A steroid monooxygenase from cells of a fungus, Cylindrocarpon radicicola ATCC 11011, grown in the presence of progesterone has been purified by affinity chromatography on a pregnenolone-Sepharose column. The obtained enzyme was gel electrophoretically homogeneous and exhibited a molecular weight of about 115,000. SDS-gel electrophoresis revealed that the enzyme consisted of two equal-sized subunits with a molecular weight of 56,000. Sedimentation equilibrium analysis at 20 degrees C indicated that the enzyme protein behaved as a mixture of monomeric and dimeric subunit species. The enzyme contained one molecule of FAD in each subunit and exhibited absorption maxima at 375 and 440 nm. The monooxygenase catalyzed a Baeyer-Villiger type oxidation, i.e., oxygenative esterification of C21-20-<em>ketosteroid</em> to form an acetate ester of C19-<em>17</em> beta-hydroxysteroid with consumptions of NADPH and molecular oxygen. The enzyme displayed a wide substrate specificity toward C21-20-<em>ketosteroids</em>, while it strictly required NADPH as the external electron donor in a ratio of 1:1:1 for <em>ketosteroid</em>:NADPH:molecular oxygen. Kinetic study showed the enzyme to have very high affinity for progesterone.

Four cases in adults of a deficiency in the 11 beta-hydroxylation of corticosteroids were investigated by both basal and dynamic biological studies. Symptoms varied from patient to patient; hirsutism, menstrual disturbance, acne, deepening of the voice, and arterial hypertension appeared post puberty. Basal testing demonstrated elevated levels of plasma androgens. These include delta 4-androstenedione (patients, 3.80-6.43 ng/ml; normal, 1.33 +/- 0.33 ng/ml), urinary <em>17</em>-<em>ketosteroids</em> (patients, 11.8-16.7 mg/24 h; normal, 5-10 mg/24 h), and urinary dehydroepiandrosterone. The basal tests were often insufficient to show the accumulation of the precursors (especially <em>17</em>-hydroxyprogesterone) which are often given as evidence for an increase in ACTH stimulation. In studying the levels of the mineralocorticoids, there was shown to be an increased basal level of tetrahydrodeoxycorticosterone (patients, 142-3<em>17</em> microgram/24 h; normal, 60-80 microgram/24 h) which was raised by ACTH stimulation. These results, therefore, confirm the characteristic partial enzyme defect and give evidence for the heterogeneity of this syndrome. Based on the above observations, we believe it is appropriate to rename this condition adult adrenocortical 11 beta-hydroxylation defect rather than late-onset congenital adrenal hyperplasia.

We have examined the suppression of urinary pregnanetriol and <em>17</em>-<em>ketosteroids</em> during treatment with cortisol, cortisone, prednisone, and dexamethasone in eight patients with congenital adrenal hyperplasia. A large individual variation in response to each agent was observed. In some individuals, cortisone is less effective than its generally accepted potency would indicate. At equivalent glucocorticoid dosage, dexamethasone was twice as effective as the other steroids in suppressing urinary <em>17</em>-<em>ketosteroids</em> and pregnanetriol. The potency of dexamethasone in suppressing adrenal function was 80 times that of cortisol, about twice its generally accepted potency as a glucocorticoid or anti-inflammatory agent.

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both <em>17</em> beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-<em>17</em>-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,<em>17</em> beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(<em>17</em>)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-<em>ketosteroids</em> and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.

The production of ACTH-like material by tumours arising in non-endocrine tissue may initiate severe adrenocortical hyperfunction. The pathogenesis and clinical and laboratory features of Cushing's syndrome associated with such tumours are characteristic. The autonomous production by the tumour of ACTH-like material cannot be suppressed by exogenous corticoids. The onset of clinical symptoms is rapid; muscle wasting, general weakness, thirst and peripheral edema predominate, and the classical signs of Cushing's syndrome may be absent. High levels of plasma <em>17</em>-hydroxycorticosteroids and urinary <em>17</em>-hydroxycorticosteroids and <em>17</em>-<em>ketosteroids</em>, usually with normal levels of urinary aldosterone, commonly occur. Hypokalemic alkalosis unresponsive to replacement therapy may cause death. In the case reported herein, the intriguing possibility exists that two hormone-like substances were produced by the primary growth and its metastases: one, ACTH-like, to account for the adrenal hyperplasia and Cushing's syndrome; and another, gastrin-like, giving rise to the ulcerogenic diathesis.

