Biological variation of total testosterone, free androgen index and bioavailable testosterone in polycystic ovarian syndrome: implications for identifying hyperandrogenaemia.

OBJECTIVE: Hyperandrogenaemia is one of the three Rotterdam consensus diagnostic criteria for polycystic ovarian syndrome (PCOS) and may be measured by estimation of total testosterone, free androgen index (FAI) or bioavailable testosterone (BioT). The aim of this study was to compare the biological variability of total testosterone with that of the biological variability of both the FAI and BioT, to determine the least variable measurement for clinical practice. DESIGN: Comparative study. PATIENTS: Blood samples were collected after an overnight fast at 4-day intervals on 10 consecutive occasions from 12 PCOS patients and 11 weight- and age-matched control women. MEASUREMENTS: Duplicate samples of stored serum were analysed for total testosterone, SHBG and BioT in a single batch. RESULTS: The PCOS group had a significantly higher median BioT, FAI and total testosterone than controls. In both the PCOS and control groups, the intraindividual variance was small and similar for BioT and FAI. There was no significant difference between the within-subject biological coefficient of variation (CV(I)) for BioT, FAI and total testosterone. The maximum and minimum critical differences were +58% and -37% for BioT and +70% and -40% for FAI, respectively. CONCLUSION: FAI appears to be the better diagnostic marker to distinguish hyperandrogenism in patients with PCOS, but once diagnosis has been made, all three methods should be equally good in monitoring further changes in the androgen status.

Hypoadrenalism is not associated with early mortality during tuberculosis treatment in Malawi.

SETTING: In the developing world, early mortality within 1 month of commencing tuberculosis (TB) treatment is high, particularly with human immunodeficiency virus (HIV) co-infection. In Malawi, 40% of those who die do so in the first month of treatment. Reasons remain unclear and may include delayed diagnosis, opportunistic infections, immune restoration inflammatory syndrome (IRIS) or malnutrition. One possible contributing factor is underlying hypoadrenalism associated with TB-HIV, exacerbated by rifampicin (RMP) induction of P450 and glucocorticoid metabolism. OBJECTIVE: To assess the prevalence of hypoadrenalism in TB patients before and after commencement of TB treatment, and relationship with early mortality. DESIGN: Prospective descriptive study assessing hypoadrenalism before and after anti-tuberculosis treatment, HIV status and outcome up to 3 months post-treatment. RESULTS: Of 51 patients enrolled, 29 (56.9%) were female (median age 32 years, range 18-62). Of 43 patients HIV-tested, 38 (88.3%) were HIV-positive and 15.7% died within the first month. At 3 months, 11 (21.6%) were known to have died. Adequate cortisol levels were found in 49/51 (95.9%) before commencing RMP. Neither of the two with reduced response died. All 34 patients revealed adequate cortisol responses at 2 weeks. CONCLUSION: No evidence of hypoadrenalism was found in this first study to assess adrenal function and outcome of anti-tuberculosis treatment.

Greater antiarrhythmic activity of acute 17beta-estradiol in female than male anaesthetized rats: correlation with Ca2+ channel blockade.

BACKGROUND AND PURPOSE: Female sex hormones may protect pre-menopausal women from sudden cardiac death. We therefore investigated the effects of the main female sex hormone, 17beta-estradiol, on ischaemia-induced cardiac arrhythmias and on the L-type Ca2+ current (ICaL). EXPERIMENTAL APPROACH: In vivo experiments were performed in pentobarbital-anaesthetized rats subjected to acute coronary artery occlusion. ICaL was measured by the whole-cell patch-clamp technique, in rat isolated ventricular myocytes. KEY RESULTS: Acute intravenous administration of 17beta-estradiol as a bolus dose followed by a continuous infusion, commencing 10 min before coronary artery occlusion, had dose-dependent antiarrhythmic activity. In female rats 300 ng kg(-1) + 30 ng kg(-1) min(-1) 17beta-estradiol significantly reduced the number of ventricular premature beats (VPBs) and the incidence of ventricular fibrillation (VF). A ten fold higher dose of 17beta-estradiol was required to cause similar effects in male rats. In vitro 17beta-estradiol reduced peak ICaL in a concentration-dependent manner. The EC50 was ten-fold higher in male myocytes (0.66 microM) than in females (0.06 microM). CONCLUSIONS AND IMPLICATIONS: These results indicate that 17beta-estradiol has marked dose-dependent antiarrhythmic activity that is greater in female rats than in males. A similar differential potency in blocking ICaL in myocytes from female and male rats can account for this effect. This provides an explanation for the antiarrhythmic activity of 17beta-estradiol and gender-selective protection against sudden cardiac death.

