The patient studied had noted the onset of virilization shortly after menopause. Urinary <em>17</em>-<em>ketosteroid</em> levels were normal, as were fractionated <em>17</em>-<em>ketosteroid</em> levels by gas liquid chromatography, but for 3 yr, serum testosterone levels had been greater than 490 ng/dl. The ovaries were found to be normal by laparoscopy. Abdominal exploration revealed a 1-cm adenoma in the right adrenal. A part of the adenoma excised from our patient was homogenized and incubated with 5 microCi [14C]androstenedione. Five percent of the 14C was converted by the tumor homogenate to a metabolite with the same mobility as testosterone on LH-20 chromatography. After thin layer chromatography, the radiolabeled material together with 3H-labeled authentic testosterone were crystallized to a constant specific activity. The net rate of testosterone synthesis by the tumor was 26 pmol/mg wet tissue wt.h vs. 0.56 pmol/mg.h by a control adrenal homogenate. Thus, the tumor demonstrated a 50-fold increase in <em>17</em> beta-hydroxysteroid dehydrogenase activity compared to normal adrenal tissue. This is the first report to identify altered activity of a specific enzyme system in this syndrome of isolated adrenal testosterone overproduction.

A tumour of the left adrenal gland was identified in a woman who presented with virilization and secondary amenorrhea. Preoperatively, the plasma levels of dehydroepiandrosterone sulphate, dehydroepiandrosterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and 5-androstene-3 beta,<em>17</em> beta-diol were elevated two- to fourfold whereas those of urinary <em>17</em>-<em>ketosteroids</em> were elevated more than tenfold. The production rate of dehydroepiandrosterone sulphate was more than 16 times that in normal women whereas those of dehydroepiandrosterone, testosterone and androstenedione were approximately twofold greater; plasma testosterone was derived almost entirely from the peripheral conversion of androstenedione. Blood was obtained by catheterization of the ovarian veins, left adrenal gland vein and inferior vena cava (at two different sites) and plasma steroid levels were determined: testosterone and cortisol levels were elevated in all blood samples whereas those of androstenedione, dehydroepiandrosterone sulphate and 11-desoxycortisol were approximately six- to eightfold, 1.5-fold and nine- to 22-fold higher in the effluent on the left adrenal gland/tumour compared with the levels in the other compartments. Blood was collected hourly for 24 h to determine steroid levels under basal conditions and, also, after ACTH treatment. Plasma cortisol levels increased markedly upon ACTH administration and fell to very low levels 11 h later, but those of androstenedione, testosterone, dehydroepiandrosterone, 5-androstene-3 beta,<em>17</em> beta-diol and dehydroepiandrosterone sulphate were not affected by ACTH treatment. A histological diagnosis of cortical adenoma of the extirpated tumour was made. Tissue explants and adenoma cells were maintained in culture to characterize the steroid-metabolizing properties of the tumour. The secretion of dehydroepiandrosterone sulphate by tissue explants was highly initially, but declined to almost undetectable levels after 5 days in culture. In the presence of ACTH, dehydroepiandrosterone sulphate secretion remained elevated throughout the entire study up to 5 days. Basal secretion of dehydroepiandrosterone sulphate, androstenedione, 11-desoxycortisol, cortisol, testosterone and 11 beta-hydroxyandrostenedione by adenoma cells was either very low or undetectable. In the presence of ACTH, dibutyryl cyclic AMP or cholera toxin the secretion of dehydroepiandrosterone sulphate, androstenedione and 11-desoxycortisol increased markedly with time in culture up to 3 days, whereas the other steroids were undetected in the medium. A homogenate of adenoma tissue metabolized testosterone to androstenedione, but the conversion of androstenedione to testosterone was minimal. The findings of this study served to establish that virilization in this woman was due at least in part, to excess testosterone--and testosterone-derived 5 alpha-dihydrotestosterone--produced at extra-adrenal tissue sites almost exclusively through metabolism of tumour-secreted androstenedione.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma follicle stimulating hormone, luteinizing hormone, testosterone, urinary 5-hydroxyindole acetic acid and <em>17</em>-<em>ketosteroids</em> were measured in patients seen at an infertility clinic. Plasma follicle stimulating hormone and luteinizing hormone levels, and urinary 5-hydroxyindole acetic acid levels were increased in patients with sperm concentrations less than 10 times 10(6) per ml. The results suggest that in patients with sperm counts less than 10 times 10(6) per ml. there is not only impaired spermatogenesis but also decreased Leydig cell function. Urinary <em>17</em>-<em>ketosteroid</em> levels were not related to sperm cell concentration.

