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SUMMARY
A radioimmunoassay method for plasma corticotrophin (ACTH) is described. ACTH is extracted from plasma by adsorption to silicic acid powder and subsequent elution by an acetone—acetic acid mixture. ACTH antiserum and 131I-labelled ACTH are added and the antigen-antibody reaction proceeds for 3 days. Activated charcoal coated with plasma is used to separate antibody-bound and unbound ACTH. Factors affecting the accuracy, sensitivity, and specificity of the assay have been assessed.
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The low plasma levels of corticotrophin (ACTH) and the variable non-specific effects of different plasma samples on the assay system make extraction and concentration desirable when ACTH is measured by radioimmunoassay. Currently available extraction procedures (Bornstein & Trewhella, 1950; Demura, West, Nugent, Nakagawa & Tyler, 1966) are time-consuming, and there is a place for a simple, rapid and reliable technique.
The affinity of ACTH for glass, talc, ion exchange resins, cellulose and charcoal was, in practice, not sufficiently reversible to be of use in the extraction of ACTH from plasma. It was found, however, that silicic acid (100-mesh Mallinckrodt) adsorbed over 90% of 131I-labelled porcine ACTH added to plasma, and yielded over 90% of the adsorbed tracer when eluted with a mixture of glacial acetic acid—acetone—distilled water in the proportion 1:25:100 (by vol.). ([131I]ACTH was prepared by J. Landon's modification of Greenwood, Hunter & Glover's 1963 technique.)
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SUMMARY
The plasma immunoreactive corticotrophin (ACTH) response to metyrapone (1 g orally at 08.00 h, and 6 hourly thereafter for 48 h) was analysed in 20 patients with a normal oxogenic steroid response. A greater than threefold rise in the mean plasma ACTH concentration was observed within 24 h. The mean plasma ACTH level at 08.00 h on the 2nd day of the test was significantly higher than the mean level at 16.00 h, indicating that the diurnal rhythm in ACTH release persists despite metyrapone administration. The degree of rhythmicity, as assessed by the ratio of the means of the 08.00 and 16.00 h ACTH values on the 2nd day of metyrapone administration, was comparable to, but slightly less than that observed in untreated normal controls.
The ACTH response to reduction of circulating cortisol levels was also studied by interrupting steroid replacement therapy for 24 h in eight patients with adrenal disease. The observed increase in plasma ACTH was more variable than that after metyrapone, the rapidity of the ACTH rise possibly being influenced by the details of previous replacement therapy and the severity of the adrenal disease.
There was no significant difference in the plasma ACTH response to metyrapone and insulin-induced hypoglycaemia in 13 patients whose pituitary function had been assessed by each of these procedures.
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Abstract
Immunoreactive corticotrophin-releasing hormone (irCRH) was present in methanolic extracts of equine peripheral blood and showed no elevation in maternal peripheral serum in late gestation (0·54 ±0·25 pmol/l; mean ± s.d.) compared with control horses (0·41 ± 015 pmol/l). The irCRH of methanolic extracts of pituitary venous plasma had a similar elution position following reverse-phase HPLC to synthetic human CRH(1–41) and to irCRH released from horse stalk-median eminence tissue incubated in vitro. Gel chromatographic studies showed no evidence for a plasma CRH-binding protein (CRHBP) analogous to that found in human plasma in either peripheral blood from normal or pregnant horses or in pituitary venous plasma sampled from a cannulated horse. CRH-binding activity was detectable in peripheral plasma from one horse, however the molecular size of this was indicative of a γ-globulin rather than the 37 kDa CRHBP.
These studies suggest that, unlike in the human, CRH does not rise to high values in late gestation nor circulate in a bound form in equine plasma.
Journal of Endocrinology (1994) 143, 455–460
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ABSTRACT
In order to assess the physiological importance of endogenous arginine vasopressin (AVP) in augmenting the ACTH response to corticotrophin-releasing factor (CRF), the response to CRF during hypertonic saline infusion in six Coopworth sheep was examined. A 4-h infusion of 5% (w/v) NaCl (3·8 ml/min) resulted in significantly (P < 0·01) greater rises in ACTH and cortisol, but not aldosterone, than were observed after CRF alone. Infusion of hypertonic saline without CRF resulted in a highly significant (P < 0·001) rise in plasma osmolality and AVP but no significant change in plasma ACTH, cortisol or aldosterone.
It is concluded that a marked but physiological increase in peripheral (and presumably central) levels of AVP does not result in any demonstrable change in plasma ACTH concentration. However, under these conditions, the ACTH and cortisol responses to CRF are considerably augmented.
