BACKGROUND

Several anabolic steroids are sold over-the-counter (OTC) in the United States, and their production is not regulated by the US Food and Drug Administration. Reports have suggested that use of these supplements can cause positive urine test results for metabolites of the prohibited steroid nandrolone.

OBJECTIVE

To assess the content and purity of OTC androstenedione and to determine if androstenedione and 19-norandrostenedione administration causes positive urine test results for 19-norandrosterone, a nandrolone metabolite.

METHODS

Randomized controlled trial of androstenedione, open-label trial of 19-norandrostenedione, and mass spectrometry of androstenedione preparations, conducted between October 1998 and April 2000.

METHODS

Outpatient facility of a university hospital.

METHODS

A total of 41 healthy men aged 20 to 44 years.

METHODS

Participants were randomly assigned to receive oral androstenedione, 100 mg/d (n = 13) or 300 mg/d (n = 11) for 7 days, or no androstenedione (n = 13); in addition, 4 patients received 10 microg of 19-norandrostenedione.

METHODS

Content of OTC androstenedione preparations; level of 19-norandrosterone in urine samples, determined by mass spectrometry, compared among the 3 randomized groups at day 1 and day 7, and among the participants who received 19-norandrostenedione from October 1998 to April 2000.

RESULTS

All urine samples from participants treated with androstenedione contained 19-norandrosterone, while no samples from the no-androstenedione group did. Urinary concentrations were averaged for day 1 vs day 7 measurements; mean (SD) 19-norandrosterone concentrations in the 100-mg/d and 300-mg/d groups were 3.8 (2.5) ng/mL and 10.2 (6.9) ng/mL, respectively (P =. 006). The 19-norandrosterone content exceeded the cutoff for reporting positive cases (>2.0 ng/mL) in 20 of 24. The androstenedione preparation used was pure at a sensitivity of 0.1%, but at 0.001% 19-norandrostenedione was found. For the 4 participants to whom 10 microg of 19-norandrostenedione was administered, 19-norandrosterone was found in all urine samples. Of 7 brands of androstenedione analyzed at the 1% level, 1 contained no androstenedione, 1 contained 10 mg of testosterone, and 4 more contained 90% or less of the amount stated on the label.

CONCLUSIONS

Our study suggests that trace contamination of androstenedione with 19-norandrostenedione is sufficient to cause urine test results positive for 19-norandrosterone, the standard marker for nandrolone use. Oral steroid doses as small as 10 microg are absorbed and excreted in urine. Some brands of androstenedione are grossly mislabeled. Careful analysis of androstenedione preparations is recommended in all studies of its biological effects. JAMA. 2000;284:2618-2621.

Aromatase, a cytochrome P<em>4</em>50, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. On the basis of a recent computer modeling of the active site of aromatase, a hydrophobic pocket, thought to be important for the binding of some aromatase inhibitors, was predicted to extend roughly in the plane of the steroid substrate, from the position that would be occupied by its C<em>4</em> and C7 atoms. Four mutants, G121A, I125N, F235N, and I<em>4</em>7<em>4</em>F, were generated to test this model. Although the mutagenesis results have shown that the current model for the active site of aromatase almost certainly contains a number of errors, the results are in general very satisfactory in that they suggest how the model should be altered by local realignments of the aromatase sequence with that of cytochrome P<em>4</em>50cam. Among the mutants, I<em>4</em>7<em>4</em>F is the most interesting one. Its Km value for <em>androstenedione</em> was found to be lower than the wild type enzyme, and the kinetic analysis exhibited a substrate inhibition-like kinetic profile through an "in-cell" assay. In addition, this mutation reduces the binding affinity of an aromatase inhibitor, <em>4</em>-hydroxy<em>androstenedione</em>, and increases the binding affinity of two aromatase inhibitors, aminoglutethimide and CGS 169<em>4</em>9. This study demonstrates a useful approach, by a combination of computer modeling, site-directed mutagenesis, and inhibitor binding studies, to examine the structure of the active site of aromatase and the binding nature of various aromatase inhibitors.

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical <em>4</em>-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and <em>androstenedione</em> levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.

