Babassu (Orbignya phalerata), a palm-tree coconut fruit, mixed with mandioca (Manihot utilissima) is the staple food of people living in the endemic goiter area of Maranhao in Brazil, where goiter prevalence among schoolchildren was still 38% in 1986 despite an adequate iodine intake in most of the population. Therefore, the question arose as to whether or not the ingestion of babassu alone or mixed with mandioca contributed to the persistence of endemic goiter in this area of Brazil. In this investigation we examined the potential antithyroid effects of babassu and mandioca by means of in vivo studies in Sprague-Dawley rats, in vitro studies in porcine thyroid slices and using a purified porcine thyroid peroxidase (TPO) system. Samples of various edible parts of babassu and mandioca flour were homogenized and extracted in goitrogen-free water (GFW) for in vivo experiments, and in methanol (100 g/l), GFW or 0.06 mol/l phosphate buffer (pH 7.0) for in vitro experiments. The edible parts of babassu produced significant in vivo antithyroid effects (p < 0.05- < 0.001) in rats on a high iodine intake (14 micrograms I- day-1.rat-1), as well as distinct and reproducible antithyroid and anti-TPO activities in both in vitro systems, their action being similar to that of the thionamide-like antithyroid drugs propylthiouracil and methimazole.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this investigation was to determine whether an intrinsic defect in thyroid hormone production is required for the development of iodide-induced hypothyroidism or does it also develop in TSH-stimulated normal thyroid tissue. To answer this question, we studied the response to iodine administration (180 mg iodide daily for 3-4 months) in eight euthyroid patients who had had partial thyroid-ectomies 2 months to 10 years previously for benign thyroid nodules, and in three euthyroid control subjects. In all 11 euthyroid patients, basal serum TSH concentrations increased during iodide administration. In six of the eight patients who had previous thyroid operations and in two of the three control patients, basal serum TSH concentrations increased into the abnormal range (greater than 6 U ml-1). Increased serum TSH concentrations were noted as early as 1 week after potassium iodide had been started and the increased levels persisted during the period of iodide administration. Although basal values for serum TSH concentration were initially within the normal range, those patients with highest basal serum TSH values developed the greatest increase in TSH in response to potassium iodine. Among the eight patients treated by partial thyroidectomy, serum T4 concentrations decreased in five, serum T3 concentration decreased in three and all five developed mild symptoms of hypothyroidism while receiving iodide. Serum T4 concentrations also decreased slightly in two of the three control patients. Serum total iodine levels increased from 7.0 +/- 0.5 to 315.7 +/- 108.6 g dl-1 (mean +/- standard error) during potassium iodide administration, but there was no correlation between the level of serum iodide concentration achieved and inhibition of thyroid function. When iodide was discontinued, symptoms disappeared and thyroid function tests returned to baseline levels within 1 month in all patients, It thus appears that a restricted thyroid mass and/or a mild increase in TSH stimulation is all that is required for the development of hypothyroidism in man when exposed to excessive iodide.

We have examined the ability of IgGs obtained from 8 endemic cretins to inhibit TSH-stimulated thyroid cell growth in culture. Clinical and laboratory evidence for hypothyroidism was present in six subjects; the two remaining patients had borderline low serum T4, normal T3 and exaggerated TSH response to TRH. In six patients 2 mg IgG exhibited an inhibitory effect in the cellular growth expressed by a diminished incorporation of 3H-thymidine into the DNA of TSH-stimulated FRTL-5 cells (range: 26-87% inhibition). Seven patients presented clinically with thyroid atrophy of relatively small thyroid enlargements for the degree of chronic iodine deficiency that was present in the area. The remaining subject had a large multinodular goiter and IgG purified from this patient had no inhibitory effect in the FRTL-5 cellular growth. A direct relationship was noted between the degree of thyroid growth inhibition (%) and the basal serum TSH concentration. We conclude that the presence of thyroid growth inhibiting immunoglobulin may be related to the absence of thyroid growth or even thyroid atrophy in endemic cretins.

