INAPPROPRIATE TRIIODOTHYRONINE (T3) AND THYROXINE (T4) RADIOIMMUNOASSAY LEVELS SECONDARY TO CIRCULATING THYROID HORMONE AUTOANTIBODIES

A 16-year-old boy with chronic lymphocytic thyroiditis was noted to have a low free thyroxine (T4) level, low triiodothyronine resin uptake (T3U), and high serum thyrotropin (TSH) values. Unexpectedly, markedly elevated T3 radioimmunoassay (RIA) and T4 (RIA) values, using a double antibody technique were obtained when performed directly on unextracted serum samples. Extremely low T4 (RIA) values were noted when polyethylene glycol (PEG) was used to separate bound from free hormone. The presence of circulating T3- and T4-binding immunoglobulins was suspected and confirmed with the following special studies. With undiluted serum in a T3 (RIA) system, using dextran-coated charcoal separation, 82% binding of ¹²⁵I-labelled T3 occurred in the absence of specific first antibody, with 55% binding retained at 1: 7 dilution with T3-free serum. Comparable results were obtained in the T4 (RIA) system using polyethylene glycol separation. Following ethanol extraction, low T4 (RIA) and low normal T3 (RIA) values were obtained, using a double antibody technique. There was ten-fold greater binding by the patient's serum to rabbit anti-human IgG in both the T3 and T4 radioassay systems as compared to controls. No preferential binding to rabbit anti-human IgM was noted. Scatchard plot analyses for the antibodies against T3 and T4 showed high affinity constants for these hormones. With adequate l -thyroxine therapy, an appropriate decline in serum TSH to normal was achieved. It is concluded that where RIA determinations of T3 and T4 are inconsistent with other laboratory and clinical indices, the presence of autoantibodies to thyroid hormones should be suspected and appropriate tests undertaken.     

Inherited abnormal thyroid hormone-binding protein causing selective increase of total serum thyroxine.

A 9-yr-old boy is described in whom increased serum T4 concentration, increased T3 uptake, and increased free T4 index were associated with a euthyroid clinical state with normal total serum T3. T4-binding globulin (TBG), measured by RIA, was decreased. Reverse flow paper electrophoresis of serum proteins after reaction with radioactively labeled T4 demonstrated increased binding of T4 to a protein with electrophoretic mobility corresponding to albumin. Displacement of serum protein-bo-nd [125I]T4 activity by increasing concentrations of T4 revealed the presence of a low affinity, high binding capacity system with an association constant similar to that of T4-binding prealbumin. This low affinity binding protein cochromatographed with TBG on a DEAE-Sephadex column which normally separates TBG from T4-binding prealbumin. At free T4 concentrations equivalent to those present in the plasma of normal individuals, the T4 bound to free ratio is higher in the patient than in normals and the total serum T4 level is increased in the presence of normal free T4 concentrations. The relative affinity of this abnormal T4-binding protein for T3 is low compared to that of TBG. The patient's father had the same abnormal binding protein, which was not found in his mother or fraternal twin brother. These data suggest an autosomal dominant mode of inheritance of an aberration leading to synthesis of a new protein instead of normal TBG. The new protein is different from TBG in electrophoretic mobility, T4 and T3 binding, and antigenic properties.     

Lower serum free thyroxine (T4) levels in painless thyroiditis compared with Graves' disease despite similar serum total T4 levels

Serum total T4 (T4), total T3 (T3), free T4 (FT4), free T3 (FT3), and T4-binding globulin concentrations and T3 resin uptake values were measured in 17 women with thyrotoxicosis due to painless thyroiditis (PT) and compared with the same parameters in 17 women with thyrotoxicosis due to Graves' disease (GD) with similar serum T4 levels. The mean serum T3 resin uptake value and T3, FT4, and FT3 concentrations in the PT patients were significantly lower than those in the GD patients. The mean serum T4-binding globulin concentration [20.2 +/- 4.2 (+/- SD) microgram/mL] in patients with PT did not differ significantly from those in patients with GD (18.0 +/- 2.6 micrograms/mL) and normal euthyroid women (21.9 +/- 4.0 micrograms/mL). The serum T3 to T4 (nanogram per microgram) ratio was higher than 20 in 14 GD patients, but lower than 20 in all patients with PT, whereas the individual serum FT3 to FT4 ratio values considerably overlapped in the 2 groups. In patients with PT, FT4 correlated well with T4 at various times during the clinical course. These findings indicate that the elevation in serum FT4 in patients with PT is mostly due to the increase in circulating T4 levels, whereas GD patients also have some diminution in T4 binding. The serum T3 to T4 ratio, but not the FT3 to FT4 ratio, may be helpful for differentiation between the two diseases.     

