According to published data the group of urinary total <em>17</em>-<em>ketosteroid</em> sulfates appears to represent an index of overall adrenal androgen production, at least before the onset of puberty. To quantify total <em>17</em>-<em>ketosteroid</em> sulfates a modified colorimetric assay based on the Zimmermann reaction was validated. <em>17</em>-<em>ketosteroid</em> sulfates were measured without previous hydrolysis (as conjugated Zimmermann chromogens against authentic dehydroepiandrosterone sulfate (DHEAS) as assay standard) after C18 reversed-phase extraction and LH-20 chromatography. Intra- and inter-assay coefficients of variation were 8.4% (15.0%) and 5.9% (<em>17</em>.6%), respectively, at urinary <em>17</em>-<em>ketosteroid</em> sulfate concentrations of 10.8 (1.9) nmol/ml. Recoveries observed in spiking and parallelism experiments varied between 88 and 102%. In a group of 4-year-old children showing a renal DHEAS output of less than 0.1 mumol/d/1.73 m2 (measured by radioimmunoassay) a relatively high median <em>17</em>-<em>ketosteroid</em> sulfate excretion of 1.29 mumol/d/1.73 m2 was found. Older children aged 8 years as well as a group aged 12-14 years demonstrated only moderately higher urinary <em>17</em>-<em>ketosteroid</em> sulfates whereas excretion of DHEAS/d/1.73 m2 more than tripled from age group to age group. For children from 8 years onwards, adolescents, and adults, linear regression analysis indicated that urinary DHEAS elevations seem to contribute with a constant proportion of approximately 70% to the increments of total urinary <em>17</em>-<em>ketosteroid</em> sulfates. These findings suggest that the attainment of such a constant relationship (between the total <em>17</em>-<em>ketosteroid</em> sulfates and their major component) from about 8 years of age onwards could represent the hormonal correlate of the completion of the continuous zona reticularis in the adrenal gland (developing around this age from a focal reticularis zone).

In a series of 146 school girls and nurses in the serum levels of growth hormone (GH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) were determined in the afternoon after 4-5 hours' fast. Blood specimens of the menstruating girls were taken on the 7th to 10th day from the beginning of the preceding menstrual bleeding. From the 24-hour urines collected on the previous day the excretion of total oestrogens, <em>17</em>-<em>ketosteroids</em> and <em>17</em>-hydrocorticosteroids was determined. The series was grouped according to skeletal age which varied from 8.0 years to the adult stage. In the girls aged 11-13 years the GH curve showed a significant rise parallel with the mean height curve. The FSH values at the early age of 8-9 years corresponded to the follicular phase of adult women. The LH level increased syteeply up to the 15th year of bone age and mean values of adults were found to be about three times those of childhood. The excretion of total oestrogens and <em>17</em>-<em>ketosteroids</em> increased steadily up to the full maturity of skeletal age. In the oldest group of girls the excretion was about four times that of the group aged 8-9 years. The hormonal maturation seems to continue until late puberty. The <em>17</em>-OHCS, on the other hand, reached the adult level in the group with a bone age of 13 years. The excretion of <em>17</em>-OHCS shows a steep rise from the age of 11 to 12 years and this acceleration of excretion perhaps exceeds the increase in surface area.

