Evaluation of a new, rapid and automated immunochemiluminometric assay for the measurement of serum intact parathyroid hormone.

The Immulite parathyroid hormone (PTH) immunochemiluminometric assay (ICMA) was evaluated by measuring serum PTH concentrations in 134 volunteers and 25 patients with various diseases affecting bone and mineral metabolism and comparing with those measured by the Nichols Allegro immunoradiometric (IRMA) assay. Although the assays were highly correlated (r = 0.99. P < 0.0001), there were discrepancies at lower PTH concentrations which may be explained by the lower limit of detection of the ICMA assay. The anti-coagulants EDTA and heparin appeared to have significant effects on PTH measurement particularly at lower concentrations. Sera assayed by ICMA diluted out more uniformly than in the IRMA and there was no cross reactivity with parathyroid hormone-related peptide in either the IRMA or ICMA. Delays of up to 8 h in specimen handling did not appear to affect PTH measurements. The enhanced sensitivity of the Immulite ICMA intact PTH assay confers an advantage in the diagnosis of both hyper- and hypocalcaemia, while the shorter incubation times, automation and lack of radioactivity are important practical advantages.

Detection of insulin antibodies in newly-diagnosed type 1 diabetic children after "acid-stripping" of sera.

A sensitive microassay using sera from which endogenous bound insulin was removed by acid-stripping was employed to screen for insulin antibodies in 48 newly-diagnosed Type 1 diabetic children and 20 age-matched controls. Using intact diabetic sera, the binding of 125I-insulin exceeded the upper control range in 16/48 (33%); when the sera were acid-stripped at pH 4.2 for 30 minutes at 4 degrees C, binding to the 16 positive sera increased further and in another five sera became significant, making a total of 21/48 positive (44%). Children 5 years of age or less had a higher prevalence of insulin antibodies. Islet cell antibodies, assayed by indirect immunofluorescence, were detected in equal fractions of insulin antibody-positive and -negative sera (76% versus 77%). Assay of insulin antibodies using acid-stripped sera under the conditions described significantly increases the sensitivity of insulin antibody detection. Insulin antibodies appear to be a specific marker of islet autoimmunity, but compared to islet cell antibodies are relatively insensitive, and may be a marker for a particular subgroup of diabetics.

Autoantibodies to thyroid hormones: association with falsely low hormone levels measured by 'Amerlex' assay.

Autoantibodies to thyroxine (T4) and tri-iodothyronine (T3) were found in a 21-yr-old thyrotoxic woman who had paradoxically low T4 and T3 as measured in the 'Amerlex' solid phase radio-immunoassay. Though rare, thyroid hormone antibodies should be suspected if anomalous thyroid function results are obtained and alternate assay procedures used.

Thyrotrophin (TSH) binding sites on Yersinia enterocolitica recognized by immunoglobulins from humans with Graves' disease.

Antibodies against the gram negative enteric bacterium Yersinia enterocolitica have been found in a high proportion of persons with autoimmune thyroid disorders, especially in those with Graves' disease or hyperthyroidism (Shenkman & Bottone, 1981). There is strong evidence that Graves' disease is caused by receptor autoantibodies which mimic the bioeffects of thyroid stimulating hormone (TSH) on the thyroid (Manley, Knight & Adams, 1982). Recently, saturable binding sites for TSH were demonstrated in Y. enterocolitica under non-physiological conditions (Weiss et al., 1983). We have characterized TSH binding sites on Y. enterocolitica under physiological conditions and studied their interaction with Graves' immunoglobulins (Ig's). Saturable and specific binding of receptor-purified 125I-TSH to lysozyme/EDTA-treated Y. enterocolitica (serotype 03) was demonstrated under both non-physiological and physiological conditions. Scatchard binding plots were linear indicating a single class of binding site (Kd 1 X 10(-7) M, maximum of 30,000 binding sites per cell). In the presence of Graves' Ig's the binding of 125I-TSH to Y. enterocolitica was significantly inhibited. Graves' Ig's also precipitated a protein of relative molecular mass (Mr) 64,000 from Triton-solubilized, 125I-labelled Y. enterocolitica, similar in size to one of the proteins precipitated by Graves' Ig's from human thyroid membranes. These findings are consistent with the hypothesis that thyroid autoimmunity may be triggered by bacterial infection via a mechanism involving crossreactivity at the level of the TSH receptor. They also suggest that elements of mammalian endocrine systems are highly conserved and have a function in prokaryotes.

