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SUMMARY
A simple and rapid method is described for labelling oxytocin with 131I at a high specific activity. This method is compared with those of previous workers. A satisfactory antiserum has been raised by direct intra-lymph node injection of oxytocin adsorbed to carbon microparticles. A number of methods for separating antibody-bound from free oxytocin are described, and reasons given for preferring a procedure using ammonium sulphate precipitation. These data form the basis for developing a radioimmunoassay intended for the determination of oxytocin in human plasma.
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SUMMARY
A radioimmunoassay for bovine neurophysin is described. The assay has a lower limit of detection of approximately 0·5 ng/ml. Bovine neurophysin extracted and purified from acetone-dried posterior pituitary lobe powder was used for standardization, radio-iodination and immunization of rabbits. Some anti-neurophysin sera were also obtained from human subjects treated with Pitressin. Most of the antisera tested did not readily distinguish between the different neurophysin components isolated from bovine neurohypophysial tissue. The antisera cross-reacted with material extracted from whole pituitaries of a number of mammalian species, including man, pig, sheep, guinea-pig and rat, but not with the pituitary peptides from the cod or dogfish, or the egret. Other pituitary peptides, including oxytocin, vasopressin, corticotrophin, growth hormone and luteinizing hormone, did not interfere with the assay.
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ABSTRACT
To study the potential role of GH-releasing hormone (GHRH) in maintaining circulating levels of GH during pregnancy, 302 maternal plasma samples were collected from non-fasted subjects at various stages of pregnancy and assayed for GHRH using a 'two-site' immunoradiometric assay. The GH and placental lactogen levels were also determined. In addition, maternal plasma samples taken during labour, amniotic fluid and cord blood were also assayed for these hormones.
Maternal plasma GHRH levels were similar to non-pregnant levels throughout gestation despite fluctuations in GH values which were always higher than non-pregnant levels. There was no significant difference between GHRH levels in maternal plasma and cord blood although high GH levels were observed in the latter. These findings suggest that peripheral GHRH levels do not play an important role in maintaining circulating GH levels during pregnancy.
Journal of Endocrinology (1990) 125, 161–167
Search for other papers by V. H. T. JAMES in
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SUMMARY
Adrenocortical suppression tests, based on the fall in urinary 17-hydroxy-corticosteroid excretion during the oral administration of dexamethasone, were found to be of value in the diagnosis of Cushing's syndrome, but less useful in differentiating bilateral adrenal hyperplasia from adrenal tumour. Such tests have the disadvantage of requiring accurate urine collections and of taking several days to perform. A test is described, based on the decrease in plasma cortisol concentration during i.v. infusion of dexamethasone at a rate of 1 mg./hr. The results obtained in 12 patients with Cushing's syndrome and bilateral adrenal hyperplasia differed from those found in control subjects in that there was a delay between the start of the infusion and the fall of plasma cortisol, and the rate of fall was less rapid. The values found after 180 min., expressed either as μg./100 ml. or as a percentage of the resting level, differed significantly (P < 0·001) in the two groups. The test proved valuable as an aid to the diagnosis of Cushing's syndrome, was easy to perform, and could be completed in 3 hr.
In some patients with Cushing's syndrome, the administration of synthetic glucocorticoids appeared to result in an increased urinary steroid excretion. A transient increase in plasma cortisol levels was also observed in some of these patients during the early period of dexamethasone infusion. It is thought that this finding reflects an alteration in steroid metabolism induced by dexamethasone and fluorocortisol.
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Search for other papers by V. H. T. JAMES in
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SUMMARY
The adrenocortical response to stress as shown by an increase of the plasma cortisol concentration during insulin-induced hypoglycaemia has been studied. The response was found to depend upon the degree and duration of the hypoglycaemia and upon the integrity of the entire hypothalamo-pituitary-adrenal axis. Thus, there was no response in subjects in whom the blood sugar did not fall below 40 mg./100 ml., nor in patients with severe hypothalamic or pituitary disorders.
The test was quick and simple to perform and did not require admission to hospital; it would seem to be of considerable value in the investigation of patients with suspected endocrine disease.
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Search for other papers by V. H. T. JAMES in
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SUMMARY
The effect of methandienone administration on urinary steroid excretion has been studied in subjects with normal pituitary-adrenal function and in patients with various endocrine diseases.
In the control subjects, a marked suppression of urinary 17-KS and 17-OHCS excretion occurred, which persisted throughout even prolonged periods of methandienone administration. Upon cessation of methandienone treatment a prompt rise in urinary steroid excretion occurred, on occasions to levels slightly higher than those seen before treatment.
