Panhypopituitarism as a model to study the metabolism of dehydroepiandrosterone (DHEA) in humans.

The physiological importance and therapeutical interest of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are still controversial. Panhypopituitarism is characterized by the absence of secretion of adrenal and gonadal steroids and thus the production of their metabolites. The conversion of DHEA given orally into delta 5 derivatives, androgens, androgen metabolites, and estrogens was studied in ten patients with complete panhypopituitarism. Sex steroid therapy was withdrawn for at least 2 months. Each patient received, at 1-month intervals and in a random order, two single oral doses of DHEA (50 mg and 200 mg) and placebo. During each treatment, urine samples were collected for 24 h, and blood samples were drawn at hourly intervals for 8 h. In patients with pituitary deficiency, plasma DHEA and DHEAS were not detectable and increased, with the 50 mg dose, up to levels observed in young adults. The administration of 200 mg of DHEA induced an increase of both steroids to supraphysiological plasma levels. A small increase of delta 5-androstenediol was observed. In contrast, the increase of plasma delta 4-androstenedione was important and dose dependent. DHEA was also converted into the potent sex steroid testosterone (T). The administration of a 50 mg dose of DHEA restored plasma T to levels similar to those observed in young women. The 200 mg dose induced an important increase of plasma T, slightly below the levels observed in normal men. The increase of plasma dihydrotestosterone levels was small at both doses of DHEA, in contrast with the large conversion of DHEA into androsterone glucuronide and androstanediol glucuronide. Finally, DHEA administration induced a significant and dose dependent increase of plasma estrogens and particularly of estradiol. In conclusion, this short term study demonstrates that: 1) panhypopituitarism is a model of interest to study the metabolism of DHEA; 2) in the absence of pituitary hormones and of adrenal and gonadal steroids, DHEA given orally is mainly converted into delta 4 derivatives, which in turn are strongly metabolized into 5 alpha-3keto-reduced steroids; 3) a significant increase of sex active hormones was observed in plasma after 200 and even 50 mg of DHEA. Thus, biotransformation of DHEA into potent androgens and estrogens may explain several of the reported beneficial actions of this steroid in aging people.

Association between plasma total testosterone and cardiovascular risk factors in healthy adult men: The Telecom Study.

The associations between androgens and cardiovascular risk factors in men are controversial. A nested case-control study was used to compare the levels of cardiovascular risk factors in two groups (n = 25 each) of healthy men contrasted by their plasma total testosterone (PTT) concentration, matched by age and ethnic origin. Compared to the men with normal PTT (mean +/- SEM, 19.8 +/- 0.7 nmol/L), the men with low PTT (10.1 +/- 0.3 nmol/L) had a significantly higher body mass index (P < 0.01), waist/hip ratio (P < 0.001), systolic blood pressure (P < 0.05), fasting and 2-h plasma glucose (P < 0.04 and P < 0.02 respectively), serum triglycerides (P < 0.001), total cholesterol (P < 0.04), low density lipoprotein cholesterol (P < 0.01), apolipoprotein B (P < 0.01), fasting and 2-h plasma insulin (both P < 0.0001), and lower values of serum high density lipoprotein cholesterol (P < 0.01) and apolipoprotein AI (P < 0.05). After adjustment for both body mass index and waist/hip ratio, fasting and 2-h plasma insulin and triglyceride levels remained significantly different between the two groups (P < 0.04, P < 0.001, and P < 0.03 respectively). Plasma sex hormone-binding globulin was markedly decreased in the low PTT group (P < 0.0001), whereas bioavailable testosterone was not significantly different. This case-control study provides further and stronger evidence of a negative association between PTT and plasma insulin in men, as suggested by cross-sectional studies. Because these are observational data, neither causality nor the direction of the associations among PTT, sex hormone-binding globulin, and insulin sensitivity can be determined. Intervention studies are needed to better assess the metabolic and cardiovascular benefits of androgen treatment that have been suggested by preliminary clinical trials.

