The effect of plasmapheresis on the concentration of certain plasma proteins: a case identified by an inaccurate LDL-cholesterol estimation.

A 35-year-old man was found to have a negative LDL-cholesterol concentration (-0.05 mmol/L) when estimated on a fasting plasma sample using the Friedewald equation. Plasma urea, electrolytes and liver function tests (LFTs) were normal except for a raised total bilirubin of 74 micromol/L. Haematological results showed both a low haemoglobin and fibrinogen concentration. It transpired that the patient had undergone daily plasmapheresis treatments on the previous four days; plasma had been exchanged with a 5% albumin solution. He had been diagnosed with Evan's syndrome previously (characterized by autoimmune haemolytic anaemia) and had been admitted with severe anaemia, which had proved unresponsive to conventional treatments. The concentration of most plasma substances is reduced by 50-60% after one standard plasmapheresis treatment, with the rate of return to steady state concentrations varying among analytes. The finding of a negative LDL-cholesterol concentration (arising primarily as a result of normal triglyceride concentrations) may reflect the more efficient removal of LDL and HDL lipoproteins during the plasmapheresis procedure (PP) than lipoproteins containing proportionally more triglycerides. Plasma lipids, total protein, immunoglobulins and transferrin had recovered to steady state concentrations by eight days post-plasmapheresis, whereas caeruloplasmin concentrations had not. This case report illustrates the difficulties of obtaining accurate information on the steady state concentrations of plasma analytes, in particular protein bound substances, when analysis is carried out on a sample from a patient that has recently undergone plasmapheresis. The normal plasma albumin in this situation did not flag the possibility of the sample being artefactually diluted.

In vitro cold activation of complement shown by an overestimation of total complement 4: a study in patients with hepatitis C virus infection.

Total complement 4 (C4) levels, when analysed on the Beckman Array nephelometer, were found to increase number of serum specimens [predominantly from patients with hepatitis C virus (HCV) infection] after overnight storage at 4 degrees C. In order to investigate whether the phenomenon of in vitro cold-dependent activation of complement (CDAC) was the explanation for this increase, paired specimens were collected from 63 patients with HCV infection in tubes with no anticoagulant (serum) and in tubes containing EDTA (which inhibits complement activation). C4 levels increased after overnight storage at 4 degrees C in 33 serum specimens (52%). In contrast, no increase in C4 levels was observed in any of the 63 EDTA specimens. Immunofixation of intact and activated C4 products confirmed that complement activation had taken place in the serum specimens in which C4 levels had increased after storage. There was a higher frequency of hepatitis C viraemia (P<0.0001), HCV antibody positivity (P<0.05) and the presence of rheumatoid factor (P<0.05) in the group of patients in whose serum samples CDAC had occurred (n = 33) than in the other group (n = 30). As a result of our findings on C4 analysis in stored serum specimens, we would recommend potassium EDTA plasma as the specimen of choice for complement analysis on the Beckman Array.

Non-ACTH POMC fragments stimulate aldosterone production by human adrenal cells in vitro.

The possibility that non-ACTH proopiomelanocortin-derived fragments may stimulate aldosterone production has previously been studied using nonhuman cells with inconsistent results. We have examined the response of aldosterone to beta-endorphin (beta-End) and joining peptide (JP) and compared these with the response to ACTH using eight cell suspensions prepared from human adrenal glands. ACTH, 10(-6), 10(-8), and 10(-10) M, consistently stimulated aldosterone accumulation above that occurring in unstimulated cells (150 +/- 83, 120 +/- 62, and 77 +/- 32 fmol/10(4) cells above basal, respectively; mean +/- SE; p < 0.05). beta-End significantly stimulated aldosterone production at 10(-6) and 10(-8) M (114 +/- 84 and 50 +/- 24 fmol/10(4) cells above basal; p < 0.05); 10(-10) M beta-End did not provide significant stimulation. Furthermore, JP stimulated aldosterone biosynthesis (41 +/- 16 fmol/10(4) cells above basal; p < 0.05), only at the highest concentration used, 10(-6) M. The addition of 10(-8) M ACTH plus 10(-6) and 10(-10) M beta-End to human adrenal cells yielded values significantly greater than those achieved with either agent alone (267 +/- 152 and 183 +/- 89 fmol/10(4) cells above basal; p < 0.05). These data indicate for the first time that beta-End and JP have the capacity to stimulate aldosterone production in human adrenal cells in vitro. The physiological and potential clinical significance of these observations has yet to be elucidated.

