1. CASE E Hyperthyroidism Hyperthyroidism Poonam Shrestha Poonam Shrestha Veronica Nou Veronica Nou Mary Tormey Mary Tormey Ainsley Macdonald Ainsley Macdonald

2. Mr TF, aged 50 years, has a history of asthma and CAL. He has recently been hospitalised for a severe chest infection. He has also lost 5kg in weight in the last 3 months, has tachycardia of 120/min and fine tremor(hands). He is currently receiving prednisolone 25mg daily & ceftriaxone 1g daily Mr TF, aged 50 years, has a history of asthma and CAL. He has recently been hospitalised for a severe chest infection. He has also lost 5kg in weight in the last 3 months, has tachycardia of 120/min and fine tremor(hands). He is currently receiving prednisolone 25mg daily & ceftriaxone 1g daily

3. Thyrotoxicosis is suspected

4. Effect of prednisolone on thyroid function Glucocorticoid group Glucocorticoid group Decrease TSH secretion Decrease TSH secretion Large dose  decrease serum T3 concentration Large dose  decrease serum T3 concentration Long term glucocorticoid therapy  decrease in serum thyroid binding globulin(TBG)  slight decrease of T4 concentration Long term glucocorticoid therapy  decrease in serum thyroid binding globulin(TBG)  slight decrease of T4 concentration

5. Thyrotoxicosis vs asthma and CAL Thyrotoxicosis can worsen asthma and CAL Thyrotoxicosis can worsen asthma and CAL Increase frequency and severity of asthma attack Increase frequency and severity of asthma attack Increase requirement for medication Increase requirement for medication Asthma attacks improve in an euthyroid state Asthma attacks improve in an euthyroid state

6. Possible cause Reactive oxygen species due to hyperthyroidism may be the contributing factor in exacerbating asthma Reactive oxygen species due to hyperthyroidism may be the contributing factor in exacerbating asthma

7. Reactive oxygen species Induce an autonomic imbalance between muscarinic receptor-mediated contraction and the beta-adrenergic-mediated relaxation of the pulmonary smooth muscle Induce an autonomic imbalance between muscarinic receptor-mediated contraction and the beta-adrenergic-mediated relaxation of the pulmonary smooth muscle Induce bronchoconstriction Induce bronchoconstriction Elevate mucus secretion Elevate mucus secretion Microvascular leakage Microvascular leakage These conditions worsens the respiratory conditions. These conditions worsens the respiratory conditions.

8. WHAT CLINICAL CHEMISTRY TESTS SHOULD BE PERFORMED TO RULE OUT HYPERTHYROIDISM & WHY?

9. HYPERTHYROIDISM? An elevation of free T4 An elevation of free T4 An elevation of free T3 An elevation of free T3 A very low TSH A very low TSH fine tremor (hands) fine tremor (hands) tachycardia tachycardia weight loss weight loss These are diagnostic signs & classic symptoms of hyperthyroidism These are diagnostic signs & classic symptoms of hyperthyroidism

10. Hyperthyroidism? Measurement of serum Thyrotropin - Thyroid Stimulating Hormone is considered the initial screening test in distinguishing hyperthyroid and primary hypothyroid states from euthyroid states Measurement of serum Thyrotropin - Thyroid Stimulating Hormone is considered the initial screening test in distinguishing hyperthyroid and primary hypothyroid states from euthyroid states

11. A suppressed TSH is the cornerstone of diagnosis of hyperthyroidism however, its secretion is influenced by many factors other than the negative feed back inhibition by t3 or t4 bmj 2000;320:1332-1334 A suppressed TSH is the cornerstone of diagnosis of hyperthyroidism however, its secretion is influenced by many factors other than the negative feed back inhibition by t3 or t4 bmj 2000;320:1332-1334 For example TSH can be reduced by fasting, glucocorticoids -exogenous & endogenous, stress, nonthyroidal illness, & false negative results For example TSH can be reduced by fasting, glucocorticoids -exogenous & endogenous, stress, nonthyroidal illness, & false negative results In the presence of the above the specificity of se TSH as a screening test is greatly reduced. In the presence of the above the specificity of se TSH as a screening test is greatly reduced.