Analytical and physiological factors affecting the interpretation of serum testosterone concentration in men.

Most hospital laboratories estimate the concentration of total circulating testosterone using a non-extraction method on an automated multi-channel immunoassay analyser supplied by a small number of multi-national diagnostic companies. Although these platforms offer advantages of quick turnaround times, small volume sampling and random access analysis, proficiency testing schemes suggest the quality of results produced remains similar to that of the early manual radioimmunoassay. An estimate of the bioavailable, non-sex hormone binding globulin (SHBG) bound fraction of circulating testosterone, be that the free or the free plus albumin-bound, may be a better index of gonadal status than total testosterone alone, especially when a borderline hypogonadal level of total testosterone is found, and may avoid misclassification of hypogonadal or eugonadal men. Free or bioavailable testosterone may be calculated or measured. The free androgen index may not give a true reflection of androgen status in men. In the interpretation of serum testosterone concentrations with results >40 nmol/L, the possibility of exogenous administration or abuse needs to be considered. The marked diurnal rhythm in total testosterone should also be taken into account. There may be a diminution of testosterone secretion with advancing age, but the great majority of older men have a circulating total testosterone concentration well within the accepted reference intervals established for younger men. As testosterone concentration may fluctuate markedly both seasonally and from day to day, it may be judicious to measure levels on more than one occasion. Provided that estimates of serum testosterone are unequivocally eugonadal (12.5-40 nmol/L) or hypogonadal (<7.0 nmol/L), results produced by routine automated immunoassays will in all probability give a satisfactory assessment of androgen status in men.Routine biochemical assessment of gonadal function in men should include measurement of early morning luteinizing hormone, follicle stimulating hormone, prolactin and SHBG together with total testosterone, and if necessary some estimate of bioactive testosterone.

Low-dose transdermal testosterone therapy improves angina threshold in men with chronic stable angina: A randomized, double-blind, placebo-controlled study.

BACKGROUND: Experimental studies suggest that androgens induce coronary vasodilatation. We performed this pilot project to examine the clinical effects of long-term low-dose androgens in men with angina. METHODS AND RESULTS: Forty-six men with stable angina completed a 2-week, single-blind placebo run-in, followed by double-blind randomization to 5 mg testosterone daily by transdermal patch or matching placebo for 12 weeks, in addition to their current medication. Time to 1-mm ST-segment depression on treadmill exercise testing and hormone levels were measured and quality of life was assessed by SF-36 at baseline and after 4 and 12 weeks of treatment. Active treatment resulted in a 2-fold increase in androgen levels and an increase in time to 1-mm ST-segment depression from (mean+/-SEM) 309+/-27 seconds at baseline to 343+/-26 seconds after 4 weeks and to 361+/-22 seconds after 12 weeks. This change was statistically significant compared with that seen in the placebo group (from 266+/-25 seconds at baseline to 284+/-23 seconds after 4 weeks and to 292+/-24 seconds after 12 weeks; P:=0.02 between the 2 groups by ANCOVA). The magnitude of the response was greater in those with lower baseline levels of bioavailable testosterone (r=-0. 455, P:<0.05). There were no significant changes in prostate specific antigen, hemoglobin, lipids, or coagulation profiles during the study. There were significant improvements in pain perception (P:=0.026) and role limitation resulting from physical problems (P:=0.024) in the testosterone-treated group. CONCLUSIONS: Low-dose supplemental testosterone treatment in men with chronic stable angina reduces exercise-induced myocardial ischemia.

The effect of free fatty acids on the in-vitro binding of testosterone in human plasma.

