Thyroid hormones: Clinical and Biochemical Insight
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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: [email protected]
Date: 15/07/2014
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Dealing with the topic
• Functions
• Biochemistry
• Physiology
• Radiographic Thyroid Testing
• Analytical Methods of the various thyroid hormones
• Clinical Correlations
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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.
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BIOCHEMISTRY OF THE HORMONES
• rT3- 3,3’,5’-L-triiodothyronine
• T3- 3,5,3’-L-triiodothyronine
• T4- tetraiodothyronine(Thyroxine)
• MIT- Monoiodotyrosine
• DIT- Diiodotyrosine
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Peripheral conversion of T4 to T3 and rT3
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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.
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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.
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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%
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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.
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Thyroid Hormone Physiology
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Thyrotropin Releasing Hormone
TRH is a tripeptide (L-pyroglutamyl-L-histidyl-L-prolinamide)
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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
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Worldwide Distribution of Iodine Nutrition
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Comparison of T4 and T3 ( properties)
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MOA of Thyroid Hormones
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Analytical Methodology
•Principal Of Chemiluminescence
•Direct Methods
•Indirect Methods
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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
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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.
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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.
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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.
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• 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.
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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
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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.
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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.
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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%.
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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.
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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.
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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)
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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)
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Clinical Correlations
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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.
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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)
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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.
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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.
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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.
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Impact of Drugs on Thyroid Hormone Levels
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Facial Features in Hypothyroidism
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See the differences after successful therapy
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Facial Features in Hyperthyroidism
• Opthalmopathy in Grave’s Disease• Lid retraction
• Periorbital edema
• Conjunctival injection
• Proptosis
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Thyroid Dermopathy over lateral aspects of the shins.Thyroid Acropachy
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