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.

We have previously demonstrated that T3 increases adenylate cyclase activity in preparations of plasma membranes from rat thymocytes. On the basis of this and other evidence, we have postulated that the increased cAMP concentration and consequent increase in 2-deoxyglucose (dGlc) uptake that T3 induces in the intact thymocyte is the consequence of a similar stimulation of adenylate cyclase activity. To obtain further evidence to this point, we have now conducted experiments with MDL 12330A [N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride], a compound that inhibits adenylate cyclase activity in several other tissues. In thymocyte plasma membrane preparations, MDL 12330A induced a concentration-dependent inhibition of both basal enzyme activity (activity in the absence of hormone) and the increase in activity induced by T3 and epinephrine. In the intact thymocyte, MDL 12330A greatly limited the marked increase in cellular cAMP concentration induced by maximally effective concentrations of the phosphodiesterase inhibitor 3'-isobutyl-1'-methylxanthine. This indicates that MDL 12330A inhibits adenylate cyclase activity in the intact thymocyte as it does in thymocyte plasma membrane preparations. Further, in intact thymocytes incubated with MDL 12330A, there occurred small but significant decreases in basal cAMP concentration and dGlc uptake, and the T3-induced enhancement of these functions was reduced or abolished. These data provide additional evidence that the increase in dGlc uptake in rat thymocytes that T3 induces is linked to an antecedent increase in cellular cAMP concentration, and that the latter results from a T3-induced enhancement of adenylate cyclase activity.

Pearl millet [Pennisetum millet (L.) leeke] is the main source of food energy for the rural poor in many areas of the semiarid tropics. Epidemiological evidence suggests that millet may play a role in the genesis of endemic goiter in these areas, and sparse experimental data in rats support this suspicion. This study was undertaken to determine in vivo in rats and in vitro using porcine thyroid slices and a thyroid peroxidase (TPO) assay the goitrogenic and antithyroid effects of millet diets, extracts of millet, and certain pure compounds contained therein. For use in these studies, whole grain millet was progressively dehulled to yield successively four bran and four flour fractions in which direct analyses revealed progressively lower concentrations of C-glycosylflavones. In vivo feeding of bran fraction 1, that richest in C-glycosylflavones, led to a significant increase in thyroid weight and antithyroid effects. Feeding of bran fraction 2, the next richest in C-glycosylflavones, produced similar, but less marked, changes. In vitro studies of 125I metabolism using porcine thyroid slices indicated that extracts of bran fractions 1 and 2 were most potent, producing changes similar to those produced by methimazole (MMI). At a concentration of 60 mumol/L, glucosylvitexin, the major C-glycosylflavone present in millet, had effects comparable to those of 1 mumol/L MMI. Similarly, in studies of porcine TPO, extracts of bran fraction 1 caused pronounced (85%) inhibition of enzyme activity, and progressively less inhibition was induced by extracts of bran fractions 2, 3, and 4. Overall, the TPO-inhibiting activities of the various millet fractions closely correlated with their C-glycosylflavone concentrations. Three C-glycosylflavones present concentrations. Three C-glycosylflavones present in millet, glucosylvitexin, glycosylorientin, and vitexin, also inhibited TPO activity. Thus, in vivo and in vitro studies revealed that millet diets rich in C-glycosylflavones produce goitrogenic and antithyroid effects similar to those of certain other antithyroid agents and small doses of MMI. We conclude that in areas of iodine deficiency in which millet is a major component of the diet, its ingestion may contribute to the genesis of endemic goiter.