The effect of supraphysiological levels of free fatty acids (FFA) on the binding of testosterone to sex-hormone binding globulin (SHBG) and on non-SHBG binding in both male plasma and plasma from pregnant women was studied. Six FFAs were added to plasma as individual acids. No alteration in testosterone binding to SHBG could be demonstrated with any of the FFAs in either male plasma or plasma from pregnant women. When the same plasma was heated to destroy SHBG binding, a highly significant (P < 0.01) increase in non-SHBG binding was seen in both male plasma and plasma from pregnant women when the unsaturated FFAs oleic, linoleic and linolenic acids were added. No significant difference was demonstrated with the saturated FFAs, palmitic, stearic and arachidic acids.

Exaggerated effects of progestogen on uterine artery pulsatility index in Turner's syndrome patients receiving hormone replacement therapy.

OBJECTIVE: To investigate the effect of estrogen and progestogen on the resistance to blood flow in the uterine arteries of Turner's syndrome patients. DESIGN: Prospective clinical study. SETTING: A tertiary infertility clinic. PATIENTS: Five Turner's syndrome patients, six patients who had surgical castration, and five patients with idiopathic primary ovarian failure. INTERVENTIONS: The patients were treated with 2 mg E2 valerate to which 500 micrograms norgesterel was added for 10 days in a 28-day cycle. Transvaginal color Doppler was used to measure pulsatility index in the uterine arteries at eight regular intervals during a single cycle. MAIN OUTCOME MEASURE: Pulsatility index of the uterine arteries. RESULTS: The administration of norgesterel to Turner's syndrome patients resulted in an increase in pulsatility index that was significantly higher than in patients who had surgical castration (confidence interval = 0.17 to 2.42). CONCLUSION: The uterine arteries of Turner's syndrome patients are more sensitive to the tonic effect of progestogen. If manifest in cardiac arteries also this phenomenon may be partly responsible for the increased incidence of cardiovascular disease and shorter life expectancy in Turner's syndrome patients. To achieve optimal protection from cardiovascular disease, Turner's syndrome patients may benefit from hormone replacement treatment containing altered doses of estrogen and progestogen.

Assays for prolactin: guidelines for the provision of a clinical biochemistry service.

This paper summarises the views of the authors on the provision of a prolactin assay service. We discuss the pathophysiology of prolactin secretion and the clinical indications that arise from that. We cover the rather complex issue of the definition of normal and elevated prolactin levels. From these considerations, certain guidelines on the analytical performance of prolactin assays and their provision in a clinical biochemistry service are given. The extent to which currently available methods and performance as revealed by the UK External Quality Assessment Scheme (EQAS) match these guidelines are described and certain conclusions are reached. Finally, probable future developments are briefly discussed. The main conclusions and recommendations are as follows: Reagents of appropriate quality are available to enable prolactin immunoassays to be provided in UK clinical biochemistry laboratories. These are provided either separately or in the form of kits from both commercial and NHS sources. There is no requirement for individual laboratories to undertake their own antiserum production or prolactin iodination. Acceptable performance (as defined using internal QC procedures and the UK EQAS) is achievable using these reagents/kits, although one commercial kit shows a consistent marked negative bias. Reference ranges, including 'normal ranges', show considerable between-centre variability. Many centres have not established their own ranges, even those using in-house methods. Reference ranges for use in clinical biochemistry laboratories are proposed in this report. Some general guidance on the provision of a prolactin service is given, although this does not differ in principle from that appropriate for other peptide hormone analytes. There is no evidence that centres with small workloads perform any worse than average, although it may be more cost-efficient for such centres to send the samples elsewhere. As with other peptide analytes, non-isotopic immunometric methodology is likely to replace current radioimmunoassay methods in the near future.

The long-term stability in whole blood of 14 commonly-requested hormone analytes.

Concentrations of 14 commonly-requested plasma hormones were measured in octuplicate in each of six subjects to determine their stability when unseparated from red cells for periods up to 1 week. Most of the analytes were stable when stored in this way and although statistically significant changes were recorded, in the great majority of cases the changes seen would have no bearing on the clinical interpretation of the result. In the light of these findings, we would confidently report results of analyses for these hormones in plasma that had remained in contact with red cells at ambient temperature for long periods of time.