The in vitro inhibition of Leydig cell microsomal steroidogenesis by ketoconazole, a potent P-450 dependent enzyme blocker, was evaluated in the human, stallion and pig. Purified Leydig cells were isolated by mechanical dispersion of teased, decapsulated whole testes and sieving through a 0.25 mm stainless steel mesh. The activity of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), <em>17</em>-hydroxylase (<em>17</em>-OHase), <em>17</em>,20-desmolase (<em>17</em>,20D), <em>17</em>-<em>ketosteroid</em> reductase (<em>17</em>-KSR) and aromatase were measured using a constant amount (50 microM) of 14C-labelled substrates in the presence of varying concentrations of pure ketoconazole. Products were isolated by thin layer chromatography and verified by derivative formation. <em>17</em>-OHase and <em>17</em>,20D activities were significantly inhibited (p less than .001) by ketoconazole at concentrations as low as 5 microM. 3 beta-HSD, <em>17</em>-KSR and aromatase activities were only significantly inhibited by ketoconazole at concentrations of 500 and 5000 microM. These data describe the specific loci of inhibition of ketoconazole on testicular steroidogenesis and confirm the observations that ketoconazole is an effective inhibitor of androgen biosynthesis in several species.

Liver metabolites and in vitro enzyme activities were measured in Sprague-Dawley rats pair-fed the standard NIH diet with or without 0.6% (wt/wt) dehydroepiandrosterone (DHEA) for 16 d. Absorption of DHEA from the gut was confirmed by a 300-fold increase in urine <em>17</em>-<em>ketosteroids</em> in DHEA-treated animals. Of the liver metabolites measured only 6-phosphogluconate was significantly changed, increasing by less than a factor of two in the DHEA-treated animals, 38.7 +/- 2.2 nmol/g, above the value in the pair-fed controls, 22.5 +/- 2.5 nmol/g. Contrary to the in vitro findings that DHEA inhibits glucose-6-phosphate dehydrogenase (EC 1.1.1.49), thus leading to the hypothesis that DHEA inhibits fat synthesis by diminishing the availability of NADPH, the [NADP+]/[NADPH] ratios calculated from the 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40) redox couples were no more oxidized in the DHEA-treated animals than in the control animals. Malic enzyme and isocitrate dehydrogenase activities were 620 and 25% higher in DHEA-treated animals than in pair-fed controls. There was no change in the measured activity of glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase. These data give no support to the hypothesis that administration of DHEA per os results in decreased cytoplasmic NADPH in liver.

To determine whether a single morning plasma level of <em>17</em>-hydroxyprogesterone (<em>17</em>OH-P), androstenedione, testosterone and progesterone reflected the degree of control of 21-hydroxylase congenital virilizing adrenal hyperplasia (CVAH) as indicated by 24-hour urinary <em>17</em>-<em>ketosteroid</em> and pregnanetriol excretion, 142 simultaneous 24-hour urine and morning blood collections were made from 65 patients with CVAH. Patients were grouped into five categories on the basis of age, skeletal age, and sex. Paired blood and urinary data were analyzed. The results suggest that androstenedione is the most reliable indicator for all patient categories. Testosterone is an excellent indicator for children of both sexes and for adolescent and adult females. Levels of <em>17</em>OH-P are difficult to interpret, as they can be several fold higher than the normal values when adrenal suppression appears adequate on the basis of urinary data. In general, progesterone is a poor indicator.

