Measurement Of ACTH Research Articles

128: Observations on experimental intrauterine growth retardation in sheep

Low birthweight in sheep is associated with increased perinatal mortality. The sheep uterus contains folds of endometrium (caruncles) which form the maternal cotyledon in pregnancy. Alexander (1964) removed caruncles & in a subsequent pregnancy observed a reduction of placental & fetal weight, increased incidence of premature labour & stillbirths. 4 small fetuses have been produced using this technique. Catheters were implanted into the ewe & the fetus between 105 & 110 d. Serial measurements of blood gases, pH, metabolites, cortisol & ACTH were made. The mean fetal carotid PaO2 & haematocrit were 15.9 ± 1.3 mm Hg & 37.4 ± 2.2% compared to 21.6 ± 1.0 mm Hg & 30.3 ± 0.6% in 7 control fetuses (P < 0.01). There were no significant changes in PaCO2 or pH. Plasma glucose (0.66 ± 0.03 μmol/ml), lactate (0.9 ± 0.02 μmol/ml) and a amino nitrogen (aNH2, 6.11 ± 0.25 μmol/ml) were lower than in control fetuses (glucose 1.19 ± 0.02 μmol/ml, lactate 1.47 ± 0.02 μmol/ml and a NH2 7.64 ± 0.02 μmol/ml)suggesting a limitation of placental transfer. We have previously observed increased ACTH with hypoxaemia or hypoglycaemia. However, the concentration of ACTH (69.2 ± 29.7 pg/ml) and cortisol (21 ± 8.2 ng/ml) in the small fetuses was not significantly different from controls.

Studies on the Corticotrophin-Releasing Activity of Vasopressin, Using ACTH Secretion by Cultured Rat Adenohypophyseal Cells

CRF activity of synthetic vasopressins and pitressin was studied in an in vitro system of cultured rat adenohypophyseal cells using direct measurement of ACTH by radioimmunoassay. Pitressin (posterior pituitary extract) induced a dose-related secretion of ACTH whereas synthetic arginine or lysine vasopressin were devoid of CRF activity, even with the largest tested dose (4 mug/ml). No potentiation of the CRF activity of hypothalamic extract was observed with any vasopressin preparation studied. We concluded that: 1) the CRF activity of posterior pituitary extract is not due to vasopressin, and 2) the ACTH secretion induced by vasopressin administration in vivo is unlikely to be due to a direct effect of vasopressin on adenohypophyseal cells.

GROWTH IN MONOLAYER CULTURE OF RAT PITUITARY CELLS WHICH SYNTHETIZE AND RELEASE ACTH

Cultures of rat pituitary gland cells were developed to study biosynthesis and release of ACTH. ACTH measurement was accomplished by radioimmunoassay. ACTH release was observed following stimulation with theophylline and cAMP in a dose-dependent manner. Biosynthesis was demonstrated by incorporation of 3-H-phenylalanine into the hormone, employing a double antibody technique.

DISSOCIATION OF IN VIVO AND IN VITRO "AUTONOMY" IN A HUMAN ADRENOCORTICAL TUMOUR

The diagnosis of Cushing's syndrome due to "autonomous adrenocortical tumour" has been established in a patient by appropriate examinations (especially dexamethasone suppression test, ACTH stimulation test, ACTH measurement by radioimmunoassay). Isolated cells have been prepared from a piece of the tumour by a trypsin technique so that the action of ACTH on steriodogenesis in these cells could be studied in vitro. Basal production of corticosteroids by the isolated tumour cells was low (0.3 mu plus or minus 0.04/2 h/10-5 cells). Addition of ACTH induced a dose-dependent stimulation of corticosteroid production. The tumour cells appeared to be unable to produce corticosteroids in the absence of ACTH; maximal production was rather low (1.38 mu plus or minus 0.07/2 h/10-5 cells). A possible explanation for the discrepancies between endocrine function tests and the results of the in vitro studies is given.

A Sensitive Bioassay for the Determination of Human Plasma ACTH Levels

A sensitive bioassay for the measurement of plasma ACTH is presented. The use of silicic acid adsorption of plasma, with a subsequent acid wash and aqueous acetone desorption, was successful in removing those substances which had interfered with the steroidogenic response of dispersed adrenal cells when unextracted plasma was employed. This extraction procedure extracted 72-76% of ACTH present in plasma. Two pg ACTH1-39 could be consistently detected. Alpha-hACTH1-39 and alpha-pACTH1-39 exhibited equal potencies. Beta-MSH was ineffective at dosage levels up to 2 x 10(8) pg. One x 10(8) pg of ACTH1-10, ACTH4-10, or alpha-MSH had a steroidogenic effect equivalent to that of 40 pg ACTH1-39. ACTH 17-39 and ACTH 11-24 were incapable of stimulating steroid production at doses of 1 x 10(8) pg. Excesses of the latter, but not of the former appeared to be able to antagonize the steroidogenic effect of ACTH1-39. Plasma from normal subjects, bioassayed by this extraction procedure, contained 12-186 pg/ml ACTH at 0400-0800: 14-93 pg/ml ACTH at 1000-1300, and less than 10-34 pg/ml ACTH at 1600-2200. Hypoglycemia and vasopressin administration were followed by increases in plasma ACTH concentratrations. Plasma ACTH concentrations in untreated patients with Cushing's disease (sampled over the period 0900-1300) ranged from 65-220 pg/ml. Three patients with Addison's disease (untreated or 12 h following replacement steroid withdrawal) had ACTH concentrations of 223, 370 and 1226 pg/ml. Markedly elevated ACTH concentrations were observed in a patient with Nelson's syndrome (391 and 835 pg/ml). Bioassayable ACTH was not detected in 2 patients with panhypopituitarism.