[Measurement of salivary cortisol with four commercial kits]. / Mesure du cortisol salivaire par quatre trousses du commerce.

Salivary cortisol was determined with four commercially available immunoassays: one enzyme-immunoassay (Cortisol Biotrol), two fluoro-immunoassays (TDX, Abbott; Stratus, Baxter) and one radioimmunoassay (Coat-A-Count, Behring). In order to improve the sensitivity of these immunoassays, it was necessary to increase sample volumes. Thus an extraction step had to be included in the procedure. It was performed with either methylene chloride or Bond Elut. However, the Coat-A-Count kit may be applied directly on salivary aliquots. The results obtained were compared to those yielded by the reference technique which includes a chromatographic step on Sephadex LH 20. According to the present data (n = 20) no kit appears to be adequate for salivary cortisol measurement at any level, at least in the conditions applied in this study. However, the introduction of a chromatographic step in the procedure improved greatly the specificity. Nevertheless, the best results were obtained with the Coat-A-Count kit. Indeed, they were generally similar to those of the reference technique, but some discrepancies were observed at low levels.

[Polycystic ovary and diazoxide. In vivo study]. / Ovaires polykystiques et diazoxide. Etude in vivo.

The suppression of hyperinsulinism with diazoxide (300 mg/d during 30 days) in a young woman with PCOS and hirsutism, hyperinsulinism and insulinoresistance was followed by a modification of plasma androgens. Testosterone (T) and free testosterone (fT) were reduced after ten days and then increased but always remained below the baseline level. DHEAS had increased 200% by day 10, and 3 alpha-adiol G to three times its basal value by day 20. These modifications were constant during the treatment. fT decrease was secondary to reduction of hyperinsulinism which was followed by an increase of TeBG and a modest and transient reduction of androgen theca cells production. DHEAS increase was due to hyperinsulinism suppression which stimulated adrenal 17-20 lyase activity. 3 alpha-adiol G increase was concomittant, and can be considered as an index of adrenal androgen secretion.

Administration of unmodified progesterone by nasal spray in fertile women.

A 11.20-mg dose of progesterone was administered by nasal spray to five healthy fertile women in the follicular phase of the menstrual cycle. Serial blood samples were collected and Cmax (the maximum progesterone concentration reached), Tmax (the time at which Cmax was reached) and the area under the curve (AUC), with the time limits of 0 and 720 min, were calculated. Serum progesterone levels were assayed by means of a non-extraction [125I]radioimmunoassay. The mean Cmax was 4.50 +/- 2.31 ng/ml at a Tmax of 30 min; levels returned to baseline after 720 min. The mean AUC value was 1180.50 +/- 613.90 ng.h/ml. The progesterone administered by nasal spray in fertile women was effective in reaching physiological progesterone levels. Even if a nasal first-pass metabolic effect is taken into account, this route allows progesterone to avoid liver first-pass metabolism and its metabolic consequences.

Tumour-associated antigens in maternal and fetal blood.

Normal levels of cancer-associated antigen (CA) 19-9, neurone-specific enolase (NSE), cancer-associated antigen (CA) 125, and mucin-like carcinoma-associated antigen (MCA) during pregnancy were determined in 87 mothers and fetuses, using a solid-phase sandwich enzyme immunoassay. CA 19-9 concentrations were higher in the fetuses, whereas the other three tumour-associated antigen levels were higher in the mothers. Only fetal NSE and MCA levels were positively correlated with those in maternal serum. Contrary to adult samples, no difference was demonstrated between male and female fetal levels of CA 125. MCA was the only maternal marker that increased significantly with gestational age between 20 and 34 weeks' pregnancy.

Plasma 17-hydroxyprogesterone determination with two commercial immunoassays.

