F. W. Chu and R. P. Ekins
Abstract. Corticosteroid binding globulin (CBG) was detected by a specific radioimmunoassay in mixed saliva (25.4 ± 4.0 μg/l, mean ± sem) and in pure, uncontaminated parotid fluids (17.4 ± 2.7 μg/l) at resting flowrates of approximately 500 μl/min and 50 μl/gland per min, respectively. In parotid fluids collected at stimulated flow-rates of between 300–1000 μl/gland per min, CBG could not be detected. This observation suggests the direct flow-rate-dependent transfer/secretion of CBG in saliva. When cortisol was measured (RIA) in dilution experiments in both mixed saliva and parotid fluids using phosphate buffer at pH 7.4 as diluent, a protein-binding effect analogous to that found in plasma samples was observed. However, this effect was abolished if a known CBG inhibitor, phosphate:citrate buffer at pH 4, was used as the diluent in the assay. A bound fraction of cortisol was found in both mixed saliva (14.0 ± 4.0%) and parotid fluid samples (12.3 ± 1.3%) by equilibrium dialysis. These findings appear to contradict the currently accepted notion that specific plasma steroid binding proteins, and hence the protein-bound steroids, are absent in uncontaminated saliva; and that their presence in mixed saliva is the consequence solely of contamination by gingival fluid and/or plasma from mouth or gum abrasions. We conclude that both protein-bound and free steroids are present in uncontaminated saliva and that salivary total and plasma free steroid concentrations are not identical.
P. A. Ealey, N.J. Marshall and R. P. Ekins
Abstract. Subsequent to the discovery of vasoactive intestinal peptide (VIP) in the thyroid gland, VIP has been shown to stimulate various thyroid functions. The site of interaction of VIP with the thyroid follicular cell is at present not known, and this study has used the ultrasensitive cytochemical bioassay (CBA) for thyroid stimulators to investigate this further. Exposure of thyroid sections for 3 min to VIP resulted in increased naphthylamidase activity, with half-maximal response observed at 3 × 10−13 m VIP. This response to such low doses of VIP is consistent with the CBA being ultrasensitive to other thyroid stimulators e.g. TSH, thyroid stimulating antibodies and forskolin. The response to VIP was abolished by rabbit anti-VIP antiserum. The dose-response curve to VIP was bell-shaped (as with the other stimulators), maximal stimulation occurring at 10−12 m VIP. In contrast, however, to other thyroid stimulators, namely TSH, LATS-B and 3 monoclonal stimulating antibodies, whose ascending limbs of the doseresponse curves extended over 3-4 orders of magnitude, the VIP curve rose rapidly from basal to maximal tissue stimulation from 10−13 to 10−12 m VIP, i.e. one order of magnitude. This unusual dose-response curve to VIP was parallel to that produced by forskolin. 11E8, a monoclonal 'blocking' antibody which is a potent inhibitor of TSH stimulation, did not 'block' forskolin stimulation, consistent with the belief that forskolin acts at a post-receptor site. However, unlike forskolin, VIP was inhibited by monoclonal 11E8, which may imply a hitherto unexpected involvement of the TSH receptor in VIP stimulation of the thyroid or, alternatively, steric inhibition by 11E8 when bound to the TSH receptor of VIP interaction with adjacent VIP-specific receptors.
M. Ballabio, A. K. Sinha and R. P. Ekins
Abstract. The presence of thyroid stimulating activity in partially purified hCG was investigated using, as bioassay system, iodide uptake in rat thyroid FRTL-5 cells. The biological responses evoked by hCG were tested after neutralisation with monoclonal and polyclonal antisera to hTSH and hCG, and after fractionation on Sephadex G-100. The molar amounts of TSH and hCG in respective preparations were calculated assuming an activity of 30 IU/mg and 19 IU/mg, respectively, for bTSH and hTSH, and of 14 000 IU/mg for hCG. A dose-dependent response, paralleling that evoked by hTSH, was observed in a concentration range of 0.1–4 μmol/l hCG; 1 μmol of hCG was equivalent to 50 pmol of bTSH and 132 pmol of hTSH. The thyrotropic activity coeluted with hCG immunoactivity on Sephadex G-100. Incubation with monoclonal anti-hTSH antibodies did not affect the stimulatory ability of hCG preparation, indicating that it was not due to hTSH contamination. Similarly, a pretreatment with monoclonal and polyclonal anti-hCG antibodies did not significantly alter the iodide uptake response induced by hCG. These results indicate that the thyrotropic activity in partially purified hCG is not due to the presence of aspecific contaminants, but to a substance structurally related to hCG in terms of molecular weight. However, it appeared to differ from hCG immunologically, suggesting the hypothesis that minor modifications in the molecular structure may confer thyrotropic activity on hCG, altering its immunoreactive potency.
