Maturation of human hypothalamic-pituitary-thyroid function and control.

Measurements of serum thyrotropin (TSH) and free thyroxine (T4) concentrations were conducted in infants, children, and adults to assess maturation of the hypothalamic-pituitary-thyroid (HPT) feedback control axis. Serum free T4 and TSH concentration data were collated for cord blood of the midgestation fetus, for premature and term infants, and for peripheral blood from newborn infants, children, and adults. Mean values were plotted on a nomogram developed to characterize the reference ranges of the normal axis quantitatively based on data from 522 healthy subjects, 2 weeks to 54 years of age; 83 untreated hypothyroid patients; and 116 untreated hyperthyroid patients. Samples for 75 patients with thyroid hormone resistance were also plotted. The characterized pattern of HPT maturation included a progressive decrease in the TSH/free T4 ratio with age, from 15 in the midterm fetus, to 4.7 in term infants, and 0.97 in adults. Maturation plotted on the nomogram was complex, suggesting increasing hypothalamic-pituitary T4 resistance during fetal development, probably secondary to increasing thyrotropin-releasing hormone (TRH) secretion, the marked, cold-stimulated TRH-TSH surge at birth with reequilibration by 2-20 weeks, and a final maturation phase characterized by a decreasing serum TSH with minimal change in free T4 concentration during childhood and adolescence. The postnatal maturative phase during childhood and adolescence correlates with the progressive decrease in thyroxine secretion rate (on a microg/kg per day basis) and metabolic rate and probably reflects decreasing TRH secretion.

Accuracy of free thyroxine measurements across natural ranges of thyroxine binding to serum proteins.

Systemic inaccuracies, proportional to the concentrations of serum proteins and the thyroxine (T4) they carry, have been reported in direct free T4 immunoassays. However, analytical recoveries of free T4 have not been carefully examined in most current methods, and they have not previously been examined across the pathophysiological range of serum T4 binding. In the present study we investigated ranges of serum T4 binding using free and total T4 measurements from 1359 individuals. Carefully characterized, gravimetrically calibrated, serum-based free T4 test solutions were then prepared with a constant normal free T4 concentration (12 ng/L) and varied serum T4 binding (approximately 300:1 to 24,000:1, ng protein bound T4: ng free T4). These standardized test solutions were analyzed using five T4 analog based free T4 methods. Analytical recoveries were calculated as ratios of actual free T4 measurements to the target value, and expressed as a percent of the target. Analytical recoveries were directly proportional to the extent of serum T4 binding and ranged 2% to 155%, 25% to 131%, 53% to 106%, 37% to 93%, and 37% to 73%, lowest to highest, in different methods. These systemic inaccuracies will confound interpretations of free T4 test results in clinical conditions with altered T4 binding. Future investigations into free T4 status must examine the analytical recovery of the free T4 method(s) used, as they relate to the extent of serum T4 binding in the clinical condition(s) studied.

Salsalate and salicylate binding to and their displacement of thyroxine from thyroxine-binding globulin, transthyrin, and albumin.

Salsalate and its metabolite salicylate are inhibitors of thyroid hormone binding to serum transport proteins and reduce serum total thyroxine (T4) and triiodothyronine (T3) in vivo. The present study is the first to examine and compare salsalate and salicylate binding to human thyroxine-binding globulin (TBG), transthyretin (TTR), albumin (ALB), and whole human serum, and displacement by salsalate and salicylate of T4 from isolated TBG, TTR, ALB. Salsalate and salicylate binding were studied by ultrafiltration at salsalate concentrations of 0.24-1.16 mM (62-300 microg/mL) and salicylate concentrations of 0.0375-2.25 mM (6.0-360 microg/mL). T4 displacement by salsalate and salicylate from TBG, TTR, and ALB was studied by equilibrium dialysis at salsalate concentrations of 0 to 1.52 mM (0-393 microg/mL) and salicylate concentrations of 0 to 60 mM (0-9600 microg/mL). In normal human serum, 95% of salsalate and 76% of salicylate were protein-bound. In the presence of isolated ALB, 89% of salsalate and 64% of salicylate were ALB-bound. Both salsalate and salicylate inhibited T4 binding to all 3 major T4 binding proteins. Both displaced proportionately more T4 from TBG compared with TTR and ALB. The principal site to which both salsalate and salicylate in serum is bound (ALB) is different from the principal site from which they displace T4 (TBG). Salsalate potency in displacing T4 from TBG and ALB was approximately 100-fold greater than salicylate potency.

