Women with polycystic ovary syndrome (PCOS) are characterized by defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis. We administered the insulin-sensitizing agent troglitazone to 13 obese women with PCOS and impaired glucose tolerance to determine whether attenuation of hyperinsulinemia ameliorates these defects. All subjects had oligomenorrhea, hirsutism, polycystic ovaries, and hyperandrogenemia. Before and after treatment with troglitazone (<em>4</em>00 mg daily for 12 weeks), all had 1) a GnRH agonist (leuprolide) test, 2) a 75-g oral glucose tolerance test, 3) a frequently sampled iv glucose tolerance test to determine the insulin sensitivity index and the acute insulin response to glucose, <em>4</em>) an oscillatory glucose infusion to assess the ability of the beta-cell to entrain to glucose as quantitated by the normalized spectral power for the insulin secretion rate, and 5) measures of fibrinolytic capacity [plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator]. There was no change in body mass index (39.9 +/- 1.<em>4</em> vs. <em>4</em>0.2 +/- 1.<em>4</em> kg/m2) or body fat distribution after treatment. Both the fasting (91 +/- 3 vs. 103 +/- 3 mg/dL; P < 0.001) and 2 h (1<em>4</em>6 +/- 8 vs. 171 +/- 6 mg/dL; P < 0.02) plasma glucose concentrations during the oral glucose tolerance test declined significantly. There was a concordant reduction in glycosylated hemoglobin to 5.7 +/- 0.1 from a pretreatment level of 6.1 +/- 0.1% (P < 0.03). Insulin sensitivity increased from 0.58 +/- 0.1<em>4</em> to 0.95 +/- 0.26 10(-5) min-1/pmol.L (P < 0.01) after treatment as did the disposition index (7<em>4</em>5 +/- 135 vs. 381 +/- 96; P < 0.05). The ability of the beta-cell to appropriately detect and respond to an oscillatory glucose infusion improved significantly after troglitazone treatment; the normalized spectral power for the insulin secretion rate increased to 5.9 +/- 1.1 from <em>4</em>.3 +/- 0.8 (P < 0.05). Basal levels of total testosterone (109.3 +/- 15.2 vs. 79.<em>4</em> +/- 9.8 ng/dL; P < 0.05) and free testosterone (33.3 +/- <em>4</em>.0 vs. 21.2 +/- 2.6 pg/mL; P < 0.01) declined significantly after troglitazone treatment. Leuprolide-stimulated levels of 17-hydroxyprogesterone, <em>androstenedione</em>, and total testosterone were significantly lower posttreatment compared to pretreatment. The reduction in androgen levels occurred independently of any changes in gonadotropin levels. A decreased functional activity of PAI-1 in blood (from 12.7 +/- 2.8 to 6.3 +/- 1.<em>4</em> AU/mL P < 0.05) was associated with a decreased concentration of PAI-1 protein (from 6<em>4</em>.9 +/- 9.1 to <em>4</em><em>4</em>.8 +/- 6.1 ng/mL; P < 0.05). No change in the functional activity of tissue plasminogen activator (from 5.3 +/- 0.<em>4</em> to 5.1 +/- 0.5 IU/mL) was observed despite a decrease in its concentration (from 9.6 +/- 0.9 to 8.2 +/- 0.7 ng/mL; P < 0.05). The marked reduction in PAI-1 could be expected to improve the fibrinolytic response to thrombosis in these subjects. We conclude that administration of troglitazone to women with PCOS and impaired glucose tolerance ameliorates the metabolic and hormonal derangements characteristic of the syndrome. Troglitazone holds potential as a useful primary or adjunctive treatment for women with PCOS.

In male patients with congenital adrenal hyperplasia, testicular tumors, or so-called adrenal rest tumors, have been described, but their presence in well controlled patients is thought to be rare. In this study, the prevalence of testicular tumors in 17 adolescent and adult male patients with congenital adrenal hyperplasia (age, 16-<em>4</em>0 yr) was investigated. In 16 of 17 patients, one or more testicular tumors, ranging in maximal length from 0.2-<em>4</em>.0 cm, were found on ultrasonography. In 6 patients, the testicular tumors were palpable. Undertreatment, defined as the presence of a salivary <em>androstenedione</em> level (mean of 6 saliva samples collected over 2<em>4</em> h with intervals of <em>4</em> h) above the upper reference morning level, was found in 5 of 17 patients at the time of investigation. The other 12 patients were treated adequately or even over treated at the time of investigation. Nevertheless, 11 of these 12 patients showed testicular tumors on ultrasonography. Neither the presence of undertreatment at the time of investigation nor characteristics of the therapeutic regimen (daily dose of hydrocortisone equivalents per body surface, the use of glucocorticoid medication either two or three times a day, or the time of taking the highest glucocorticoid dose either in the morning or the evening) could predict tumor size (maximal diameter of largest tumor). In patients who were heterozygous or homozygous for the deletion or conversion of the CYP21 gene, tumor size was significantly larger than in patients who did not have this genotype. Impairment of Leydig cell function as manifested by decreased plasma levels of T was found in 6 of 17 patients. Semen analysis in 11 patients revealed azoospermia in 3 patients and poor semen quality in <em>4</em> patients. We conclude that, when carefully sought for, testicular adrenal rest tumors are frequently present in adolescent and adult males with congenital adrenal hyperplasia and are often accompanied by impaired spermatogenesis and Leydig cell failure.