Decline in insulin-like growth factor I levels after clomiphene citrate does not correct hyperandrogenemia in polycystic ovary syndrome.

It is widely accepted that the action of clomiphene citrate (CC) is mediated through its antiestrogenic properties on the hypothalamic-pituitary axis. Although insulin-like growth factor I (IGF-I) enhances the thecal cell response to LH, and estrogen treatment is associated with a reduction in IGF-I levels, CC is known to decrease circulatory IGF-I levels in polycystic ovary syndrome (PCOS) patients. The impact of lowering IGF-I levels on androgen levels in PCOS is unknown. This study was designed to examine the impact of CC treatment on the interrelationships of IGF-I, androgens, and estrogens in normal subjects and patients with PCOS. IGF-I, gonadotropin, androgen, estrogen, and sex hormone-binding globulin levels were measured in 8 PCOS patients and 10 normal subjects before and after treatment with the antiestrogen CC. Studies were performed in the early follicular phase, days 4-6 of the menstrual cycle in normal subjects. In normal subjects, CC treatment led to a significant increase in estradiol (84 +/- 10 to 234 +/- 62 pmol/L, untreated and CC treated; P < 0.05) and estrone (125 +/- 14 to 257 +/- 29 pmol/L; P < 0.05) levels with a significant lowering of IGF-I levels (297 +/- 25 to 230 +/- 17 micrograms/L; P < 0.05). Similarly, in PCOS patients a significant increase in estradiol (110 +/- 11 to 245 +/- 58 pmol/L; P < 0.05) and estrone (301 +/- 32 to 401 +/- 90 pmol/L; P < 0.05) levels and a significant lowering of IGF-I levels (330 +/- 43 to 214 +/- 27 micrograms/L; P < 0.05) were observed after CC treatment. However, no significant correlation was observed between changes in IGF-I and changes in estradiol in either group. Compared to pretreatment levels, no significant changes in the following parameters were observed after 5 days of CC treatment in either study group: testosterone, testosterone/sex hormone-binding globulin ratio, and androstenedione. The relationship among CC treatment, gonadotropin, estrogen, and IGF-I levels is complex. Changes in blood IGF-I levels are not associated with changes in androgen levels, although paracrine and or autocrine effects cannot be excluded.

Intra-adrenal factors are not involved in the differential control of cortisol and adrenal androgens in human adrenals.

The differential control of adrenal androgens and cortisol may be due to intra-adrenal factors, which may be age- or sex-related, or due to extra-adrenal factors, such as circulating hormones. The purpose of this study was to identify any intrinsic differences that may exist in steroidogenic production occurring within adrenals obtained from males and females, and any maturational differences that may evolve with age. Using human adrenals from 48 transplant donors (32 males, 16 females; ages 5-60 years), the influences of age and sex on basal production of and ACTH-stimulated cortisol, androstenedione and dehydroepiandrosterone (DHEA) were examined in freshly prepared adrenal cell suspensions. Basal and ACTH-stimulated cortisol, androstenedione and DHEA production were similar in adrenals from males and females and did not correlate significantly with age when the whole group was examined. When steroidogenesis in male and female adrenals was examined separately against age, a significant correlation was observed only for basal and ACTH-stimulated androstenedione in adrenals from males in the younger age group, 5-30 years (basal: r=0.84, P=0.0001; ACTH-stimulated: r=0.52, P=0.007). Examination of the relationships between the steroids disclosed that the basal and ACTH-stimulated cortisol/androgen ratios did not correlate significantly with age, but the androstenedione/DHEA ratio showed a significant direct relationship with age in males only (basal: r=0.53, P=0.006; ACTH-stimulated: r=0.5, P=0.01). These data suggest that the influences of sex and age are minor in the modulation of adrenal steroidogenesis and support the concept that extra-adrenal factors dominate in the differential modulation of adrenal androgens and cortisol. The relationship between the androstenedione/ DHEA ratio and increasing age in men is consistent with the recently reported stimulatory effect of testosterone on adrenal steroidogenesis by induction of the conversion of DHEA to androstenedione.