An 18-month-old girl with virilization was found to have an encapsulated right adrenal carcinoma (2 x3 cm) with great variation in nuclear size, frequent mitoses, and possible blood vessel invasion. Preoperative urinary excretions of <em>17</em>-<em>ketosteroids</em>, androsterone, etiocholanolone, dehydroepiandrosterone, testosterone, pregnanetriol, 3alpha-androstenol, and 3 beta-androstadienol were elevated; all showed a noticeable decrease postoperatively. Cortisol acetate, given preoperatively, produced a definite decrease in the urinary excretion of <em>17</em>-<em>ketosteroids</em> and dehydroepiandrosterone; administration of corticotropin resulted in an increase in levels of urinary <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxycorticosteroids, and pregnanetriol. Urinary testosterone and 3beta-androstadienol may have diagnostic value since neither was suppressed by cortisol therapy. The behavior of both 3alpha-androstenol and 3beta-androstadienol in this study suggests that they are of adrenal origin.

The presence of an androgen-secreting tumor in a 29-year-old woman was confirmed and its location was determined by computerized axial tomographic (CAT) scanning. The hormone production from this virilizing adrenal adenoma was studied in vivo and in vitro. The major secretory products of the tumor (as compared to normal adrenal tissue) were testosterone (24-fold) and <em>17</em> beta-estradiol (five-fold). Although the adenoma produced lesser amounts of dehydroepiandrosterone sulfate (DHEAS), the demonstration of elevated serum testosterone and DHEAS in serial samples was a better marker for an androgen-secreting adrenal tumor than were the urinary <em>17</em>-<em>ketosteroids</em>, which remained in the upper limit of normal. The hormone production from the tumor depended neither on adrenocorticotropic hormone nor on human chorionic gonadotropin. The conclusions were that: (1) on the basis of serial measurements of serum testosterone and DHEAS, virilizing adrenal adenomas may be suspected when the concentrations of these hormones reach or exceed 200 ng/dl and 6,600 ng/ml, respectively; (2) the high-resolution CAT scanner can accurately localize these tumors; (3) cosmetic and menstrual dysfunction regressed after resection of the tumor; and (4) virilizing adrenal adenomas may produce both androgens and estrogens.

Improvements in the Zimmermann reaction are effected by using methanol as a solvent for the reagents employed in the reaction in place of ethanol or aqueous ethanol. The advantages are as follows. Considerably lower blanks are obtained; extraneous chromogens are negligible at the wavelength of measurement, so that no correction procedure or extraction process is required; high concentrations of potassium hydroxide may be used at an elevated temperature leading to good sensitivity and a considerably shortened reaction time; and finally, the potassium hydroxide is stable for long periods under good storage conditions. A steroid with keto (oxo) groups at positions 3, 11, and 20, but not at <em>17</em>, failed to give any coloured product.

3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P)⁺-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, <em>17</em>- and 20-<em>ketosteroids</em> and prostaglandin D₂, which were converted to 3α-, <em>17</em>β- and 20α-hydroxysteroids and 9α,11β-prostaglandin F₂, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low K(m) values (0.1-5.9 μM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5β-androstan-3α-ol-<em>17</em>-one were metabolized into 3-hydroxyhexobarbital and 5β-androstane-3α,<em>17</em>β-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D₂, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.