Cytochrome P-<em>4</em>50-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, <em>androstenedione</em>, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-<em>4</em>% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-<em>4</em>50 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-<em>4</em>50NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine <em>4</em>-hydroxylase, phenacetin O-deethylase, or S-mephenytoin <em>4</em>-hydroxylase activities or the specific contents of their respective associated P-<em>4</em>50 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-<em>4</em>50NF or a rat homolog, P-<em>4</em>50 PB-2a. Anti-P-<em>4</em>50NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-<em>4</em>50 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-<em>4</em>50 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-<em>4</em>50NF gene subfamily (P-<em>4</em>50 IIIA subfamily). These observations establish that P-<em>4</em>50NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-<em>4</em>50 form.

We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/2<em>4</em> h and about <em>4</em>-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, <em>androstenedione</em>, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.

Human and some other primates are unique since their adrenals secrete large amounts of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), which are converted into <em>androstenedione</em> (<em>4</em>-dione) and then into potent androgens and estrogens in peripheral tissues, therefore providing autonomous intracrine control to target tissues that can adjust the formation and metabolism of active sex steroids according to local requirements. Knowledge in this area has recently made rapid progress with the elucidation of the structure of most of the tissue-specific cDNAs and genes that encode the steroidogenic enzymes responsible for the transformation of these inactive precursor steroids into androgens and/or estrogens. It is estimated that 30 to 50% of total androgens in men are synthesized in peripheral intracrine tissues from inactive adrenal precursors while, in women, peripheral estrogen formation is even more important, the best estimate being 75% before menopause and 100% after menopause. The marked reduction in the formation of DHEA-S by the adrenals during aging, especially before the age of 50 years, results in a dramatic fall in the formation of active sex steroids in peripheral target tissues, a situation which is thought to be associated with a long series of age-related decreases such as insulin resistance, obesity, osteoporosis, cardiovascular diseases, loss of muscle mass, cancer and other diseases. We have demonstrated for the first time a series of medically important beneficial effects of DHEA administered for 12 months to post-menopausal women. Most interestingly, the bone mineral density significantly increased. This relatively rapid change was associated with an increase in plasma osteocalcin, a marker of bone formation, while a decrease in bone resorption reflected by a decrease in urinary hydroxyproline excretion was observed in parallel. In addition, the estrogenic stimulation of vaginal cytology in the absence of any sign of stimulatory effect on the endometrium is also of potentially major interest for the prevention and management of menopause. Furthermore, the inhibitory effect of DHEA on the growth of human breast cancer xenografts in vivo in nude mice supports the beneficial use of DHEA as hormone replacement therapy in women.

The present study examined the effects of the androgen steroid, dihydrotestosterone (DHT), on murine T-cell production of a number of lymphokines. Direct exposure of murine T cells to DHT in vitro was found to reduce the amount of interleukin-<em>4</em> (IL-<em>4</em>), IL-5, and gamma-interferon (gamma IFN) produced after activation with anti-CD3 without affecting the production of IL-2. Exposure of T cells to either <em>androstenedione</em> or testosterone (the metabolic precursors of DHT) affected no change in the biosynthesis of either of these lymphokines. We have determined that macrophages possess 5 alpha-reductase, and are thus competent to metabolize testosterone to DHT. This physicochemical information is complemented by a functional analysis of macrophage metabolism of testosterone. By incubating bone marrow macrophages with testosterone, before their use as accessory cells, the IL-<em>4</em> and IL-5 producing potential of the activated T cells cocultured with them was depressed. That the observed effect was mediated by the conversion of testosterone to DHT was further corroborated by illustrating that the inhibition of IL-<em>4</em> production was abrogated if <em>4</em>MA, a specific 5 alpha-reductase inhibitor, was added to macrophage cultures containing testosterone. The biologic role of DHT in lymphokine and immune response regulation in vivo was addressed using several lines of investigation. First, transdermal delivery of DHT to groups of mice altered the capacity of T cells residing in the draining lymph nodes, only, to produce lymphokines. Second, treatment of either aged mice or the T cells isolated from them with a combination of dehydroepiandrosterone and DHT restored the capacity of their T cells to produce IL-2, IL-<em>4</em>, and gamma IFN to levels equivalent to that of younger mice. Finally, we observed a difference between males and females of a given age to produce IL-2, IL-<em>4</em>, and gamma IFN, with both IL-<em>4</em> and gamma IFN production being elevated in females. Collectively, our findings indicate that DHT, similar to other steroid hormones, may play an important role in lymphokine regulation in vivo.