A critical review of the origin and control of adrenal androgens.

The reticularis and fasciculata zones of the adrenal cortex are the predominant sources of dehydroepiandrosterone (DHEA) and DHEA-sulphate and contribute directly or indirectly 60-75% of androstenedione and testosterone in women. The specific control of adrenal androgens remains unclear. While ACTH stimulates adrenal androgen secretion, the dissociation of cortisol and androgens occurring during adrenarche and under pathological conditions suggests other factors are involved. Recent studies using human adrenal cells in vitro have demonstrated that the ratio of androgen to cortisol produced is substantially independent of the age and gender of the adrenal, indicating that extra-adrenal factors are of greater importance. beta-Endorphin and joining peptide have been shown to stimulate androgen production in human adrenal cells and to influence ACTH-stimulated steroidogenesis in a manner that promotes adrenal androgen production. The activity of these pro-opiomelanocortin-derived peptides may explain the physiological and pathological dissociations of androgens and cortisol.

Effects of nifedipine treatment on the renin-angiotensin-aldosterone axis.

Nifedipine is a commonly used agent in treating hypertension and angina because of its vasodilator properties. An inhibitory role of nifedipine on aldosterone (Aldo) biosynthesis has been documented in in vitro studies. This study was designed to examine the impact of a sustained release nifedipine formulation on Aldo biosynthesis and its clinical consequences. Early and late effects of nifedipine on Aldo, PRA, and Aldo/PRA ratio levels were studied in a single blind, placebo-controlled, 10-day pilot study. Ten normotensive subjects and 10 patients with hypertension were studied. Blood samples for the measurement of Aldo and PRA were obtained at 2-h intervals for 10 h on a control day and on days 1 and 8 of nifedipine treatment for the determination of baseline, early, and late values. Placebo was administered at 0800 h on the first and second days of the study, whereas nifedipine (60 mg/day) was given for the following 8 days. The Aldo/ PRA ratio was used as a sensitive indirect index of the responsiveness of Aldo secretion to adrenal stimulation with angiotensin. Compared to those on the control day, a significant rise in the integrated PRA levels occurred on the first day of nifedipine treatment, with a further rise observed on the eighth day of the treatment in the normotensive subjects (1.1 +/- 0.6, 1.7 +/- 1.2, and 2.5 +/- 1.8 ng/mL.h on the control day and the first and eighth days of treatment, respectively; P < 0.05) and by the eighth day in the hypertensive subjects (2.2 +/- 2.8 and 4.0 +/- 4.1 ng/mL.h; P < 0.05). A significant rise in integrated Aldo levels occurred in the normotensive subjects on the eighth day of nifedipine treatment (control day, 319 +/- 187; eighth day of nifedipine, 363 +/- 167 pmol/L; P < 0.05) and in the hypertensive subjects (426 +/- 219 and 535 +/- 284 pmol/L; P < 0.05). This was associated with a significant lowering of the Aldo/PRA ratio on the first day of the treatment, with further lowering on the eighth day in the normotensive (435 +/- 454, 269 +/- 209, and 182 +/- 107; P < 0.05) and by the eighth day in the hypertensive subjects (716 +/- 833 and 305 +/- 315; P < 0.05). When individual time points were examined in the normotensive subjects, Aldo/PRA levels were significantly lower on day 8 of nifedipine treatment at 1000, 1200, and 1400 h than corresponding values on the control day. The fall in the Aldo/PRA ratio during nifedipine treatment indicates that the previously reported in vitro inhibition of Aldo biosynthesis in adrenal cells is reproduced in vivo. In the absence of nifedipine, it is likely that Aldo levels would be higher for any given level of PRA. It is probable that the Aldo inhibition and the vasodilatatory effect of nifedipine combine to bring about the lowering of blood pressure. Drugs that inhibit renin-angiotensin axis activity are likely to be particularly effective when additional lowering of blood pressure is required.

The steroidogenic effects of beta-endorphin and joining peptide: a potential role in the modulation of adrenal androgen production.

