Measurements of Iodine-Containing Hormone Precursors and Products of Degradation

The last two decades have witnessed the development of RIAs for the measurement of a number of naturally occurring, iodine-containing substances that possess little if any thyromimetic activity. Some of these substances are products of T4 and T3 degradation in peripheral tissues. Others are predominantly, if not exclusively, of thyroidal origin. Since they are devoid of significant metabolic activity, measurement of their concentration is of value only in the research setting in detecting abnormalities in the metabolism of thyroid hormone in peripheral tissues, as well as defects of hormone synthesis and secretion.

3,3',5'-Triiodothyronine or Reverse T3 (rT3). rT3 is principally a product of T4 degradation in peripheral tissues (see Chapter 3). It is also secreted by the thyroid gland, but the amounts are practically insignificant.¹²⁶ Thus, measurement of rT3 concentration in serum reflects both tissue supply and metabolism of T4 and identifies conditions that favor this particular pathway of T4 degradation.

When total rT3 (TrT3) is measured in unextracted serum, a competitor of rT3 binding to serum proteins must be added.¹²⁷ Several chemically related compounds may cross-react with the antibodies. The strongest cross-reactivity is observed with 3,3'-T2 but this does not present a serious methodologic problem because of its relatively low levels in human serum. Though cross-reactivity with T3 and T4 is lesser, these compounds are more often the cause of rT3 overestimation due to their relative abundance, particularly in thyrotoxicosis.¹²⁸ Free fatty acids interfere with the measurement of rT3 by RIA.¹²⁹

The normal range in adult serum for TrT3 is 14-30 ng/dl (0.22 - 0.46 nmol/L) although varying values have been reported. It is elevated in subjects with high TBG and in some individuals with FDH.¹³² Serum TrT3 levels are normal in hypothyroid patients treated with T4, indicating that peripheral T4 metabolism is an important source of circulating rT3.^126,133 Values are high in thyrotoxicosis and low in untreated hypothyroidism. High values are normally found in cord blood and in newborns.^133,134

With only a few exceptions, notably uremia, serum TrT3 concentrations are elevated in all circumstances that cause low serum T3 levels in the absence of obvious clinical signs of hypothyroidism. These conditions include, in addition to the newborn period, a variety of acute and chronic nonthyroidal illnesses, calorie deprivation, and the influence of a growing list of clinical agents and drugs (see Table 5-3).

Current clinical application of TrT3 measurement in serum is in the differential diagnosis of conditions associated with alterations in serum T3 and T4 concentrations when thyroid gland and metabolic abnormalities are not readily apparent.

The dialyzable fraction of rT3 in normal adult serum is 0.2 - 0.32%, or approximately the same as that of T3. The corresponding serum FrT3 concentration is 50 - 100 pg/dl (0.77 - 1.5 pmol/L). In the absence of gross TBG abnormalities, variations in serum FrT3 concentration closely follow those of TrT3.¹⁰¹

3,5-Diiodothyronine (3,5-T2). The normal adult range for total 3,5-T2 in serum measured by direct RIAs is 0.20 - 0.75 ng/dl (3.8 - 14 pmol/L).¹³⁵ That 3,5-T2 is derived from T3 is supported by the observations that conditions associated with high and low serum T3 levels have elevated and reduced serum concentrations of 3,5-T2, respectively.¹³⁶ Thus, high serum 3,5-T2 levels have been reported in hyperthyroidism, and low levels in serum of hypothyroid patients, newborns, during fasting, and in patients with liver cirrhosis.

3,3'-Diiodothyronine (3,3'-T2). Normal concentrations in adults probably range from 1 to 8 ng/dl (19 - 150 pmol/L).¹³⁷ Levels are clearly elevated in hyperthyroidism and in the newborn. Values have been found to be either normal or depressed in nonthyroidal illnesses,¹³⁷ in agreement with the demonstration of reduced monodeiodination of rT3 to 3,3'-T2.¹³⁸ In vivo turnover kinetic studies and measurement of 3,3'-T2 in serum after the administration of T3 and rT3 have clearly shown that 3,3'-T2 is the principal metabolic product of these two triiodothyronines.