Polycystic ovary syndrome (PCOS) is associated with hyperinsulinemia, insulin resistance (IR), increased risk of glucose intolerance, and type 2 diabetes. Family studies have indicated a genetic susceptibility to PCOS. The aims of this study were 1) to assess glucose tolerance status, gonadotropins, and androgens in first degree relatives of patients with PCOS; and 2) to assess IR in normal glucose tolerant (NGT) family members. One hundred two family members of 52 patients with PCOS [Mothers(PCOS) (n = 3<em>4</em>; mean age, <em>4</em>6.5 yr; mean body mass index (BMI), 28.8 kg/m(2)), Fathers(PCOS) (n = 2<em>4</em>; mean age, 50.<em>4</em> yr; mean BMI, 27.5 kg/m(2)), Sisters(PCOS) (n = 19; mean age, 25.1 yr; mean BMI, 22.9 kg/m(2)), and Brothers(PCOS) (n = 25; mean age, 23.7 yr; mean BMI, 22.5 kg/m(2))] and 82 unrelated healthy control subjects without a family history of diabetes or PCOS (<em>4</em> age- and weight-matched subgroups, i.e. Control(MothersPCOS), Control(FathersPCOS), Control(SistersPCOS), and Control(BrothersPCOS)) were studied. Glucose and insulin (at baseline and during a 75-g, 2-h oral glucose tolerance test) were measured. IR was assessed by fasting insulin (FI), fasting glucose to insulin ratio (FGI), homeostatic model assessment (HOMA IR), and area under the curve for insulin during the oral glucose tolerance test (AUC(insulin)) in NGT Mothers(PCOS), Fathers(PCOS), Sisters(PCOS), Brothers(PCOS), and matched control subgroups. Including the prestudy-diagnosed 3 mothers and 2 fathers with diabetes, diabetes and impaired glucose tolerance (IGT) were noted in 16% and 30% of Mothers(PCOS) and 27% and 31% of Fathers(PCOS), respectively. There was no diabetes in Sisters(PCOS) and Brothers(PCOS). IGT was found in 5% of Sisters(PCOS). Impaired fasting glucose was found in 3% of Mothers(PCOS) and <em>4</em>% of Brothers(PCOS). The analysis of NGT family members showed that Mothers(PCOS) had higher FI (P < 0.05), HOMA IR (P < 0.05), and AUC(insulin) (P < 0.01) and lower FGI (P < 0.05) than Control(MothersPCOS), whereas all IR parameters were comparable between Fathers(PCOS) and their matched control subgroup. Sisters(PCOS) had higher FI (P < 0.05), HOMA IR (P < 0.01), and AUC(insulin) (P < 0.05) and lower FGI (P < 0.01), and Brothers(PCOS) had higher AUC(insulin) (P < 0.01) than their matched control subgroups, respectively. Mothers(PCOS) had higher testosterone levels than Control(MothersPCOS) (P < 0.01 and P < 0.05 for pre- and postmenopausal women, respectively). Sisters(PCOS) had higher LH (P < 0.01), testosterone (P < 0.001), <em>androstenedione</em> (P < 0.01), and dehydroepiandrosterone sulfate (P < 0.05) levels than Control(SistersPCOS). There was no difference in gonadotropin and androgen levels in Fathers(PCOS) compared with Control(FathersPCOS) or in Brothers(PCOS) compared with Control(BrothersPCOS). Our results suggest that 1) first degree relatives of patients with PCOS may be at high risk for diabetes and glucose intolerance; 2) NGT female family members have insulin resistance; and 3) mothers and sisters of PCOS patients have higher androgen levels than control subjects. We propose that the high risks of these impairments warrant screening in first degree relatives of patients with PCOS.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) play an essential role in the formation of active intracellular sex steroids. Six types of 17betaHSD have been described to date, which only share approximately 20% homology. Human type 5 17betaHSD complementary DNA is unique among the 17betaHSDs because it belongs to the aldo-keto reductase family, whereas the others are members of the short chain alcohol dehydrogenases. The characteristics of human type 5 17betaHSD were investigated in human embryonic (293) cells stably transfected with human and mouse type 5 17betaHSD, as well as human type 3 3alphaHSD. Using intact transfected cells, type 5 17betaHSD shows a substrate specificity pattern comparable to those of human type 3 17betaHSD and mouse type 5 17betaHSD. These enzymes catalyze more efficiently the transformation of <em>androstenedione</em> (<em>4</em>-dione) to testosterone, whereas the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol is much lower. In contrast, type 3 3alphaHSD catalyzes more efficiently the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol, whereas the transformation of <em>4</em>-dione to testosterone represents only 7% of the 3alphaHSD activity. However, upon homogenization, human type 5 17betaHSD activity decreases to approximately 10% of the activity in intact cells and remains stable at this level together with the 3alphaHSD activity. Under the same conditions, however, the mouse enzyme is not altered by homogenization. Indeed, using purified human 17betaHSD overexpressed in Escherichia coli, we could confirm that a much greater amount of protein is required to produce activity similar to the enzymatic activity measured in intact transfected cells. The present data provide the answer to the question of why previous researchers could hardly detect type 5 17betaHSD activity. Indeed, all previous publications used cell or tissue homogenates or purified enzymes. Under such conditions, only the low level, but stable, 3alphaHSD and 17betaHSD activities could be measured, whereas the high level, but highly unstable, 17betaHSD activity could not be measured. As type 5 17betaHSD shares 8<em>4</em>%, 86%, and 88% amino acid identity with types 1 and 3 3alphaHSD and 20alphaHSDs, respectively, Northern blot analysis used in previous studies could not provide unequivocal information. In this report, we used a more specific ribonuclease protection assay and could thus show that human type 5 17betaHSD is expressed in the liver, adrenal, and prostate; in prostatic cancer cell lines DU-1<em>4</em>5 and LNCaP; as well as in bone carcinoma (MG-63) cells. By analogy with type 3 17betaHSD, which is responsible for the formation of androgens in the testis, the expression of type 5 17betaHSD in the prostate and bone cells suggests that this enzyme is involved in the formation of active intracellular androgens in these tissues.