Similar results were obtained in subjects with acromegaly and Cushing's syndrome, but in patients with anorexia nervosa and a low basal steroid excretion, the suppressive effect of methandienone was less marked.
During treatment with methandienone, pituitary response to metopirone was depressed, but adrenal response to corticotrophin was unaltered.
It was concluded that methandienone diminishes the rate of production of adrenocortical steroid by inhibiting corticotrophin production or release. Unlike the inhibition observed during treatment with glucocorticoids, it was not associated with atrophy of the adrenal glands.
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Search for other papers by LESLEY H. REES in
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SUMMARY
The pars intermedia of the rat pituitary contains a peptide resembling the 18–39 portion of adrenocorticotrophic hormone (ACTH), which has been termed 'corticotrophin-like intermediate lobe peptide' (CLIP). It can be detected by its cross-reaction with an antiserum directed against the CO2H-terminal portion of the ACTH molecule; it has an amino acid composition identical to the 18–39 portion of human ACTH, except for one less glycine and an extra valine residue, and it is rapidly released from neurointermediate lobes maintained in organ culture. The pars intermedia also contains a peptide with an amino acid composition and biological potency identical to that of melanocyte-stimulating hormone (α-MSH) isolated from other mammals, and which accounts for the bulk of melanocyte-stimulating activity in the pituitary. Rat ACTH resembles human ACTH in amino acid composition, except for an extra valine and one less glycine residue. On the basis of these data it is proposed that ACTH is the precursor of α-MSH and CLIP, which are both present in the cells of the pars intermedia.
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SUMMARY
A radioimmunoassay for oxytocin in aqueous solution is described, with a sensitivity comparable with the best current bioassays. It is highly specific; arginine-vasopressin and lysine-vasopressin interfere only at 1000-fold greater concentration, while bradykinin, histamine, acetycholine and many other substances, which interfere with some bioassays, have no effect. In certain circumstances, there is a dissociation between loss of biological and immunological activity. Thus reducing agents had no effect on immunological activity, in contrast to their effect on biological activity. In late pregnancy plasma, the biological activity of oxytocin is destroyed more rapidly than the immunological activity. Radioimmunoassays have considerable advantages over bioassays both in convenience and specificity. However, bioassays should be employed for reference purposes because of the dissociation between biological and immunological activity that may occur.
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Search for other papers by MURIEL J. KITAU in
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SUMMARY
A radioimmunoassay for vasopressin was developed using antibodies produced against conjugated and non-conjugated arginine vasopressin. Despite the fact that the vasopressin molecule has only eight amino acids, cross reactivity studies showed that these antibodies were specific for different amino acid sequences.
Labelled hormone of high specific activity (350–800 μCi/μg) was produced by a modification of the chloramine-T method. Unreacted iodide was removed by the batchwise addition of an ion-exchange resin. Other techniques of purification produced no advantage over this simple method.
Several methods of separating antibody-bound and free hormone were studied. All except chromatoelectrophoresis proved satisfactory. Ammonium sulphate or ethanol precipitation of bound hormone was chosen because of simplicity, speed and reproducibility.
The lower limit of detection of the assay was 80 pg arginine vasopressin/ml diluent buffer. Therefore an extraction and concentration procedure is necessary for the measurement of basal circulating levels of the hormone.
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SUMMARY
The plasma sugar, 11-hydroxycorticosteroid, and growth hormone responses to insulin have been studied in patients with Cushing's disease. They showed an impaired or absent plasma 11-hydroxycorticosteroid and growth hormone rise during the test, as compared with control subjects, despite the injection of amounts of insulin which produced a similar degree of hypoglycaemia. This test proved of value in differentiating between these patients and those with 'simple ' obesity since the latter usually showed a normal growth hormone and adrenal response provided an adequate amount of insulin was administered.
The patients with Cushing's disease also had an impaired adrenal response to pyrogen and to dexamethasone administration and failed to show a normal plasma 11-hydroxycorticosteroid circadian rhythm. Their response to corticotrophin, lysine vasopressin, and metyrapone, however, was normal or enhanced. It is suggested that these findings imply an abnormality of hypothalamic or cerebral control and not a primary defect of pituitary function as proposed originally by Harvey Cushing.
The growth hormone response to insulin remained impaired in four out of six patients totally adrenalectomized for Cushing's disease but was normal in three patients adrenalectomized for other reasons. It is suggested that the defect which impairs the adrenal response to insulin may, on occasions, also impair the mechanism normally operative for growth hormone secretion.