Four human aldo-keto reductases (AKRs) that belong to the AKR1C subfamily function in vitro as 3-keto-, <em>17</em>-keto- and 20-<em>ketosteroid</em> reductases or as 3alpha-, <em>17</em>beta- and 20alpha- hydroxysteroid oxidases to varying degrees. By acting as <em>ketosteroid</em> reductases or hydroxysteroid oxidases these AKRs can either convert potent sex hormones (androgens, estrogens and progestins) into their inactive metabolites or they can form potent hormones by catalyzing the reverse reaction. In this manner they may regulate occupancy and trans-activation of steroid hormone receptors. Tissue distribution studies previously indicated that AKR1C2 (type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD)) and AKR1C3 (type 2 3alpha-HSD) are highly expressed in human prostate. An assessment of the directionality of these AKR1C isozymes in a cellular environment would help identify which isozymes are responsible for 5alpha-dihydrotestosterone (5alpha-DHT) formation or its elimination in the prostate. An imbalance in 5alpha-DHT levels has been implicated in development of prostate carcinoma and benign prostatic hyperplasia. We focused our attention on AKR1C2 since this is the isoform that will oxidize 3alpha-androstanediol (3alpha-diol) to 5alpha-DHT in vitro, suggesting it could elevate 5alpha-DHT levels. To determine whether AKR1C2 preferentially functions as a reductase or an oxidase in a cellular context, we transiently transfected AKR1C2 (pcDNA3-AKR1C2) into COS-1 cells and stably transfected pcDNA3-AKR1C2 and pLNCX-AKR1C2 constructs into PC-3 and LNCaP cells, respectively. COS-1 is a monkey kidney cell line, while PC-3 and LNCaP cells are androgen receptor (-) and (+) prostate adenocarcinoma cell lines, respectively. In transient COS-1-AKR1C2 and in stable PC3-AKR1C2 transfectants, AKR1C2 functioned as a 3-<em>ketosteroid</em> reductase inactivating 5alpha-DHT. In androgen dependent human prostate cancer cells LNCaP, it was not possible to ascertain the preferred direction of AKR1C2 by stable transfection due to the high rate of 5alpha-DHT and 3alpha-diol glucuronidation. Based on these findings AKR1C2 may diminish 5alpha-DHT and prevent this ligand from activating the androgen receptor in situ.

Conversion of soybean sterols to <em>17</em>-<em>ketosteroids</em> by thawed cells of Mycobacterium fortuitum is strongly inhibited by organic solvents. Use of natural oils instead of the solvents leads to enhanced activity, even after 4 h.

Bioconversion of sterols to <em>17</em>-<em>ketosteroids</em> by an Arthrobacter species occurred in the presence of hydrophobic metal-chelating agents but the production of <em>17</em>-<em>ketosteroids</em> (<em>17</em>-KS) was seriously limited by the rapid loss of the viability of cells in the presence of these inhibitors. Besides, the conversion was inhibited by <em>17</em>-KS at concentrations of 500 ppm or more. The <em>17</em>-KS formed consisted exclusively of 1,4-androstadiene-3,<em>17</em>-dione (ADD) and 4-androstene-3,<em>17</em>-dione (AD) and these were found in the extracellular medium predominantly in bound form or as molecular aggregates which may limit their accumulation. It was concluded that enhanced production of <em>17</em>-KS could be achieved by protecting the viability of cells and by removing the steroid metabolites from the site of inhibition.

