• T4- tetraiodothyronine(Thyroxine)

• MIT- Monoiodotyrosine

• DIT- Diiodotyrosine

Peripheral conversion of T4 to T3 and rT3

Thyroid Hormone Receptor Sizes

Amino Acids kDa

• TRα1 410 47

• TRα2 490 55

• TRβ1 461 53

• TRβ2 514 58

• TRβ is encoded by 11 exons on THRB gene on chromosome 3p24.3.

• Three isoforms of TRβ exist: TRβ1, TRβ2, TRβ3

• TRβ1 found in Heart, Kidney, Liver and Brain.

• TRβ2 found in Adenohypophysis, Retina, Cochlea and Developing Brain.

• Defects in TRβ1 can produce resistance to thyroid hormone.

• Defects in TRα is not been reported.

The log-linear relationship between TSH and FT4. A two fold change in TSH is associated with

approx. 100-fold change in circulating FT4 concentration.

Thyroid Hormones in Circulation

• TBP TBG TTR Albumin

• Concentration 4-5.4 µg/dL 10-20 µg/dL 3.5-5 g/dL

• Affinity for T4 High Modest Low

• T4 capacity, µ/dL 22 120 1000

• Distribution

• T4 67% 20% 13%

• T3 53% 1% 46%

Salient Points

• Protein Bound T4 and T3 serve as thyroid hormone reservoirs within plasma.

• FT4 concentrations correlate more closely to clinical status of the patients than total T4 conc.

• Low FT3 do not always correlate with clinical hypothyroidism as evidenced in sick euthyroid syndrome.

• Similarly elevated FT4 and FT3 don’t always correlate with hyperthyroidism coz of possibility of peripheral thyroid hormones resistance syndrome and rare MCT8 loss of function mutations.

Thyroid Hormone Physiology

Thyrotropin Releasing Hormone

TRH is a tripeptide (L-pyroglutamyl-L-histidyl-L-prolinamide)

Radioiodine uptake (RAIU)

• Radioactive iodine(I123 or I131) or 99mTc-pertechnetate

• Reference interval is usually 5 to 25% in 24hrs.

• In most endogenous hyperthyroid states the RAIU is

• In hypothyroid, RAIU is decreased.

• In thyrotoxicosis, measurement of peak at 6hrs seen.

• Anatomic disorders:

• Hemithyroid (toxic hyperthyroid nodule)

• Cold nodule

• Ectopic thyroid

Worldwide Distribution of Iodine Nutrition

Comparison of T4 and T3 ( properties)

MOA of Thyroid Hormones

Analytical Methodology

•Principal Of Chemiluminescence

•Direct Methods

•Indirect Methods

Chemiluminescence

• Emission of light when an electron returns to a lower energy level from a higher energy level.

• The excitation is caused by oxidation of organic compound such as luminol, isoluminol, acridinium esters or luciferin by an oxidant like H2O2, HOCl-, O2

• Reaction accurs in presence of catalyst enzymes( Alkaline Phosphatase, Horse radish Peroxidase, Microperoxidase), metal ions etc.

• Ultrasensitive assays- 10-18 to 10-21

Sample collection and storage:

• For all hormonal parameters done in J.J. Hospitals, Mumbai with the use of IMMULITE 1000 Immunoassay( Chemiluminescence):

• Sample is collected in Plain vaccutainers or Plain Bulb.

• The samples are centrifuged at around 2500-4000 rpm for 2-3 mins.

• The serum can stored at 2-8ºC maximum for 7 days and when stored at ―20ºC it can be stored for 2 months.

Determination of TSH

• Immunoassay is the method of choice.

• We use Immulite 1000 Rapid TSH kit with incubation cycle of 1 X 30 mins.

• Volume required: 75 µL (Sample = 100 µL more)

• Analytical Sensitivity: 0.01 µIU/mL

• Callibration Range: 75 µIU/mL

• Euthyroid: 0.4-4 µIU/mL

• True Hyperthyroidism: <0.01 µIU/mL

• Secretion of TSH is in circadian fashion:• Highest between 2:00 am to 4:00 am

• Lowest between 5:00 pm to 6:00 pm

• Low amplitude oscillations occur throughout the day.