This study examines the androgen-stimulating properties of pro-opiomelanocortin-derived peptides, ACTH, beta-endorphin (beta-End) and joining peptide (JP). Ten different cell suspensions were prepared from ten human adrenal glands. ACTH and JP stimulated cortisol, androstenedione (delta 4) and dehydroepiandrosterone (DHEA) production (P < 0.05); beta-End stimulated only delta 4 and DHEA production. beta-End brought about significant increases in the delta 4 or DHEA to cortisol ratios. The addition of beta-End (10(-10) M) suppressed ACTH-stimulated cortisol production from 7573 +/- 2960 to 5994 +/- 2654 pmol/10(6) cells (means +/- S.E.M.; P < 0.05). The addition of beta-End did not affect ACTH-stimulated delta 4 production (210 +/- 88 and 236 +/- 105 pmol/10(6) cells). JP (10(-10) M) inhibited ACTH-stimulated cortisol production so that the mean values fell to 5186 +/- 2588 and also inhibited DHEA production, from 240 +/- 48 to 180 +/- 33 pmol/10(6) cells. These results suggest that the relative production of androgen to cortisol is greater in response to beta-End and JP than in response to ACTH. If blood levels of these peptides rise to herald adrenarche as reported for beta-End, suppression of cortisol production may result in an increase in ACTH to correct cortisol levels resulting in an increase in delta 4 and DHEA levels. This may explain the occurrence of increasing androgen levels at adrenarche.

The role of plasma renin activity in evaluating the adequacy of mineralocorticoid replacement in primary adrenal insufficiency.

BACKGROUND: Elevation of plasma renin activity (PRA) is a feature of mineralocorticoid deficiency in patients with primary adrenal insufficiency. This study was designed to assess the usefulness of PRA as an index of adequacy of fludrocortisone (FC) replacement in patients with primary adrenal failure, paying particular attention to the variability in PRA levels during FC and glucocorticoid treatment. METHODS: Twenty-two patients with mineralocorticoid deficiency due to primary adrenal diseases were studied at 3 time points: 8, 24 and 32 hours following the administration of FC replacement. Body weight, blood pressure while supine and erect, PRA, and plasma or serum levels of aldosterone, urea, sodium and potassium were measured at each time. The clinical and biochemical consequences of adjusting the FC dose were monitored in 5 patients with PRA levels above the range seen in normal subjects and in one hypokalaemic patient with normal PRA levels. RESULTS: At 8 and 32 hours following FC administration, PRA levels were not significantly different. PRA levels were significantly higher at 32 hours following FC administration (4.7 +/- 1.1 nmol/l/h) than at 24 hours (4.2 +/- 1.1 nmol/l/h, mean +/- SEM, P < 0.05). At 8 and 32 hours following FC administration, potassium levels were similar. Potassium levels were significantly higher at 32 hours following FC administration (3.9 +/- 0.1 mmol/l) than at 24 hours (3.6 +/- 0.1 mmol/l, P < 0.05). No changes in measurements of sodium, urea, mean supine and erect arterial pressure or body mass index were noted at the different study points. Attempted lowering of elevated PRA in 5 normokalaemic subjects by raising the dose of FC led to normalization of PRA in all of these patients but 2 developed hypokalaemia and oedema. Lowering of FC dose in one hypokalaemic patient with normal PRA levels led to the PRA levels rising to a supranormal value while the hypokalaemia was corrected. CONCLUSIONS: These results indicate that when plasma renin activity is estimated in patients with primary adrenal insufficiency replaced with daily doses of fludrocortisone, the time of day of blood sampling is not critical. Lowering elevated plasma renin activity levels to normal in patients who were considered to be otherwise normal may lead to over-treatment in some patients. Therefore, optimal fludrocortisone replacement may be associated with mildly elevated plasma renin activity levels. The information obtained by monitoring plasma renin activity adds little to the assessment of patients based on clinical evaluation and measurement of urea and electrolyte levels in blood.

Role of progesterone deficiency in the development of luteinizing hormone and androgen abnormalities in polycystic ovary syndrome.