12. Hyperthyroidism? In at least 90% of patients with hyperthyroidism T4 & T3 are elevated In at least 90% of patients with hyperthyroidism T4 & T3 are elevated In 5% of hyperthyroid patients T3 is exclusively elevated. In 5% of hyperthyroid patients T3 is exclusively elevated. In developing hyperthyroidism fT4 & fT3 are elevated before tT4 & tT3 In developing hyperthyroidism fT4 & fT3 are elevated before tT4 & tT3 Increased fT4 & decreased TSH is seen in conditions other than hyperthyroidism therefore need to measure fT3 as well Increased fT4 & decreased TSH is seen in conditions other than hyperthyroidism therefore need to measure fT3 as well

13. changes in TSH, T3 & T4 during systemic illness are poorly understood bmj 2000;320:1332-1334 changes in TSH, T3 & T4 during systemic illness are poorly understood bmj 2000;320:1332-1334 In very ill patients both T3 &T4 (free & total) are suppressed In very ill patients both T3 &T4 (free & total) are suppressed Free hormone assays are preferable to total levels as there is decreased protein binding of thyroid hormone - relevant to this patient : Free hormone assays are preferable to total levels as there is decreased protein binding of thyroid hormone - relevant to this patient : acute severe illness, acute severe illness, protein malnutrition protein malnutrition with the ingestion of steroids with the ingestion of steroids

14. Free T3 index levels can be measured to compensate for altered binding levels Free T3 index levels can be measured to compensate for altered binding levels These levels are derived from the total hormone levels & measurement of the distribution of radiolabelled t3 between unoccupied protein binding sites in the sample and an absorbent resin These levels are derived from the total hormone levels & measurement of the distribution of radiolabelled t3 between unoccupied protein binding sites in the sample and an absorbent resin Expect Free Thyroxine Index decreased in NTI & steroid administration. Expect Free Thyroxine Index decreased in NTI & steroid administration. Expect Reverse T3 normal / increased in NTI Expect Reverse T3 normal / increased in NTI

15. Very systemically ill Patients with low T4 levels have a poor prognosis/high morbidity Very systemically ill Patients with low T4 levels have a poor prognosis/high morbidity TFTs cannot be interpreted in patients with systemic illness bmj 2000;320:1332-1334 TFTs cannot be interpreted in patients with systemic illness bmj 2000;320:1332-1334 Doing more indiscriminate biochemical tests will lead to confusion not clarity bmj 2000;320:1332-1334 Doing more indiscriminate biochemical tests will lead to confusion not clarity bmj 2000;320:1332-1334

16. Significant false positive & negative TFT results seen in the presence of NTI as well as during the administration of glucorticoids Significant false positive & negative TFT results seen in the presence of NTI as well as during the administration of glucorticoids Repeat TSH, fT3 & fT4 after recovery from systemic illness. Repeat TSH, fT3 & fT4 after recovery from systemic illness. TSH can remain suppressed for months after starting treatment for hyperthyroidism even when T4 & T3 are normal TSH can remain suppressed for months after starting treatment for hyperthyroidism even when T4 & T3 are normal

17. Prolonged thyrotoxicosis can cause a number of non-specific biochemical abnormalities oxford textbook of medicine 2003 Abnormal LFTs Abnormal LFTs Hypercalcuria Hypercalcuria Elevated levels of serum ferritin Elevated levels of serum ferritin less common - less common - se calcium & phosphate raised se calcium & phosphate raised glucose intolerance glucose intolerance microcytic aneamia or thrombocytopenia microcytic aneamia or thrombocytopenia

18. How to measure Total and Free T3?

19. Overview Over 99% of Triiodothyronine (T3) circulates in blood bound to carrier proteins: thyroxine- binding globulins (TGB) Over 99% of Triiodothyronine (T3) circulates in blood bound to carrier proteins: thyroxine- binding globulins (TGB) Only the free (unbound) portion of T3 is responsible for its biological action. Only the free (unbound) portion of T3 is responsible for its biological action. The concentration of the carrier proteins may be altered but the total concentration of T3 will change so that the concentration of free T3 wil stay relatively constant. The concentration of the carrier proteins may be altered but the total concentration of T3 will change so that the concentration of free T3 wil stay relatively constant.

20. Overview (2) Thus, the concentration of free T3 correlates more reliabily than total T3 levels. Thus, the concentration of free T3 correlates more reliabily than total T3 levels. Serum T3 measurement has little specificity or sensitivity for diagnosing hypothyroidism, since enhanced T4 to T3 conversion maintains normal T3 concentrations until hypothyroidism becomes severe. Serum T3 measurement has little specificity or sensitivity for diagnosing hypothyroidism, since enhanced T4 to T3 conversion maintains normal T3 concentrations until hypothyroidism becomes severe.

21. Approaches to the measurement of total and free T3. Physical techniques: Equilibrium Dialysis, Ultrafiltration and Gel FiltrationOR Physical techniques: Equilibrium Dialysis, Ultrafiltration and Gel FiltrationOR Assay or Index Approaches: Estimate the free hormone concentration in the presence of protein-bound hormone. Assay or Index Approaches: Estimate the free hormone concentration in the presence of protein-bound hormone.