Recent measurements of circulating inhibin A and inhibin B concentrations indicate that inhibin B may play an important role in the selection of dominant follicles. The concentrations of inhibin A, inhibin B and activin A were measured in the follicular fluids of 61 individual follicles (<em>4</em>.8-20 mm in diameter) from <em>4</em>7 regularly cycling women using specific two-site enzyme-linked immunosorbent assays. The microenvironment of each follicle was characterized by measuring follicular fluid <em>androstenedione</em> and oestradiol concentrations. The mean activin A concentrations were < 8 ng/ml for follicles of all sizes (<em>4</em>-17 mm). Inhibin A concentrations were < 1 ng/ml in follicles < 6 mm, and progressively increased to concentrations>> 50 ng/ml in follicles>> or = 13 mm. Follicles with <em>androstenedione</em>/oestradiol ratios < or = <em>4</em> had higher concentrations of inhibin A than follicles with <em>androstenedione</em>/oestradiol ratios>> <em>4</em>. Inhibin B concentrations were higher than inhibin A concentrations in all follicles, increasing from 19.2 +/- 8.3 ng/ml in <em>4</em> mm follicles to <em>4</em>09 +/- 9.6 ng/ml in 13 mm follicles and then declining to 275 +/- <em>4</em>7 ng/ml in 17 mm follicles. These results support the hypothesis that inhibin B may play a more important paracrine role in developing follicles and a greater regulatory role with respect to follicle stimulating hormone (FSH) secretion than inhibin A.

To evaluate the influence of an hydrophilic statin, pravastatin, on adrenal and testicular steroidogenesis and spermatogenesis, eight male hypercholesterolemic patients were studied. All patients observed a hypocholesterolemic diet and received placebo for <em>4</em> weeks followed by pravastatin (20 mg/die) for 6 months. Before, during (<em>4</em>th-5th week) and at the end (23th-2<em>4</em>th week) of active treatment, CRH (1 microgram i.v.), ACTH (Synacthen 250 micrograms i.v.) and human CG (HCG 3000 IU i.m.) tests were performed in addition to semen analysis. Pravastatin significantly reduced total cholesterol (20.3%), calculated LDL-cholesterol (2<em>4</em>.6%) and apolipoprotein B (10.5%, increased apolipoprotein A1 (16.1%) and did not influence plasma HDL-cholesterol and triglycerides. Basal plasma cortisol, aldosterone, <em>androstenedione</em>, testosterone and oestradiol did not change under active treatment. Pravastatin administration affected neither adrenal hormone responses to CRH and ACTH or testicular response to HCG nor spermatogenesis in respect of motility, morphology and sperm count. In conclusion, long-term pravastatin treatment, at doses effective in improving lipid profile, did not influence testicular reproductive and endocrine function and did not interfere with basal and stimulated adrenal activity of male hypercholesterolemic patients.