Patricia Banks, R. P. Ekins and J. D. H. Slater
S. P. Bidey, K. Ryder, R. Gaines-Das, N.J. Marshall and R. P. Ekins
Abstract. A clonal strain of thyrotrophin (TSH)-dependent rat thyroid cells (FRTL-5) has been used to evaluate the biological activity of reference preparations of both human and bovine TSH. Using the accumulation of intracellular cyclic AMP as a response parameter, the widely used bovine TSH preparation. Armour 'Thytropar', was calibrated against the First International Standard of Thyrotrophin (pituitary TSH), bovine, for immunoassay. Log dose – log response curves were parallel, and a relative potency of 2.4 IU/unit of 'Thytropar' was obtained. Subcultures of FRTL-5 cells were more responsive to both bovine and human TSH than were human thyroid follicular cells maintained as primary monolayer cultures. Dose-response curves for cyclic AMP accumulation were parallel for a single cell type differentially incubated with human TSH (the First International Reference Preparation) and bovine TSH (Armour 'Thytropar') preparations. The relative potencies (units: IU) of bovine-human TSH were of the order of 2.0 when tested on both FRTL-5 cultures and primary human thyroid monolayers. This suggests that in the spectrum of structural differences between TSH receptors of different species, the discriminatory powers of the human and FRTL-5 cell TSH receptor are similar. Thus FRTL-5 cells form the basis of a bioassay system of considerable value in the study of human thyroid stimulators, as we demonstrate in an evaluation of two recent preparations of human TSH.
R. F. Harvey, E. S. Williams, S. Ellis and R. P. Ekins
Thyroxine-binding globulin (TBG) levels were found to be significantly lower in 40 thyrotoxic patients than in 70 healthy subjects. A similar mean fall in TBG of 18% was produced by administration of 'physiological' doses of triiodothyronine to healthy volunteers. However, intramuscular injection of thyroid-stimulating hormone (TSH) had no consistent effect on the level of TBG as measured up to 4 days after injection. These findings are believed to explain the anomalous results described by earlier workers. It is suggested that TBG concentration is regulated by unknown mechanisms subject to the influence of many hormones, including triiodothyronine.
J. D. M. Albano, R. P. Ekins, G. Maritz and R. C. Turner
A radioimmunoassay method for the measurement of plasma insulin is described relying on activated charcoal for the separation of free and bound fractions. The technique illustrates the application of theoretical precepts designed to maximise assay precision in all radioimmunoassay and other saturation assay techniques.
In addition, because particular emphasis has been placed on ensuring that, as far as is possible, all incubation mixtures are similar as possible other than in hormone concentration, non-specific effects appear to have been essentially eliminated. The technique yields a mean normal fasting value of 5.3μU/ml (range 2–14 μU/ml). Its sensitivity is such that 10 μl samples of serum (or plasma) may be assayed.
Maria Santos, V. Poshyachinda, B. L. Brown, A. Marisa Sgherzi and R. P. Ekins
David B. Dunger, Janet A. Perkins, Terence P. Jowett, Philip R. Edwards, Leslie A. Cox, Michael A. Preece and Roger P. Ekins
Thyroid hormones are essential for normal pubertal growth, yet the changes in total and, especially, free thyroid hormones and thyroxine-binding globulin during puberty have not been adequately defined. Serum from 39 normal children (20 girls, 19 boys) between the ages of 10 and 15 years were assayed for total T4, free T4, free T3 and thyroxine-binding globulin at 6-monthly intervals; the free hormone assays were valid, non-analogue methodologies. In the girls, free T4 levels fell from 15.7±0.6 pmol/l at 10 years to 13.0±0.6 (p<0.001) at 12.5 years before rising to 15.9±0.7 at 15 years; this nadir occurred at puberty stages 3-4. Changes in total T4 followed a similar pattern with a slight delay in the nadir (13 years, puberty stage 4). In the boys, free T4 fell from 16.3±0.6 pmol/l at 10 years to 14.3±0.3 at 13.5 years, then rising to 15.4±0.5 at 15 years; the nadir again occurred at puberty stages 3-4. The corresponding nadir in total T4 which occurred at puberty stages 4-5 was not apparent by age analysis. Thyroxine-binding globulin concentrations remained unchanged in the girls, but fell slightly in the boys during later puberty. Free T3 concentrations in the girls showed a progressive fall after 12.5 years which was significant by the age of 14 when most had been in puberty stage 5 for more than 1 year. The boys showed no change of free T3 concentration throughout the study. These data demonstrate important differences in the levels of free thyroid hormones in males and females during puberty. Normal ranges for girls and boys between the ages of 9.5 and 15.5 years are presented.