Circulating antitriiodothyronine autoantibodies in two euthyroid patients: apparent lack of interference in total T3 radioimmunoassay based on second antibody or solid phase separation techniques

Two clinically euthyroid patients were noted to have low total T3 levels as assessed by RIA using either dextran-charcoal (DC) or polyethylene glycol (PEG) for separation of bound from unbound T3, in spite of normal free T3, total and free T4 and basal and TRH-stimulated TSH concentrations. The presence of circulating substances binding T3 was suggested by high nonspecific binding in total T3 RIA system using either DC or PEG separation. The presence of anti-T3 autoantibodies was then suspected and confirmed by the presence of [125]-T3 bound to patients' gammaglobulins, precipitated with rabbit anti-human immunoglobulins. Serum T3 concentration determined by extracting T3 from patients' sera with methanol was 166 and 226 ng/dl. Similar or even lower values were unexpectedly obtained in RIA systems with solid phase or second antibody (anti-rabbit) separation and with competitive protein binding assay. To face this paradoxical finding, simulated experiments were carried out by incubating T3- and T4-free sera added with various amounts of stable T3 and T4 in the presence of goat anti-T3 or anti-T4 serum. These samples were then radioimmunoassayed. The DC separation caused a consistent underestimation of the actual T3 and T4 concentration. The second antibody separation caused a T3 and T4 overestimation for actual levels below 200 ng/dl and 10 micrograms/dl, respectively, while at the higher T3 or T4 concentrations, an overlap or, even, an underestimation of actual T3 or T4 levels were found. These data provide evidence that, with second antibody or solid phase separation methods, there could be an apparent lack of interfering effect of endogenously occurring antibodies.     

Antibody binding serum T3 in a patient with hepatocarcinoma

Thyroid hormone levels were studied in a euthyroid patient with hepatocellular carcinoma. The thyroid gland was normal at autopsy and both antithyroglobulin and antimicrosomal antibodies were undetectable in serum. Serum triiodothyronine (T3) values as measured by different RIA procedures, showed striking discrepancies suggesting the presence of an endogenous T3 binding antibody. The preincubation of the patient's serum with 125I-T3, followed by a precipitation with polyethyleneglycol showed a 74.8% of binding, confirming the presence of an endogenous factor interfering with T3 assays. Agarose electrophoresis of the patient's serum showed that 125I-T3 migrated mainly with the gammaglobulin fraction (60%). When immunoprecipitation tests with different antihuman antiimmunoglobulins were carried out, a positive binding for immunoglobulin G (11.9%), Fab (8.5%) and lambda chain (9.3%) was noted. Scatchard plot analysis showed a binding affinity of 0.77 X 10(9) liter/mol and a binding capacity of 1.02 nmol/liter. These data suggest that the abnormal serum T3 binding was caused by the presence of a T3 antibody which was shown to be an immunoglobulin G specific only for the lambda chain.     

Specific methods to identify plasma binding abnormalities in euthyroid hyperthyroxinemia

Methods to identify the plasma T4-binding abnormalities that can cause euthyroid hyperthyroxinemia were evaluated in patients with excess T4-binding globulin, familial dysalbuminemic hyperthyroxinemia, prealbumin-associated hyperthyroxinemia, and autoantibody binding of T4. Familial dysalbuminemic hyperthyroxinemic serum showed a unique persistence of abnormal [125I]T4 binding when diluted 1:100 in phosphate buffer with added 1000-fold excess of unlabeled T4 (10(-6) M T4). Immunoprecipitation of [125I]T4 by antibody to prealbumin, precipitation of [125I]T4 by polyethylene glycol 6000 19%, and in vitro resin uptake of T3 were specific for prealbumin-associated hyperthyroxinemia, autoantibody binding of T4, and T4-binding globulin excess, respectively. These simple methods facilitate investigation of patients with euthyroid hyperthyroxinemia and will identify individuals and families at risk of misdiagnosis by standard methods. Use of these techniques rules out the known binding abnormalities in hyperthyroxinemic patients and may make the diagnosis of generalized hormone resistance more specific.     