The adrenal production of the delta 5-androgens, dehydroepiandrosterone (DHEA) and its sulfate ester dehydroepiandrosterone sulfate (DHEAS), declines linearly with aging. The evidence that DHEA or DHEAS administration may alleviate some of the problems related to aging has opened new perspectives for clinical research. The present study aims to investigate the effects of a 6-month DHEA supplementation in early and late postmenopausal women, with normal or overweight body mass index (BMI), on the level of circulating steroids, sex hormone binding globulin (SHBG), beta-endorphin and gonadotropins, and on the adrenal gland response to dexamethasone suppression and adrenocorticotropic hormone (ACTH) stimulation. Early postmenopausal women (50-55 years) both normal weight (BMI 20-2<em>4</em>, n = 9) and overweight (BMI 26-30, n = 9) and late postmenopausal women (60-65 years) both of normal weight and overweight, were treated with oral DHEA (50 mg/day). Circulating DHEA, DHEAS, 17-OH pregnenolone, progesterone, 17-OH progesterone, allopregnenolone, <em>androstenedione</em>, testosterone, dihydrotestosterone, estrone, estradiol, SHBG, cortisol, luteinizing hormone, follicle stimulating hormone and beta-endorphin levels were evaluated monthly and a Kupperman score was performed. The product/precursor ratios of adrenal steroid levels were used to assess the relative activities of the adrenal cortex enzymes. Before and after 3 and 6 months of therapy, each women underwent an ACTH stimulating test (10 micrograms i.v. in bolus) after dexamethasone administration (0.5 mg p.o.) to evaluate the response of cortisol, DHEA, DHEAS, <em>androstenedione</em>, 17-OH pregnenolone, allopregnanolone, progesterone and 17-OH progesterone. The between-group differences observed before treatment disappeared during DHEA administration. Levels of 17-OH pregnenolone remained constant during the 6 months. Levels of DHEA, DHEAS, <em>androstenedione</em>, testosterone and dihydrotestosterone increased progressively from the first month of treatment. Levels of estradiol and estrone significantly increased after the first/second month of treatment. Levels of SHBG significantly decreased from the second month of treatment only in overweight late postmenopausal women, while the other groups showed constant levels. Progesterone levels remained constant in all groups, while 17-OH progesterone levels showed a slight but significant increase in all groups. Allopregnanolone and plasma beta-endorphin levels increased progressively and significantly in the four groups, reaching values three times higher than baseline. Levels of cortisol and gonadotropins progressively decreased in all groups. The product/precursor ratios of adrenal steroid levels at the sixth month were used to assess the relative activities of the adrenal cortex enzymes and were compared to those found before therapy. The 17,20-desmolase, sulfatase and/or sulfotransferase, 17,20-lyase and 5 alpha-reductase activities significantly increased, while the 3 beta-hydroxysteroid-oxidoreductase activity did not vary. On the contrary, the 11-hydroxylase and/or 21-hydroxylase activities showed a significant decrease after 6 months of treatment. In basal conditions, dexamethasone significantly suppressed all the adrenal steroids and this suppression was greater after 3 and 6 months of treatment for DHEA, DHEAS and allopregnanolone, while it remained unchanged for other steroids. Before treatment, ACTH stimulus induced a significant response in all parameters; after the treatment, it prompted a greater response in delta 5- and delta <em>4</em>-androgens, progesterone and 17-OH progesterone, while cortisol responded less in both younger and older normal-weight women. The endometrial thickness did not show significant modifications in any of the groups of postmenopausal women during the 6 months of treatment. Treatment with DHEA was associated with a progressive improvement of the Kupperman score in all groups, with major effects on the vasomotor symptoms in

Results of supraphysiological adrenocorticotropic hormone (ACTH) stimulation of biosynthetic pathways of adrenal zona fasciculata indicate that a deficiency of 11-hydroxylase exists in patients with essential hypertension. The deficiency is suggested by the much greater stimulus of synthesis of deoxycorticosterone (DOC) and deoxycortisol in hypertensive subjects than in controls (p less than 0.001). No significant difference in the synthesis of cortisol, corticosterone, progesterone, 17-hydroxyprogesterone (17-OHP), and delta-<em>4</em>-<em>androstenedione</em> (D<em>4</em>) was observed between the two groups. The ratios for synthesis of DOC and corticosterone and for deoxycortisol and cortisol found in hypertensive patients were significantly higher than those found in controls (p less than 0.001); no significant difference was observed in the synthesis of 17-OHP and progesterone. The synthesis of DOC and deoxycortisol was not significantly correlated with either blood pressure or plasma renin activity. Plasma renin activity was significantly lower in hypertensive subjects than in normotensive subjects (p less than 0.0001), while no difference was found in aldosterone secretion between the two groups. The 11-hydroxylase deficiency in the adrenal zona fasciculata may be one of the genetic factors causing hypertension together with environmental factors (particularly salt intake and work-related stress). The investigation performed in our study may be useful for the evaluation of adrenal zona fasciculata enzymatic activities during the study of hypertensive patients.

OBJECTIVE

Relationships between regional body fat distribution and sex hormones as well as changes in sex hormones after weight loss were evaluated.

METHODS

All subjects were hospitalized in the Institute of Internal Medicine of the University of Verona.

METHODS

Twenty-six premenopausal (age 33.7 +/- 10.2 years) and 15 postmenopausal (age 57.9 +/- 5.9 years) obese women.

METHODS

Body weight, body-mass index, waist and hip circumferences, visceral fat by computed tomography and sex hormones were evaluated before and after <em>4</em> weeks on a very low energy diet.