Plasma 17-hydroxyprogesterone (17-OH-P) was determined by two commercially available immunoassay kits, a radioimmunoassay (RIA) (OHP-CT, CIS) and an enzyme-immunoassay (EIA) (Serozyme 17 alpha-OH-progesterone, Serono). The determination by RIA was performed according to two procedures, directly on plasma or on a crude plasma extract, whereas that by EIA used only the second procedure. These determinations were carried out in 27 infants below 1 year of age and in 33 women in the follicular phase of the menstrual cycle. The results were compared to those obtained by an in-home RIA (RIA-FRH) which includes an extraction step followed by chromatography on Sephadex LH 20 column. The levels observed were overestimated by both kits. In infants, interference from 17-hydroxy-pregnenolone (17-OH-5P) sulfate occurred when the RIA (CIS) kit was used directly on plasma samples. Using plasma extracts, 17-OH-5P interfered with EIA (Serono) in the infant group and with the RIA (CIS) in the second group. The two kits do not appear to be adequate for 17-OH-P determination at least in infants and in women in the follicular phase of the menstrual cycle.

Nasal spray administration of unmodified progesterone: evaluation of progesterone serum levels with three different radioimmunoassay techniques.

A 11.20 mg dose of progesterone was administered by nasal spray (NS) to six healthy postmenopausal women. Serial blood samples were collected and plasma progesterone was assayed by radioimmunoassay (RIA) according to three different procedures. In the first, RIA was carried out directly on plasma aliquot (Method A), in the second after diethyl ether extraction (Method B) and the third, after diethyl ether extraction and Celite column chromatography (Method C). The mean serum peak level (CMax) calculated with Method A (2.87 +/- 1.14 ng/ml) was higher than that obtained with both Method B (2.24 +/- 0.76 ng/ml) and C (1.58 +/- 0.76 ng/ml; P < 0.05); similarly the area under the curve (AUC) measured with Method A (695.79 +/- 348.24 ng h/ml) was higher than that obtained with both Method B (390.12 +/- 95.16 ng h/ml) and C (243.71 +/- 82.97 ng h/ml; P < 0.02). On the other hand, progesterone serum levels measured with Method C peaked earlier than those observed with Methods B and A (21.67 +/- 19.40, 25.83 +/- 18.55 and 35 +/- 20.70 min, respectively). These data are consistent with the high specificity of Method C for progesterone whereas the other methods could overestimate the progesterone serum levels probably measuring also progesterone metabolites particularly 5 alpha- and 5 beta-dihydroprogesterone. This study confirmed the rapid absorption of progesterone across the nasal mucosa avoiding the first-pass liver metabolism; however, a 'first-pass effect' of the nasal mucosa should be taken into consideration when progesterone is delivered by the nasal route because probably a significant portion of progestational effects are due to its active metabolites.

Plasma 3 beta-hydroxy-delta 5-steroids in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

There is little information about the plasma concentrations of 3 beta-hydroxy-delta 5-steroids (delta 5-steroids) in untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. To further study the delta 5 pathway, we measured plasma levels of delta 5- and delta 4-steroids in 21 adult patients with different degrees of 21-hydroxylase deficiency (11 salt-wasters, 5 simple virilizers, and 5 patients with the nonclassical form of the disease). In all patients, investigations were performed after withdrawal of steroid treatment for at least 10 days. In addition, catheterization of gonadal and adrenal veins was performed in two salt-wasting male patients displaying bilateral testicular tumors to study adrenal secretion of delta 5- and delta 4-steroids. In one of them, surgical resection of the intratesticular adrenal rests gave the opportunity to measure 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity. In all untreated patients, an increase in plasma delta 4-steroids was observed. In contrast, although plasma levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were not significantly modified in simple virilizers, a paradoxical decrease in all delta 5-steroids was observed in salt-wasters. Catheterization of the adrenal veins confirmed the decrease in delta 5-steroids, particularly DHEA and DHEAS. The androstenedione/DHEA ratio was increased in all patients proportionally to the severity of the disease, suggesting an increase in adrenal 3 beta HSD. In vitro analysis of 3 beta HSD activity showed a 4-fold increase in intratesticular adrenal tissue compared to that in normal adrenals. A positive correlation between the androstenedione/DHEA ratio and plasma ACTH levels was observed, suggesting a long term stimulatory effect of ACTH on 3 beta HSD. Angiotensin-II could have an additive effect on ACTH-induced 3 beta HSD activity.

