Production of a highly specific monoclonal antibody progesterone.

A progesterone monoclonal antibody which may be used in the development of a sensitive and specific immunoassay is described. The preparation involved isolation of spleen cells from Balb/c mice immunized with progesterone 11 alpha-hemisuccinate-bovine serum albumin conjugate, which were then fused with two different mouse myeloma cell lines. Hybridomas secreting antibody to progesterone were detected by immunoassay and cloned in soft agar. Five percent of the resulting clones secreted antibody; these clones originated from cultures producing very little antibody, and so might easily have been discarded. THe monoclonal antibody obtained is highly specific and has a binding affinity of approximately 1.7 X 10(9) M-1, so it may be advantageously used in the detection of progesterone in body fluids.

Effect of human plasma on the bioactivity of adrenocorticotrophin.

The effect of plasma on the bioactivity of ACTH was investigated with the cytochemical section bioassay. Levels of ACTH in normal human plasma were underestimated if the plasma concentration in the incubation medium exceeded 2% and if the plasma ACTH level was less than 2 ng/l. A retrospective analysis of 250 plasma ACTH assays revealed that the ACTH concentration was consistently underestimated in the 1:100 dilution compared with the 1:1000 dilution, although not to the extent that any assay would be rejected for non-parallelism. The extent of the underestimation increased as the ACTH concentration increased. Guinea-pig adrenal sections responded faster to 1:10 or 1:50 dilutions of human plasma (containing 2 ng ACTH/l) than to either 1:100 or 1:1000 dilutions of that same plasma, or to ACTH (over the range 0.005-5 ng/l)in the absence of plasma. This faster response could also be seen in the absence of plasma by increasing the ACTH concentration above 5 ng/l. It seems that plasma contains factors which potentiate the biological effect of ACTH in the cytochemical assay system.U

Negative rate-sensitive feedback effects on adrenocorticotrophin secretion by cortisol in normal subjects.

Plasma ACTH and corticosteroid levels were measured in normal subjects during constant infusion of either 0.9% (W/V) NaCl solution or cortisol, and during insulin-induced hypoglycaemia. During infusions of 0.9% NACl solution the secretion of ACTH and corticosteroids was episodic. Fast, rate-sensitive, negative feedback inhibition of ACTH secretin was observed during cortisol infusions, when the corticosteroid levels were within the physiological range (200-750 nmol/l) and were rising at a rate of between 5 and 10 nmol/l per min for 30 min or longer. When plasma corticosteroid levels were in a steady state, the initial fast feedback effects were abolished and ACTH secretion resumed. However, this recovery of ACTH secretion was not seen when the corticosteroid levels were persistently above 800 nmol/l. It appears that corticosteroid-induced negative feedback in man may be both rate- and level-sensitive. During insulin stress test ACTH secretion fell at time when the plasma corticosteroid level was rising rapidly (greater than 5 nmol/1 per min) despite persistent hypoglycaemia.

Hypophosphataemic osteomalacia associated with a malignant tumour of the tibia: report of a case.

A case of vitamin D resistant hypophosphataemic osteomalacia with glycosuria and aminoaciduria is reported. The course of the disease was dramatically altered by the removal of a bone sarcoma which probably had been growing slowly since the onset of the patient's symptoms five years previously. The histology and electron microscopy of the tumour are described. Three years after the operation the patient remains free of symptoms.

A simple method for the determination of free cortisol in plasma, with normal levels in men and women, including women taking low oestrogen contraceptives.

A small scale equilibrium dialysis method for the measurement of the non-protein bound cortisol in plasma is described. Dialysis is carried out in PTFE cells against an isosmolar Hepes buffer, equilibration occurring within 18 hours. Inter-assay variation was found to be approximately 6%. Samples taken from normal males and females and from oestrogen-treated females were analysed and the results compared with published levels. A significant difference in both the total and free cortisol levels was found between the normals and oestrogen-treated females.

The effect of oral contraceptives on plasma cortisol and cortisol binding capacity throughout the menstrual cycle in normal women.