RESULTS

Body-mass index was higher in pre-than in postmenopausal women, although the difference was not significant. Total and free testosterone were significantly higher in the pre- than in the postmenopausal group (P < 0.001). Significant negative correlations were found between age and total testosterone (r = -0.65; P < 0.001), free testosterone (r = -0.5<em>4</em>; P < 0.001), androstenedione (r = -0.<em>4</em>6; P < 0.01) and urinary cortisol excretion (r = -0.50; P < 0.01). A negative correlation was found between visceral fat and total testosterone (r = -0.<em>4</em>1; P < 0.01). After adjusting for age, the negative correlation between total testosterone and visceral fat encountered both in the subject group as a whole and in premenopausal women was no longer significant, whilst a significant negative association between visceral fat and sex hormone binding globulin (SHBG) (r = -0.56; P < 0.001) was always found. When step-down regression analysis was used to evaluate the joint effect of age, menopausal status, and anthropometric and metabolic variables on sex hormones, age was the most powerful independent variable for predicting total testosterone, free testosterone and androstenedione levels, whilst menopausal status was the most powerful predictor of FSH and LH levels. Changes in hormones after VLED were analysed separately in pre- and postmenopausal women. None of the hormones changed significantly after VLED in the postmenopausal group, except for FSH values. LH, free testosterone and urinary cortisol excretion values decreased significantly after VLED in the premenopausal group.

CONCLUSIONS

Our data show that age, to a greater extent than visceral fat, seems to be negatively associated with steroid sex hormones. Weight loss seems to be associated with changes in sex hormones only in premenopausal women.

Patients with the genetic liver disease, acute intermittent porphyria (AIP), have a defect in the reductive transformation of steroid hormones that is manifest by the disproportionate generation of 5beta-steroid metabolites from precursor hormones. 5beta-steroid metabolites were earlier shown to be potent inducers experimentally of delta-aminolevulinate synthetase (ALAS), the mitochondrial enzyme that is rate-limiting in porphyrin synthesis, and that is found at high levels of activity in the livers of AIP patients. In this report, the basis for the defective steroid metabolism in AIP has been shown, through studies with the (1<em>4</em>)C-labeled adrenal hormone 11beta-hydroxy-Delta(<em>4</em>)-<em>androstenedione</em>, to reside in a substantial deficiency of hepatic steroid Delta(<em>4</em>)-5alpha-reductase activity. This enzymic deficiency was found in all seven AIP patients studied, and ranged from 3<em>4</em>% to as much as 70% below the mean enzyme activity characterizing normal subjects. The functional consequence of the low levels of 5alpha-reductase activity in AIP is to divert the reductive transformation of certain natural hormones from the 5alpha- to the 5beta-pathway; the latter is the metabolic route through which endogenous steroids having the potential for inducing hepatic ALAS are generated. It is not presently known whether the 5alpha-reductase deficiency in AIP is acquired in some fashion or whether it has partial genetic determinants. It seems probable, however, that this enzymatic abnormality, coupled with the dramatic increase in hormone synthesis that occurs at puberty, may be of major importance in determining clinical expression of the latent gene defect for AIP in many individuals. The 5alpha-reductases for steroid hormones are known to be localized in the endoplasmic reticulum of hepatic cells and the present findings in AIP thus represent the first demonstration that an enzymic component of these membranous structures is functionally abnormal in this hereditary liver disease.

BACKGROUND

The determination of steroids is important for the diagnosis and monitoring of endocrine diseases and infertility workup. We developed a rapid and reliable mass spectrometric method for the simultaneous quantification of steroid patterns in human serum.

METHODS

An on-line solid phase extraction (SPE)-liquid chromatography-triple quadrupole linear ion trap (LC-QTrap) method utilizing atmospheric pressure chemical ionization was developed. Following protein precipitation of 100 microL serum, on-line SPE and chromatographic separation was performed for 13 steroids in 1.8 min. Analytes were confirmed by the characteristic fragment patterns.

RESULTS

The total run time of the method was <em>4</em> min. Detection limits ranged between 0.02 microg/L (testosterone) and 9 microg/L (dehydroepiandrosterone sulfate). The method was linear up to 7000 microg/L for dehydroepiandrosterone sulfate, 500 microg/L for cortisol, 125 microg/L for 11-deoxycortisol, and 25 microg/L for aldosterone, 17-hydroxyprogesterone, progesterone, testosterone, <em>androstenedione</em> and beta-estradiol, respectively. Accuracy ranged between 80 and 11<em>4</em>%. Between-day variance at three different concentration levels was <15%. Excellent correlations with immunoassays were observed for testosterone, cortisol and beta-estradiol with Pearson's correlation coefficient r=0.967, 0.963, and 0.998, respectively.