• TSH surges immediately after birth to around 25-160 µIU/mL

• Reach back to cord blood levels by 3 days and then to adult value by 7 days.

Determination of Thyroxine (Total T4)

• Immunoassays measure both free and bound form.

• Therefore dissociation is required as 99.97% of T4 is bound to TBG, TBPA and Albumin.

• Volume required: 15 µL(Sample = 100 µL more)

• Incubation cycles: 1 X 30 mins

• Association constant:• T4 to Albumin: ~1.6 X 106 L/mol• T4 to TBG: ~2 X 1010 L/mol• T4 to TBPA: ~2 X 108 L/mol• T4 to Antibody in test: ~109 L/mol

• Association Broken by:• T4 to TBPA: Barbital Buffers as they do this selectively.• T4 to TBG: Agent of choice is 8-anilino-1-naphthalene-sulfonic (ANS) acid.

• Normal Range: 4.5-12.5 µg/dL

• Calibration range: 1.0-24 µg/dL

• Analytical Sensitivity: 0.4 µg/dL

• At birth, serum total T4 are higher in neonatal period

because of maternal estrogen-induced increase in serum

TBG while FT4 values are near adult concentrations.

• Total T4 values rie abruptly in the firsts few hours after

birth and decline gradually until the age of 15 yrs.

Determination of Triiodothyronine (Total T3)

• Antiserum has been produced using T3 enriched Tg, T3-human serum albumin (HAS) or T3-bovine serum albumin (BSA) conjugates.

• Monoclonal T3 antibodies have also been produced using hybridomatechnique.

• Method of choice now is Chemiluminescence.

• Levels of T3 depends on age.

• Volume required: 25 µL( Sample = 100 µL more)

• Normal Range: 81-178ng/dL

• Analytical sensitivity: 35 ng/dL

• Calibration range: 40-600 ng/dL

Determination of reverse Triiodothyronine (rT3)

• It’s a biologically inert and is a catabolite of T4.

• rT3 estimation is usually not required clinically, hence generally hospital labs refer this to other large reference labs.

• In J.J. Hospitals, rT3 is not done.

• Formed by 5´-deiodinases when acting on T4 peripherally.

Determination of Free Thyroid Hormones

• Technical challenge as FT4 is 0.03% and FT3 is 0.3% of T4 & T3.

• Most reliable methods are:• Direct Equilibrium Dialysis

• Ultrafiltration

• These are extremely time consuming and hence have been replaced by Chemiluminescence for all practical purposes.

Direct Equilibrium Dialysis

• Undiluted serum samples are dialyzed for 16 to 18 hrs at 37ºC in a reusable dialysis chamber.

• Dialysis buffer provides for minimal changes in the serum matrix

• Dialysate is then analyzed directly using a sensitive (RIA).

• The range of expected results is 2 to 128 ng/L.

• Interassay CV is <10%.

Ultrafiltration

• Significantly less time consuming.

• Serum specimen is adjusted to a pH of 7.4.

• Then incubated for 20 mins at 37ºC.

• Applied to an ultracentrifugation device for 30 mins at 37ºC and 2000 X g.

• Later ultrafiltrate is analyzed for T4 by immunoassay.

Indirect methods for Free Thyroid Hormones

• In these methods, the basic principle is Estimation of FT4 and FT3 by antibody extraction techniques.

• Two Step Immunoassays:• The free hormones is made to react with solid phase antibodies and the other serum protein

bound hormones are washed away.

• Tracer(labelled) T4 & T3 is made to react with the left over antibodies(back titration).

• The quantity of bound tracer then is compared with calibration curve generated from secondary calibrators that have had target values assigned to them by reference method.

• One step Immunoassays:• Unlike two step methods, analogue assays rely on simultaneous rather than sequential back

titration of unoccupied antibody binding sites.