The present studies were undertaken to explore further the mechanism by which T3 increases adenylate cyclase activity and the uptake of the sugar analog 2-deoxyglucose (2-DG) in freshly isolated rat thymocytes. In studies of cells preloaded with the fluorescent probe quin-2, whose fluorescence intensity increases linearly with increases in cytoplasmic free calcium concentration [( Ca2+]i), we have now demonstrated that T3 increases [Ca2+]i in thymocytes suspended in buffer containing 1 mM Ca2+. This effect was extremely prompt, becoming evident much less than 1 min after the addition of T3 and reaching maximal values in about 5-8 min. The subsequent time course of the T3 effect was obscured by an increase in fluorescence intensity in control thymocytes not exposed to T3, beginning after about 8 min of incubation. However, the T3 effect, after reaching its peak, appeared to remain stable for about 5 min and then to decline, abating completely in 18-30 min. No effect of T3 on [Ca2+]i was observed when thymocytes were suspended in Ca2+-free medium. The effect of T3 on [Ca2+]i was concentration dependent, and as with its actions on thymocyte adenylate cyclase activity, cAMP concentration and 2-DG uptake, the lowest effective concentration of T3 was 1 nM. Among several thyronine analogs studied, L-T3 was the most potent, followed in decreasing order of potency by L-T4, D-T3, 3,5-diiodo-3'-isopropyl-L-thyronine, and D-T4. rT3, 3,5-diiodo-L-thyronine, and D,L-thyronine were without effect. l-Alprenolol alone (10 microM) produced a modest increase in thymocyte [Ca2+]i, but, as it does with the effect of T3 on cellular cAMP concentration and 2-DG uptake, it markedly inhibited or abolished the stimulatory effect of T3 on [Ca2+]i. From these observations we conclude that T3 initiates the increase in thymocyte [Ca2+]i by enhancing the influx of extracellular calcium, though the possibility that it also releases calcium from an intracellular calcium pool cannot be excluded. Since the effects of T3 on thymocyte adenylate cyclase activity, cAMP concentration, and 2-DG uptake occur subsequent to these effects on calcium metabolism and require the presence of Ca2+ in the extracellular fluid, we suggest that an increase in [Ca2+]i, due at least partly to an influx of extracellular calcium, is the initiating event in these plasma membrane-mediated responses of the rat thymocyte to T3.

We have previously demonstrated that in the rat thymocyte T3 produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2-DG) uptake. We have also demonstrated the presence of specific receptors for T3 in the rat thymocyte plasma membrane. From these and other observations we have suggested that T3 initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T3 to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T3 on thymocyte cAMP concentration and 2-DG uptake. Mild trypsin treatment of rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T3 to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T3-induced increase in both 2-DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T3, but decreased significantly the increase in sugar uptake induced by T3, whereas a higher concentration of trypsin (5 mg/ml) decreased basal 2-DG uptake by only 23%, but abolished the response to T3 altogether. T3, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T3 or epinephrine. Trypsinization also resulted in a progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase by trypsin could explain these results was rendered unlikely by experiments with prostaglandin E1 (PGE1). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGE1 was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE1 was much higher than that in cells treated with trypsin alone.(ABSTRACT TRUNCATED AT 400 WORDS)