Triiodothyronine(T3) autoantibodies in a woman with nonthyroidal disorder: a study on preparation of serum IgG fraction employing protein A column chromatography

A 48-year-old non-goitrous woman, who had undergone cardiac surgery for mitral stenosis under the extracorporeal circulation, showed high levels of serum T3 and free T3 in a recent follow-up study, employing antibody coated-bead RIA for T3 and -Amerlex M particle RIA for free T3. However, other thyroid function tests (T4, free T4, TSH and TBG) were normal. We suspected that thyroid hormone autoantibodies (THAA) in her serum interfered with T3 and free T3 analyses. The presence of THAA was demonstrated by the use of various procedures as follows. Firstly, the patient's serum was directly incubated with 125I-T3 or -T4 analog which did not bind to TBG, followed by B/F separation with polyethyleneglycol, counting the precipitates. Secondly, after the serum was treated with an acid-charcoal solution to remove circulating thyroid hormone, the measurement of THAA was made as stated above. Normal sera were used as controls. Both the non- and acid-charcoal-treated sera showed much higher percentages of 125I-T3 analog precipitation as compared with controls. In the case of 125I-T4 analog, there was no difference between them. In the third study, the presence of IgG antibodies that bound T3 but not T4 was investigated. The IgG fraction of the patient's serum was separated employing a Protein A-Sepharose CL-4B column chromatography. Then, the prepared IgG fraction was purified by a technique of gel filtration chromatography (Sephacryl S 200). Non-purified and purified-IgG fractions both revealed higher binding percentages of 125I-T3 analog than the control IgG fraction and non-IgG fraction of the patient. Furthermore, a good dose response was observed between the binding percentage of 125I-T3 analog and each dose of the patient's serum or IgG fraction. From these observations, it was clarified that this woman had anti-T3 IgG autoantibodies using a Protein A column chromatography with confirmation of gel filtration chromatography.     

Thyroid disease with monoclonal (immunoglobulin G lambda) antibody to triiodothyronine and thyroxine

A man with previous Graves' disease spontaneously developed hypothyroidism. He became euthyroid with T4 therapy, but developed inappropriately elevated serum levels of T3 and, to a lesser extent, T4. Gel filtration analysis (Sephadex G-150) of serum trace-labeled with [125I]T3 revealed binding to a high molecular weight fraction, distinct from normal T3-binding proteins. This abnormal activity cochromatographed with serum immunoglobulin G (IgG) by DEAE-cellulose chromatography and gel filtration, and was retained by the F(ab)2 fragment of IgG, indicating its true antibody nature. By isoelectric focussing, there was restricted heterogeneity of the [125I]T3-antibody complex (pI 9.0-9.1), and the antibody was identified as an IgG (lambda) monoclonal Ig by immune precipitation. Antigenic cross-reactivity with T4 was demonstrated by inhibition of hapten binding. The affinity of the antibody for T3 was high (Ka = 0.9 x 10(9) liter mol-1), and the T3 binding capacity of the antibody in serum was estimated as 1132 ng/dl, equivalent to 1.39 mg T3-specific IgG/liter (0.014% of the total serum IgG). This binding capacity was similar to the serum T3 values (1100-1300 ng/dl) at which transition from hypothyroid to euthyroid states was observed, as judged by clinical examination and measurement of serum TSH levels.     

Dependence of the mitochondrial uptake of triiodothyronine (T3) in rat kidney on cytosolic T3-binding protein