3',5'-Diiodothyronine (3',5'-T2). Reported concentrations in serum of normal adults have a mean overall range of 1.5 - 9.0 ng/dl (30 - 170 pmol/L).^139,140 The substances that principally cross react in the assay are rT3, 3,3-LT2 and 3-T1. Values are high in hyperthyroidism and in the newborn.^139,140 Being the derivative of rT3 monodeiodination,¹³⁹ 3',5'-T2 levels are elevated in serum during fasting^140,141 and in chronic illnesses¹³³ in which the level of the rT3 precursor is also high. Administration of dexamethasone also produces an increase in the serum 3',5'-T2 level.¹³⁹

3'-Monoiodothyronine (3'-T1). The concentration of 3'-T1 in serum of normal adults, measured by RIA, has been reported to range from 0.6 to 2.3 ng/dl (15 - 58 pmol/L)¹³³ and from <0.9 to 6.8 ng/dl (<20 - 170 pmol/L). Its two immediate precursors, 3,3,'-T2 and 3',5'-T2 are the main cross-reactants in the RIA. Serum levels are very high in hyperthyroidism and low in hypothyroidism. The concentration of 3'-T1 in serum is elevated in all conditions associated with high rT3 levels, including newborns, nonthyroidal illness, and fasting.¹³⁴ This finding is not surprising since the immediate precursor at 3'-T1 is 3',5'-T2,¹⁴² a product of rT3 deiodination, which is also present in serum in high concentration under the same circumstances. The elevated serum levels of 3'-T1 in renal failure are attributed to decreased clearance since the concentrations of its precursors are not increased.

3-Monoiodothyronine (3-T1). Experience with the measurement of 3-T1 in serum is limited. Normal values in serum of adult humans using 3H labeled 3-T1 in a specific RIA ranged from <0.5 - 7.5 ng/dl (<13 - 190 pmol/L).¹⁴³ The mean concentration of 3-T1 in serum of thyrotoxic patients and in cord blood was significantly higher. 3-T1 appears to be a product of in vivo deiodination of 3,3'-T2.

Tetraiodothyroacetic Acid (TETRAC or T4A) and Triiodothyroacetic Acid (TRIAC or T3A). The iodoamino acids T4A and T3A, products of deamination and oxidative decarboxylation of T4 and T3, respectively, have been detected in serum by direct RIA measurements.^21,76,144 Reported mean concentrations in the serum of healthy adults have been 8.7 ng/dl¹⁴⁴ and 2.6 ng/dl (range, 1.6 - 3.0 ng/dl or 26 - 48 pmol/L))²¹ for T3A and 28 ng/dl (range <8 - 60 mg/dl or <105 - 800 pmol/L)⁷⁶ for T4A. Serum T4A levels are reduced during fasting and in patients with severe illness,¹⁴⁵ although the percentage of conversion of T4 to T4A is increased.^20,146 The concentration of serum T3A remains unchanged during the administration of replacement doses of T4 and T3.²¹ It has been suggested that intracellular rerouting of T3 to T3A during fasting is responsible for the maintenance of normal serum TSH levels in the presence of low T3 concentrations.¹⁴⁷

3,5,3'-T3 Sulfate (T3S). A RIA procedure to measure T3S in ethanol extracted serum samples is available.²² Concentrations in normal adults range from 4-10 ng/dl (50-125 pmol/L). Although the principal source of T3S is T3, and the former binds to TBG, values are high in newborns and low in pregnancy. This suggests different rates of T3S generation or metabolism in mother and fetus. T3S values are high in thyrotoxicosis and in nonthyroidal illness.

Diiodotyrosine (DIT) and Monoiodotyrosine (MIT). Although RIA methods for the measurement of DIT and MIT have been developed, due to limited experience, their value in clinical practice remains unknown. Early reports gave a normal mean value for DIT in serum of normal adults of 156 ng/dl (3.6 nmol/L),¹⁴⁸ with progressive decline due to refinement of techniques to values as low as 7 ng/dl with a range of 1 - 23 ng/dl (0.02 - 0.5 nmol/L).¹⁴⁹ Thus, the normal range for MIT of 90 - 390 ng/dl (2.9 - 12.7 nmol/L)¹⁵⁰ is undoubtedly an overestimation. Iodotyrosine that has escaped enzymatic deiodination in the thyroid gland appears to be the principal source of DIT in serum. Iodothyronine degradation in peripheral tissues is probably a minor source of iodotyrosines since administration of large doses of T4 to normal subjects produces a decline rather than an increase in the serum DIT level.¹⁴⁹ DIT is metabolized to MIT in peripheral tissues. Serum levels of DIT are low during pregnancy and high in cord blood.

Thyroglobulin (Tg). RIA methods were those first used routinely for measurement of Tg in serum,¹⁵¹, although other assays methods employing IRMA, ICMA, and ELISA technology have been reported ^151a-d and are gaining increasing popularity. They are specific and, depending upon the sensitivity of the assay, capable of detecting Tg in the serum of approximately 90% of the euthyroid healthy adults. When antisera are used in high dilutions, there is virtually no cross-reactivity with iodothyronines or iodotyrosines. Results obtained from the analysis of sera containing Tg autoantibodies may be inaccurate, depending upon the antiserum employed.¹⁵² The presence of thyroid peroxidase antibodies does not interfere with the Tg RIA. Despite the relaibility of measurements of serum Tg, it is clear that different assay methods may result in values discrepant by up to 30%, even though refernce preparations are available.^152a Typically, IMA methods underestimate the serum Tg value, while RIA methods overestimate it, so it is essential that clinical decisions are based upon serial measurements using the same assay.