Salsalate administration--a potential pharmacological model of the sick euthyroid syndrome.

This study examined salsalate ingestion as a model of the sequelae of acute inhibition of thyroid hormone binding to serum protein. One dose of salsalate (60-65 mg/kg) was administered to healthy volunteers. Serum salsalate concentrations peaked at 2 h (82 micrograms/mL), then declined at 8 h to 1.2 micrograms/mL. Serum total T4 (TT4) and total T3 (TT3) concentrations declined for 4 h, then recovered by 96 h, while T4 binding protein concentrations remained unchanged. TT3 was reduced to a greater extent than TT4 between 2 h and 72 h, and serum total reverse(r)T3 (TrT3) was transiently increased at 8 h. TSH concentrations fell while TT4 and TT3 fell, then recovered while TT4, TT3, and free T3, but not free T4, were still reduced. Subsequently, TSH overshot basal levels and continued to rise after 96 h while TT4, TT3, free T4, free T3, and TrT3 were all at basal levels. We postulate that an acute release of T4 and T3 from circulating transport proteins, induced by an inhibitor of binding, can result in large and rapid redistribution of T4 and T3 into tissue compartments associated with transiently reduced peripheral tissue 5'-monodeiodination and deranged TSH regulation.

Analytical performance of free and total thyroxine assays.

Excessive bias and imprecision are major analytical problems associated with some assays for free and total thyroxine (T4). Bias in free T4 methods is largely proportional to variations in serum T4 binding. In direct methods, this is attributable to requirements for substantial quantities of protein-bound T4 to replace analytical losses of free T4. In some total T4 methods, bias is inversely proportional to the amount of serum T4 binding and is attributable to the incomplete release of serum protein-bound T4. In others, bias is fixed and attributable to inaccurate calibration. Manufacturers should report the bias in their methods. Calibrations should be standardized. Imprecision varies widely among methods, but is generally less for total T4 methods than for free T4 methods. A consensus on quantitative analytical performance goals for free and total T4 methods would be helpful. Here, performance goals are proposed, based in part on the best achievements of current methods.

Variable underestimates by serum free thyroxine (T4) immunoassays of free T4 concentrations in simple solutions.

The immunoassays used to measure free T4 in serum frequently underestimate free T4 concentrations. These underestimates of serum free T4 are often greater than can be attributed to sample dilution. Unrecognized T4 sequestration by assay materials could cause such underestimates, but T4 sequestration during free T4 measurements has not been well studied. To study T4 sequestration, we used simple solutions containing gravimetrically determined quantities of T4 without T4 binding proteins. T4 sequestration was calculated as the differences between the actual quantities of free T4 required to obtain a specific free T4 measurement and the quantities that would, in theory, have been required, assuming zero T4 sequestration. Sequestration occurred in every serum free T4 assay studied, was often in the nanomolar range, and varied from 26-99+% of the actual amount of free T4 required to obtain free T4 measurements. We conclude that unrecognized T4 sequestration is a major confounding variable in free T4 assay performance, and that it explains the underestimates of serum free T4 concentrations by free T4 immunoassays which cannot be explained by sample dilution.

Underestimates of serum free thyroxine (T4) concentrations by free T4 immunoassays.