The identification of several steroid-transforming enzymes within human breast cancers has led to speculation that the growth of some hormone-responsive tumors might be mediated in part by intracellularly derived estrogens. Reports that MCF-7 human breast cancer cells can transform both estrone (E1)1 to estradiol (E2) and dehydroepiandrosterone (DHEA) to the estrogenic steroid 5-androstenediol (AED), have prompted us to investigate the <em>17</em>-<em>ketosteroid</em> reductase activities (<em>17</em>-KSR's) which mediate these potentially important reactions. Enzyme assays were performed by quantifying the amounts of [3H]AED or [3H]E2 former from [3H]DHEA or [3H]E1, respectively, by various subcellular preparations from MCF-7 cells under a variety of experimental conditions. DHEA <em>17</em>-KSR was found to be localized exclusively within cytosol, whereas the E1 <em>17</em>-KSR activity appeared to be nearly equally divided between the soluble and particulate cytoplasmic subfractions. The particulate E1 <em>17</em>-KSR appeared capable of utilizing NADH or NADPH, whereas both the cytosolic form of this enzyme and the soluble DHEA <em>17</em>-KSR activity showed a strict requirement for NADPH. Although both of the soluble <em>17</em>-KSR's also showed similar pH optima, several other features suggested that they are different enzymes in MCF-7. E1 did not inhibit the conversion of DHEA to AED, and DHEA did not interfere with the transformation of E1 to E2, indicating that major differences in substrate specificity exist between the two cytosolic activities. Furthermore, DHEA <em>17</em>-KSR activity within cytosol stored at -20 degrees C deteriorated almost completely over twelve weeks of storage, whereas E1 <em>17</em>-KSR activity remained stable. Finally, although both enzymes were found to be subject to product inhibition, AED inhibited DHEA <em>17</em>-KSR competitively, whereas cytosolic E1 <em>17</em>-KSR activity was inhibited by E2 in noncompetitive fashion. Studies of the oxidation of E2 to E1 by MCF-7 cells showed that this transformation is catalyzed by both soluble and particulate <em>17</em>-hydroxysteroid oxidases which utilize either NAD or NADP as cofactor. Having previously reported the presence of a particulate NADP(H)-linked androstenedione (AE) <em>17</em>-<em>ketosteroid</em> oxidoreductase in MCF-7, we now suggest that at least three different enzymes, one particulate and two soluble forms, participate in the conversion of <em>17</em>-<em>ketosteroids</em> to their hormonally active <em>17</em>-hydroxysteroid derivatives within this cell line. The restricted substrate requirements of each enzyme provide a rationale for developing selective enzyme inhibitors which could provide important investigational tools and potentially effective therapeutic agents.

Dehydroepiandrosterone (DHEA) and androstenedione are weak androgens, which need conversion to more potent testosterone in order to enhance anabolic action. Consequences of oral dosing at 1 mg/kg on the urinary and plasma androgen profile of mare and gelding have been evaluated with an analytical method involving conjugate fractionation and selective hydrolysis, group separation, and quantitation by gas chromatography-mass spectrometry with selected ion monitoring of trimethylsilyl ethers. Peak levels of testosterone total conjugates in urine (range 300-6000 microg/L) were attained a few hours after dosing. Renal clearance was fast, so the testosterone detection period lasted only 20 to 33 h, the longest time being generated by androstenedione. The urinary testosterone/epitestosterone ratio for detection of exogenous testosterone in the mare was inoperative after DHEA administration because there was a concomitant increase of epitestosterone, which thereby acted as a masking agent. Androstanediols and androstenediols, as well as some <em>17</em>-<em>ketosteroids</em>, were additional markers. A transient increase of circulating free testosterone has been evidenced, and this would support possible anabolic/androgenic action by supplementation with DHEA and androstenedione along the oral route.

Unilateral cryptorchidism was induced in newborn rats and when the rats reached adult age Leydig cell morphology and steroid biosynthesis were compared in scrotal and abdominal testes. The cell profile area of abdominal Leydig cells was reduced, the cytoplasm contained an increased number of lipid droplets and the endoplasmatic reticulum was more sparse than in scrotal Leydig cells. The intratesticular concentrations of progesterone, 20 alpha-dihydroprogesterone, <em>17</em> alpha-hydroxyprogesterone and testosterone were measured both basally and after LH stimulation. The synthesis of testosterone was subnormal in the abdominal testes. The data on basal steroid concentrations suggest a block at the <em>17</em> alpha-hydroxylase level, but observations after LH stimulation suggest that earlier steps are also involved. When studied in vitro the steroid biosynthesis in abdominal testes was also disturbed. The conversion mediated by <em>17</em> alpha-hydroxylase and <em>17</em> beta-<em>ketosteroid</em> reductase was lower and the conversion mediated by 20 alpha-dehydrogenase was higher in the abdominal testis. The reason for the disturbed steroid biosynthesis in abdominal testes is unknown. It may be related to the increased concentration of oestradiol in abdominal testes. Other possibilities are altered paracrine influence from the damaged tubules or changes in the microcirculation of the testis.