A recently described method for the prevention and treatment of endemic iodine deficiency and goiter, introduction of iodine into a public water supply, was tested in Troina, a town of about 13,000 inhabitants in northeast Sicily. There, before initiation of the program, a goiter endemic of moderate severity was present, as evidenced by goiter prevalence of 55% in school children. Iodine deficiency in nongoitrous adults was indicated by daily urinary iodine excretion of 40.7 +/- 2.6 micrograms (mean +/- SE) and 24-h thyroid radioiodine uptake of 50.8 +/- 2.4%. Iodination of the water supply was initiated in November 1979 using a stream-splitting device that diverts a controlled fraction of the total water flow to a canister containing iodine crystals, where the water becomes saturated with iodine (approximately 300 mg/liter) before returning to the main stream. Except for a 15-month interruption during which governmental authorization of the program was being reconfirmed, treatment of the water has continued to the present time, initially at a level of 81 +/- 25 micrograms/liter (mean +/- SD) and since resumption at a level of 46.5 +/- 5 micrograms/liter. Iodination of the water was followed by a prompt and marked reduction in goiter prevalence, and by improvement in biochemical indices of iodine deficiency. By April 1983, overall goiter frequency in school children had declined to 6.1%, and large goiters (WHO Grade 2) had virtually disappeared. By January 1984, daily urinary iodine excretion had increased to 85.6 +/- 6.5 (SEM) micrograms and radioiodine uptake had decreased to 40.7 +/- 4.7%. Serum thyroid-related hormone concentrations were as follows (pretreatment vs. November-December 1983): T4, 5.8 +/- 0.3 vs. 8.4 +/- 0.3 microgram/dl; T3, 1.6 +/- 0.05 vs. 1.2 +/- 0.06 ng/ml; TSH, 3.7 +/- 0.2 vs. 2.2 +/- 0.1 microU/ml; all changes being statistically significant. By late 1983, serum T4, T3, and TSH values in Troina were almost identical to those in Catania, a community in which iodine deficiency is not present (goiter prevalence in school children, 2.2%). In contrast, in Troina serum T4 concentrations were significantly higher and serum TSH concentrations were significantly lower than those in Maniaci, a iodine-deficient town near Troina, in which the water was not iodinated. Iodinated water was well tolerated by the population of Troina, and no adverse effects of water iodination, including any increase in the frequency of hyperthyroidism, was observed. At present prices, the cost of the water iodination program in Troina would be approximately 4 cents (U.S.) per person per year.(ABSTRACT TRUNCATED AT 400 WORDS)

In female and male Sprague-Dawley CD rats, both total mass and thymocyte content of thymus gland peaked during the second month of life and then declined with age. Values of both 2-deoxyglucose (2-DG) uptake and thymidine incorporation in thymocytes were higher in cells from females than from males in all age groups studied, these differences being significant only at 3 months of age and onward. In cells from both sexes, thymocyte 2-DG uptake and thymidine incorporation were at maximum at the first and second months of age, respectively. Thereafter, in cells from males, 2-DG uptake and thymidine incorporation declined rapidly with advancing age; whereas in cells from females, these functions started to decline only after 6 months. In both freshly isolated and cultured cells, the concanavalin A (Con A)-induced increase in thymidine incorporation was also affected by age, peaking at about 3 months. The proportionate effect of Con A on thymidine incorporation was similar in cells of both sexes, but because of differences in basal values, the absolute increase in thymidine incorporation produced by Con A was higher in cells from females than from males. Since thymocytes are the progenitors of T-cells, these changes in the number and function of thymocytes may be the forerunner of similar functional abnormalities that are seen in circulating T-cells, but at a later age.

In previous studies we have demonstrated that 3,5,3'-triiodothyronine (T3) in vitro produces a prompt increase in the uptake of the sugar analogue 2-deoxyglucose (2-DG) by freshly isolated rat thymocytes. This effect is prompt, being evident at 20 min after addition of T3, is independent of new protein synthesis, and can be elicited by physiologic concentrations of the hormone. In the present studies, we have sought to determine whether physiologic doses of T3 are capable of inducing an increase in 2-DG uptake in the thymocytes of the living animal. Therefore, 26-28-d-old female rats were injected with increasing doses of i.v. T3, followed 60 min later by 3H-labeled 2-DG. 30 min later, animals were killed, thymocytes were isolated, and their 3H content determined. Uptake of [3H]2-DG was increased by T3 in a dose-dependent manner. The lowest effective dose was 10 ng/100 g of body weight (30% above control) and the maximally effective dose 1 microgram/100 g of body weight (116% above control). The effect of T3 was independent of new protein synthesis in that it was not blocked by a dose of cycloheximide that inhibited the incorporation of [3H]leucine into thymocyte protein by 92-95%. Comparable studies with various thyronine analogues revealed the following rank order of potency: L-T3 greater than L-3,5,3'5'-tetraiodothyronine (L-T4) greater than D-T3 greater than or equal to D-T4 greater than L-3,3'5'-triiodothyronine greater than 3'-isopropyl-3,5-L-diiodothyronine (T2) = 3,5-L-T2. DL-thyronine was without effect. These studies indicate that T3 in physiologic doses acts in vivo to increase the uptake of sugar by rat thymocytes by a mechanism that is extranuclear in origin, in that it is independent of new protein synthesis. The findings support the conclusion that the previously demonstrated effects of T3 on thymocyte sugar uptake in vitro, which seem clearly to be mediated at the level of the plasma membrane, have physiologic relevance.