In the rhesus monkey and ovine fetus in utero, aldosterone concentrations do not rise in response to surgical stress, ACTH, or angiotensin-II, all of which are secretagogues for this mineralocorticoid in the adult. To assess the mechanism of this phenomenon in the human fetus, metabolism of pregnenolone and corticosterone by second trimester human fetal adrenal definitive zone and fetal zone tissue was studied. After incubation of fresh tissue with trace amounts of [3H]pregnenolone or [3H]corticosterone, the products of metabolism were separated using high performance liquid chromatography and quantified. The delta 5-3 beta-hydroxysteroids <em>17</em>-hydroxypregnenolone and dehydroepiandrosterone and their sulfates comprised 85-90% of metabolized pregnenolone. In the fetal zone, cortisol was the predominant secreted delta 4-3-<em>ketosteroid</em>, accounting for 6-8% of the metabolized pregnenolone. In the definitive zone, progesterone and corticosterone were the predominant secreted delta 4-3-<em>ketosteroids</em>, each accounting for about 2% of the metabolized pregnenolone. 11-Dehydrocorticosterone and sulfates were the only metabolites detected after incubation of fetal adrenal tissue with corticosterone. 11-Dehydrocorticosterone accounted for more than 80% of the metabolized corticosterone in the definitive zone and 50% in the fetal zone. Incubations with secretagogues or antioxidants (10 nmol/L ACTH, 10 nmol/L angiotensin-II, 21 mmol/L potassium, 100 mmol/L dimethylsulfoxide, 5 mumol/L metyrapone, or 100 mumol/L butylated hydroxyanisole) did not change the pattern or extent of precursor metabolism. No aldosterone, 18-hydroxycorticosterone, or 18-hydroxydeoxycorticosterone was detected in baseline or stimulated incubations of human fetal tissue. In contrast, adult human zona glomerulosa metabolized corticosterone to aldosterone, 18-hydroxycorticosterone, and 11-dehydrocorticosterone under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

A 58-yr-old man presented with gynecomastia and elevated serum estrogens. The diagnosis of an estrogen-secreting adrenal tumor was made based upon the finding of a 4-cm left adrenal mass, elevated levels of estradiol in peripheral and left adrenal venous blood, and increased urinary <em>17</em>-<em>ketosteroids</em>. In addition to marked elevations in estradiol and <em>17</em>-<em>ketosteroids</em> there was an increased baseline level of 11-deoxycorticosterone and a slightly decreased level of 18-hydroxycorticosterone, suggesting the possibility of impaired P450c11 activity. The effect of ketoconazole administration (600 mg/day) for 4 weeks was studied. Urinary free cortisol and <em>17</em>-<em>ketosteroid</em> excretion and serum testosterone levels fell acutely (1 week). Serum estradiol levels decreased gradually over the 4-week course. Plasma aldosterone levels were essentially unaltered and 18-hydroxcorticosterone levels fell gradually, but there were marked increases in 11-deoxycorticosterone and corticosterone. Coincident with the increase in 11-deoxycorticosterone there was an increase in blood pressure and a transient fall in serum potassium. We conclude that ketoconazole administration may result in a hypermineralocorticoid state. Therefore, the usefulness of ketoconazole therapy for steroid hormone-producing neoplasms will depend upon the individual tumor's steroidogenic profile.

Ketoconazole (K) is an antifungal imidazole derivative which has been shown to be a potent inhibitor of testosterone (T) biosynthesis in rodents and humans. To study the effect of K on rat testicular steroidogenesis we measured the activities of five testicular microsomal steroidogenic enzymes in K-treated rats and controls. Thirty male adult rats were given either 2 mg K or water every 12 hours by mouth during 5 days. Mean testicular weight was similar in both groups of animals. The K-treated group had a T serum concentration of 83 +/- 14 ng/dL whereas it was 94 +/- 16 ng/dL in the control group (NS). The K-treated animals had decreased activities of the 3 beta-hydroxysteroid dehydrogenase (830 +/- 48 vs 2,245 +/- 109 pmol/mg protein/min, P less than 0.001), <em>17</em>-hydroxylase (243 +/- 5 vs 676 +/- <em>17</em> pmol/mg protein/min, P less than 0.001), <em>17</em>-<em>ketosteroid</em> reductase (31 +/- 2 vs 169 +/- 7 pmol/mg protein/min, P less than 0.001), and aromatase enzymes (92 +/- 6 vs 123 +/- 7 pmol/mg protein/min, P less than 0.01). The <em>17</em>,20-desmolase activity was similar in both groups of animals (210 +/- 4 vs <em>17</em>1 +/- 18 pmol/mg protein/min). We conclude that K given orally to rats inhibits the activity of several testicular steroidogenic enzymes.