A 12.9 year-old girl, genotypically 46, XY, and considered to have a testicular feminization syndrome, developed signs of virilization and gynaecomastia. Very high androstenedione concentrations (10-fold the mean of the reference interval in boys) in relation to low normal testosterone in peripheral serum indicated a <em>17</em>-<em>ketosteroid</em> reductase deficiency. In addition to androstenedione, the basal peripheral levels of <em>17</em>-hydroxyprogesterone and estrone were increased, being 5- and 3-fold the mean of the reference interval, respectively, whereas pregnenolone, progesterone, dehydroepiandrosterone, 5 alpha-dihydrotestosterone and estradiol concentrations were within pubertal stage-appropriate reference intervals. The total spermatic vein serum steroid concentrations were about 5-fold the mean in old men, and androstenedione, estrone and dehydroepiandrosterone were particularly elevated, whereas estradiol was normal and testosterone subnormal by a factor of 1/8. In the testis tissue, the concentration of androstenedione was extremely high, whereas that of testosterone tended to be relatively low. Our patient was obviously producing testicular steroids at her maximal rate, because no response to hCG administration was observed. This state was associated with a high-normal circulating LH concentration. The concentration of testicular LH/hCG receptors was only one-fifth of that seen in old men, which may have resulted from receptor down-regulation associated with a high degree of stimulation.

To investigate the changes of testosterone (T) secretion under sustained hypoxia, we determined basal levels of urine T, <em>17</em> <em>ketosteroid</em>, luteinizing hormone releasing hormone (LHRH), luteinizing hormone (LH), follicle stimulating hormone (FSH) and response to LHRH and HCG (human chorionic gonadotropin) in male patients with respiratory failure. After evaluating blood gas data, we also measured serum T, LH, FSH, plasma progesterone (P) and <em>17</em> hydroxyprogesterone (<em>17</em>OH-P). The subjects were divided into 3 groups according to PaO2; Group 1 with a PaO2 under 60 Torr, Group 2 with a PaO2 between 60 Torr and under 70 Torr, Group 3 was an age-matched control group. Urine T and serum T were significantly lower in Group 1 compared with those of Group 3. In the LHRH test, augmented relative responsiveness and delayed peak value in LH secretion were observed in Group 1, compared with those of Group 3. As for the HCG test, no differences were observed among the 3 groups. The ratio of <em>17</em>OH-P to P, which indicates activity of <em>17</em>-hydroxylase, was observed to be diminished with increasing degrees of hypoxia. These data suggest that in male patients with respiratory failure there was depression in T secretion as well as <em>17</em>-hydroxylase activity due to hypothalamic-pituitary hypofunction.

The circadian periodicity of urinary <em>17</em>-ketogenic steroids (<em>17</em>-KGS), <em>17</em>-<em>ketosteroids</em> (<em>17</em>-KS) and creatinine (Cr) was studied preoperatively and on the 9th postoperative day in 25 histopathologically proved breast cancer patients and in 15 healthy Indian women under tropical conditions. A statistically significant rhythm was observed in healthy participants for all three variables. Urinary corticoids were markedly elevated in breast cancer patients irrespective of the stage of the disease in comparison with healthy controls. The degree of elevation was more pronounced preoperatively in advanced stage breast cancer in comparison with other groups. After mastectomy, the values of all three variables declined markedly, approaching usual values with a circadian rhythm resembling the pattern found in clinical health.

Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5->>delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,<em>17</em>-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, <em>17</em>-hydroxysteroid dehydrogenase (<em>17</em>-OH SDH) and 3-<em>ketosteroid</em>-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-<em>17</em>-<em>ketosteroid</em> producing mycobacteria were hitherto unreported.

Radiation induced changes in testicular activity were studied by estimating sialic acid content in plasma and testis and <em>17</em>-<em>ketosteroids</em> in 24 hr urine samples of male Sprague Dawley rats following 8 Gy whole body gamma ray exposure with and without pretreatment with 2-aminoethylisothiuronium bromide hydrobromide (AET) or with a combination of 5-hydroxy L-tryptophan (5-HTP) and AET. Combination of 5-HTP with AET or AET alone in optimum radioprotecting dose has significantly modified the radiation damage to the testis.

Pituitary-gonadal function was studied in 50 male diabetic patients under 53 years of age. Forty-three had normal sexual activity and 7 were impotent. Plasma testosterone levels and urinary <em>17</em> <em>ketosteroids</em>, androsterone and dehydroepiandosterone levels were measured. LH and FSH levels before and after LHRH, and prolactin levels before and after TRH were also measured in plasma. No significant changes in pituitary-gonadal function were detected, irrespective of the patient's sexual activity. Neither the type and degree of control of diabetes nor the presence of absence of microangiopathy had any influence on the results. Basal LH and FSH levels were slightly higher in older patients. Prolactin levels after TRH were significantly higher in the later stages of the test in patients with microangiopathy.