In previous studies we have shown that the thyroid hormone T3 induces a prompt increase in the cAMP concentration of rat thymocytes in vitro. This is followed by and very likely leads to an increase in cellular uptake of the glucose analog 2-deoxyglucose (2-DG). Since these effects of T3 were shown to require the presence of Ca2+ in the suspending medium, it seemed reasonable to determine whether T3 would influence the metabolism of calcium itself. In standard medium containing 1 mM Ca2+ and 45Ca as a tracer, T3 induced a very prompt, dose-related, but transient, increase in thymocyte calcium accumulation. This effect was evident within the first minute after the addition of T3 (including processing time) and is to our knowledge the most rapid effect of T3 yet demonstrated. The effect reached a maximum very shortly thereafter and then abated within a few minutes. Uptake of 45Ca from Ca2+-free medium (5 microM Ca2+ as a contaminant) was also increased by T3, but under these conditions, the increase was sustained for the entire 120-min period of study. At a concentration of 25 microM, lanthanum (La3+) unexpectedly produced a rapid and marked (4- to 6-fold) increase in 45Ca accumulation in the thymocytes. The increase in cellular calcium accumulation produced by La3+ was not associated with any effect on thymocyte cAMP concentration or 2-deoxyglucose (2-DG) uptake. However, La3+ potentiated the response of the cell to T3 in respect to calcium accumulation, cAMP concentration, and 2-DG uptake, shifting the dose-response curve of the latter two effects to the left. In view of the calcium dependence of the T3 effect on thymocyte cAMP concentration and 2-DG uptake, and the effect of La3+ to concomitantly enhance the sensitivity of the cell to the effects of T3 on calcium accumulation and cAMP and 2-DG metabolism, we suggest that the prompt increase in cellular Ca2+ uptake induced by T3 that we demonstrated is causally related to its subsequent effect on cellular cAMP concentration and 2-DG uptake.