Longitudinal study of maternal plasma bioavailable testosterone and androstanediol glucuronide levels during pregnancy.

OBJECTIVE: The aim of this study to evaluate during normal pregnancy plasma bioavailable testosterone and androstanediol glucuronide levels. MEASUREMENTS: Bioavailable testosterone, androstanediol glucuronide and SHBG levels were evaluated every 4 weeks from week 6 to week 38 in 10 normal pregnant women. We also measured plasma oestradiol, oestriol, delta 4-androstenedione, 17-hydroxyprogesterone, progesterone and testosterone. RESULTS: The mean bioavailable testosterone levels were within the range of non-pregnant women but with an increasing trend until delivery. Androstanediol glucuronide had increased at weeks 6 and 8, decreased at week 14, remained low at week 30, and increased again at week 34. SHBG was significantly correlated with testosterone, oestradiol and oestriol. No correlation could be established between androstanediol glucuronide and any other parameter. DISCUSSION: Bioavailable testosterone (non-SHBG bound testosterone) represents the sum of free testosterone plus albumin bound testosterone. The increase in testosterone concentrations with decreased albumin levels during pregnancy, could suggest reduced metabolic clearance of testosterone throughout pregnancy. No correlation was established between the decrease in androstanediol glucuronide and increase in progesterone, suggesting that the decrease in androstanediol glucuronide is not a consequence of the inhibitory effect of progesterone on 5 alpha-reductase activity.

[Mid-term and long-term outcome in Leydig cell estrogen tumors]. / Devenir à moyen et à long terme des tumeurs estrogéniques à cellules de Leydig.

A study has been realized in six patients with estrogen secreting Leydig cell tumors from 18 to 120 months after hemicastration. Testicular contralateral volume is normalized, gynecomastia can be completely reduced, sex steroids are normalized while gonadotropins can be temporary increased. Spermogram which is abnormal can be normalized. These results confirm the heterogeneity of these tumors.

Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism.

In men with hypogonadotropic hypogonadism, prolonged treatment with LH and FSH induces spermatogenesis. To compare the respective role of exogenous testosterone and intratesticular testosterone on the induction and maintenance of spermatogenesis, 10 men with hypogonadotropic hypogonadism and without history of cryptorchidism were studied. They were treated with human gonadotropins (hMG; 150 IU FSH and LH and 1500 IU hCG, im, three times weekly) or pure FSH (150 IU, im, three times a week) and testosterone (T: 250 mg, im, once a week). Five men were treated first with hMG-hCG and then with pure FSH plus T. The other five men started with pure FSH plus T. Each treatment period lasted 24 months. In all men, hMG-hCG induced spermatogenesis after 24 months, with normal motility and quality. The combination of pure FSH and T was not able to induce spermatogenesis after 24 months. In addition, sperm count dropped dramatically to 0.3 +/- 0.1 x 10(6)/mL within 3 months and to 0 after 6 months when pure FSH and T followed [corrected] hMG-hCG. Plasma T levels were increased by both treatments, but significantly more after pure FSH and T (35.3 +/- 5.2 nmol/L) than after hMG-hCG (20.4 +/- 5.2 nmol/L; P < 0.05). Plasma estradiol levels after treatment with pure FSH and T were also increased, but the difference from those obtained during hMG-hCG treatment was not significant. In conclusion, in men with complete gonadotropin deficiency, FSH and exogenous T are not able to induce spermatogenesis. Furthermore, spermatogenesis induced by LH plus FSH (hMG-hCG) cannot be maintained when exogenous T replaced LH in the regimen. Thus, exogenous T is unable to replace LH (and intratesticular T) to induce spermatogenesis. These data are noteworthy in the prospect of male contraception after a complete blockade of gonadotropin activity.

Profiles of plasma estrogens, progesterone and their metabolites after oral or vaginal administration of estradiol or progesterone.