CONCLUSIONS

The novel on-line SPE-LC-MS/QTrap platform offers a very fast, reliable, and sensitive quantification of steroid patterns and fulfils the quality criteria for routine laboratory application.

Using stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS), a physicochemical method, we have profiled the plasma steroids 17 alpha-hydroxyprogesterone, <em>4</em>-<em>androstenedione</em>, and testosterone in normal children of various age groups. Comparison of our values with those obtained by direct immunologic assays and those using an extraction or purification step showed that immunoassays in general overestimate steroid concentrations. This was especially true for plasma samples in the neonatal period and was most expressed for the concentrations of 17 alpha-hydroxyprogesterone. Our study demonstrated the applicability of ID/GC-MS to routine clinical steroid analysis. The application of ID/GC-MS is recommended whenever problems from matrix effects or cross-reactivity are likely to arise or suspicious results by immunoassays need to be rechecked.

Testosterone metabolism by cytochrome P-<em>4</em>50 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and <em>4</em> of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot <em>4</em>. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-<em>4</em>50. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-<em>4</em>50 acts as a peroxidase in the formation of <em>androstenedione</em>.

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-<em>4</em>50 activity was studied in adult male rat liver using P-<em>4</em>50 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-<em>4</em>50 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-<em>4</em>50 forms designated P-<em>4</em>50 2c (P-<em>4</em>50 gene IIC11), P-<em>4</em>50 2a (gene IIIA2), and P-<em>4</em>50 RLM2 were decreased by 70-90% after 7-1<em>4</em> days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-<em>4</em>50 3 (gene IIA1) and P-<em>4</em>50j (gene IIE1) were elevated approximately 2-<em>4</em>-fold. The female-specific microsomal enzyme <em>androstenedione</em> 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-<em>4</em>50 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-<em>4</em>50 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-<em>4</em>50 enzyme expression in adult male rats.

OBJECTIVE

Adiposity, insulin resistance (IR), and hyperandrogenism are features of polycystic ovary syndrome (PCOS). Retinol-binding protein <em>4</em> (RBP<em>4</em>) secreted from adipose and liver tissues has been linked to IR. The impact of RBP<em>4</em> on IR in PCOS and its usability to identify women with metabolic syndrome (MS) or impaired glucose tolerance ((IGT) or diabetes) were investigated.

METHODS

Plasma RBP<em>4</em> was determined in 115 consecutive PCOS women. Associations with IR, body composition, and hyperandrogenemia were investigated by correlation and multiple linear regression analyses in 110 non-diabetics. Receiver operating characteristic curve analysis was used to evaluate RBP<em>4</em> as a parameter for identifying MS and IGT or diabetes.

RESULTS

RBP<em>4</em> increased over tertiles of IR (P=0.009). RBP<em>4</em> correlated with HOMA %S (R=-0.286, P= 0.002), waist-to-hip ratio (WHR) (R=0.233, P=0.03<em>4</em>), and dual energy X-ray absorptiometry (DEXA)-lean body mass (R=0.282, P=0.016) but not with body mass index (BMI), DEXA-total or -trunk fat mass, hsCRP, free testosterone, DHEAS, androstenedione, and 17beta-estradiol. Adjusted for age, BMI, smoking, and IGT, the association between RBP<em>4</em> and HOMA %S remained significant (P=0.032). RBP<em>4</em> explained <em>4</em>.6% of the variation in HOMA %S. RBP<em>4</em> was higher in MS and IGT or diabetes, but its ability to identify these women was low (area under the curve, AUC=0.631, P=0.0<em>4</em>1 or AUC=0.660, P=0.016).

CONCLUSIONS

In PCOS, RBP<em>4</em> has a small independent impact on IR. It is not correlated with hyperandrogenemia, 17beta-estradiol, other adrenal steroids, or with markers of adiposity in general. Furthermore, RBP<em>4</em> does not appear suitable for screening MS or impaired glucose metabolism (IGT or diabetes).