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor.

Glucocorticoids decrease in conversion of thyroxine into 3, 5, 3'-tri-iodothyronine by isolated rat renal tubules.

1. The effect of glucocorticoids on the deiodination of thyroxine (T4) to 3,5,3'-tri-iodothyronine (T3) was studied in rat renal tubules prepared by collagenase digestion. 2. In short-term (6h) experiments, cortisol and dexamethasone inhibited the conversion of T4 into T3 at concentrations of 2 X 10-4 mol/l and 2 X 10-5 mol/l respectively. The inhibition by cortisol and dexamethasone was time dependent and was prevented by actinomycin D and progesterone, suggesting that the inhibition is mediated by an effect on nuclear transcription dependent on binding to glucocorticoid receptors. 3. In long-term (16 h) experiments, cortisol and dexamethasone inhibited T4 to T3 conversion by the tubules at concentrations of 1 X 10-12 mol/l and above. In addition, physiological concentrations of corticosterone (1 X 10-8 mol/l) were able to decrease T3 generation from T4. 4. Our data provide strong evidence that physiological concentrations of glucocorticoids are able to affect T3 production from T4 directly and suggest that they may be important regulators of T4 deiodination.U

The effect of iodothyronines on the conversion of thyroxine into 3,3'-5-tri-iodothyronine in isolated rat renal tubules.

Isolated rat renal tubules prepared by collagenase digestion were used to study the effects of 3,3',5'-tri-iodothyronine ('reverse T3', rT3) and other iodothyronines on the formation of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4). rT3 inhibited the conversion with a dose response over the concentration range 1.5nM-1.5microM. The inhibition was competitive in nature. Both 3,3'-di-iodothyronine and 3',5'-di-iodothyronine also inhibited the production of T3 and T4 in isolated rat renal tubules, but tetraiodothyroacetic acid and 3,5-di-iodothyronine were found to have no effect. These experiments demonstrate in an intact cell system that some naturally occurring iodothyronines have significant effects on T4 deiodination.

D-propranolol and DL-propranolol both decrease conversion of L-thyroxine to L-triiodothyronine.

The effects of propranolol (DL-propranolol) and D-propranolol on thyroid hormone metabolism were studied in six euthyroid volunteers receiving L-thyroxine (T4) and six hypothyroid patients receiving T4 replacement. D-propranolol as well as propranolol decreased L-triiodothyronine (T3) concentrations and the ratio of T3 to T4 in the euthyroid subjects, and D-propranolol decreased these variables in the subjects with hypothyroidism (propranolol was not given to this group). It is concluded from this study and from parallel invitro investigations that the effect of propranolol on the conversion of T4 to T3 is unrelated to its beta-adrenergic blocking activity, and that at low therapeutic doses propranolol may exert appreciable "membrane-stabilising" effects in vivo.

The effect of long-term diphenylhydantoin therapy on glucose tolerance and insulin secretion: a controlled trial.

The influence of long-term administration of diphenylhydantoin (DPH) on glucose tolerance and insulin secretion was studied in a random controlled trial in non-epileptic patients receiving the drug for 2 years following recovery from myocardial infarction. While receiving DPH, insulin response to glucose was less than that in the control group, both in absolute terms and when related to the blood glucose level. Despite this, glucose tolerance did not differ from the control group. One month after cessation of DPH, the plasma insulin response had returned to the levels found in the control group, and glucose tolerance had improved to be significantly better than that found in the control group. Thus, the tendency of DPH to impair the insulin response to glucose has been confirmed in this controlled study. However, this does not result in significantly impaired glucose tolerance; it is suggested that the decreased insulin secretion is accompanied by improved insulin sensitivity.