We have recently reported a method for the receptor purification of Graves' disease-specific immunoglobulin G (IgG) using plasma membranes from guinea pig adipocytes as a source of TSH receptor. The preparations of receptor-purified Graves'-IgG (RPG-IgG) thereby obtained were highly enriched (40- to 150-fold) in TSH binding inhibitory activity and were comparably enriched in human thyroid adenylate cyclase-stimulating activity. Unlike the less active preparations obtained by others who have attempted receptor purification but have used human thyroid membranes, our preparations were devoid of the antimicrosomal antibodies and, presumably, the antithyroglobulin antibodies common to the IgG of patients with Graves' and Hashimoto's diseases. We have now succeeded in preparing 125Ilabeled RPG-IgG (RPG-IgG*) by a lactoperoxidase method that permits retention of the original TSH binding inhibitory activity. Such preparations have been employed to study both the direct binding of Graves'-specific IgG to human thyroid membranes and several of the properties of this interaction. The binding of RPG-IgG* to human thyroid membranes exceeded that of 125I-labeled receptor-purified normal IgG and was saturable, since it could be inhibited almost entirely by sufficient concentrations of unlabeled Graves'-IgG. The binding of RPG-IgG* prepared in the standard manner (i.e. with adipocyte membranes) was inhibited by Graves'-IgG, but not by Hashimoto's IgG, when tested in either human thyroid membranes or guinea pig adipocyte membranes. RPG-IgG* prepared with the use of human thyroid membranes exhibited the same differential inhibition of binding when tested in adipocyte membranes, but was inhibited by both Graves'- and Hashimoto's-IgG when tested in human thyroid membranes. We interpret these findings to indicate that, in general, human thyroid membranes contain antigens that are complementary to antibodies present in the serum in both Graves' and Hashimoto's diseases, while adipocyte membranes contain antigens complementary to antibodies found in the serum in Graves' disease, but not in Hashimoto's disease. The binding of RPG-IgG* prepared with adipocyte membranes and the binding of 125I-labeled bovine TSH (bTSH*) to human thyroid membranes had several features in common. Like the binding of bTSH*, that of RPG-IgG* was temperature dependent, had a pH optimum of about 6.0, and was progressively inhibited by increasing concentrations of NaCl. In membranes from a variety of tissues, saturable binding of RPG-IgG* was greatest in those with the greatest binding of bTSH*. Moreover, the ability of individual specimens of Graves' IgG to inhibit the binding of RPG-IgG* to human thyroid membranes was closely correlated with their ability to inhibit the binding of bTSH*. Finally, the studies appeared to provide the first demonstration of the ability of unlabeled bovine and human TSH to inhibit the binding of RPG-IgG*, though inhibition was only partial and relatively high concentrations were required. Even higher concentrations of other peptide hormones were without effect, however. The studies have provided, therefore, both the first direct demonstration of saturable binding of Graves'-specific IgG to human thyroid membranes and strong evidence that such binding occurs at the TSH receptor, though perhaps not exclusively so. The preparations of RPG-IgG* that we have described could possibly be used as a reagent in radioreceptor assays for Graves'-specific IgG.

Studies of the influence of age and sex on the concentrations of total thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3) in serum and on the free T4 and free T3 indices, were conducted in Sprague-Dawley rats of the CD strain varying in age between 10 days and 12 months. Both sex- and age-related differences were found. In all age-groups studied, serum T4 concentrations were higher in the male than in the female, whereas serum T3 concentrations were higher in the female. In both sexes, concentrations of T4 and T3 in serum reached a peak early in life, between the first and second month of age, and declined thereafter. In addition, in both sexes the intensity of thyroid hormone binding, as judged from values of the in-vitro uptake of T3, did not change with age, suggesting that free T4 and T3 concentrations in the serum display the same sex differences and age-related changes as do the concentrations of total T4 and T3. It remains to be determined whether these sex- and age-related alterations in serum thyroid hormone concentration are expressed in differences in the activity of various thyroid hormone-dependent processes.

In the fetuses of some mammalian species, including man and possibly sheep, serum TSH concentrations are higher than those in the adult, and TSH secretion is less readily inhibited by exogenous T3. In an effort to obtain information concerning the factors that might contribute to these differences, comparative studies of the metabolism of 125I-labeled T4 and its conversion to 125I-labeled T3 were carried out in homogenates of pituitary glands from pregnant sheep (125 days of gestation) and their own fetuses. Homogenates of livers from the same animals were studied concomitantly. All specimens were enriched with the stimulatory agent dithiothreitol (20 RIM). Under these conditions of study, in all tissues, only three 125I-labeled products of [125I]T4 metabolism were observed: T3, iodide, and chromatographically immobile origin material. The T4-degrading and T3- generating activities of fetal and maternal pituitary homogenates were virtually equal, and were far greater than those in the corresponding homogenates of liver. In both fetal and maternal pituitaries, net generation of [125I]T3 accounted for by far the greatest fraction (-90%) of overall [125I]T4 degradation. The comparable fraction was significantly higher in maternal than in fetal liver, but was far lower in both than in the corresponding pituitaries. Iodide, origin materal, and 3,3′-diiiodothyronine were the only labeled products of [125I]T3 metabolism detected. Degradation of [125I]T3 was much less rapid than that of [125I]T4 in all tissues studied, especially in pituitaries, but that in fetal pituitary was significantly more rapid than that in maternal pituitary. Thus, as judged from the data obtained under these conditions of study, 5′-monodeiodination leading to the generation of T3 is by far the major pathway of T4 metabolism in the ovine fetal and maternal pituitary gland. Since differences in the activity of this pathway in fetal and maternal pituitaries were not seen, they cannot account for the differing activities of the TSH secretory mechanism in mother and fetus. The more rapid degradation of T3 in fetal than in maternal pituitary tissue that was observed might possibly contribute, however, to the relative resistance of TSH secretion in the fetus to inhibition by exogenous T3. These possible physiological implications of the present findings have validity only to the extent that the characteristics of thyroid hormone metabolism by the mixed cell types present in the whole pituitaries studied accurately reflect the corresponding characteristics in the thyrotroph per se.