Tg concentrations in serum of normal adults range from <1 to 25 ng/ml (<1.5 - 38 pmol/L), with mean levels of 5 - 10 ng/ml.^151,153-155 On a molar basis, these concentrations of Tg are minute relative to the circulating iodothyronines; 5,000-fold lower than the corresponding concentration of T4 in serum. Values tend to be slightly higher in women than in men.¹⁵¹ In the neonatal period and during the third trimester of pregnancy, mean values are approximately 4- and 2-fold higher.^154,156 They gradually decline throughout infancy, childhood and adolescence.¹⁵⁷ The positive correlation between the levels of serum Tg and TSH indicates that pituitary TSH regulates the secretion of Tg.

Elevated serum Tg levels reflect increased secretory activity by stimulation of the thyroid gland or damage to thyroid tissue, whereas values below or at the level of detectability indicate a paucity of thyroid tissue or suppressed activity. Tg levels in a variety of conditions affecting the thyroid gland have been reviewed¹⁵⁸ and are listed in Table 6-6.

Interpretation of a serum Tg value should take into account the fact that Tg concentrations may be high under normal physiologic conditions or altered by drugs. Administration of iodine and antithyroid drugs increase the serum Tg level, as do states associated with hyperstimulation of the thyroid gland by TSH or other substances with thyroid-stimulating activity. This is due to increased thyroidal release of Tg rather than changes in its clearance.¹⁵⁹ Administration of TRH and TSH also transiently increases the serum level of Tg.¹⁶⁰ Trauma to the thyroid gland, such as that occurring during diagnostic and therapeutic procedures including percutaneous needle biopsy, surgery, or 131I therapy, can produce a striking, although short-lived, elevation in the Tg level in serum.^154,161,162 Pathological processes with destructive effect on the thyroid gland also produce transient, though more prolonged increases.¹⁶³ Tg is undetectable in serum after total ablation of the thyroid gland as well as in normal persons receiving suppressive doses of thyroid hormone.¹⁵⁸ It is thus a useful test in the differential diagnosis of thyrotoxicosis factitia,¹⁶⁴ especially when transient thyrotoxicosis with a low RAIU or suppression of thyroidal RAIU by iodine are alternative possibilities.

The most striking elevations in serum Tg concentrations have been observed in patients with metastatic differentiated nonmedullary thyroid carcinoma even after total surgical and radioiodide ablation of all normal thyroid tissue.^154,165 It usually persists despite full thyroid hormone suppressive therapy, suggesting excessive autonomous release of Tg by the neoplastic cells. The determination is thus of particular value in the follow-up and management of metastatic thyroid carcinomas, particularly when they fail to concentrate radioiodide.^153,165 Follow-up of such patients with sequential serum Tg determinations helps the early detection of tumor recurrence or growth and the assessment of the efficacy of treatment. Measurement of serum Tg is also useful in patients with metastases, particularly to bone, in whom there is no evidence of a primary site and thyroid malignancy is being considered in the differential diagnosis.^154,165 On the other hand, serum Tg levels are of no value in the differential diagnosis of primary thyroid cancer because levels may be within the normal range in the presence of differentiated thyroid cancer and high in a variety of benign thyroid diseases.^153,155,165 Whether early detection of recurrent thyroid cancer after initial ablative therapy could be achieved by serum Tg measurement without cessation of hormone replacement therapy is debated because Tg secretion by the tumor is modulated by TSH and is suppressed by the administration of thyroid hormone.^166-168 Detectable serum thyroglobulin during thyroid hormone suppression reliably indicated the presence residual or recurrent disease.

Tg levels are high in the early phase of subacute thyroiditis.¹⁶³ Declining serum Tg levels during the course of antithyroid drug treatment of patients with Graves' disease may indicate the onset of a remission.^162,169 Tg may be undetectable in the serum of neonates with dyshormonogenetic goiters due to defects in Tg synthesis¹⁷⁰ but are very high in some hypothyroid infants with thyromegaly or ectopy.¹⁷¹ Measurement of serum Tg in hypothyroid neonates is useful in the differentiation of infants with complete thyroid agenesis from those with hypothyroidism due to other causes, and thus in most cases obviates the need for the diagnostic administration of radioiodide.^171,172