The aetiology of polycystic ovary syndrome (PCOS) is unknown. It is uniquely characterized by oligomenorrhoea or amenorrhoea associated with normal or high oestrogen levels. This prospective clinical study was designed to examine the possible role of the lack of cyclical exposure to progesterone in the development of gonadotrophin and androgen abnormalities in PCOS. Gonadotrophin, androgen and oestrogen levels were measured in 15 PCOS patients and 10 normal subjects untreated and following treatment with the progestogen medroxyprogesterone acetate (MPA). When compared to control subjects, PCOS patients had significantly higher luteinizing hormone (LH) pulse height, pulse amplitude, integrated LH levels, LH response to gonadotrophin-releasing hormone (GnRH) and LH/FSH ratio; LH pulse frequency was similar in the two groups. In addition, the testosterone/sex hormone binding globulin ratio (T/SHBG), androstenedione and oestrone concentrations in the plasma were significantly higher in PCOS than in control subjects. When PCOS patients were treated with MPA for 5 days, there were significant decreases (p < 0.02-0.001) to values no longer different from normal: from 8.7 +/- 1.2 to 5.6 +/- 0.8 IU/l for integrated LH levels (untreated and MPA-treated PCOS); from 31.2 +/- 3.5 to 12.9 +/- 1.5 IU/l for LH response to GnRH; from 2.4 +/- 0.26 to 1.3 +/- 0.2 for LH/FSH ratio; and from 10.4 +/- 0.63 to 8.5 +/- 0.7 nmol/l for androstenedione. Significant decreases (p < 0.05-0.005) to values that still remained significantly higher than in normal subjects occurred for: LH pulse height, 11.05 +/- 1.3 to 6.88 +/- 0.79 IU/l (untreated and MPA-treated PCOS); LH pulse amplitude, 2.8 +/- 0.5 to 1.8 +/- 0.2 IU/l; total testosterone, 2.5 +/- 0.2 to 2.0 +/- 0.2 nmol/l; T/SHBG ratio, 14.1 +/- 1.7 to 11 +/- 1.5; and oestrone, 265 +/- 24 to 208 +/- 29 pmol/l. These results are consistent with the concept that ovulation failure and progesterone deficiency play a facilitatory role in the development of the hypothalamic-pituitary abnormality giving rise to disordered LH secretion in PCOS.

beta-Lipotropin-stimulated adrenal steroid production.

The present study was undertaken to examine the mechanism whereby beta-lipotropin stimulates adrenal steroidogenesis. In guinea pig adrenal cells, beta-lipotropin (10(-8) M) increased basal steroid production 6-, 4-, and 5-fold for cortisol, androstenedione, and dehydroepiandrosterone (DHEA), respectively, whereas the corresponding responses to adrenocorticotropin (ACTH) (10(-9) M) were 12-, 8-, and 7-fold. The conversion of cholesterol to pregnenolone was studied in cells treated with trilostane, an inhibitor of 3 beta-hydroxysteroid delta 4-5 isomerase. beta-Lipotropin (10(-10) and 10(-8) M) and ACTH (10(-9) M) stimulated pregnenolone production in trilostane-treated cells. The production of cortisol and androgens from precursor steroids was also studied in cells treated with aminoglutethimide, an inhibitor of cholesterol side chain cleavage, after addition of exogenous pregnenolone, 17-hydroxypregnenolone, progesterone, or DHEA. Neither ACTH nor beta-lipotropin stimulated cortisol, androstenedione, or DHEA production in the presence of exogenous precursors in aminoglutethimide-treated cells. No inhibition of the beta-lipotropin- or ACTH-stimulated cortisol or androstenedione responses was demonstrated with the opioid receptor antagonist naloxone (10(-11) to 10(-5) M). The results suggest that beta-lipotropin stimulates steroidogenesis by acting on the conversion of cholesterol to pregnenolone and that its effects are not mediated via an opioid receptor but may be mediated via an ACTH receptor.

Adrenal androgen production in polycystic ovary syndrome.

Secondary PCOS may occur in association with disorders characterized by adrenal androgen excess, e.g. congenital adrenal hyperplasia. Primary PCOS is associated frequently with more subtle abnormalities in adrenal androgen status. However, it has not been established that the mild adrenal androgen occurring in PCOS is causally involved in the development of PCOS, although adrenal hyperresponsiveness to stimulation appears to be characteristic of PCOS. It remains to be clarified whether this is due to excess stimulation of the adrenal by the putative CASH, which with ACTH probably coordinates adrenal androgen steroidogenesis, or whether adrenal hyperresponsiveness occurs as a consequence of increased cortisol clearance with compensatory hypersecretion of ACTH, which is associated with excessive adrenal androgen production. The possibility also exists that the enzyme system responsible for 17-hydroxyprogesterone production and its conversion to androgens is excessively active and may occur as a common defect in the adrenal and ovaries as a consequence of a congenital disorder. For at least some patients, treatment with a nocturnal low-dose glucocorticoid is an effective form of treatment. Indeed, this is the only hormonal form of treatment for hirsutism that also facilitates fertility and pregnancy. It is possible that PCOS may occur as a consequence of any disorder in which anovulation is associated with normal or elevated oestrogen levels. For some patients with PCOS, mild adrenal androgen excess is probably primary to development of the disorder. Thus, a trial of treatment with low-dose glucocorticoid at night appears to be a reasonable option in susceptible patients who can probably be recognized by demonstration of an excessive androgen response to ACTH or metyrapone.