The effect of cytosolic T3-binding protein (CTBP) on mitochondrial T3 uptake was investigated by using rat kidney tissue in vitro. [125I]T3 uptake into mitochondria was time dependent in the absence of CTBP. Addition of CTBP to the incubation medium significantly increased mitochondrial [125I]T3 uptake. When mitochondria were incubated with a [125I]T3-CTBP complex, [125I]T3 uptake into mitochondria continued to be time dependent, but the amount of [125I]T3 incorporated was greater than in mitochondria incubated with free [125I]T3. Although [125I]T3 uptake was not significantly inhibited by an excess of unlabeled T3, it was markedly inhibited by an excess of unlabeled T3-CTBP complex. The degree of inhibition was related to the concentration of T3-CTBP complex. However, [125I]T3 uptake produced by incubation of mitochondria with [125I]T3-CTBP complex was not inhibited by unsaturated CTBP. [125I]T3 binding to outer mitochondrial membranes was observed when the membranes were incubated with free [125I]T3 or [125I]T3-CTBP complex; the amount of [125I]T3 bound was greater in the membranes incubated with [125I]T3-CTBP complex. [125I]T3 binding to the membranes incubated with [125I]T3-CTBP complex was displaced by the addition of unlabeled T3-CTBP complex. These results suggest that mitochondrial T3 uptake is mediated by previous binding of T3 to CTBP, and that the uptake is possibly regulated by T3-CTBP complex binding to its receptor in the outer mitochondrial membrane.     

THE RADIOIMMUNOASSAY OF 3,3',5'-TRHODOTHYRONINE (REVERSE T3) IN UNEXTRACTED HUMAN SERUM

A specific, sensitive and simple double antibody radioimmunoassay for total serum 3,3′,5′,-triiodothyronine (reverse T3, rT3) in small volumes of unextracted human serum is described. High titre antisera were raised in rabbits using dl -rt 3 or l -rT3 conjugated to bovine serum albumin. The selected antisera cross reacted less than 0–003% with triiodothyronine (T3) and 0–14% with thyroxine (T4). A stable high activity rT3 tracer was prepared by iodination of 3,3′-diiodo-l -thyronine by the chloramine-T method, and purified by column chromatography on Sephadex LH-20. Binding of rT3 to endogenous serum proteins was blocked by including 8-anilino-l-naphthalene sulphonic acid (ANS) in the assay. Mean rT3 levels in healthy euthyroid adults are 0.27 nmol/1 (range 015–0.42); in euthyroid patients 0.29 nmol/1 (range 0.11–0.80); in thyrotoxic subjects 1.26 nmol/1 (range 0.41–4.66); in T3 thyrotoxic subjects 0.47 nmol/1 (range 0.21–1.18); in cord sera 3.67 nmol/1 (range 2.30–7.45) and in amniotic fluid taken during the second trimester 4.70 nmol/1 (range 2.22–8.00). In approximately half the hypothyroid subjects, rT3 levels were undetectable (<0.05 nmol/1) and in the remaining subjects the mean rT3 level was 0.10 nmol/1 (range 0.05–0.25).     

Three novel mutations causing complete T(4)-binding globulin deficiency

Inherited T(4)-binding globulin deficiency is caused by mutations in the T(4)-binding globulin gene located on the X chromosome. We describe herein three novel mutations in three different families producing complete T(4)-binding globulin deficiency. The proposita of a family from Harwichport is a female with XO Turner's syndrome who expressed only the mutant T(4)-binding globulin allele. Her T(4)-binding globulin sequence has a 19-nucleotide deletion in the distal portion of exon 4. This causes a frameshift and a premature stop at codon 384 of the mature protein. Structure analysis with the Swiss PDB-Viewer revealed that this mutation removes beta-strand s5B from the core of the T(4)-binding globulin molecule, leading to a severe folding defect that is likely to prevent synthesis and secretion. The propositi of complete T(4)-binding globulin deficiency 7 and 8 were 7-month-old and 3-wk-old male infants who were identified because of low serum T(4) levels detected during neonatal screening. Sequencing of complete T(4)-binding globulin deficiency 7 revealed a single nucleotide deletion, a G at position 2690 in exon 3. This leads to an alteration of the amino acid sequence starting at codon 283 and a premature stop at codon 301. Complete T(4)-binding globulin deficiency 8 also has a deletion of the first nucleotide of exon 4, a G at position 3358. This leads to a frameshift and a premature stop at codon 374. As in the case of complete T(4)-binding globulin deficiency J, which has also a nucleotide deletion but downstream (position 3421) and a stop at codon 374, these two T(4)-binding globulin mutants undoubtedly have a defect in intracellular transport and therefore fail to be secreted. This explains the lack of T(4)-binding globulin in the hemizygous affected subjects.     