The 2<em>4</em>-h profiles of plasma cortisol (F), 11-beta-hydroxy<em>androstenedione</em> (11OHAD), <em>androstenedione</em> (AD), dehydroisoandrosterone (DHEA) and testosterone (T) were obtained simultaneously in 11 normal males sampled at 15-min intervals. The data were submitted to a detailed quantitative analysis including the estimation of the circadian rhythm and of the episodic variations as well as the evaluation of the concomitance of episodic pulses of different hormones. A bimodal circadian rhythm was detected in the various individual profiles. The major acrophase occurred in the morning earlier for T (around 0<em>4</em>:00 h) than for the hormones of totally or partially adrenal origin (around 07:00 h); the secondary acrophase (around 17:00 h) and the main midnight nadir were common to all hormones. The amplitude of the rhythm was highest for purely adrenal hormones (F and 11OHAD), averaging 79 and 75%, respectively, lower for hormones of mixed origin (DHEA and AD), averaging <em>4</em><em>4</em> and <em>4</em>2%, respectively, and minimal for T (22%). The possible relationship between the circadian and pulsatile variations of the various steroids was estimated in each individual by calculating Pearson's standard coefficient of variation on all pairs of hormonal profiles. A very tight relationship (r greater than 0.75; p less than 0.001) was found between the <em>4</em> adrenal hormones in each individual; a looser but significant correlation (r greater than 0.30; p less than 0.001) was also detected between T and its partial precursors (AD and DHEA) and between T and the purely adrenal hormones: F and 11OHAD (r greater than 0.30; p less than 0.01). The pulsatility of the corticotrophic axis was readily transmitted to the secretory pattern of 11OHAD, DHEA and AD. Ninety-six percent of the F pulses were reflected in at least one other hormonal profile. Finally, we showed that concomitant pulses common to the five adrenal and gonadal patterns were more frequent than would be expected on the basis of chance. These results: demonstrate a total parallelism between the long-lasting secretory events and the episodic bursts of the <em>4</em> adrenal hormones showing that the reticular and fascicular zones of the adrenal respond to pituitary control as an homogeneous structure; demonstrate the existence of a partial synchronization of adrenal and testicular pulsatile variations; suggest that, throughout the afternoon, a common mechanism may influence the slow variations of adrenal hormones and of testicular testosterone.

OBJECTIVE

Prolonged period of amenorrhoea are regarded as a risk factor for the appearance of osteoporosis. Amenorrhoea is a feature of different pathological conditions with heterogeneous endocrine profiles. We evaluated bone mineral metabolism in patients with polycystic ovary syndrome (PCOS), hypothalamic amenorrhoea and idiopathic hirsutism in order to establish the relative importance for the maintenance of normal bone mass of ovulatory cycles and androgen and oestrogen production.

METHODS

Bone mineral density (BMD), bone turnover markers and endocrine profile were evaluated in 51 patients with PCOS, 2<em>4</em> patients with idiopathic hirsutism, 26 patients with hypothalamic amenorrhoea and 35 healthy women. Body mass index (BMI) ranged between 20.1 and 31.0 kg/m2, and age from 17 to 33 years. Thirty-eight of the PCOS patients were amenorrhoeic (< <em>4</em> menstrual cycles/year).

RESULTS

Spine and femoral BMD were significantly decreased and bone markers (serum osteocalcin, and urinary excretion of free deoxypyridinoline, cross-linked N-telopeptide and hydroxyproline) significantly increased in the patients with hypothalamic amenorrhoea, when compared to control subjects and the other two patient groups. In the sub-group of PCOS patients with amenorrhoea, spine and femoral neck BMD was significantly lower than in patients with idiopathic hirsutism and the non-amenorrhoeic PCOS patients. In all PCOS patients, spine and neck BMD were positively correlated (P < 0.05) with serum androstenedione and free testosterone levels.

CONCLUSIONS

The results of this study suggest that in patients with polycystic ovary syndrome the deleterious effect on bone of amenorrhoea is balanced by androgen overproduction.

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a <em>4</em>7% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less <em>androstenedione</em> and <em>4</em>0% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta <em>4</em> pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.

Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 μm were cultured on Millicell membrane inserts, with culture medium supplemented with <em>4</em>% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml <em>androstenedione</em>, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 1<em>4</em>-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Oral contraceptive (OC) treatment has an inhibiting effect on protein synthesis in tendon and muscle connective tissue. We aimed to investigate whether OC influence myofibrillar protein turnover in young women. OC-users (2<em>4</em>±2 years; Lindynette® n=7, Cilest® n=<em>4</em>) and non-OC-users (controls, 2<em>4</em>±<em>4</em> years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3-methyl-histidine (3-MH), was measured in the interstitial fluid of the vastus lateralis. Measurements were performed in both legs. In general, myofibrillar protein FSR was lower in OC-users (two-way analysis of variance, P<0.05), although the difference seemed to depend on the OC type. Interstitial 3-MH in the skeletal muscle was not different between groups and did not vary by OC type. Exercise did not change myofibrillar protein FSR or 3-MH concentrations. Serum <em>androstenedione</em> and bioavailability of testosterone were lower in OC-users. In conclusion, the results indicate that the use of OC has an inhibiting effect on myofibrillar protein synthesis and the magnitude of the effect may depend on the type of OC. In contrast, there was no effect of OC on myofibrillar protein breakdown in the fed state.

The effects of <em>4</em>-hydroxy<em>androstenedione</em> (<em>4</em>-OHA) and other aromatase inhibitors, 10-propargylestr-<em>4</em>-ene-3,17-dione and imidazo[1,5-alpha]-3,<em>4</em>,5,6-tetrahydropyrin-6-yl-(<em>4</em>-benzonitrile), as well as 5 alpha-reductase inhibitors N,N-diethyl-<em>4</em>-methyl-3-oxo-<em>4</em>-aza-5 alpha-androstane-17 beta-carboxyamide and <em>4</em>-methyl-3-oxo-<em>4</em>-aza-androsta-5-ene-17-ol were investigated in prostatic tissue from six patients with benign prostatic hypertrophy and seven patients with prostatic cancer, and from normal men at autopsy. We attempted to measure aromatase activity in the tissue incubations by quantitating 3H2O released from <em>androstenedione</em> or testosterone labeled at the C-1 position. High performance liquid chromatography and thin layer chromatography were used to isolate steroid products. Although the amount of 3H2O released was at least twice that of the heat-inactivated tissue samples, no estrone or estradiol was detected on high performance liquid chromatography. The 3H2O release was significantly inhibited by <em>4</em>-OHA and N,N-diethyl-<em>4</em>-methyl-3-oxo-<em>4</em>-aza-5 alpha-androstane-17 beta-carboxyamide, but not by the other aromatase inhibitors. <em>4</em>-OHA also inhibited 5 alpha-reductase in both benign prostatic hypertrophy and cancer tissue, although to a lesser extent than N,N-diethyl-<em>4</em>-methyl-3-oxo-<em>4</em>-aza-5 alpha-androstane-17 beta-carboxyamide. The other aromatase inhibitors were without effect on 5 alpha-reductase. Our results indicate that 3H2O released from [1 beta-3H]<em>androstenedione</em> and [1,2,6,7-3H]<em>androstenedione</em> does not correlate with estrogen formation and may be the result of other metabolic reactions. Although it appears that the prostate lacks aromatase, <em>4</em>-OHA may be of benefit in patients with benign prostatic hypertrophy or prostatic cancer by inhibiting this enzyme in peripheral tissue.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C<em>4</em>-C5 double bond found in Delta<em>4</em>-3-ketosteroids, including steroid hormones and bile acids precursors such as 7alpha-hydroxy-<em>4</em>-cholesten-3-one and 7alpha,12alpha-dihydroxy-<em>4</em>-cholesten-3-one. In order to elucidate the binding mode and substrate specificity in detail, biochemical and structural studies on human 5beta-reductase (h5beta-red; AKR1D1) have been recently undertaken. The crystal structure of a h5beta-red binary complex provides a complete picture of the NADPH-enzyme interactions involving the flexible loop B, which contributes to the maintenance of the cofactor in its binding site by acting as a "safety belt". Structural comparison with binary complexes of AKR1C enzymes, specifically the human type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and the mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21), also revealed particularities in loop B positioning that make the steroid-binding cavity of h5beta-red substantially larger than those of the two other enzymes. Kinetic characterization of the purified recombinant h5beta-red has shown that this enzyme exerts a strong activity toward progesterone (Prog) and <em>androstenedione</em> (Delta<em>4</em>) but is rapidly inhibited by these substrates once their concentrations reach 2-times their K(m) value. A crystal structure of the h5beta-red in ternary complex with NADPH and Delta<em>4</em> has revealed that the large steroid-binding site of this enzyme also contains a subsite in which the Delta<em>4</em> molecule is found. When bound in this subsite, Delta<em>4</em> completely impedes the passage of another substrate molecule toward the catalytic site. The importance of this alternative binding site for the inhibition of h5beta-red was finally proven by site-directed mutagenesis, which demonstrated that the replacement of one of the residues delineating this site (Val(309)) by a phenylalanine completely abolishes the substrate inhibition. The results of this report provide structural insights into the substrate inhibition of h5beta-red by C19- and C21-steroids.