Dissociation of adrenal androgen and cortisol secretion in Cushing's syndrome.

OBJECTIVES: While ACTH may modulate adrenal androgen production, there is evidence that other factors are required. Cushing's disease and ectopic ACTH secretion provide a little utilized opportunity to examine adrenal androgen levels in conditions of ACTH excess. We have compared plasma cortisol values with plasma levels of androstenedione, dehydroepiandrosterone (DHEA), DHEA-sulphate (DHEAS), testosterone and an index of free testosterone, the testosterone/sex hormone binding globulin ratio, prior to treatment in patients with Cushing's syndrome. PATIENTS AND MEASUREMENTS: Plasma from 15 adult patients with Cushing's disease and three adults with the ectopic ACTH syndrome was obtained prior to treatment and submitted to specific immunoassays for the measurement of the above steroids. RESULTS: Plasma cortisol values of 15 patients with Cushing's disease (range 326-1140 nmol/l, normal range 190-690 nmol/l) were elevated in 9; in contrast, plasma androstenedione (4.1-11.3 nmol/l, normal range, men 2.1-7.7, women 3.3-9.9 nmol/l) was elevated in only two patients, plasma DHEAS (3.3-17.8 mumol/l, normal range, men 4.5-18.4, women 3.5-11.8 mumol/l) was elevated in only 4 patients and plasma DHEA (4.8-45.2 nmol/l, normal range 11-48 nmol/l) was normal or low in all 15 patients. Plasma androstenedione was markedly elevated (74 nmol/l) in one of three patients with ectopic ACTH syndrome, moderately elevated in another, and normal in the third patient. In contrast, plasma DHEA and DHEAS levels were suppressed in the patient with the highest androstenedione level and low or normal in the other two patients. CONCLUSIONS: These data suggest that ACTH alone does not control adrenal androgen secretion. The data also suggest that variability in the processing of proopiomelanocortin (the precursor of ACTH and related peptides) occurring in Cushing's disease and ectopic ACTH syndrome may account for differences in the relation of cortisol to androgens observed between the disorders and when compared to that in normal subjects.

The relationship of blood glucose and haemoglobin A1 levels in diabetic subjects.

Recommendations exist that fasting plasma glucose (FPG) levels can be substituted for glycosylated haemoglobin A1 (HbA1) in Type II diabetic subjects (DM II), which have potential important financial implications. The present study was designed to expand this examination and to include Type I DM (DMI) patients and random blood glucose (RBG) values. Data were obtained from 234 DM II and 104 DM I patients, over 3 years. Correlation of HbA1 with FPG levels in DM II yielded an r value of 0.61. Correlation of HbA1 with RBG and 2 h post prandial glucose measurements yielded r values of 0.59 and 0.51 respectively, p < 0.001. In DM I, similar correlations gave r values ranging between 0.27 and 0.38, p < 0.01-0.001. Thus while significant correlations exist between HbA1 and FPG and RBG measurements in both DM I and DM II, clinically applicable information on long-term diabetic control can only be achieved from glucose measurements in DM II but the correlation is not sufficiently tight to recommend substitution of plasma glucose for HbA1 determinations, despite the cost advantages.

The overnight single-dose metyrapone test is a simple and reliable index of the hypothalamic-pituitary-adrenal axis.