J. Endocr. (1986) 108, 309–312
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SUMMARY
The coated charcoal immunoassay method was applied to the measurement of the corticotrophin (ACTH) response to insulin hypoglycaemia, lysine-vasopressin, metyrapone and surgical stress in female piglets. The maximum ACTH responses average 392 picograms (pg.)/ml. (insulin hypoglycaemia), 111 pg./ml. (lysine-vasopressin) and exceeded 350 pg./ml. after metyrapone. The time relationships between blood sugar, 11-hydroxycorticosteroid and ACTH levels were also examined. Despite constant infusion rates, the ACTH response to lysine-vasopressin was not sustained. Previous administration of dexamethasone suppressed the response to insulin hypoglycaemia and lysine-vasopressin.
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A sensitive and specific radioimmunoassay developed for measuring the met-enkephalin analogue d-ala2-met(0)5-ol-enkephalin (DAMME) was used to study the pharmacokinetics of DAMME in the circulation of sheep. Plasma concentrations of DAMME were measured at varying time-intervals after an intravenous bolus injection or following a constant intravenous infusion of the analogue. The mean metabolic clearance rate of DAMME was 2·8 ml/min per kg, the mean circulating half-life was 52 min and the mean volume of distribution was 190 ml/kg. The longer circulating time of the analogue when compared with that of naturally occurring met-enkephalin would appear to explain its prolonged analgesic effect.
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We have investigated whether sexual maturation in female rats is affected by repeated flurothyl-induced convulsions. This treatment had no effect on the normal age-related increase in body weight though puberty (vaginal opening) was significantly delayed when compared with non-convulsed control rats. In an attempt to probe the mechanism of this delaying effect we observed that (1) anterior pituitary response to gonadotrophin releasing hormone in vitro was normal in terms of LH release but FSH secretion was impaired and (2) progesterone injection in oestrogen-primed convulsed rats failed to generate an ovulatory-type surge of LH or FSH. Basal serum levels and basal in-vitro secretion of LH and FSH were normal. We conclude that repeated convulsions adversely affect the hypothalamo-pituitary-gonadotrophin system of immature female rats.
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Urine for the analysis of pregnanediol, oestrogens, FSH and LH, was collected weekly from 50 normal menstruant women. Twenty of these women were aged ≥ 40 years and had a history of regular menstrual cycles; they are termed premenopausal. The other 30 reported a recent break in regular cyclicity and are termed perimenopausal. All menstrual cycles observed in the premenopausal women were ovulatory in type and 25–30 days in length. The 124 cycles observed in the perimenopausal women were 18–260 days in length (median, 29 days), with 52% of the ovulatory type. To describe this diversity, a systematic classification is proposed based on (1) the excretion of pregnanediol in the 12 days preceding menstruation (classes I–IV), (2) gonadotrophin output (categories A–E, and L), and (3) the length of the menstrual cycle in days. The premenstrual surge of pregnanediol was greatest in class I cycles and diminished progressively until it became undetectable in class IV. Gonadotrophin excretion was lowest in category A cycles and increased progressively until all levels were within the postmenopausal range by category E. In cycles of category L only LH (and not FSH) was raised.
In the perimenopausal women 37 cycles included episodes of high gonadotrophin excretion (categories C–L), a phenomenon which was not seen in the premenopausal women. These cycles were usually longer than 50 days and were often anovulatory in type (classes II—IV). Typically they began with the high gonadotrophin levels and the low oestrogens which characterize the postmenopausal state, and ended after a rise in oestrogen output to levels ≥ 70 nmol/24 h. It is concluded that 'anovulatory' cycles and cycles in which there are 'postmenopausal' levels of FSH and LH are common in the perimenopause and that they are rare in premenopausal women.
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The Medical Unit, The Princess Margaret Hospital, Christchurch 2, New Zealand
(Revised manuscript received 21 August 1978)
The frequent clinical and research requirement for measurement of both plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) has prompted the development of a simultaneous radioimmunoassay for these two hormones. The considerable advantages of a simultaneous method include an economy of plasma volume, assay reagents, test-tubes and, more importantly, the time required for radioactive counting and performance of the assay by technical staff. The latter two factors comprise a significant proportion of radioimmunoassay operating costs.
This report describes a simultaneous radioimmunoassay based on the use of 131I-labelled FSH, 125I-labelled LH, anti-FSH serum M93 6873 (a generous gift from Professor W. R. Butt, Birmingham), anti-human chorionic gonadotrophin (HCG) serum for LH measurement (Donald, 1972) and donkey anti-rabbit precipitating serum (Wellcome Reagents, U.K.) for separation of antibody-bound and free hormones. Pituitary gonadotrophin standard (LER 907)