We describe the clinical presentation, biochemical features, diagnostic criteria, clinical course and differential diagnosis in three cases of feminizing adreno-cortical carcinoma (FACC) with a review of the literature.

METHODS

From 1970 throughout December 2003 among a series of 801 adrenalectomies, three had been performed for FACC.

RESULTS

Age at presentation was 7<em>4</em>, 63 and 23 years. Estradiol hypersecretion was observed in 3/3 patients, 17 OH progesterone was elevated in 2/3 patients and both of them had a diminution of testosterone, delta <em>4</em> <em>androstenedione</em> was elevated in 1/3 patients. Imaging studies suggested malignancy in 3/3 patients by the presence of necrosis, heterogeneity, calcifications, size of the tumor and compression of adjacent organs. All patients were stage III at presentation and had a Weiss score>>or=6. Size and weight of the tumors were 30, 20, 15cm and 3750, <em>4</em>80 and 275g respectively. All 3 patients received mitotane and cortisone post-operatively and at follow up (7, 3 and 2 years) all 3 died of the disease.

CONCLUSIONS

Feminizing adreno-cortical carcinomas in adults are exceedingly rare (1-2% of adreno-cortical carcinomas). Tumors are huge and even after surgery for cure their prognosis is worse than for other varieties of adreno-cortical carcinomas either secreting or non secreting. Early diagnosis and treatment may improve overall prognosis.

Oestrone and dihydrotestosterone had no significant action. Other steroids inhibited maturation. The stage of maturation most affected and the median effective concentration (MEC) at this stage varied with different steroids. The predominant effect of pregnenolone (MEC = 6.<em>4</em> microM), progesterone (MEC = 5.3 microM), <em>androstenedione</em> (MEC = 28.0 microM) and oestradiol-17 beta (MEC = 23.0 microM) was to block maturation after the resumption of meiosis but before completion of the first meiotic division. Testosterone was also effective at this stage (MEC = 23.5 microM) but at higher concentrations it prevented germinal vesicle breakdown (MEC = <em>4</em>0 microM) without causing oocyte degeneration. The inhibitory actions of all steroids were reversible in oocytes exposed for <em>4</em> or 18 h.

BACKGROUND

Androstenedione, a steroid hormone and the major precursor to testosterone, is available without prescription and is purported to increase strength and athletic performance. The hormonal effects of androstenedione, however, are unknown.

OBJECTIVE

To determine if oral administration of androstenedione increases serum testosterone levels in healthy men.

METHODS

Open-label randomized controlled trial conducted between October 1998 and April 1999.

METHODS

General clinical research center of a tertiary-care, university-affiliated hospital.

METHODS

Forty-two healthy men aged 20 to 40 years.

METHODS

Subjects were randomized to receive oral androstenedione (either 100 mg/d [n = 15] or 300 mg/d [n = 14]) or no androstenedione (n = 13) for 7 days.

METHODS

Changes in serum testosterone, androstenedione, estrone, and estradiol levels, measured by frequent blood sampling, compared among the 3 treatment groups.

RESULTS

Mean (SE) changes in the area under the curve (AUC) for serum testosterone concentrations were -2% (7%), -4% (4%), and 34% (14%) in the groups receiving 0, 100, and 300 mg/d of androstenedione, respectively. When compared with the control group, the change in testosterone AUC was significant for the 300-mg/d group (P<.001) but not for the 100-mg/d group (P = .48). Baseline testosterone levels, drawn 24 hours after androstenedione administration, did not change. Mean (SE) changes in the AUC for serum estradiol concentrations were 4% (6%), 42% (12%), and 128% (24%) in the groups receiving 0, 100, and 300 mg/d of androstenedione, respectively. When compared with the control group, the change in the estradiol AUC was significant for both the 300-mg/d (P<.001) and 100-mg/d (P = .002) groups. There was marked variability in individual responses for all measured sex steroids.

CONCLUSIONS

Our data suggest that oral androstenedione, when given in dosages of 300 mg/d, increases serum testosterone and estradiol concentrations in some healthy men.