Different free T4 (FT4) assays often give different FT4 measurements, and conflicting measurements have been striking in nonthyroidal illness. Because FT4 immunoassays depend upon serum protein-bound T4 (PBT4) dissociation to stabilize the FT4 concentration during assay perturbations, interassay differences in perturbations combined with variation in serum PBT4 concentrations could produce discordant FT4 measurements. This study examined the effects of PBT4 on FT4 measurements obtained by direct immunoassay methods. Standard solutions with constant FT4 levels and varying PBT4 concentrations were prepared and analyzed by direct equilibrium dialysis, two-step immunoextraction, one-step labeled T4 antibody, and one-step labeled T4 analog FT4 methods. Direct equilibrium dialysis results were independent of PBT4 concentrations and gave correct measurements of serum FT4 when the PBT4 concentration was above 8 nmol/L or 0.6 micrograms/dL, but were PBT4 dependent and underestimated serum FT4 at lower PBT4 concentrations. The other three methods were PBT4 dependent and variably underestimated serum FT4 at all levels of PBT4 up to 256 nmol/L (19.9 micrograms/dL), the highest level studied. Thus, PBT4-dependent underestimates of serum FT4 occurred with all four methods, whereas the measured FT4 level at each PBT4 concentration varied widely between methods. A serum PBT4 dependent bias causes discordant FT4 measurements and probably explains the observed underestimates of FT4 in nonthyroidal illness.

Heterogeneity in affinities of serum proteins for thyroxine among patients with non-thyroidal illness as indicated by the serum free thyroxine response to serum dilution.

UNLABELLED: Some patients with non-thyroidal illness (NTI) have sera with characteristics suggesting decreased functional affinity of thyroxine-binding globulin (TBG) for T4: increased T4 free fraction, decreased total T4 to TBG ratio, and a fall in free T4 with serum dilution greater than theoretical predictions assuming normal affinity. These studies aimed to further characterize this phenomenon. According to the law of mass action binding protein affinity for T4 can be estimated from the response of free T4 to progressive serum dilution. Free T4 responses in 25 normal controls and in 7 congenital TBG deficient controls resembled the predictions from the law of mass action. Among 25 of 38 medical intensive care unit patients, responses were intermediate between normals and TBG deficiency. Responses in the other 13 NTI were consistent with the presence of a dissociable inhibitor. In this subgroup, total T4 was depressed, both free T4 and TSH were elevated, and there was a positive correlation between TSH and free T4 (r2 = 0.41, p < 0.02). CONCLUSIONS: (i) decreased affinities of serum proteins for T4 consistent with binding inhibitor(s) are found in some patients with NTI; (ii) there is an association of these severely altered affinities with reversal of the expected free T4-TSH relationship.

Dependence of free thyroxine estimates obtained with equilibrium tracer dialysis on the concentration of thyroxine-binding globulin.

Some equilibrium dialysis determinations of free thyroxine (T4) vary directly with thyroxine-binding globulin (TBG) concentration. This apparent TBG dependence has been limited to methods involving radiolabeled T4 added to the dialysis system (tracer dialysis). In this study we compared tracer dialysis with direct dialysis for determining free T4 and obtained the following results (mean +/- SD) for patients with hypothyroxinemia of nonthyroidal illness (23.8 +/- 10.7 vs 24.2 +/- 10.9 pmol/L, P greater than 0.8), patients with congenital TBG deficiency (11.4 +/- 2.2 vs 16.2 +/- 7.1 pmol/L, P greater than 0.05), normal control subjects (32.7 +/- 6.5 vs 18.5 +/- 5.8 pmol/L, P less than 0.001), and pregnant women (31.2 +/- 8.7 vs 12.1 +/- 2.6 pmol/L, P less than 0.001). Direct dialysis determinations were independent of TBG and total T4. Tracer determinations were greater than direct determinations in normals, a discrepancy that increased in pregnancy. Tracer determinations correlated significantly with total T4 and TBG concentrations (P less than 0.001). TBG and total T4 dependence in the tracer method was attributable to small overestimations of the free fraction of T4. Similar overestimations multiplied by increasing total T4 concentrations resulted in greater errors. Relative to results for normal sera, the tracer method overestimated free T4 when total T4 was increased and underestimated free T4 when total T4 was decreased.

Temporal shaping of third-harmonic pulses on the Nova laser system.

We demonstrate temporal shaping of 0.35-microm-wavelength pulses produced by a third-harmonic conversion of the output from the Nova Nd:phosphate glass-laser amplifier system for use in inertial confinement fusion experiments. We describe the computer models used to calculate the pulse shape that is required as the input to the amplifier system, the experimental apparatus used to produce these pulses, and the high-power 0.35-microm shaped pulses produced in recent experiments.