22. Equilibrium Dialysis Separates bound from free hormone. Separates bound from free hormone. Time-consuming, expensive, technically demanding and unavailable in most commercial laboratories. Time-consuming, expensive, technically demanding and unavailable in most commercial laboratories. Measuring serum free T 3 using overnight equilibrium dialysis of serum containing 125 I-T 3 Measuring serum free T 3 using overnight equilibrium dialysis of serum containing 125 I-T 3 The percentage of free T 3 is calculated by determining the total counts in the dialysate divided by the total 125 I-T 3 added to the serum multiplied by the total T 3 concentration. The percentage of free T 3 is calculated by determining the total counts in the dialysate divided by the total 125 I-T 3 added to the serum multiplied by the total T 3 concentration.

23. Index / Immunoassays The free hormone methods used by most clinical laboratories (indexes and immunoassays) do not employ physical separation of bound from free hormone and do not measure free hormone concentrations directly! The free hormone methods used by most clinical laboratories (indexes and immunoassays) do not employ physical separation of bound from free hormone and do not measure free hormone concentrations directly! These tests are typically binding protein dependent to some extent and should more appropriately be called "Free Hormone Estimate" tests, abbreviated FT4E and FT3E. These tests are typically binding protein dependent to some extent and should more appropriately be called "Free Hormone Estimate" tests, abbreviated FT4E and FT3E.

24. Index Methods: FT4I and FT3I Require two separate measurements:1. One test is a total hormone measurement (TT4 or TT3) the other 2. Is an assessment of the thyroid hormone binding protein concentration using either an immunoassay for TBG or a T4 or T3 "uptake" test called a Thyroid Hormone Binding Ratio (THBR). Require two separate measurements:1. One test is a total hormone measurement (TT4 or TT3) the other 2. Is an assessment of the thyroid hormone binding protein concentration using either an immunoassay for TBG or a T4 or T3 "uptake" test called a Thyroid Hormone Binding Ratio (THBR).

25. Indexes Using a Thyroid Hormone Binding Ratio (THBR) or "Uptake" Test "Classical" uptake tests add a trace amount of radiolabeled T3 or T4 to the specimen and allow the labeled hormone to distribute across the thyroid hormone binding proteins in exactly the same way as endogenous hormone. "Classical" uptake tests add a trace amount of radiolabeled T3 or T4 to the specimen and allow the labeled hormone to distribute across the thyroid hormone binding proteins in exactly the same way as endogenous hormone. The distribution of the tracer is dependent upon the saturation of the binding proteins. The distribution of the tracer is dependent upon the saturation of the binding proteins.

26. Indexes ctd. The free T 3 index is then calculated using the total T 3 and the TBG level. The free T 3 index is then calculated using the total T 3 and the TBG level. The index is directly proportional to the free T 3 level. The index is directly proportional to the free T 3 level.

27. Immunoassays The two most commonly used methods are a two-step and a one-step immunoassay method. The two most commonly used methods are a two-step and a one-step immunoassay method. These assays are not completely free of the influence of binding proteins or substances in serum that may result in false increases or decreases in the free T 4 / 3 levels These assays are not completely free of the influence of binding proteins or substances in serum that may result in false increases or decreases in the free T 4 / 3 levels

28. Two Step Immunoassay Two-step assays use a physical separation of free from protein-bound hormone before free hormone is measured by a sensitive immunoassay, or alternatively, an antibody is used to immunoextract a proportion of ligand out of the specimen before quantitation. Two-step assays use a physical separation of free from protein-bound hormone before free hormone is measured by a sensitive immunoassay, or alternatively, an antibody is used to immunoextract a proportion of ligand out of the specimen before quantitation.

29. One Step Immunoassay One-step ligand assays attempt to quantify free hormone in the presence of binding proteins. One-step ligand assays attempt to quantify free hormone in the presence of binding proteins. Important to maintain free to protein- bound equilibrium. Important to maintain free to protein- bound equilibrium.

30. Solid Phase competitive ELISA. The samples, assay buffer and T3 enzyme conjugate are added to the wells coated with anti- T3 monoclonal antibody. The samples, assay buffer and T3 enzyme conjugate are added to the wells coated with anti- T3 monoclonal antibody. FT3 in the patients serum competes with a T3 enzyme conjugate for the binding sites. FT3 in the patients serum competes with a T3 enzyme conjugate for the binding sites. Unbound enzyme conjugate is washed off by washing with buffer. Unbound enzyme conjugate is washed off by washing with buffer. Upon the addition of the substrate, the intensity of the colour is inversely proportional to the concentration to the FT3 Upon the addition of the substrate, the intensity of the colour is inversely proportional to the concentration to the FT3