Analysis of fecal neutral steroids and bile acids in humans on constant fat diet.

A method is described for the separation and quantification of fecal neutral steroids and fecal bile acids. The fecal extract is separated into the neutral steroid fraction and bile acid fraction with ionexchange resin columns. The principal neutral steroids and bile acids are then separated and quantitated by thin-layer chromatography. Values for the fecal neutral steroids, cholesterol, coprostanol and coprostanone and fecal bile acids, deoxycholic acid and lithocholic acid of 5 subjects on a constant fat diet for a 3-week period are presented.

Sex steroids and bone turnover markers in men with symptomatic vertebral fractures.

Sex steroids play an important role in the maintenance of bone density in men and women, but the circulating, biologically active unbound fraction is influenced by the concentration of sex hormone binding globulin (SHBG). SHBG increases with advancing age in men and leads to a reduction in serum free testosterone and oestradiol, which may then affect bone turnover, bone mineral density (BMD) and the risk of fractures. We have therefore measured total and unbound sex steroids, SHBG, bone turnover markers and BMD in 57 men with symptomatic low trauma vertebral fractures and 57 age-matched male control subjects. Fasting blood and urine samples were collected from all subjects, who also underwent BMD measurement of the lumbar spine and hip. Serum testosterone, oestradiol, SHBG, bone specific alkaline phosphatase (bone ALP) and urine free deoxypyridinoline/creatinine ratio (fDPD/Cr) were measured. Free sex steroid concentrations were calculated using their ratio with SHBG and albumin and bioavailable testosterone was measured using radioimmunoassay. The two groups were then compared and regression models developed to determine the best predictors of BMD and fracture. Men with vertebral fractures had significantly lower weight and BMD at all sites than control subjects (p<0.0001). Serum total testosterone and oestradiol did not differ between the two groups, but calculated free androgen and free oestradiol indices were lower in the fracture group than the control subjects (p=0.04), due to higher SHBG (46.6 versus 36.1 nmol/L: p=0.005). The men with vertebral fractures had significantly higher mean bone ALP (15.8 versus 11.8 microg/L: p=0.002) and fDPD/Cr (5.5 versus 4.0 nmol/mmol: p=0.03). Stepwise multiple regression analysis in both fracture and control groups found body weight to be the best predictor of BMD. In the fracture group weight predicted between 19.7 and 30.7% of the variance in BMD and in control subjects this was between 12.3 and 13.2%. SHBG contributed to the model for hip BMD in the fracture group alone, so that weight and SHBG together accounted for 32 to 42.9% of the variance. A model combining BMD at the spine, total femur and femoral neck with height loss best predicted fracture. In conclusion, men with symptomatic vertebral fractures have higher SHBG and lower calculated free sex steroid indices, increased bone turnover and lower BMD. Whilst body weight was the best predictor of BMD, symptomatic vertebral fracture was best predicted by BMD and height loss.

Plasma aldosterone and renin activity.

Eight infants had paired measurements of plasma aldosterone and plasma renin activity while being treated for congestive heart failure. There is parallelism with aldosterone and renin activity in the presence of a hyperactive renin-angiotensin-aldosterone system. Six patients had plasma renin activity and plasma aldosterone measured after commencing captopril and we have shown biochemical blockade of the renin-angiotensin-aldosterone system.

Plasma renin activity in infants with congenital heart disease.

Plasma renin activity was estimated in 11 infants with severe congestive heart failure. The infants had congenital heart disease with left to right shunts and were receiving diuretic treatment. Plasma renin activity was measured by radioimmunoassay of generated concentrations of angiotensin I. The mean (SD) plasma renin activity was 84 (21) ng angiotensin I/ml/hour, which is considerably above normal infant values. A hyperactive renin-angiotensin system may be detrimental in these patients. Angiotensin converting enzyme inhibitors may be of value in treating infants with severe congestive heart failure.

Plasma progesterone concentration in women with and without adenocarcinoma of the endometrium.