Effects of exogenous monoiodotyrosine on the serum levels of anterior pituitary hormones.

Monoiodotyrosine is a tyrosine hydroxylase inhibitor. Ingestion of one gram monoiodotyrosine caused a 10,000-fold increase of serum monoiodotyrosine from basal levels of 0.69 +/- 0.20 nmol/l to a peak of 10.6 +/- 1.7 mumol/l in women and 7.1 +/- 2.3 mumol/l in men 30 min later, and the t1/2 was 45 min. Monoiodotyrosine stimulated PRL to a peak of 170 +/- 51 micrograms/l in women and 90 +/- 6 micrograms/l in men 30 min after the monoiodotyrosine peak, or 60 min after the ingestion. Other anterior pituitary hormones were unchanged. Dopamine infusion or L-dopa pretreatment attenuated the monoiodotyrosine effect. TRH exaggerated the PRL peak, and chlorpromazine did not increase but prolonged the hyperprolactinemia. These results suggest that dopamine synthesis inhibition may be the mechanism of PRL stimulation.

Extrathyroidal physiology of monoiodotyrosine in humans.

Normal serum monoiodotyrosine (MIT) levels (n = 152) were 0.69 +/- 0.20 nmol/l. There was wide variation of MIT levels in a 24-hour period without diurnal pattern, and there was no change throughout the menstrual cycle. MIT levels declined upon aging, but levels in hypo- and hyperthyroidism were not significantly different. MIT levels were detected in athyrotic patients (0.32 +/- 0.08 nmol/l). Desiccated thyroid raised the athyrotic MIT levels to the normal range, while levothyroxine did not. Diiodotyrosine (DIT) infusion caused an MIT rise which paralleled but lagged 1 h behind the DIT rise. These data suggest thyroidal as well as nonthyroidal sources of MIT, one of which is deiodination of DIT. Ingestion of 1 g MIT increased serum MIT to 10.6 +/- 1.7 mumol/l in women, and 7.1 +/- 2.3 mumol/l in men 30 min after ingestion; the serum half-life was 45 min.

Interactions of putative estrogens with the intracellular receptor complex in mouse Leydig cells: relationship to preneoplastic hyperplasia.

The interaction of 14 steroidal and nonsteroidal estrogen agonists and antagonists with the intracellular estrogen receptor system was examined in cell suspensions prepared from the testes of mice that develop malignant Leydig cell tumors after prolonged estrogen administration. The ability of these substances to stimulate DNA synthesis in short-term (3-day) studies and to provoke Leydig cell hyperplasia with prolonged (3-mo) administration was also measured. Our data were consistent with the proposal that, in Leydig cells, the carcinogenic effects of estrogens are mediated through the intracellular receptor complex that results in a localization of hormone bound to chromatin and nuclear matrix. All tested compounds displaced 17 beta-[3H]estradiol from the cytosolic estrogen receptor, but to varying degrees; and there was no discernible relationship between their ability to compete for this receptor and their efficacy in stimulating DNA synthesis. The effect of the test compounds on the levels of estrogen receptor in cytosol and in nuclei was measured by [3H]estradiol exchange. 17 beta-Estradiol, equilin, 17 alpha-ethinylestradiol, diethylstilbestrol, hexestrol, dienestrol, coumestrol, and nafoxidine provoked a complete estrogen receptor response: acutely a decrease in the level of cytosolic estrogen receptor and an increase in the nuclear estrogen receptor. All of these substances acutely stimulated DNA synthesis. Tamoxifen, clomiphene, and nitromifene provoked a decrease in cytosolic receptor but no increase in demonstrable nuclear estrogen receptor. 17 alpha-Estradiol, mestranol, and estriol did not significantly alter the levels of estrogen receptor in cytosol or nuclei. Only those substances that increased measurable nuclear estrogen receptor also acutely stimulated DNA synthesis. Chronic (3-mo) treatment of 2-mo-old male BALB/c mice with diethylstilbestrol, 17 beta-estradiol, ethinylestradiol, and nafoxidine led to Leydig cell hyperplasia. Chronic mestranol treatment also provoked Leydig cell hyperplasia; this is most probably due to induction of liver metabolism of mestranol to ethinylestradiol. Chronic treatment with 17 alpha-estradiol, tamoxifen, and clomiphene failed to produce significant histologic al changes in the testes. Only chronic administration of those substances that exhibited a complete estrogen receptor response and acutely stimulated DNA synthesis produced Leydig cell hyperplasia.