The deficiency of ovarian <em>17</em>-<em>ketosteroid</em> reductase (<em>17</em>-KSR) was recently discovered to be a possible cause of polycystic ovarian disease (PCOD) in hirsute women. Forty three patients with PCOD (age range, 18-38 yr) were reevaluated to search for a hormonal pattern that might suggest an ovarian <em>17</em>-KSR deficiency. Androstenedione, testosterone, FSH, LH, <em>17</em>-hydroxyprogesterone, and dehydroepiandrosterone sulfate were evaluated basally on the day <em>17</em> of the menstrual cycle, when present, and after dynamic tests (ACTH stimulation, 1 mg im for 2 consecutive days; dexamethasone inhibition, 0.5 mg four times a day for 14 days; and cyproterone acetate treatment, 50 mg each day for 14 days) in three successive menstrual cycles or at 30-day intervals. All patients studied presented with hyperestronemia, abnormal gonadotropin pattern, and hyperandrogenism, but showed different responses of androstenedione and testosterone to dynamic tests. In two patients the hormonal pattern suggested an ovarian <em>17</em>-KSR deficiency: in fact they showed plasma values of androstenedione (22 and 31.3 nmol/L, respectively) and estrone (628 and 849 pmol/L, respectively) that were greatly increased compared with other patients and with controls. Androstenedione did not increase after ACTH stimulation (21.5 and 32.1 nmol/L, respectively) and did not decrease after dexamethasone inhibition (21 and 29 nmol/L, respectively), but only decreased after cyproterone acetate treatment (8 and 10.8 nmol/L, respectively). An hCG test, performed during dexamethasone suppression, confirmed the diagnosis of ovarian <em>17</em>-KSR defect in one of these two patients (patient 1). Two of three brothers of patient 1 (aged 25 and 34 yr) presented with persistent important pubertal gynecomastia; one brother also had severe oligospermia. These clinical findings and the high values of androstenedione/testosterone (0.85) and estrone/estradiol (4.1) ratios of baseline plasma levels compared with controls (0.18 and 2.1, respectively) suggested a partial testicular <em>17</em>-KSR deficiency. Five other patients showed PCOD secondary to nonclassic 21-hydroxylase defect diagnosed on the basis of high <em>17</em>-hydroxyprogesterone plasma values and highly responsive to ACTH. The remaining 36 patients showed increased values of androstenedione and testosterone after ACTH stimulation and a decrease of these two parameters after both dexamethasone inhibition and cyproterone acetate treatment. The discovery of the <em>17</em>-KSR deficiency in men and women in the same family demonstrates genetic control of this enzyme similar in both sexes, confirming the hypothesis that this disorder is inherited as an autosomal recessive character. Finally, it is strongly supported that ovarian <em>17</em>-KSR defect may cause a syndrome closely resembling PCOD.

A 24 years old male with pseudohermaphroditism due to a deficiency in <em>17</em>-<em>ketosteroid</em> reductase activity is described. Plasma delta 4 is 21 times higher than normal for an adult male, delta 4/T is greater than 6, both E1 and F2 are elevated and E1/E2 = 3. There is very slight modification of delta 4 on administration of ACTH, dexamethasone, hCG and fluoxymesterone. Steroid concentrations in the spermatic veins and arteries confirm the testicular origin of the increased secretion of delta 4 and E1 and show a lower secretion by the cryptorchidic testis. In vitro testicular tissue incubation and fibroblast studies confirm the <em>17</em>-<em>ketosteroid</em> reductase deficiency and rule out any other anomaly as the cause of the ambiguous genitalia. Psychologically the patient seemed to be identified with a female social and sexual role in spite of her advanced degree of virilization.