A case report of Hashimoto's disease with anti-thyroxine autoantibody (author's transl)]

In one case of untreated Hashimoto's disease, serum thyroxine (T4) value by radioimmunoassay (RIA) was significantly lower than that by competitive protein binding analysis (CPBA). The discrepancy was found to be due to the presence of antithyroxine autoantibody in the serum. This phenomenon was considered to be of practical importance in interpreting the T4 value by RIA in cases with autoimmune thyroid diseases. The patient was 59-year-old woman with a 30-year history of goiter. A diagnosis of Hashimoto's thyroiditis had been established by open biopsy of the thyroid ten years ago. The patient was judged to be euthyroid on the basis of clinical and laboratory evaluation (mean serum T4 by CPBA (Tetrasorb and Tetratab kit), 5.0 mug/100 ml; serum T3, 165 ng/100 ml; T3 resin uptake, 31.8%; and serum TSH, 2.0 muU/ml). TBG binding capacity was 24 mug/100 ml. Anti-thyroglobulin antibodies (anti-Tg), once positive ten years before, was negative at this time. But the mean T4 in the serum measured by T4 RIA and RIA-Mat T4 kit were 1.7 and 2.9 mug/100 ml, respectively. Recovery of the T4 added to the patient's serum evaluated by RIA-Mat T4 kit, was 71.2%, although the recovery using a control serum was 108%. Binding of 125I-T4 to the serum or fractions of the serum was studied by using polyethylene glycol (PEG) method, column chromatography, and double antibody precipitation. The results were as follows: 1) The binding of 125I-T4 to the patient's serum was detected by using RIA kit system without addition of anti-T4 serum. 2) On Sephadex G-200 chromatography of 125I-T4 incubated with the serum or the rabbit anti-T4 antibody in the presence of ANS, an early radioactive peak was observed by using the patient's serum as in the case of the anti-T4 antibody. When the serum after thermal inactivation of TBG, was incubated with 125I-T4, and was applied to the Sephadex G-200 column, a radioactive peak was observed in the area where 7S fraction was detected by protein peak. 3) The binding of 125I-T4 to the patient's IgG was 9.0% by using double antibody method when the binding to a control IgG was 0.5%. 4) The binding of 125I-T4 to IgG fractions was also proved by PEG method. 5) The binding of 125I-T4 was competitively inhibited by the addition of unlabeled T4. The affinity constant was 1.9 X 10(8) L/mol and its binding capacity was 0.8 mug/100 ml serum. From these data this T4 binding IgG was considered to be anti-T4 autoantibody. The cross reaction with T3 was approximately 8.3%. MIT and DIT did not displace labeled T4 when tested in amounts varying from 0.1 to 100 ng/assay. By using the paper electrophoresis, the binding of 125IT4 to the serum or IgG was not detectable. Therefore this method was considered unsuitable for detecting such anti-T4 antibody. As we couldn't find any significant binding of 125I-T4 to sera in 37 other patients with Hashimoto's disease by using the PEG method, the incidence of this phenomenon was considered to be low...     

A new case of familial partial generalized resistance to thyroid hormones: study of 3,5,3'-triiodothyronine (T3) binding to lymphocyte and skin fibroblast nuclei and in vivo conversion of thyroxine to T3

A clinically euthyroid 30-yr-old man with high serum levels of both total (T4, 14.5 micrograms/dl; T3, 272 ng/dl) and free (FT4, 33 pg/ml; FT3, 9.7 pg/ml) thyroid hormones and inappropriately normal TSH levels, both basally and after TRH stimulation, is described. Peripheral indices of thyroid hormone action and the patient's clinical status were not modified by the prolonged administration of supraphysiological doses of both T4 (up to 900 micrograms/day) and T3 (up to 80 micrograms/day), which decreased but did not completely abolish the TSH response to TRH. However, the TSH response to TRH was normally blunted by dexamethasone administration, which also reduced serum T4 and T3 levels to normal. T3 binding to nuclei of mononuclear leukocytes and cultured skin fibroblasts was normal. The overall pattern demonstrates that the patient was affected by partial peripheral resistance to thyroid hormone action. Study of the patient's family revealed the same hormone pattern in the patient's father, suggesting an autosomal dominant mode of inheritance. An in vivo study performed after the iv injection of tracer doses of [125I]T4 and [131I]T3, demonstrated increased production rates (PR) of both T4 [PR, 113.0 micrograms/day X m2; normal subjects, 55.4 +/- 12.3 (mean +/- SD); n = 13] and T3 (PR, 41.1 micrograms/day X m2; normal subjects, 16.3 +/- 2.7). In vivo conversion of T4 to T3 was also evaluated in the patient; a nearly normal T4 to T3 conversion factor was found (0.3108 vs. 0.2576 +/- 0.0422 in normal subjects). In four hyperthyroid patients, the T4 to T3 conversion factors were similar (0.2932 +/- 0.0600), while the PRs of T4 and T3 were increased (PR of T4, 308.6 +/- 85.6; PR of T3, 110.3 +/- 35.0 micrograms/day X m2) compared to those in the normal subjects.     