Immunoglobulin G (IgG) fractions prepared from the serum of patients with Graves' disease (Graves'-IgG) are generally capable of inhibiting the binding of 125I-labeled bovine TSH ([125I]bTSH) to crude preparations of human thyroid membranes [TSH binding inhibitory (TBI) activity]. In current TBI assays, membranes, [125I]bTSH, and IgG are incubated together, and the extent of inhibition of [125I]bTSH binding produced byGraves'-IgG is compared with that produced by specimens of normal IgG. Such direct TBI assays, as we term them, have only moderate sensitivity, positive results being reported in approximately 50-75% of the actively thyrotoxic patients with Graves' disease. This appears to be owing to the wide ranging and often substantial TBI activity displayed by preparations of normal IgG. Reasoning that the TBI activity of normal IgG might be more readily dissociable from the thyroid membranes than the IgG specific for Graves' disease would be, we modified the TBI assay by first incubating the membranes with or without IgG, washing them thoroughly with buffer, and then incubating them with [125I]bTSH. We term this a residual TBI assay, since it tests the extent of TBI activity that remains associated with the membranes despite the washing procedure. This procedure greatly reduced or eliminated the TBI activity of normal IgG and yielded a 94% frequency of positive responses in studies of 50 specimens of Graves' -IgG. In 31 specimens so tested, values of the residual TBI assay correlated significantly with their ability to increase the cAMP concentration in human thyroid slices. Ensuing experiments were conducted to test the feasibility of applying the principleof the residual TBI assay to the assay of whole serum, rather than IgG. A modification of the washing procedure used in residual TBI assays of IgG was shown to greatly decrease the almost complete inhibitory activity of serum seen in direct TBI assay. In residual TBI assays of 35 specimens of whole serum from patients with Graves' disease, a 77% frequency of positive responses was observed. In 27 samples so studied, a significant correlation was observed between the TBI activity of Graves' -IgG and that of the sera from which they were prepared. Application of the residual assay principle affords promise of greatly simplifying and enhancing the sensitivity of TBI assays.

Studies were performed to determine the effects of erythrosine (2′,4′,5′,7′-tetraiodofluorescein; FD&C red no. 3), a widely used coloring agent for foods and pharmaceutical preparations, on the metabolism of 125I-labeled T4 and T3 by rat liver homogenates. When added in vitro to either buffer or homogenates, erythrosine produced a prompt and marked, nonenzymatic deiodination of [125I]T4. In buffer, this effect of erythrosine was shown to be decreased by the exclusion of light or the addition of serotonin. In view of these findings, the remaining studies examined the effects of administering erythrosine to rats in vivo on the metabolism of [125I]T4 and [125I]T3 by homogenates of their livers in vitro. Over a wide range of doses (2.5–250 mg/ kg BW./day), erythrosine induced a dose-dependent inhibition of the deiodination of [125I]T4 and the generation of [125I]T3 therefrom, effects that were uninfluenced by the exclusion of light from the incubation system. At higher doses of erythrosine, the proportionate reduction of the deiodination of [125I]T4 exceeded that of the generation of [125I]T3, indicating that the pathways of T4 metabolism other than that leading to T3 formation were also inhibited. Evidence was adduced that erythrosine may inhibit the 5-monodeiodination of T4 that generates rT3, especially since the administration of erythrosine also led to the inhibition of the 5-monodeiodination of T3 that leads to the generation of 3,3′-diiodothyronine. Fluorescein, the noniodinated analog of erythrosine, did not induce a photoactivated deiodination of [125I]T4, in buffer in vitro, and had no effect on the in vitro hepatic metabolism of T4 when administered in vivo The foregoing findings indicate that erythrosine, like other iodinated aromatic dyes, is capable of inhibiting the 5′-monodeiodination of T4 and very likely inhibits other pathways of hepatic T4 metabolism as well. The possibility that erythrosine produces similar effects in man in the doses normally ingested is discussed, but is considered unlikely.