We studied metabolic, endocrine, and environmental factors in 59 women who had delivered a child with cleft lip with or without cleft palate (CL +/- CP) and compared these values with those of 56 mothers of unaffected children. There was no significant difference between the two groups with respect to race, age, weight, height, education, parity, menstrual history, medical illnesses, or the use of contraceptives, tobacco, alcohol, or caffeine. All patients had a normal XX karyotype confirmed by the fluorescent banding technique. The two groups demonstrated no significant difference in test results of serum chemistries, glucose tolerance, serum or erythrocyte folate, vitamin A, carotene, corticoids, prolactin T4, free T4, urine <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxysteroids, total estrogens, or pregnanediol. Urinalyses revealed no group differences in the presence of barbiturates, amphetamines, salicylates, or benzodiazepines. The percentage of immunologic studies reflecting susceptibility to toxoplasmosis, rubella, cytomegalic inclusion disease, and herpes was not different between the two groups. The only statistically significant metabolic differences between the two groups were serum alkaline phosphatase, creatinine, creatinine clearance, and creatinine clearance/m2. Phenytoin pharmacokinetics and urinary metabolic patterns were compared in a subgroup of ten mothers of affected children and ten mothers from the control group. No significant differences were observed. However, a brief course of phenytoin treatment induced a greater inhibition of the folate tolerance test in controls than in mothers of children with clefts.

Two distinct enzyme defects affecting androgen production and resulting in male pseudohermaphroditism were found in a Turkish kindred from a small isolated village in the Taurus mountains of southern Turkey. Pedigree analysis revealed the inter-relationships of 9 male pseudohermaphrodites. Six affected subjects had adequate steroid hormone analysis. Two adult male pseudohermaphrodites had <em>17</em>-<em>ketosteroid</em> reductase deficiency with elevated concentrations of plasma androstenedione relative to testosterone, and elevated concentrations of urinary androsterone (A) and etiocholanolone (E) relative to tetrahydrocortisol (THF), 5 alpha-tetrahydrocortisol (5 alpha-THF) and tetrahydrocortisone (THE). Four affected males (three adults, one child) had 5 alpha-reductase deficiency (elevated ratios of plasma testosterone/dihydrotestosterone and urinary 5 beta/5 alpha C19 and C21 steroid metabolites). The homozygous state for both enzyme deficiencies was not demonstrable in the same affected subject, suggesting that the enzyme deficiencies are segregating separately within this kindred. Whether the mutant genes are segregating on allelic chromosomes or other autosomes cannot be determined from this study.

The isozymes of the <em>17</em> beta-hydroxysteroid dehydrogenase (<em>17</em> beta-HSD) gene family are responsible for the formation of the <em>17</em> beta-hydroxysteroids delta 5-androstene-3 beta,<em>17</em> beta-diol, testosterone, <em>17</em> beta-estradiol, and dihydrotestosterone from their corresponding <em>17</em>-<em>ketosteroid</em> precursors, thus playing a pivotal role in the formation of active sex steroids in both steroidogenic and peripheral target tissues. To clone the type II <em>17</em> beta-HSD gene, the full-length cDNA type II <em>17</em> beta-HSD was used as probe to screen a human leukocyte genomic DNA library. The type II <em>17</em> beta-HSD gene contains seven exons and spans>> 40 kbp. The type II <em>17</em> beta-HSD gene encodes two alternatively spliced mRNAs that give rise to the previously identified type IIA <em>17</em> beta-HSD protein of 387 amino acids, as well as to a related 291-amino-acid type IIB <em>17</em> beta-HSD protein of unknown function. RNA blot analysis revealed the presence of a major 1.45-kb transcript that is abundant in placenta and endometrium. The mRNA cap site has been localized in a region between <em>17</em>9 and 167 nucleotides upstream of the ATG start codon by RNase protection and S1 nuclease mapping analyses. Cloning of the <em>17</em> beta-HSD type II gene provides us with the tools to study its transcriptional expression.

BACKGROUND

The rapid development of modern imaging techniques, has led to an increase in accidentally discovered adrenal masses without clinically apparent hormonal abnormalities. Such tumours have been termed "incidentalomas". The diagnostic work-up in patients with adrenal incidentalomas is aimed at the determination of hormonal activity of the tumour and identification of patients with potentially malignant tumours. The aim of our study was a retrospective analysis of selected clinical characteristics and hormonal studies in accidentally discovered adrenal tumours.