A 12 year old child (46,XY) with <em>17</em>-<em>ketosteroid</em> reductase deficiency was investigated. The patient, reared as a female, was first noted to have clitoromegaly at 10 years of age. Increased facial hair, deepening of the voice, acne, increased body hair and minimal breast development were noted at 12 years. delta4-Androstenedione (delta4) in peripheral blood was markedly elevated (1913 ng/100 ml) whereas testosterone (T) was in the male range of Tanner III puberty (240 ng/100 ml). Thus, delta4/T in this patient was 9.4, compared to a normal ration of 0.15 to 0.25. T/DHT was normal (10.5). Oestrone (Oe1) level was slightly elevated (6 ng/100 ml, normal: 2.5-4.5 ng/100 ml). Oestradiol (Oe2) was normal (1.7 ng/100 ml, normal: 1.5-3 ng/100 ml. Oe1/Oe2 was slightly elevated (3.6, normal: 1-2). At laparotomy, testes were found and spermatic vein blood was obtained prior to castration. Androgen determinations of spermatic vein blood demonstrated extremely high delta4 levels (283 microgram/100 ml) and low levels of T (16 microgram/100 ml). delta4/T in spermatic vein was <em>17</em>, higher than in the peripheral blood, suggesting intact peripheral conversion of delta4 to T. Incubation of testes slices with delta4 demonstrated severely impaired conversion to T. Conversion of Oe1 to Oe2 was impaired to a lesser degree.

CONCLUSIONS

<em>17</em>-<em>ketosteroid</em> reductase deficiency was documented in vivo by impaired conversion of precursor hormones resulting in higher than normal delta4T and Oe1/Oe2 ratios in blood. In vitro studies with testes slices confirmed the enzymatic defect.

We report a 14 year-old male with severe, long-lasting gynecomastia. Baseline serum androstenedione levels were elevated compared to testosterone levels (330 ng/dl vs 28 ng/dl). In order to evaluate testosterone biosynthesis by this patient in more detail, androstenedione, testosterone, dehydroepiandrosterone (DHEA) and estradiol responses to a single dose of hCG were measured. The responses observed were different from those reported in normal males in two respects: 1) there was no immediate rise in testosterone two to four hours after the injection of hCG, and 2) levels of androstenedione and estradiol at 24, 36 and 48 hours after injection were much higher than expected. We postulate that a partial defect in testicular <em>17</em>-<em>ketosteroid</em> reductase activity was responsible for the abnormal androstenedione to testosterone ratio in our patient. This, in turn, lead to an increased peripheral synthesis of estrogens and marked gynecomastia.

BACKGROUND

It has been reported recently that fluvoxamine (a selective serotonin reuptake inhibitor) is effective and safe for children with monosymptomatic nocturnal enuresis (MNE). However, the exact mechanism by which fluvoxamine is beneficial in the treatment of MNE remains unknown. One possibility is that it controls emotional stress.

METHODS

We divided children with MNE into primary MNE (n = 40) and secondary MNE (n = 7). We measured urinary <em>17</em>-hydroxycorticosteroids (<em>17</em>-OHCS) and <em>17</em>-<em>ketosteroid</em> sulfates (<em>17</em>-KS-S) as a stress barometer in children with MNE to evaluate adaptation to emotional stress before and during fluvoxamine treatment. We initially administered fluvoxamine at a dose of 25 mg at bedtime. If patients remained incontinent after 3 weeks, we increased the dose to 50 mg.

RESULTS

Fluvoxamine was effective in 26 of 28 children (93%) with primary MNE and an abnormality of the stress barometer and in six of six children (100%) with secondary MNE and an abnormality of the stress barometer. Fluvoxamine was effective in only six of 12 children (50%) with primary MNE and normality of the stress barometer and was not effective in one child with secondary MNE and normality of the stress barometer.

CONCLUSIONS

The stress barometer is useful clinically for evaluating the therapeutic effect of fluvoxamine for children with MNE.