OBJECTIVE: The ACTH stimulation test examines adrenal responsiveness but may not examine the entire hypothalamic-pituitary-adrenal (HPA) axis and requires parenteral administration. The cortisol response to hypoglycaemia provides an index of activity of the entire HPA axis but is demanding for patients and medical staff. The aim of the present study was to examine the performance of the overnight single-dose metyrapone test as it provides a simple alternative test for HPA axis function. DESIGN: Audit of the overnight metyrapone test performed in one centre between 1979 and 1991. PATIENTS: Three hundred and ninety-eight patients underwent 576 tests. Comparisons between the responses to metyrapone and the ACTH stimulation test and of the responses to metyrapone and insulin induced hypoglycaemia test were possible in 87 and 17 patients respectively. MEASUREMENTS: Following the midnight administration of metyrapone tablets, 30 mg/kg orally, blood samples were obtained between 0800 and 0930 h for radioimmunoassay of both 11-deoxycortisol and cortisol. RESULTS: Five hundred and seventy-six metyrapone tests were performed on 398 patients with no serious side-effects encountered. Adrenal insufficiency was diagnosed in 105 patients. Of these, 18 had a primary adrenal disorder and 87 had a disorder of the hypothalamic-pituitary unit. One hundred per cent concordance between the metyrapone, the ACTH and the hypoglycaemia test was seen in patients with primary adrenal insufficiency. In 19 patients with secondary adrenal insufficiency, who underwent both the metyrapone and the ACTH tests, discord between these two tests was observed in 10 patients (53%). Nine of these patients demonstrated a normal response to ACTH and a subnormal response to metyrapone. In only one patient was an abnormal cortisol response to ACTH associated with a normal response to metyrapone. In contrast, in 17 patients discord between the metyrapone and the hypoglycaemia test was seen in only 1 patient who demonstrated a normal response to the metyrapone test and a subnormal response to hypoglycaemia. CONCLUSION: Since the metyrapone test gives similar information about hypothalamic-pituitary axis function as does the hypoglycaemia test, we recommend the use of the overnight metyrapone test as a safe, simple and reliable index of the hypothalamic-pituitary axis integrity. The ACTH stimulation test should not be used for patients suspected of having secondary adrenal insufficiency.

The effect of prolactin, human chorionic gonadotropin, insulin and insulin-like growth factor 1 on adrenal steroidogenesis in isolated guinea-pig adrenal cells.

The controlling mechanism for adrenal androgen production has not been elucidated. The presence of receptors for prolactin, human chorionic gonadotropin (hCG), insulin and insulin-like growth factor 1 (IGF-1) in the adrenal cortex raises the possibility of their involvement in the control of adrenal steroidogenesis. This study was undertaken to investigate the effects of prolactin, hCG, insulin and IGF-1 in the presence and absence of ACTH on cortisol and androgen production using isolated guinea-pig adrenal cells. hCG 10(-7) and 10(-6) M significantly increased cortisol (P < 0.05) production. hCG 10(-6) M significantly increased androstenedione (A4) (P < 0.05) production. In the presence of ACTH, 10(-12) M, hCG 10(-6) M significantly increased the cortisol (P < 0.01) and A4 (P < 0.01) responses. Although the mean cortisol and A4 response to ACTH 10(-9) M was reduced in the presence of hCG 10(-6) M, this was not statistically significant. Prolactin 10(-8) M increased cortisol (P < 0.01), A4, and dehydroepiandrosterone (P < 0.05) production. In the presence of ACTH 10(-12) M, prolactin 10(-8) M increased the cortisol and A4 (P < 0.05) responses. However, the maximally ACTH-stimulated cortisol and A4 responses were not significantly altered in the presence of prolactin 10(-8) M. Insulin 10(-11)-10(-8) M and IGF-1 10(-10)-10(-7) M resulted in no significant increase in cortisol, A4 or dehydroepiandrosterone production. This study suggests that prolactin and hCG could play a role in modulation of adrenal steroidogenesis, particularly when ACTH levels are low. However, there was no evidence that prolactin or hCG is the specific cortical androgen stimulating hormone.

The pathogenesis of adrenal and extra adrenal hyperandrogenism.