Inhibition by propranolol of 3,5,3'-triiodothyronine formation from thyroxine in isolated rat renal tubules: an effect independent of beta-adrenergic blockade.

Recent studies in man have shown a decrease in serum L-T3 (T3) levels in subjects treated with DL-propranolol, but the mechanism of this effect is unknown. Isolated rat renal tubules were used to study the effect of propranolol and related drugs on the formation of T3 from L-T4 (T4). Racemic (DL) propranolol at 100 microM inhibited the net formation of T3 from T4 by 35% (P less than 0.01). The D- and L-isomers of propranolol were also potent in inhibiting T3 formation, but the beta-blockers atenolol and sotalol, which have no significant membrane-stabilizing activity, had no effect at similar concentrations. Quinidine inhibited T3 formation, with a dose-response curve which was similar over the concentrations studied to that of DL-propranolol. L-Epinephrine and L-isoproterenol had no effect on T3 formation, and equimolar amounts of L-isoproterenol did not prevent the inhibition of T3 formation by DL-propranolol. cAMP production was stimulated by 200 micro M L-isoproterenol, and this was blocked by an equimolar concentration of DL-propranolol but not of D-propranolol. It is concluded that DL-propranolol directly inhibits net T3 formation from T4 in this system by a direct membrane-stabilizing or quinidine-like action and not by specific beta-blockade.

The formation of tri-iodothyronine and reverse tri-iodothyronine from thyroxine in isolated rat renal tubules.

1. Conversion of thyroxine into tri-iodothyronine and reverse tri-iodothyronine in intact cells was studied with isolated renal tubules prepared by collagenase digestion. 2. Conversion of thyroxine into tri-iodothyronine and reverse tri-iodothyronine increased progressively for at least 90 min. 3. Studies of tri-iodothyronine production from increasing amounts of thyroxine revealed that the thyroxine to tri-iodothyronine conversion is saturable. 4. Iodine and carbimazole had no effect on the thyroxine to tri-iodothyronine conversion. 5. 6-Propyl-2-thiouracil had a direct non-competitive inhibitory effect on the conversion of thyroxine into tri-iodothyronine with a 75% inhibition of the conversion at a propylthiouracil concentration within the therapeutic range in vivo. Propylthiouracil also inhibited the net formation of reverse tri-iodothyronine from thyroxine at a similar propylthiouracil concentration, as well as inhibiting the subsequent degradation of reverse tri-iodothyronine.

Thyroid hormone levels and protein binding in patients on long-term diphenylhydantoin treatment.

The effect of long-term diphenylhydantoin (DPH) treatment on thyroid hormone concentrations and protein binding was determined in a randomized controlled trial. As has been demonstrated previously, total thyroxine (T4) concentrations were significantly depressed in patients on DPH. There was no significant effect on indirect indices of protein binding of thyroid hormones, and the free thyroxine index (FTI) was also significantly depressed. Triiodothyronine (T3) and thyrotrophin (TSH) concentrations were either unaffected, or only very slightly affected by DPH. Significant effects on the FTI were still apparent 4 weeks after discontinuing treatment. It is concluded that the depression of total T4 levels observed in vivo is not due solely to diminished protein binding, but may instead be largely explained by reports suggesting enhanced degradation of T4 following DPH therapy.

Down syndrome and thyroid function in adults.

Thyroid function has been studied in 11 middle-aged and elderly subjects with proven Down syndrome. Unequivaocal biochemical evidence of hypothyroidism was found in 6, and of thyrotoxicosis in 1. It is concluded that, in contrast to the situation in children with Down syndrome, thyroid dysfunction is exceedingly common in adults with the syndrome.