Our patient had a left suprarenal mass. His blood pressure was normal, but his urinary catecholamines (CA), vanillylmandelic acid (VMA), total metanephrines (TMn) and 5-hydroxyindolacetic acid (5HIAA) were elevated. In addition, he had elevated, nonsuppressible urinary <em>17</em>-<em>ketosteroids</em> (<em>17</em>KS) and androsterone, but his urinary <em>17</em>-hydroxycorticoids (<em>17</em>OHCS) and free cortisol were normal, as were his plasma cortisol and ACTH. After resection of the suprarenal mass, the patient's urinary hormone values reverted to normal. The mass contained a pheochromocytoma and an adrenocortical adenoma. The pheochromocytoma was unusual in that it contained very little norepinephrine (NE) and dopamine (DA) and an abundance of epinephrine (E) despite normal enzyme concentrations. Electron micrographs showed primarily E granules with few of the NE-type. The immunoperoxidase histochemical stains for vasoactive intestinal peptide (VIP) and serotonin (S) were strongly positive. The patient's blood pressure may have been normal because his pheochromocytoma secreted E, VIP, or S. The associated adrenocortical adenoma produced no symptoms and was probably coincidental.

A delta 5-3-<em>ketosteroid</em> isomerase (EC 5.3.3.1) has been isolated from Pseudomonas putida Biotype B and purified to homogeneity. This previously undescribed steroid isomerase resembles that isolated from Pseudomonas testosteroni (Talalay, P., and Wang, V.S. (1955) Biochim. Biophys. Acta 18, 300-301). The enzyme is induced by various steroids, has a subunit molecular weight of 13,750 +/- 250, a pI of 4.8 +/- 0.1 has a specific activity of 40,000 units/mg, using 5-androstene-3,<em>17</em>-dione as the substrate. The amino acid composition of the enzyme subunit is Lys 2, His 2, Arg 8, Asp 11, Thr 5, Ser 4, Glu <em>17</em>, Pro 8, Gly 12, Ala 12, Val 10, Met 5, Ile 6, Leu 8, Tyr 4, Phe 4, and Cys 4. The amino acid sequence has been determined for the NH2-terminal 50 residues. This portion of the polypeptide chain is approximately 47% homologous with the amino acid sequence of the first 50 residues of the delta 5-3-<em>ketosteroid</em> isomerase from P. testosteroni. The amino acid sequence of residues 33 to 41 of the P. putida isomerase is identical with the region Ala 31 through Pro 39 in the P. testosteroni enzyme. Residue 40 of the P. putida enzyme is aspartic acid and corresponds in the sequence to Asp 38 of the P. testosteroni isomerase, which has been shown to be essential for enzymatic activity (Ogez, J.R., Tivol, W.F., and Benisek, W.F. (1977) J. Biol. Chem. 252, 6151-6155).

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-<em>ketosteroid</em> delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-<em>17</em>beta-hydroxy-4,4,<em>17</em>alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-<em>17</em>-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-<em>ketosteroid</em>.

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-<em>ketosteroids</em> to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-<em>Ketosteroid</em>-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of <em>17</em> nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-<em>Ketosteroid</em>-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.

Dispersed mouse testicular interstitial cells were treated with the transglutaminase inhibitor monodansylcadaverine (500 microM) for 30 min. Subsequent incubation of the cells with [3H]pregnenolone increased formation of steroidogenic intermediates, tentatively identified as progesterone, <em>17</em> alpha-hydroxyprogesterone, and androstenedione, but decreased testosterone formation by monodansylcadaverine-treated cells. Measurement of <em>17</em>-<em>ketosteroid</em> reductase activity (the enzyme that converts androstenedione to testosterone) demonstrated that monodansylcadaverine treatment caused a reversible, noncompetitive inhibition of this enzyme. These results suggest that transglutaminase catalyzed protein cross-links may influence the activity of <em>17</em>-<em>ketosteroid</em> reductase.