Dietary and hormonal interrelationships in premenopausal women: evidence for a relationship between dietary nutrients and plasma prolactin levels.

To investigate possible differences in tissue exposures to reproductive hormones and to determine hormone-nutrient interrelationships, we studied 10 vegetarian and 10 nonvegetarian premenopausal Seventh-day Adventist women. Over 3 d in each of three consecutive menstrual cycles, we collected diet records, fasting midluteal phase blood, and 24-h urine samples. During each study period, we measured plasma and urinary estrogens, plasma free and protein bound-estradiol-17 beta, the binding capacity of sex-hormone-binding globulin (SHBG), androgens, progesterone, and prolactin levels. The omnivores consumed significantly more protein, total and saturated fatty acids, oleic and linoleic acids, and cholesterol than did the vegetarians. Hormonal status and binding capacity of SHBG were similar in both groups. However, for nonvegetarians, prolactin levels were positively correlated with dietary energy, protein, total and saturated fatty acids, and oleic acid. Further study delineating the effects of specific dietary nutrients on the basal level of prolactin secretion is warranted.

Serum diiodotyrosine.

Serum diiodotyrosine (DIT) was measured by radioimmunoassay in healthy subjects patients with thyroid disease and a variety of laboratorty animals. Ninety-two healthy adults had a mean level of 101 ng/100 ml. There was no sex difference in DIT levels but DIT fell with aging. There was no change with short term oral SSKI administration. Athyrotic subjects had measurable but reduced levels (mean = 52 ng/100 ml). Hyperthyroid subjects had levels slightly, but not significantly, higher than controls (mean = 149 ng/100 ml). Treatment of hyperthyroidism was followed by a small but significant fall in DIT levels, but there was no change in DIT levels with thyroid hormone therapy of hypothyroidism. A large species variation in serum DIT levels was found among laboratory animals with mean levels ranging from 17 ng/100 ml in mice to 428 ng/100 ml in dogs.

A multiple ligand-binding radioimmunoassay of diiodotyrosine.

A radioimmunoassay has been developed for the measurement of 3,5-diiodo-L-tyrosine (DIT) in serum. DIT was coupled to porcine thyroglobulin (PTg) with a molar ratio of 205:1. Rabbits were immunized with 1 mg of immunogen emulsified in complete Freund's adjuvant. Sera were screened for their ability to bind trace amounts of [125I]DIT. A serum that bound 40% of the tracer at a final dilution of 1:1,750 was used in the assay. Assay specificity was improved by the use of thyroxine (T4)-binding globulin as a second ligand-binding protein to decrease T4 and triiodothyronine (T3) cross-reactivity with the antibody. Double antibody and polyethylene glycol radioimmunoassays were compared. DIT present in the second antiserum shifted the double antibody assay standard curve and altered estimates of assay specificity and assay sensitivity. By using the polyethylene glycol system and butanol:ethanol extracts of serum, DIT was measured in human serum. In 35 apparently healthy young adult controls DIT levels averaged 156 ng/100 ml. Random DIT levels averaged 158 ng/100 ml in 11 untreated hyperthyroid patients and 84 ng/100 ml in 15 untreated primary hypothyroid patients. No diurnal pattern in DIT levels could be demonstrated. Thyroid-stimulating hormone administration led to a variable but small rise in DIT levels, but short term T3 suppression was not associated with a measurable fall in DIT concentrations. Paired serum samples from the carotid artery and thyroid vein of 10 euthyroid goiter patients and one patient with a toxic solitary adenoma all showed a positive transthyroidal gradient indicating the thyroidal release of DIT in each patient. Measurable DIT levels of 45, 47, 68, and 80 ng/100 ml, respectively, were found in four fasting athyrotic patients indicating that the thyroid is not the only source of serum DIT.