Studies of the pathways of peripheral metabolism of iodothyronines in vitro are often obscured quantitatively and qualitatively, both by the rapid degradation of products of interest and by the generation of a single product from more than one precursor. To overcome these problems, we tested the ability of rabbit immunoglobulin G containing antibodies against particular iodothyronines to inhibit the enzymatic degradation of their respective antigens. Such inhibition was indeed demonstrable in the case of all the iodothyronines tested, i.e. T3, rT3, and 3,3'-diiodothyronine. The general method was then applied to studies of the ontogeny of peripheral iodothyronine metabolism in homogenates of chick embryo liver. By appropriate selection of antibodies and 125I-labeled substrates, it was shown that livers from immature embryos (<19–20 days of age) deiodinate T4 rapidly and almost entirely to rT3, with very little T3 being formed. In livers from embryos that had matured spontaneously or had been injected with hydrocortisone, T4 was deiodinated more slowly, but almost exclusively to T3. The findings indicate that the maturation of hepatic T4 metabolism involves an increase in the rate of 5’-monodeiodination coupled with a proportionately greater decrease in 5-monodeiodination, so that formation of T3 from T4 is increased and formation of rT3 is greatly retarded. The new technique, which we have termed immunosequestration, thus provided proof of conclusions reached only by inference in earlier studies that employed the in vitro techniques used in the past. The technique should prove valuable in studies of other problems related to peripheral iodothyronine metabolism and in other metabolic studies as well. (Endocrinology108: 1665, 1981)

The present experiments were designed to explore the mechanism whereby 3,5,3'-triiodothyronine (T3) stimulates the uptake of 2-deoxy-D-glucose (2-DG) in rat thymocytes in vitro. Addition of T3 evoked a transient, dose-related increase in cellular cyclic (c) AMP concentrations, evident within 5 min. followed soon by an increase in 2-DG uptake. The effects of T3 on both cAMP concentration and 2-DG uptake were dependent upon the presence of extracellular calcium. Epinephrine also induced a sequential increase in thymocyte cAMP concentration and 2-DG uptake. These responses were more prompt than those to T3, but were calcium independent. As with their combined effects on 2-DG uptake, T3 and epinephrine produced synergistic or additive effects on cellular cAMP concentration. Dibutyryl cAMP also stimulated 2-DG uptake, an effect that was more prompt than that of epinephrine, and like that of epinephrine, was calcium independent. Prior or simultaneous addition of L-alprenolol (10 microM), which, we have previously shown, blocks the effect of both T3 and epinephrine on 2-DG uptake, also blocked the increase in thymocyte cAMP concentration induced by these agents. In contrast, L-alprenolol failed to block the increase in 2-DG uptake produced by dibutyryl cAMP. On the basis of these observations we suggest that T3 increases 2-DC uptake in the rat thymocyte by increasing the cellular concentration of cAMP, which then acts to enhance sugar transport. The increase in 2-DC uptake induced by epinephrine is also mediated by an increase in cAMP concentration. The greater response of cellular cAMP concentration to T3 and epinephrine when added together than to either agent added alone may explain their synergistic action to increase 2-DG uptake. We suggest that these actions of T3 and epinephrine are both initiated at the level of the plasma membrane.