METHODS

Fourty hundred sixty-three patients with serendipitously discovered adrenal masses, diagnosed and treated in the Department of Endocrinology and Internal Diseases, Medical University of Gdansk as well as in the affiliated Endocrinology Clinic between 1993 and October of 2009 were included in the analysis. Out of all patients, 245 were referred for adrenalectomy.

RESULTS

We found that clinically "silent" tumours often demonstrate subclinical hormonal activity. In our report, increased 24-h urinary excretion of cortisol correlated positively with tumour size (p < 0.001). Moreover, a statistical relationship was demonstrated between tumour size and serum cortisol concentration assessed in the 1 mg dexamethasone suppression test (p < 0.001). Increased values of dehydroepiandrosterone/dehydroepiandrosterone sulphate were more often found in malignant than in benign tumours (p < 0.01). Urinary concentrations of <em>17</em>-<em>ketosteroids</em> correlate positively with diagnosis of adrenocortical cancer (p = 0.02).

CONCLUSIONS

We found that clinically "silent" tumours often demonstrate subclinical hormonal activity (subclinical Cushing syndrome, subclinical pheochromocytoma, low-symptomatic adrenocortical cancer).

The <em>17</em>beta-hydroxysteroid dehydrogenases (<em>17</em>betaHSD) gene family comprises different enzymes involved in the biosynthesis of active steroid hormones. The <em>17</em>betaHSD type 3 (<em>17</em>betaHSD3) isoenzyme catalyzes the reductive conversion of the inactive C19-steroid, Delta4-androstenedione (Delta4- A), into the biologically active androgen, testosterone (T), in the Leydig cells of the testis. It is encoded by the <em>17</em>beta-hydroxysteroid dehydrogenase type 3 (HSD<em>17</em>B3) gene, which maps to chromosome 9q22. Mutations in the HSD<em>17</em>B3 gene are associated with a rare form of 46,XY disorder of sex development referred to as <em>17</em>betaHSD3 deficiency (or as <em>17</em>-<em>ketosteroid</em> reductase deficiency), due to impaired testicular conversion of Delta4-A into T. 46,XY patients with <em>17</em>betaHSD3 deficiency are usually classified as female at birth, raised as such, but develop secondary male features at puberty. Diagnosis, and consequently early treatment, is difficult because clinical signs from birth until puberty may be mild or absent. Biochemical diagnosis of <em>17</em>betaHSD3 deficiency requires measurement of serum T/Delta4-A ratio after hCG stimulation test in pre-pubertal subjects, while baseline values seem to be informative in early infancy and adolescence. However, low basal T/Delta4-A ratio is not specific for <em>17</em>betaHSD3 deficiency, being sometimes also found in patients with other defects in T synthesis or with Leydig cells hypoplasia. Mutational analysis of the <em>17</em>HSDB3 gene is useful in confirming the clinical diagnosis of <em>17</em>betaHSD3 deficiency. This review describes clinical findings, diagnosis, and molecular basis of this rare disease.

The effects of garlic supplementation on protein metabolism were investigated by measuring testis testosterone and plasma corticosterone in rats fed diets with different protein levels. In Experiment 1, rats were fed experimental diets with different protein levels (40, 25 or 10 g/100 g casein) with or without 0.8 g/100 g garlic powder. After 28 d of feeding, testosterone contents in the testis were significantly higher and plasma corticosterone concentrations were significantly lower in rats fed 40 and 25% casein diets with garlic powder than in those fed the same diets without garlic powder. Urinary excretion of <em>17</em>-<em>ketosteroid</em> (an index of testosterone), nitrogen balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with garlic powder than in the 40% casein controls. In Experiment 2, the effect of diallyldisulfide (a major volatile sulfur-containing compound in garlic) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates testosterone production in the testis, was investigated in anesthetized rats. Plasma LH concentration increased dose dependently after administration of diallyldisulfide (P < 0.01, r = 0.558). These results suggest that dietary supplementation with 0.8 g/100 g garlic alters hormones associated with protein anabolism by increasing testicular testosterone and decreasing plasma corticosterone in rats fed a high protein diet.