31. The Application Of Total And Free T3 Levels In Evaluation Of Patient Status

32. Total And Free T3 Only 0.3% of the total T3 is freely available. Only 0.3% of the total T3 is freely available. Most of the T3 in circulation is bound to and transported by TBG (thyroxine binding globulin). Most of the T3 in circulation is bound to and transported by TBG (thyroxine binding globulin). Therefore abnormalities in levels of TBG can cause erroneous results in total and free T3. Therefore abnormalities in levels of TBG can cause erroneous results in total and free T3. Excess or lower TBG levels can be passed down as a hereditary trait. It causes no problems aside from causing false test increases or decreases in thyroid hormones. Excess or lower TBG levels can be passed down as a hereditary trait. It causes no problems aside from causing false test increases or decreases in thyroid hormones. People with this trait are often diagnosed as having a thyroid dysfunction in the absence of any real problem or need for treatment. People with this trait are often diagnosed as having a thyroid dysfunction in the absence of any real problem or need for treatment.

33. Serum free T3 levels are generally considered more reliable as they are less affected by carrier proteins (as the free T3 concentration is preserved by equilibrium.) Smaller amounts of T3 are bound to albumin and prealbumin. Smaller amounts of T3 are bound to albumin and prealbumin. Normal Levels Normal Levels Total T3: 80-180 ng/dl (nanograms/decilitre) Total T3: 80-180 ng/dl (nanograms/decilitre) Free T3: 230-619 pg/dl (picograms/decilitre) Free T3: 230-619 pg/dl (picograms/decilitre)

34. Measurement of Serum Thyroid Hormones Measurement of Serum Thyroid Hormones an indicator of thyroid function. an indicator of thyroid function. The most frequently used test is T4 by radioimmunoassay. This is referred to as a T7 The most frequently used test is T4 by radioimmunoassay. This is referred to as a T7 meaning that a resin T3 uptake or RT3u has been done to correct for certain medications such as birth control pills, other hormones, seizure medication, cardiac drugs, or even aspirin that may alter the routine T4 test. meaning that a resin T3 uptake or RT3u has been done to correct for certain medications such as birth control pills, other hormones, seizure medication, cardiac drugs, or even aspirin that may alter the routine T4 test. The T4 reflects the amount of thyroxine in the blood. If the patient does not take any type of thyroid medication, this test is usually a good measure of thyroid function. The T4 reflects the amount of thyroxine in the blood. If the patient does not take any type of thyroid medication, this test is usually a good measure of thyroid function. However thyroxine (T4) only represents 80% of thyroid hormone produced. However thyroxine (T4) only represents 80% of thyroid hormone produced.

35. The other 20% is tri-iodothyronine or T3. T3 levels are elevated in most patients with thyroid dysfunction and are therefore unreliable as an exclusive screen. The other 20% is tri-iodothyronine or T3. T3 levels are elevated in most patients with thyroid dysfunction and are therefore unreliable as an exclusive screen. T3 may be measured in cases where there is some doubt about whether the patient has hyperthyroidism or hypothyroidism after measuring T4 and RT3u. T3 may be measured in cases where there is some doubt about whether the patient has hyperthyroidism or hypothyroidism after measuring T4 and RT3u. where symptoms of hyperthyroidism are apparent but the thyroid gland is still producing normal levels of T4. where symptoms of hyperthyroidism are apparent but the thyroid gland is still producing normal levels of T4. They are also used to monitor response to therapy. They are also used to monitor response to therapy. Therefore T3 is measured as part of a thyroid function evaluation to provide a more accurate picture. Therefore T3 is measured as part of a thyroid function evaluation to provide a more accurate picture.

36. T3 Elevation Greater-than-normal levels may indicate: Hyperthyroidism (e.g., Graves’ disease) Hyperthyroidism (e.g., Graves’ disease) Serum T3 elevation parallels serum T4 in 90-95% of cases Serum T3 elevation parallels serum T4 in 90-95% of cases Isolated serum T3 elevation in ~ 5% of cases Isolated serum T3 elevation in ~ 5% of cases T3 thyrotoxicosis (rare) T3 thyrotoxicosis (rare) Isolated T3 elevation Isolated T3 elevation Thyroid Cancer (rare) Thyroid Cancer (rare) Thyroiditis Thyroiditis

37. T3 Reduction Lower-than-normal levels may indicate: - Chronic illness - Hypothyroidism (e.g., Hashimoto’s disease) - Starvation

38. Other Factors That May Affect T3 Measurement Drugs that can increase T3 measurements include: - clofibrate, estrogens, methadone, and oral contraceptives. Drugs that can decrease T3 measurements include: - anabolic steroids, androgens, antithyroid drugs (for example, propylthiouracil), lithium, phenytoin, and propranolol.