The data reviewed in this paper suggest that a factor other than ACTH which is suppressible by treatment with glucocorticoid, plays an essential role in the regulation of adrenal androgen production. Adrenal androgen biosynthesis probably takes place exclusively in specific androgen-secreting cells. That availability of androgen substrate alone e.g. 17OH-progesterone, is not sufficient to lead to hyperandrogenaemia is clear from data which was obtained from treated patients with the 21 hydroxylase deficiency type of congenital adrenal hyperplasia. In pituitary ACTH excess, cortisol production is relatively greater than that of androgens. In contrast, in some patients with ectopic ACTH production, the excess production of androgens is relatively greater than that of cortisol. Taken together, these observations suggest that a factor closely related to ACTH, i.e. a POMC fragment other than ACTH, plays an important role in the regulation of adrenal androgen production, that in Cushing's disease the ratio of ACTH to the androgen-stimulating fragment increases, and that in some patients with ectopic ACTH syndrome the ratio of ACTH to the alternative fragment may be decreased. In addition, the data reviewed are consistent with a model for the pathogenesis of idiopathic hirsutism and polycystic ovary syndrome whereby mild adrenal androgen excess is primary to the development of these disorders. However, the identity of the putative adrenal androgen stimulating hormone has yet to be established.

Effects of pro-opiomelanocortin-derived peptides on adrenal steroidogenesis in guinea-pig adrenal cells in vitro.

The specific control of adrenal androgen secretion is unclear. This study was undertaken to investigate the effects of peptides derived from the ACTH precursor molecule pro-opiomelanocortin (POMC) on cortisol and androgen production using isolated guinea-pig adrenal cells. ACTH 10(-13)-10(-9)M, stimulated steroid production in a dose dependent manner, reaching a maximum of 12, 10 and 7 times basal levels for cortisol, androstenedione (A4) and dehydroepiandrosterone (DHEA), respectively, measured by specific radioimmunoassays. beta-Lipotropin (beta-LPH), 10(-10)-10(-8) M, also stimulated steroid production, reaching 6, 5 and 5 times basal levels of cortisol, A4 and DHEA, respectively. The N-terminal 16K fragment, gamma 3- and beta-MSH stimulated steroid production which reached statistical significance (P < 0.05) only in the case of cortisol. Joining peptide, alpha-, beta- and gamma-endorphin resulted in no significant change in steroid production. Met- and leu-enkephalin resulted in significant inhibition of DHEA production. POMC-derived peptides altered the steroid response to ACTH. beta-LPH and 16K fragment significantly increased the cortisol and A4 responses to a low concentration of ACTH. beta-LPH reduced the maximum cortisol and A4 responses to a high concentration of ACTH. This study suggests that beta-LPH may have a role in modulation of adrenal steroidogenesis but yielded no evidence to support a role for POMC-derived peptides in preferential stimulation of adrenal androgen production in guinea-pig adrenal cells.

A role for a non-androgenic anovulant in the management of hirsutism.

Sixty-nine patients who had Ferriman/Gallwey hirsutism scores (FG) of greater than or equal to 8 were treated with Diane, an anovulant containing cyproterone acetate, 2 mg, an anti-androgenic progesterone and ethinyl oestradiol, 5.0 micrograms. Twenty-one of these had been previously treated with dexamethasone (DEX) and did not respond, i.e. FG greater than 50% of pre-treatment value. Prior to Diane treatment, plasma total testosterone (T) values, 1.4 +/- 0.5 nmol/l, mean +/- S.D., were similar to those in 43 normal women, 1.23 +/- 0.3 nmol/l, as were plasma androstenedione levels, 6.8 +/- 2.5 and 6.0 +/- 1.7 nmol/l respectively. However, plasma sex hormone-binding globulin (SHBG) values were suppressed being 36.2 +/- 16 nmol/l in hirsute women and 45.8 +/- 15 nmol/l in normal women, p less than 0.01. The T/SHBG ratio, an index of free testosterone, was elevated in hirsute women, 4.8 +/- 4.1, compared to values in normal women, 2.9 +/- 1.0, p less than 0.001. Following Diane therapy (2-24 months), 73% of patients responded clinically. There was no change in T, but SHBG was increased to 181 +/- 54 nmol/l, p less than 0.001 and T/SHBG was decreased markedly to 0.9 +/- 0.4, p less than 0.001. Androstenedione fell also to 4.8 +/- 1.7 nmol/l, p less than 0.001. The clinical and hormone response to Diane was similar in both DEX-resistant and previously untreated groups. We conclude that Diane is an effective agent in the treatment of hirsutism while it avoids the adverse effects of androgenic progesterone and of high dose cyproterone acetate therapy.