Continuous infusions of Delta(<em>4</em>)-<em>androstenedione</em>-7-(3)H and testosterone-7-(3)H have been used to demonstrate that these androgens are converted to estrone and 17beta-estradiol, and contribute to the circulating blood levels of these estrogens in normal males and females. The conversion ratio (ratio of concentrations of radioactivity of free product steroid [chi(-PRO)] and free precursor steroid [chi(-PRE)], both corrected for recoveries, after an infusion of radioactive precursor steroid) for <em>androstenedione</em> (precursor) to estrone (product) is 0.013 in males and 0.007 in females, and the conversion ratio for testosterone (precursor) to estradiol (product) is 0.0018 in males and 0.005 in females. The transfer constant, [rho](BB) (AE1), for <em>androstenedione</em> conversion to estrone ([rho](BB) (AE1) = per cent of infused <em>androstenedione</em>, precursor, converted to estrone, product, when infusion and measurement are both in blood) is 1.35% in males and 0.7<em>4</em>% in females, and the transfer constant, [rho](BB) (TE2), for testosterone conversion to estradiol is 0.39% in males and 0.15% in females. Whether measured as conversion ratio or transfer constant, the peripheral aromatization of <em>androstenedione</em> takes place to a greater degree than that of testosterone, and, for the respective androgens, both the conversion ratio and [rho](BB) value are greater in males than females. For the androgen interconversions, [rho](BB) (AT) is <em>4</em>.5% in males and 2.2% in females; [rho](BB) (TA) is 8.2% in males and 12.0% in females. Studies on the distribution coefficients (effective concentration in red cells/plasma) for precursor radioactivity were also made. In both males and females the distribution coefficient for <em>androstenedione</em> is 0.16-0.17 while that of testosterone is 0.01-0.03.

Current evidence suggests that estrogen plays a dominant role in determining bone mineral density (BMD) in men, and inactivating mutations in the aromatase CYP19 gene have been associated with low bone mass in young males. We previously reported an association between a TTTA repeat polymorphism in intron <em>4</em> of the CYP19 gene and osteoporotic risk in postmenopausal females. Here we explore the role of this polymorphism as a genetic determinant of BMD in a sample of elderly males who were recruited by direct mailing and followed longitudinally for 2 (n = 300) and <em>4</em> (n = 200) yr. Six different allelic variants, containing seven, eight, nine, 10, 11, and 12 TTTA repeats, were detected. There was a bimodal distribution of alleles, with two major peaks at seven and 11 repeats and a very low distribution of the nine-repeat allele. Men with a high-repeat genotype >>nine repeats) showed higher lumbar BMD values, lower bone turnover markers, higher estradiol levels, and a lower rate of BMD change than men with a low-repeat genotype (<nine repeats). The association with BMD was not significant in the subgroup of patients with high body mass index (>25), suggesting that the effect of CYP19 genotypes on bone may be masked by the increase in fat mass. Moreover, the high-repeat genotype was less represented, although not significantly, in the vertebral fracture group with respect to the nonvertebral fracture group. Functional in vitro analysis after incubation with [3H]-<em>androstenedione</em> showed a higher aromatase activity in fibroblasts from subjects with a high-repeat genotype than in fibroblasts from subjects with a low-repeat genotype. In conclusion, differences in estrogen levels due to polymorphism at the aromatase CYP19 gene may predispose men to increased age-related bone loss and fracture risk.

BACKGROUND

Measurement of serum androgens is important in adult, geriatric, pediatric endocrinology, and oncology patients. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of androstenedione, dehydroepiandrosterone (DHEA), and testosterone in these patients.

METHODS

We spiked 200 muL of serum or plasma with isotope-labeled internal standards and performed extraction with methyl t-butyl ether. We then derivatized the extracts with hydroxylamine and analyzed them by LC-MS/MS using a 2-dimensional chromatographic separation with a 3.5-min analysis time.

RESULTS

Total imprecision for each analyte was <11.2%. Limits of quantification were 10, 50, and 10 ng/L for androstenedione, DHEA, and testosterone, respectively. Reference intervals were established for children (age 6 months to 17 years), men, and women. Androstenedione and DHEA concentrations were lowest in 2- to 3-year-old children. Adult concentrations were achieved in girls at Tanner stage 3 and in boys at Tanner stage 4-5. In premenopausal and (postmenopausal) women the median concentrations of androstenedione, DHEA, and testosterone were 810 (360), 3000 (1670), 270 (180) ng/L, respectively. In postmenopausal women, concentrations of testosterone were age independent, whereas androstenedione and DHEA concentrations decreased with age. In men the median concentrations of androstenedione, DHEA, and testosterone were 440, 2000, and 3700 ng/L, respectively. In men older than 40 years, median concentrations decreased at rates of 5%, 10%, and 20% per decade for androstenedione, DHEA, and testosterone, respectively.

CONCLUSIONS

This LC-MS/MS method has the required lower limit of quantification and specificity for analysis of endogenous concentrations of androgens in all groups studied. Reference intervals were established for healthy children and adults.

[7-3HA1Androstenedione and [<em>4</em>-1<em>4</em>C]estrone or [7-3H]testosterone and [1<em>4</em>C]estradiol were infused at constant rates into brachial arm veins of 15 normal men. During the infusions blood samples were obtained from the brachial artery, a deep vein draining primarily muscle, and a superficial vein draining primarily adipose tissue of the arm contralateral to the infusion. In seven men the mean +/- SE value for the fractional conversion of androstene tissue. In eight men the mean +/- SE value for the fractional conversion of testosterone to estradiol was 0.0007 +/- 0.0001 for muscle and 0.0012 +/- 0.0002 for adipose tissue. Both of these values were significantly (P less than 0.01) less than the respective values of <em>androstenedione</em> aromatization to estrone. If constancy of tissue aromatization throughout the body is assumed, the muscle accounts for 25-30% and adipose tissue for 10-15% of the total extragonadal aromatization of androgens to estrogens.

Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P<em>4</em>50c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to <em>androstenedione</em> by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P<em>4</em>50c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P<em>4</em>50c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P<em>4</em>50 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta <em>4</em> and the delta 5 steroidogenic pathways can lead to testosterone production.

The postpubertal outcome of a group of girls diagnosed of premature pubarche during childhood was assessed 1) to determine the incidence of functional ovarian hyperandrogenism (FOH) through the ovarian-steroidogenic response to the GnRH agonist leuprolide acetate, 2) to validate leuprolide acetate stimulation in FOH diagnosis, and 3) to ascertain whether FOH-predictive biochemical markers exist at the diagnosis of premature pubarche. Of 35 patients (age, 15.<em>4</em> +/- 1.5 yr), 16 showed hirsutism, oligomenorrhea, and elevated baseline testosterone and/or <em>androstenedione</em> (delta <em>4</em>-A) levels. Subcutaneous administration of leuprolide acetate (500 micrograms) produced similar increases in gonadotropin levels in oligomenorrheic patients, regularly menstruating patients (n = 19), and controls (n = 12; age, 15.3 +/- 1.3 yr) when tested at 6 h. Of all of the steroids measured, 17-hydroxyprogesterone (17-OHP) and delta <em>4</em>-A levels 2<em>4</em> h postleuprolide acetate stimulation were significantly higher in oligomenorrheic patients than in the other two groups (P < 0.0001). No overlapping in 17-OHP responses occurred between oligomenorrheic patients and the other groups. Baseline dehydroepiandrosterone sulfate and delta <em>4</em>-A levels at the diagnosis of premature pubarche correlated with 17-OHP values postleuprolide acetate challenge (r = 0.<em>4</em>7; P < 0.005 and r = 0.67; P < 0.0001, respectively). These results show a distinct leuprolide acetate challenge response in <em>4</em>5% of the postpubertal premature pubarche girls studied, suggestive of an increased incidence of FOH, and support the need for continued routine postmenarcheal evaluation of this group of patients. Responses of 17-OHP to leuprolide acetate challenge facilitate the identification of FOH patients, establish this test as a reliable diagnostic tool in FOH diagnosis, and confirm the ovaries as the source of hyperandrogenemia in most patients with androgen excess. Although increased 17-OHP responses after leuprolide acetate stimulation seem to occur more frequently in girls with elevated dehydroepiandrosterone sulfate and/or delta <em>4</em>-A levels at the diagnosis of premature pubarche, specific biochemical markers predictive of FOH in this group of patients are still lacking.

The objective of the study was the comparison of anti-Müllerian hormone (AMH) levels among obese or overweight and normal-weight women with the four different polycystic ovary syndrome (PCOS) phenotypes and healthy control subjects. AMH levels were evaluated in four age- and body mass index (BMI)-matched groups of 25 normal-weight and 25 obese or overweight women each, belonging to the four main subsets of the syndrome resulting from combinations of the three diagnostic criteria [group 1: oligo- and/or anovulation (ANOV), hyperandrogenemia (HA), and polycystic ovaries (PCO) on ultrasonographic evaluation; group 2: ANOV and HA; group 3: HA and PCO, group <em>4</em>: ANOV and PCO], and in 50 (25 obese or overweight and 25 normal weight) age- and BMI-matched healthy control subjects. Age, BMI, waist circumference, FSH, LH, prolactin, testosterone, Delta(<em>4</em>)-<em>androstenedione</em>, dehydroepiandrosterone-sulfate, 17alpha-OH-progesterone, fasting insulin, glucose, AMH, free androgen index, and homeostasis model assessment for insulin resistance index were analyzed. AMH levels were significantly higher in PCOS groups 1 and 2 compared with groups 3 and <em>4</em> and the control group and higher in PCOS groups 3 and <em>4</em> compared with the control group. AMH levels were significantly increased in normal-weight compared with obese and overweight women. AMH concentrations were independently predicted, in order of significance, by LH and testosterone levels, BMI (negatively), and the total number of follicles 2-9 mm in diameter. The differences in circulating AMH levels between the main phenotypic groups of PCOS women appear to reflect the severity of the syndrome, but are negatively affected by obesity. Increased LH levels might be the most significant independent link between PCOS-associated disorders of ovulation and the observed increase in circulating AMH concentration.