Subcutaneous glucagon as a test of the ability of the pituitary to secrete GH and ACTH.

SUMMARYThe response to the subcutaneous injection of glucagon (1 mg) has been studied as a test of the ability of the pituitary to secrete ACTH and growth hormone (GH). Tests have been performed on fifteen normal subjects or patients with unrelated conditions, nine patients with pituitary tumours and seventeen with acromegaly. It is suggested that a normal response is a peak level of 5 ng/ml or more of GH (standard NIH GH 1216C) and either a rise of plasma cortisol measured by competitive protein binding of at least 4 μg/100 ml or a peak level of 7 μg/100 ml. In seventeen patients the responses were compared with those obtained in the insulin hypoglycaemia test and there was good agreement. Measurement of plasma ACTH showed that the response is at pituitary and not adrenal cortical level. Peak responses of ACTH and GH were obtrained at 150 min and of cortisol at 180 min. Plasma glucagon concentrations were falling steadily by this time although still above the control level. The mechanism of action of glucagon is not clear but does not appear to be related to fall of blood sugar. The subcutaneous injection of glucagon is not without side effects; about half the patients were nauseated and nine vomited. Nevertheless we believe that a subcutaneous glucagon test is of value for testing the pituitary reserve of ACTH and GH in situations where insulin hypoglycaemia cannot be used.

The measurement of acth in plasma

An ACTH radioimmunoassay is described which provides a method for measuring ACTH concentrations in plasma over the range of 5-500pg/ml (1.1-111 pmoles/1). The ability of ACTH to adsorb strongly to glass is exploited by using a solid phase similar to glass but in a finely divided form to extract ACTH from plasma. ACTH is assayed by a classical radioimmunoassay procedure. The extraction process by removing proteolytic enzymes eliminates the need for mercapto-ethanol in the incubation medium and so enables the use of a second antibody reaction to separate free from antibody bound hormone. Studies of a commercially labelled preparation of ACTH indicated quantities of damage fragments. A much cleaner product was prepared by labelling human ACTH (1-39) by chloramine-T oxidation. Gel filtration experiments indicated the need for repurification of the tracer at fortnightly intervals.

Studies on Ectopic ACTH-Producing Tumors: I. Measurement of ACTH in Tumor Tissue

Studies on Ectopic ACTH-Producing Tumors: I. Measurement of ACTH in Tumor Tissue

EXTRACTION OF ACTH FROM PLASMA FOR USE IN RADIOIMMUNOASSAY

The radioimmunologie system for measurement of ACTH is disturbed by a plasmatic factor which binds ACTH in a manner similar to an antibody (1). In our hands the measurement of ACTH in untreated plasma yielded no valid results. Therefore, ‘free’ ACTH hat to be extracted from plasma.

The half-life of porcine corticotrophin in pigs.

ABSTRACT The plasma half-life of exogenous unlabelled porcine ACTH (Organon) and porcine (synthetic 1–39) ACTH 131I has been compared with that of endogenous ACTH in the domestic pig. A sensitive radioimmune assay was used for measurement of exogenous and endogenous ACTH. With ACTH 131I, free undamaged hormone in the plasma was assessed by a technique using antibody binding and charcoal separation. The estimated half-life of porcine ACTH 131I and exogenous unlabelled porcine ACTH were found to be 13 and 28 min respectively. The half-life of endogenous ACTH measured following the abrupt termination of an insulin hypoglycaemic stimulus by intravenous glucose and dexamethasone was 7 min. Radioimmune assay of endogenous ACTH produced in physiological amounts was considered to be the best method of assessing the half-life of the hormone.

Experience with a rapid oral metyrapone test and the plasma ACTH content in determining the cause of Cushing's syndrome

A rapid oral metyrapone test (0.5 Gm. every hour for six doses) and a 4 p.m. plasma ACTH bioassay were performed in 20 consecutive patients with Cushing's syndrome. At surgery, 11 had adrenocortical hyperplasia, six had an adrenocortical adenoma, and three had an adrenocortical carcinoma. None had clinical findings of the ectopic ACTH syndrome and all have been followed at least two years postoperatively. All but one patient with hyperplasia doubled their urinary 17-hydroxycorticosteroids the day of the rapid oral metyrapone test; those with adrenocortical tumors showed no significant rise. Eight patients with hyperplasia had 4 p.m. ACTH measurements and all showed detectable plasma ACTH. Six of those with tumors also had a bioassay and in none was the ACTH detectable. The rapid oral metyrapone test and measurement of the plasma ACTH level are extremely useful in determining the cause of Cushing's syndrome, and considerably reduce the number of hospital days required.

A sensitive biologic assay for ACTH.

Fluorometric determination of corticosteroids in adrenal venous plasma of the acutely hypophysectomized rat has led to the development of 2 new techniques for the measurement of ACTH. The first, consisting of jugular injection of test material, with adrenal venous bleeding 5 minutes later, gives linearity of response to log doses of ACTH ranging from 0.05 to 1.0 milliunits (mU). The second method, consisting of retrograde injection of ACTH up the adrenal vein, with adrenal venous bleeding 90 seconds later, gives linearity of response over a range from 0.005 to 0.5 mU ACTH. The retrograde technique, while less precise, provides greater adrenal sensitivity to minute doses of ACTH than heretofore described. These methods are adaptable to the measurement of ACTH in biologic fluids.