Interference of T4 in different serum T3 radioimmunologic determination kits

The cross reactivity of T3 antibodies for T4 was studied with five T3 RIA kits. T4 used was provided from T4 RIA Kits (Abbot Beckman, Corning). The results are shown on the table indicating the concentration of T3 or T4 required to displace 50% of the T3 125 from anti T3. The cross reactivity for T4 of T3 antibodies from Lepetit and Phadebas was the same and was smaller than cross reactivity with the other laboratories. In clinical practice serum T4 levels of hypo and euthyroid subjects are not so high to false T3 determination. In hyperthyroid subjects the diagnosis cannot be influenced by this cross reactivity. However, the study of the kinetic and monodeiodination of T4 into T3 may be wrong when T4/T3 ratio increases.     

Seasonal changes in serum thyroid hormone binding proteins in the woodchuck (Marmota monax)

To evaluate the role of serum T4- and T3-binding proteins in the elevation of serum T4 and T3 concentrations in the woodchuck in the fall and winter, blood was collected from woodchucks during the four seasons of the year (seasonal study) and from 2-week fasted woodchucks in the summer (fasting study) and the serum concentrations of total T4 and T3 were measured. The distribution of [125I]4 and [125I]T3 tracers among the serum binding proteins and the serum T4-binding globulin (TBG) binding capacity for T4 was determined by polyacrylamide gel electrophoresis. Plasma concentrations of both T4 and T3 were highest in the winter. The major T4-binding protein in the woodchuck is TBG. There was an increase in both [125I]T4 and [125I]T3 tracer binding to serum TBG in fall and winter, and TBG binding capacity for T4 was 2-fold higher in winter than in summer. There were increases in TBG binding and in the TBG T4 binding capacity in the 2-week starved animals. The increased binding of T4 and T3 by TBG in the fall and winter may be partially responsible for the increased serum concentrations of T4 and T3 in the fall and winter in the woodchuck, a time when secretion of T4 and T3 by the thyroid gland is very low. This may be facilitated by the low or absent food consumption at these times of the year.     

Hyperthyroxinemia due to the coexistence of two raised affinity thyroxine-binding proteins (albumin and prealbumin) in one family

The T4-binding proteins of a euthyroid subject with persistent hyperthyroxinemia (T4, greater than 20 micrograms/dl) were present in normal concentrations. Abnormal transport of both T4 and rT3 was demonstrated by reverse flow paper electrophoresis; excess T4 was bound to albumin and prealbumin, while increased binding of rT3 was confined to prealbumin. The three T4-binding proteins in the serum of the subject were isolated by affinity chromatography and characterized. Equilibrium dialysis experiments demonstrated a 20-fold increase in affinity of the albumin for T4 (Ka, 5.1 X 10(6) M-1) and a 4-fold increase in affinity of prealbumin for T4 (Ka, 3.0 X 10(8) M-1); T4-binding globulin affinity was normal. Nine other members of the family were also studied. Two sisters of the propositus have both the abnormal albumin and the variant prealbumin, while a brother has normal T4-binding proteins. The mother has the abnormal albumin alone. The father, his sister, and one of his three brothers have the variant prealbumin only. Despite the presence of the variant prealbumin in some of the paternal relatives of the propositus, their total iodothyronine concentrations were within the normal ranges; the condition may, therefore, often go undetected. The characteristics of the albumin found in the affected members of this kindred are those we have defined for familial dysalbuminemic hyperthyroxinemia type I, which is inherited as an autosomal dominant trait. The pattern of inheritance of the variant prealbumin is also consistent with a dominant mode with strong penetrance. The presence of two separately inherited abnormal T4 transport proteins in the same family suggests that both conditions may be more common than has been thought.     