Women with polycystic ovary syndrome (PCOS) have high circulating androgens, thought to originate from ovaries and adrenals, and frequently suffer from the metabolic syndrome including obesity. However, serum androgens are positively associated with body mass index (BMI) not only in PCOS, but also in simple obesity, suggesting androgen synthesis within adipose tissue. Thus we investigated androgen generation in human adipose tissue, including expression of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isozymes, important regulators of sex steroid metabolism. Paired omental and subcutaneous fat biopsies were obtained from 27 healthy women undergoing elective abdominal surgery (age range 30-50 years; BMI 19.7-39.2 kg/m(2)). Enzymatic activity assays in preadipocyte proliferation cultures revealed effcient conversion of <em>androstenedione</em> to testosterone in both subcutaneous and omental fat. RT-PCR of whole fat and preadipocytes of subcutaneous and omental origin showed expression of 17beta-HSD types <em>4</em> and 5, but no relevant expression of 17beta-HSD types 1, 2, or 3. Microarray analysis confirmed this expression pattern (17beta-HSD5>17beta-HSD<em>4</em>) and suggested a higher expression of 17beta-HSD5 in subcutaneous fat. Accordingly, quantitative real-time RT-PCR showed significantly higher expression of 17beta-HSD5 in subcutaneous compared with omental fat (P<0.05). 17beta-HSD5 expression in subcutaneous, but not omental, whole fat correlated significantly with BMI (r=0.51, P<0.05). In keeping with these findings, 17beta-HSD5 expression in subcutaneous fat biopsies from six women taking part in a weight loss study decreased significantly with weight loss (P<0.05). A role for 17beta-HSD5 in adipocyte differentiation was further supported by the observed increase in 17beta-HSD5 expression upon differentiation of stromal preadipocytes to mature adipocytes (n=5; P<0.005), which again was higher in cells of subcutaneous origin. Functional activity of 17beta-HSD5 also significantly increased with differentiation, revealing a net gain in androgen activation (<em>androstenedione</em> to testosterone) in subcutaneous cultures, contrasting with a net gain in androgen inactivation (testosterone to <em>androstenedione</em>) in omental cultures. Thus, human adipose tissue is capable of active androgen synthesis catalysed by 17beta-HSD5, and increased expression in obesity may contribute to circulating androgen excess.

To explore the relation between androgens and prostatic hypertrophy in man, the concentrations of testosterone, dihydrotestosterone, and <em>androstenedione</em> and the rate of conversion of testosterone to dihydrotestosterone have been measured in normal and hypertrophic prostate tissue. First, a double isotope derivative technique was adapted for the measurement of tissue androgen content in 15 normal and 10 hypertrophic prostates. Although there was no significant difference in the content of <em>androstenedione</em> and testosterone between the two types of tissue, the content of dihydrotestosterone was significantly greater in the hypertrophic tissue (0.60 +/-0.10 mug/100 g) than in the normal glands (0.13 +/-0.05 mug/100 g). Second, a regional study was performed in three normal prostates and four glands with early hypertrophy, and it was demonstrated that the dihydrotestosterone content was two and three fold greater in the periurethral area where prostatic hypertrophy usually commences than in the outer regions of the gland. Finally, the rate of conversion of testosterone to dihydrotestosterone has been measured under standardized conditions in tissue slices from <em>4</em> normal and 20 hypertrophic prostates. There was no significant difference in the rate of dihydrotestosterone formation between the two types of gland (6.0 +/-0.8 and 7.8 +/-0.5 mumumoles/15 mg of tissue per hr). While the mechanism by which dihydrotestosterone accumulation occurs remains unexplained, it is possible that the local accumulation of dihydrotestosterone may be involved in the pathogenesis of prostatic hypertrophy in man.

We report the features of a new syndrome of aromatase deficiency due to molecular defects in the CYP19 (P<em>4</em>50arom) gene in a <em>4</em>6,XX female. At birth, the patient presented with a nonadrenal form of female pseudohermaphrodism. At 17 months of age, laparotomy revealed normal female internal genital structures; the histological appearance of the ovaries was normal. FSH concentrations were markedly elevated at 9.<em>4</em> ng/mL LER 869, and estrone and estradiol levels were undetectable (< 37 pmol/L). By 1<em>4</em> yr of age, she had failed to exhibit breast development. The clitoris had enlarged to <em>4</em> x 2 cm, and pubic hair was Tanner stage IV. The plasma concentration of testosterone was elevated at 329<em>4</em> pmol/L, as was <em>androstenedione</em> at 9951 pmol/L. Plasma estradiol levels were below 37 pmol/L. ACTH and dexamethasone tests indicated a nonadrenal source of testosterone and <em>androstenedione</em>. Plasma gonadotropin levels were in the castrate range. Pelvic sonography and magnetic resonance imaging showed multiple <em>4</em>- to 6-cm ovarian cysts bilaterally. Despite increased circulating androgens and clitoral growth, the bone age was 10 yr at chronologic age 1<em>4</em> 2/12 yr. Estrogen replacement therapy resulted in a growth spurt, breast development, menarche, suppression of gonadotropin levels, and resolution of the cysts. The clinical findings suggested the diagnosis of P<em>4</em>50arom deficiency. Analyses of genomic DNA from ovarian fibroblasts demonstrated two single base changes in the coding region of the P<em>4</em>50arom gene, one at 1303 basepairs (C-T), R<em>4</em>35C, and the other at 1310 basepairs (G-A), C<em>4</em>37Y, in exon 10. The molecular genetic studies indicate that the patient is a compound heterozygote for these mutations. Expression of these mutations showed that the R<em>4</em>35C mutation had 1.1% the activity of the wild-type P<em>4</em>50arom enzyme, whereas the C<em>4</em>37Y mutation demonstrated no activity. The cardinal features of this syndrome are a consequence of P<em>4</em>50arom deficiency: 1) the fetal masculinization in this syndrome can be ascribed to defective placental conversion of C19 steroids to estrogens, leading to exposure of the female fetus to excessive amounts of testosterone; 2) the pubertal failure, mild virilization, multicystic ovaries, and hyperstimulation of the ovaries by FSH and LH are the result of the inability of the ovary to aromatize testosterone and <em>androstenedione</em> to estrogens; and 3) the striking delay in bone age at 1<em>4</em> 2/12 yr supports the notion that estrogens, in contrast to androgens, are the major sex steroid driving skeletal maturation during puberty. Familial P<em>4</em>50arom deficiency, although rare, may be more common than previously suspected.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The enzyme delta <em>4</em>-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and <em>4</em>,5 beta-dihydrocortisone: NADP+ delta <em>4</em>-oxidoreductase) catalyzes the reduction of the delta <em>4</em> double bond of bile acid intermediates and steroid hormones carrying the delta <em>4</em>-3-one structure in the A/B cis configuration. Human delta <em>4</em>-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y. Noshiro, M., Shimosato, T. & Okuda, K.-I. (1991) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-positive clone predicted the human delta <em>4</em>-3-oxosteroid 5 beta-reductase to contain 326 amino acids. The amino acid sequence of the human delta <em>4</em>-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence. It also showed 5<em>4</em>% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single delta <em>4</em>-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-<em>4</em>-cholesten-3-one and 7 alpha-hydroxy-<em>4</em>-cholesten-3-one. In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta <em>4</em>-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta <em>4</em>-androsten-3-one (testosterone) whereas no activity was observed toward delta <em>4</em>-pregnene-3,20-dione (progesterone) or delta <em>4</em>-androstene-3-17-dione (<em>androstenedione</em>). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.