Doses of 100 mg of micronized progesterone (P) and of 0.5 mg of micronized estradiol (E2) were administered vaginally and orally, respectively, in the early follicular phase of the menstrual cycle in six premenopausal women. In the second cycle, the same doses were administered in the same subjects, orally for P and vaginally for E2. Serial blood samples were collected and the following steroids were assayed by highly reliable techniques: P, E2, estrone (E1), deoxycorticosterone (DOC), 5 alpha- and 5 beta-pregnanolone and the sulfates of E1, E2, and DOC. Circulating P and E2 levels were higher after vaginal than after oral administration, while those of E1 were similar after either route. Metabolites of P (DOC, DOCS and pregnanolone) were higher after oral administration. Concerning estrogen sulfates, E1S concentrations were similar whichever the route, while those of E2S were lower after oral than after vaginal administration. This study has confirmed that metabolism of ingested P and E2 occurs mainly in the intestine. Moreover, P was predominantly metabolized to 5 alpha-reduced derivatives, whatever the route of administration. In view of the metabolic pathways which are operative and of the peripheral plasma levels which were found, the vaginal route appears to be more adequate than the oral one for hormone replacement therapy.

Interrelation between plasma sex hormone-binding globulin and plasma insulin in healthy adult women: the telecom study.

In order to study the relationship between plasma sex-hormone-binding globulin (SHBG) and insulin levels in healthy women, we investigated the association between plasma SHBG and insulin in an occupational sample of 786 nonhormone-using women. Levels of plasma SHBG showed a stepwise decrease with increasing fasting plasma insulin in premenopausal as well as in postmenopausal women. In these cross-sectional data, this significant negative relationship between SHBG and insulin was shown to be independent of age, body mass index, subscapular skinfold, fasting and 2-h plasma glucose in both groups. The etiology and the consequences of this inverse association between SHBG and insulin are unclear. Prospective and clinical studies in women will be necessary to determine the direction and causal nature of the association between SHBG and insulin, as well as its mechanism and its physiological and/or pathophysiological consequences.

Effects of metyrapone infusion on corticotropin-releasing factor and arginine vasopressin secretion into the hypophysial portal blood of conscious, unrestrained rams.

The effects of rapid changes of circulating cortisol levels on ACTH secretion and on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) concentrations into hypophysial portal blood were studied in six adult rams. Pharmacological adrenalectomy was obtained by 3 h metyrapone infusion (100 mg.kg-1.h-1). Blockade of cortisol synthesis induced a tenfold increase of plasma ACTH levels accompanied by a moderate increase of CRF secretion (150% vs preinjection levels) and a large increment of AVP secretion (535% vs preinjection levels). ACTH levels remained high during the 3 h following the end of metyrapone infusion. During the same period, CRF secretion was still elevated (231% vs preinjection levels), while AVP secretion was further stimulated (2,151% vs preinjection levels). Subsequent hydrocortisone infusion (66 micrograms.kg-1.h-1) for 2 h induced a rapid decrease of both ACTH and AVP secretion, while CRF levels in hypophysial portal blood still remained elevated. These data suggest that changes in ACTH secretion induced by acute modifications of the negative glucocorticoid feedback are, in addition to the well documented direct effect of cortisol on the corticotropes, mainly mediated by variations of hypothalamic AVP secretion.

Determination of urinary cortisol with three commercial immunoassays.