In the search for the postulated but elusive "estrogenic bias" in patients developing endometrial cancer, several authors have suggested lack of progesterone as the common denominator. In this study 21 patients at the time of diagnosis of Stage I disease are compared to stringently matched healthy control subjects. No significant difference was found in plasma progesterone concentrations between the two groups. These results, in conjunction with other calculated estrogenic indices based on these patients, provide no evidence of lack of progesterone at the onset of clinical disease. However, the effects of long-term progesterone lack related to chronic anovulation are not excluded by these findings.

The relationship between plasma prolactin and testosterone levels in male hypogonadism.

Fifty seven male patients either complaining of poor sexual development, gynecomastia or dwarfism and signs of sexual infantilism were studied. Plasma prolactin (PRL) and testosterone (T) were estimated in all patients while 33 of them were also subjected to full pituitary function tests. Twelve of the latter had an elevated basal plasma cortisol or growth hormone which suggested the patient may have been "stressed"; the results were analyzed both excluding and including these patients. The remaining patients were divided into those with a plasma T less than 8.0 nmol/l (Group A, 25 patients) and those with a plasma T greater than 8.0 nmol/l (Group B, 20 patients). The results were compared with those from 18 normal men (Group C). The mean plasma PRL in group A (108.1 mU/l) was significantly lower than that in group B (181.5 mU/l, p less than 0.005) or group C (255.7 mU/l, p less than 0.001). The difference between groups A and B became much less (p less than 0.01) when results from the "stressed" patients were included but this did not affect the difference with group C. The mean plasma PRL in group B was also significantly lower (p less than 0.05) than that in group C but the significance of the difference disappeared when all the patients were included (p less than 0.2). In the patients there was a significant correlation between plasma and PRL plasma T (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Undiagnosed morbidity in adult women with Turner's syndrome.

OBJECTIVE: Adult patients with Turner's syndrome are rarely followed up at specialist clinics after discharge from paediatric care but do have a predisposition to several long-term medical problems. We have assessed the undiagnosed morbidity that exists among adult women with Turner's syndrome. PATIENTS: A group of 32 women (age range 17-52 years; mean 25 years) attending a specialist out-patient clinic. MEASUREMENTS: Blood samples were obtained at the initial visit for lipid assessment, thyroid function, gonadal status and routine biochemical profile. Bone mineral density (BMD) was measured in 31 of the women. RESULTS: Thirty-one women were receiving some form of oestrogen replacement. Two were receiving T4 therapy. In 50%, total cholesterol was greater than 5.2 mmol/l (range 3.4-9.3 mmol/l, mean 5.8 mmol/l) and 28% had an abnormality of thyroid function tests. Two women were newly diagnosed as hypothyroid, 6 had compensated hypothyroidism and one was under-replaced with T4. Lumbar spine BMD was below 100% of age matched reference range in 84% and below 75% in 26% of patients. Femoral neck BMD was below 100% of age matched reference range in 90% and below 75% in 10% of patients. CONCLUSIONS: There is a high incidence of undiagnosed lipid, thyroid and bone mineral density abnormalities in the adult population with Turner's syndrome. Doctors caring for these women need to be aware of and look for the potential problems. Appropriate long-term treatment should be commenced to help prevent the development of lipid, skeletal and thyroid abnormalities which may cause these patients major problems in the future.

Virilization due to ovarian hyperthecosis in a postmenopausal woman.

A 51-year-old woman presented with hirsutism and virilization of gradual onset. The serum gonadotropin concentrations were in the postmenopausal range, the serum testosterone concentration was markedly elevated (9.8 nmol/l) and the serum estradiol concentration (220 pmol/l) was elevated above the postmenopausal range. A selective venous catheterization study demonstrated raised serum testosterone and androstenedione levels in ovarian veins and suggested the presence of a left ovarian tumor. The raised peripheral estradiol level was shown to be due to ovarian hypersecretion. After bilateral oophorectomy the serum testosterone became normal. Ovarian histology revealed bilateral stromal hyperthecosis. Ovarian hyperthecosis is a rare but important cause of serum testosterone levels in the neoplastic range. This is the third case reported of postmenopausal virilization due to ovarian hyperthecosis and the first report of a selective venous catheterization study in such a patient.