FT4 through Chemiluminescence

• Ranges:• Euthyroid: 0.89-1.76 ng/dL

• Hypothyroid: <0.89 ng/dL

• Hyperthyroid: >1.76 ng/dL

• Reportable range: 0.3-6 ng/dL

• Volume required: 10 µL ( Sample = 100 µL more)

• Analytical Sensitivity: 0.13 ng/dL (Limit of Blank)

• Functional Sensitivity: 0.30 ng/dL(conc. with 20% CV)

• Incubation Cycles: 1 X 30 mins (Time to first result: 42 mins)

FT3 through Chemiluminescence

• Normal Range: 1.5-4.1 pg/mL

• Calibration range: 1-40 pg/mL

• Analytical Sensitivity: 1.0 pg/mL

• Incubation cycles: 2 X 30 mins

• Volume required: 100 µL (Sample = 250 µL more)

Clinical Correlations

Thyroid Stimulating Hormone (TSH)

• In Primary Hypothyroidism: TSH levels are typically high

• In secondary and tertiary hypothyroidism: TSH levels low.

• In Primary Hyperthyroidism: TSH levels are very low

• In Secondary and tertiary hyperthyroidism: TSH levels are very high.

• Measurement of circulating TSH has been used as a primary test for DD of Hypothyroidism and as an aid in monitoring hormone replacement therapy.

• TSH >2.0 µIU/mL have increased risk to develop thyroid diseases in next 20yrs.

Total Thyroxine (Total T4)

• Hyperthyroidism: Increased

• Hypothyroidism: Decreased

• Elevated T4 levels may be seen when TBG levels are high as in pregnancy, Acute intermittent porphyria, hyperproteinemia, hereditary TBG elevation, pts. Undergoing estrogen therapy or taking OCPs.

• Total T4 levels are low when TBG is low as in Nephrotic, Hepatic, Gastrointestinal and neoplastic disorders, acromegaly, hypoproteinemia, hereditary TBG deficiency, pts. Undergoing androgen, testosterone or anabolic steroid therapy.

• Diphenylhydantoin and large doses of salicylates and liothyroninemay also cause low T4 values (Competition for binding TBG)

Total Triiodothyronine (Total T3)

• T3 represents approx. 5% of total thyroid hormones in system

• T3 has greater intrinsic metabolic activity, faster turnover and larger volume of distribution than circulating T4.

• Reports do suggest that thyrotoxicosis may be caused by abnormally high conc. of T3 rather than T4.

• T3- Imp. Tool for monitoring patients receiving sodium liothyroninetherapy.

• Reports suggest, T3 can differentiate well between Euthyroid and Hyperthyroid but provide a less clear-cut separation between hypothyroid and euthyroid subjects.

Free Thyroxine (FT4)

• Altered TBG result in false T4 levels and FT4 often remain under a very tight range.

• For this reason Total T4 do not always reflect the thyroid status.

• Total T4 levels even though increased, the FT4 levels may be normal.

• Alternatively, patients with dysfunctional thyroid gland and altered TBG levels can have normal total T4 levels masking the illness.

• Therefore its FT4 that highly correlate with clinical scenario.

Free Triiodothyronine (FT3)

• Free T3 conc. Constitutes only about 0.25% of total T3 in circulation.

• T3 measurements on top of T4/FT4 helps to confirm hyperthyroidism diagnosis.

• Abnormal elevations of total T3 may occur when total T4 conc. is normal- “T3- toxicosis”

• But we see that Free T3 correlates more closely with the actual thyroid status of the patient than total T3.

Impact of Drugs on Thyroid Hormone Levels

Facial Features in Hypothyroidism

See the differences after successful therapy

Facial Features in Hyperthyroidism

• Opthalmopathy in Grave’s Disease• Lid retraction

• Periorbital edema

• Conjunctival injection

• Proptosis

Thyroid Dermopathy over lateral aspects of the shins.Thyroid Acropachy

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