Urinary cortisol determination was performed with three commercially available immunoassays: one enzyme-immunoassay (Cortisol Biotrol) (EIA) and two radioimmunoassays: Quanticoat Cortisol (Kallestad Diagnostics) (KD-RIA) and GammaCoat Cortisol (Clinical Assays) (CA-RIA). Four procedures were carried out. Procedure I (methylene chloride extraction) was applied to EIA and CA-RIA and procedure II (ethyl acetate extraction) to KD-RIA. Procedure III combining procedure I and column chromatography on Sephadex LH 20 in methylene chloride was applied to the three kits. Procedure IV consisting of carbon tetrachloride preextraction and extraction with cyclohexane-ethyl acetate (50:50, v/v) was applied to CA-RIA. The results obtained were compared with those of the reference technique, "on-line" HPLC with u.v. detection. Two groups of results were arbitrarily considered, those below (n = 28) and those above (n = 6) 270 nmol/l. In the first group, the results were markedly overestimated when the procedure was limited to solvent extraction. Conversely, the third procedure proved the efficiency of the chromatographic step since specificity was greatly improved in the three cases, the levels obtained with either kits being similar to those of the reference technique. The second group of results (above 270 nmol/l) yielded by the three kits were not always higher than those of HPLC when the procedure was limited to solvent extraction. When column chromatography was included in the procedure, the results were comparable to those of HPLC in three cases and lower in the three others. Since, the latter samples were collected after cortisol administration, and overestimated cortisol values obtained by HPLC might be due to the interference of some cortisol metabolites.

Oestrogen secreting Leydig cell tumour and GnRH agonist in-vivo and in-vitro studies.

OBJECTIVE: The purposes of our study concerning two patients with oestrogen secreting Leydig cell tumour were to determine whether endogenous LH levels are involved in testicular tumour steroidogenesis and whether aromatase activity of oestrogen secreting Leydig cell tumours is directly or indirectly dependent on LH levels. MEASUREMENTS: E2 and T were evaluated after hCG injection (5000 IU) during 96 hours. Bio and immuno LH, T, E2, were determined at the basal state and after administration of D-Trp-6-GnRH agonist (3.75 mg) every 3 weeks. The abnormal testis was removed after the third injection and testicular venous blood was collected during the operation. Testicular tumour was incubated with 4-14C-T. RESULTS: Oestradiol (E2) response to hCG injection (5000 IU) was prolonged and exaggerated while that of testosterone (T) was similar to that of the controls. The aromatase index (E2/T) remained elevated even 96 hours after hCG. Intramuscular injection of the GnRH agonist, D-Trp-6-GnRH (3.75 mg) resulted in a reduction of immunoreactive and bioactive LH. T was decreased to about 10% of baseline levels and E2 fell from 240 to 36 pmol/l. In the blood of the spermatic veins collected in the course of surgery, E2 levels were found to be lower in comparison with the controls. E2 was found to be twofold higher in the spermatic vein draining the tumoral side than in that of the contralateral testis. Incubation of the testicular tumours with 4-14C-T, displayed a reduced aromatase activity (conversion of T to E2: 0.3 and 0.1% in patients 1 and 2 respectively). CONCLUSIONS: The kinetics of E2 response to hCG administration would suggest a modification of the regulation of the aromatase activity in this type of oestrogen secreting tumour. A certain endogenous LH level may be necessary to supply a sufficient quantity of T substrate, and to maintain aromatase activity of such Leydig cell tumours secreting oestrogens. These tumours seem to be responsive to endogenous LH levels.

Hemodynamic, neurohumoral, and metabolic responses to amino acid infusion in patients with cirrhosis.

In patients with cirrhosis the renal response to amino acid infusion is controversial. In addition, the renal and systemic metabolic effects of amino acids are unknown. Therefore, the present study examined the effects of amino acids on renal hemodynamics, renal and systemic oxygen (O2) consumption, and hormones in patients with cirrhosis. Twelve patients received an 8% amino acid solution for 30 minutes at a rate providing 250 mg of amino acids/kg body wt. Renal blood flow increased by 45% (P less than 0.05) and the glomerular filtration rate by only 9% (P greater than 0.05). Renal vascular resistance decreased by 23% (P less than 0.05), and renal perfusion pressure did not change significantly. Renal and systemic O2 consumption and pulmonary artery plasma glucagon level significantly increased. There were no significant changes in plasma osmolality, plasma volume, and plasma atrial natriuretic peptide concentrations. In conclusion, the results show that amino acid-induced renal vasodilation caused hyperperfusion but not renal hyperfiltration in patients with cirrhosis. In addition, renal hyperemia was associated with renal and systemic hypermetabolism.