It is well recognized that there are two androgens, namely testosterone (T) and dihydrotestosterone (DHT); T plays an important role in the testis and muscle, and DHT is crucial for the development, function and pathology of the prostate. It is generally thought that DHT is produced from the 5alpha-reduction of circulating T before being inactivated by 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) that converts DHT into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol). However, the presence of various steroidogenic enzymes in the prostate as well as the availability at high levels of various steroid precursors such as dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and <em>4</em>-<em>androstenedione</em> (<em>4</em>-dione) strongly suggest the existence of additional pathways involved in the biosynthesis and metabolism of DHT. Because steroidogenesis could be different in different species, data from the literature obtained from various human, dog, rat and mouse prostate tissues, as well as primary cells and prostatic cancer cell lines, provide a somewhat confusing picture. In the present chapter, we review the data in order to provide a clearer picture of the pathways involved in DHT biosynthesis and metabolism in the human prostate.

<em>4</em>-Hydroxy<em>androstenedione</em> (<em>4</em>-OHA), a potent new aromatase inhibitor, was given i.m. (500-1000 mg) to 58 patients with advanced postmenopausal breast cancer. Of 52 assessable patients 1<em>4</em> responded (27%), in 10 (19%) the disease stabilized, and in 28 (5<em>4</em>%) the disease progressed. Sterile abscesses occurred at the injection site in 6 patients and painful lumps were found in a further 3 patients. Two patients developed allergic-type reactions and <em>4</em> developed lethargy, suspected to be treatment induced. Plasma estradiol levels were suppressed from a mean of 7.2 +/- 0.8 (SE) pg/ml before treatment to 2.6 +/- 0.2, 2.7 +/- 0.2, and 2.8 +/- 0.3 pg/ml after 1, 2, and greater than <em>4</em> months, respectively, of treatment and remained suppressed in patients whose disease relapsed. No significant fall in estrone levels was seen. Similarly, dehydroepiandrosterone sulfate, sex hormone binding globulin, and gonadotrophin levels were unaltered after 6 months of treatment. Plasma <em>4</em>-OHA levels were measured in a radioimmunoassay for <em>androstenedione</em> after chromatographic separation of <em>4</em>-OHA from <em>androstenedione</em>. Drug concentrations ranged from 0.7 to 23.2 (7.8 +/- 1.1) ng/ml after 2 months on treatment. <em>4</em>-OHA is an effective drug in the management of postmenopausal patients with breast cancer and does not produce notable systemic side effects.

PBDEs have been synthesized in large quantities as flame retardants for commercial products, such as electronic equipment and textiles. The rising in levels of PBDEs in tissues in wildlife species and in human milk and plasma samples over the past several years have raised concerns about possible health effects. Recently, we showed that the PBDE mixture, DE-71, delayed puberty and suppressed the growth of androgen-dependent tissues in male Wistar rat following a peri-pubertal exposure. These effects suggested that DE-71 may be either inducing steroid hormone metabolism or acting as an androgen receptor (AR) antagonist. To elucidate the potential anti-androgenic effects of this mixture, we evaluated DE-71 in several in vivo assays, which are responsive to alterations in androgen activity. In a pubertal exposure study designed to further evaluate the delay in preputial separation (PPS), we observed a dose-dependent delay in PPS with 60 and 120 mg/kg/day of DE-71 (<em>4</em> and 5 days) and a corresponding suppression of ventral prostate (VP) and seminal vesicle growth at both doses. Adult males exposed to 60 mg/kg DE-71 for 3 days resulted in a significant increase in luteinizing hormone and a non-significant increase in testosterone, <em>androstenedione</em> and estrone. DE-71 also tested positive for anti-androgenic activity in an immature rat Hershberger assay, with decreases in mean VP and seminal vesicle weight following doses of 30-2<em>4</em>0 mg/kg. DE-71 and the individual BDE congeners which comprise the mixture (BDE-<em>4</em>7, -99, -100, -153, -15<em>4</em>) were also evaluated in vitro. First, AR binding was evaluated in a competitive binding assay using rat VP cytosol. In addition, we evaluated gene activation in a transcriptional activation assay using the MDA-kb2 cell line which contains an endogenous human AR and a transfected luciferase reporter. DE-71 and BDE-100 (2, <em>4</em>, 6-pentaBDE) both inhibited AR binding, with IC50s of approximately 5 microM. In addition, DE-71 and two of the congeners (BDE-100 and BDE-<em>4</em>7) inhibited DHT-induced transcriptional activation. The pattern of inhibition shown in the double-reciprocal plot for BDE-100 and the linear slope replot confirmed that the in vitro mechanism is pure competitive inhibition, with a inhibition constant (Ki) of 1 microM. The delay in puberty in the male rat and decreased growth of androgen-dependent tissues observed previously following exposure to DE-71 were likely due to this inhibition of AR binding by several of the congeners which make up this mixture.