RAPID COMMUNICATION: A COMPARISON OF FREE T4 AND THE RATIO OF TOTAL T4 TO T4-BINDING GLOBULIN IN SERUM THROUGH PREGNANCY

Serum free T4 concentrations have been variously reported as either constant or falling in pregnancy. In this study, 122 serum samples from apparently normal pregnancies were used to derive euthyroid ranges throughout pregnancy for T4 and free T 4 (measured by the Amerlex kits) and for T4-binding globulin (TBG). The mean free T4 concentration fell with increasing gestational age, the range of values in the third trimester being narrower than the non-pregnant normal range. Evidence is reviewed which shows that the Amerlex free T4 assay is unaffected by the elevation of TBG in non-pregnant subjects. The decline in mean free T4 as pregnancy proceeds is in good agreement with the change in free T4 predicted by the falling T4/TBG ratio.     

A radioimmunoassay for 3',5'-diiodothyronine.

The present report describes a RIA for 3',5'-diiodothyronine (T2) that can be performed on unextracted serum and which has a lower limit of detectability of 2 ng/dl. Cross-reactivity with other iodothyronines was negligible, except for rT3 which began to demonstrate cross-reactivity when rT3 levels were elevated to 180 ng/dl. Employing this RIA for T2, we have determined that 83 healthy individuals had a mean (+/-SE) serum T2 concentration of 5.0 +/- 0.3 ng/dl, thyrotoxic subjects (n = 12) had a mean T2 level that was elevated to 10.8 +/- 0.8 ng/dl, and each of 6 hypothyroid subjects had undetectable (less than 2 ng/dl) concentrations. Athyreotic patients (n = 8), receiving 0.4 mg T4 daily, had serum T2 concentrations of 15.0 +/- 3.0 ng/dl. Fasting in obese subjects was associated with an increase in serum T2 to 6.9 +/- 0.6 ng/dl from a basal level of 4.4 +/- 0.4 ng/dl in the fed state (P less than 0.01). Despite the fact that rT3 levels may be elevated in amniotic fluid and that rT3 is expected to represent the major source from which extrathyroidal T2 arises, T2 levels were low in amniotic fluid, being undetectable (less than 2 ng/dl) in 9 of 19 samples; the mean (+/-SE) T2 concentration in the 10 detectable samples was 5.4 +/- 1 ng/dl. These data indicate T2 is a normal component of serum and that the majority of serum T2 is probably derived from peripheral conversion. Furthermore, these observations suggest that situations associated with elevated rT3 levels (e.g. thyrotoxicosis and fasting) may also have increased T2 values.     

Immersion of rainbow trout in 3,5,3'-triiodo-L-thyronine (T3): effects on plasma T3 levels and hepatic nuclear T3 binding

An immersion protocol was established for chronically administering thyroid hormone to fish. Rainbow trout were immersed in solutions of 3,5,3'-triiodo-L-thyronine (T3) at 11 degrees C for various periods. Most plasma T3 adjustment occurred by 24 hr and within 5 days consistent plasma T3 levels were observed. Availability of unoccupied hepatic nuclear T3-binding sites was assessed in two experiments by uptake of nuclear radioactivity in T3-immersed trout injected 12 hr previously with [125I]T3. At low ambient T3 levels over 92% of nuclear radioactivity was identified chromatographically and immunologically as T3, but at high ambient levels (25 micrograms/100 ml) biliary [125I]T3 metabolites may contaminate the nuclear fraction. Displacement of [125I]T3 from nuclei was first detected at 0.25 micrograms T3/100 ml water, corresponding to a plasma T3 level of 0.55 ng/ml; maximum displacement, depending on experiment, occurred at 1.25 and 3.8 micrograms T3/100 ml, corresponding to plasma T3 levels of 14 and 9 ng/ml. In conclusion, chronic physiologic T3 treatment of these trout can be achieved by immersion in T3 solutions of 0.1-5 micrograms T3/100 ml water.