Plasma cortisol and cortisol binding capacity (CBC) was measured throughout the menstrual cycle in 25 healthy women aged between 20 and 35 years. Ten women were taking an oral contraceptive containing 50 mug oestrogen and progestogen ("combined pill"), one patient took a progestogen-only contraceptive and 14 served as controls. Despite the low oestrogen content of the combined pill the level of plasma cortison and CBC were signifcantly higher than in controls at all stages of the cycle, and the cortisol/CBC ratio greater than unity during most of it. Levels in the women taking a progestogen-only contraceptive were indistinguishable from controls. The plasma cortisol and CBC levels were significantly higher in the last week of the cycle than in the first in the combined pill group, but there was no significant variation in the controls.

Negative feedback effects on ACTH secretion by cortisol in Cushing's disease.

Bioassayable plasma ACTH and corticosteroid levels were measured during constant infusions of low doses of cortisol (3-15 mg/h) into normal subjects and into two patients with Cushing's disease. Plasma ACTH levels decreased significantly in all subjects within 60 min from the start of the infusion. The rate of rise of plasma corticosteroid levels concomitant with this feedback suppression were considerably higher in the patients with Cushing's disease (greater than 13 nmol/l/min) than in normal subjects (3-6 nmol/l/min). In one of the Cushing's patients, ACTH secretion resumed in spite of the corticosteroid levels rising at a rate of greater than 20 nmol/l/min. It is concluded that cortisol-induced suppression of ACTH secretion may be operational in Cushing's disease but that the mechanism is less sensitive than in normal subjects.

The effect of short-and long-term corticosteroid treatment on sleep-associated growth hormone secretion.

Eight healthy medical studients and four renal transplant patients had blood sampled two or three times hourly throughout EEG monitored nocturnal sleep. This was carried out on the healthy subjects for a total of 12 nights without medication (control nights asleep), a total of 12 nights following 40 mg of flucortolone the previous morning, and a total of 6 nights with similar blood sampling when sleep was prevented (control nights awake). Four renal transplant patients who were receiving long-term therapy with prednisolone were similarly studied (total of 7 nights asleep). Circulating corticosteroid and growth hormone (GH) levels were determined. A peak of GH was seen during the first 2 h of sleep on the control nights when slow-wave sleep predominated. The GH peak was absent on the control nights awake. The pattern of plasma corticosteroid levels was identical during control nights asleep and awake. Both single-dose and chronic corticosteroid administration inhibited the GH peak associated with slow-wave sleep. Chronic corticosteroid therapy, but no single-dose administration in the morning, suppressed the circadian rise of plasma corticosteroids which normally occurs late in sleep.

Single daily dose corticosteroid treatment.

Thirteen patients with rheumatoid or psoriatic arthritis who had not previously received corticosteroids were treated with prednisolone in a single-dose each morning. Insulin-hypoglycaemia tests were performed before starting steroids in each patient, and again at the conclusion of the study in twelve of the thirteen (duration of steroid treatment 8-40 m). There was no difference in the mean basal or peak levels of corticosteroids, or the mean peak of growth hormone (GH) in the tests done before or during treatment, although one patient lost GH responsiveness. There was thus no evidence of hypothalamo-pituitary-adrenal (HPA) suppression in any of the twelve patients, and there was a good therapeutic response in twelve out of thirteen. One patient was dropped from the trial because treatment failed. In contrast, of seven patients who had received a similar total dose of prednisolone twice daily, three showed HPA suppression and two had lost GH responsiveness.

Association between exaggerated responsiveness to thyrotrophin-releasing hormone and hypercholesterolaemia.

Thyroid-function tests were performed on one hundred patients with suspected premyxoedema and the results compared with those in twenty age and sex matched controls. The increase in thyroid-stimulating hormone (T.S.H.) in response to thyrotrophin-releasing hormone (T.R.H.) was exaggerated in eighty-eight of the hundred patients with suspected premyxoedema. These included twenty-five with normal basal T.S.H. and sixty-three with a raised basal T.S.H. Serum-cholesterol concentration was significantly higher in those in whom premyxoedema was suspected than in the controls, even when patients presenting with degenerative arterial disease were excluded.