There is an increasing trend to apply gas chromatography combined with mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) assay methods to large-scale epidemiologic studies for the measurement of serum sex steroids. These methods are generally considered the gold standard for sex steroid measurements because of their accuracy, sensitivity, turnaround time, and ability to assess a more complete panel of steroid metabolites in the same run. In this report, we evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility, of steroid hormone measurements determined by GC-MS and LC-MS/MS assays and RIA and compared measurements among these methods. Specifically, 282 overnight fasting serum samples from 20 male volunteers were analyzed for 12 steroid metabolites by GC-MS or LC-MS/MS in one lab over a <em>4</em>-month period. Six of the analytes were also measured by RIA in another lab. Unconjugated hormones, including testosterone, dihydrotestosterone, dehydroepiandrosterone, <em>androstenedione</em>, androst-5-ene-3beta,17beta-diol, estrone, and estradiol, were measured by GC-MS, whereas conjugated hormones, including DHEA sulfate, androsterone glucuronide, 5alpha-androstane-3alpha,17beta-diol 3-glucuronide, 5alpha-androstane-3alpha,17beta-diol 17-glucuronide, and estrone sulfate, were measured by LC-MS/MS. A subset of these hormones, including testosterone, dihydrotestosterone, <em>androstenedione</em>, 5alpha-androstane-3alpha,17beta-diol 17-glucuronide, estrone, and estradiol, were also measured by RIA following extraction and chromatography. We used the coefficient of variation (CV) and the intraclass correlation coefficient (ICC) to assess within- and between-batch assay variations. For the 12 analytes measured by GC-MS or LC-MS/MS, CVs and ICCs for within- and between-batch measurements were similar, with CVs ranging from 6.1% to 21.<em>4</em>% and ICCs ranging from 87.6% to 99.2%. The six analytes measured by RIA had good CVs and ICCs, with CVs <10% and ICCs >70% (range, 71.7-99.7%). For the six metabolites that were measured by both methods, the CVs were similar, whereas the ICCs were generally higher with the GC-MS method. The absolute values for each analyte measured by RIA and GC-MS differed, with RIAs usually yielding markedly higher levels than GC-MS, although the Pearson and Spearman correlation coefficients for these six analytes were near one and all were significant (P < 0.001). Our results show that RIA, GC-MS, and LC-MS/MS assays for androgens and estrogens in the two labs included in the study have good reproducibility, as measured by small CVs (<15%) and high ICCs (>80%), with the exception of estradiol (71.7%) when measured by RIA. Despite substantial differences in absolute measurements of sex steroid hormones by RIA and MS methods, correlations between the two assays for the six sex steroids measured in the two labs were high (>0.9). However, it is important for future large epidemiologic studies to incorporate MS with high reproducibility and specificity to measure a more complete profile of androgen and estrogen metabolites to clarify the role of sex steroids in prostate cancer.

The efficacy of recombinant human LH (rhLH) for supporting human (rhFSH)-induced follicular development was investigated in hypogonadotropic hypogonadal women (WHO group I anovulation). Patients (n = 38) were randomized to receive rhLH (0, 25, 75, or 225 IU/day) in addition to a fixed dose of rhFSH (150 IU/day). rhLH was found 1) to promote dose-related increases in estradiol (E2) and <em>androstenedione</em> secretion by rhFSH-induced follicles, i.e. serum concentrations on the last day of FSH administration were 65 +/- <em>4</em>, 195 +/- 9<em>4</em>, 1392 +/- 585, and 2<em>4</em><em>4</em>1 +/- 90<em>4</em> pmol/L for E2 and 3.6 +/- 0.9, 5.1 +/- 1.3, 6.<em>4</em> +/- 1.3, and 6.7 +/- 1.3 nmol/L for <em>androstenedione</em> for patients treated with 0, 25, 75, and 225 IU rhLH, respectively; 2) to increase ovarian sensitivity to FSH, as demonstrated by the proportion of patients who developed follicles after the administration of a fixed dose of FSH, i.e. 1 of 8, 3 of 7, 7 of 9, and 8 of 10 in patients treated with 0, 25, 75, and 225 IU rhLH, respectively; and 3) to enhance the ability of these follicles to luteinize when exposed to hCG. A daily dose of 75 IU rhLH was effective in the majority of women in promoting optimal follicular development (defined as>> or = 1 follicle>> or = 17 mm; E2,>> or = <em>4</em>00 pmol/L; midluteal phase progesterone,>> or = 25 nmol/L) and maximal endometrial growth. A minority of patients may require up to 225 IU/day. rhLH, given sc at a dose up to 225 IU/day, was not immunogenic and was well tolerated.