Altered glucocorticoid metabolism in girls with central obesity.

BACKGROUND: Dysregulation of glucocorticoid metabolism is known to be a causative factor of obesity. However, only a few studies have evaluated the enzymatic activities involved in glucocorticoid metabolism in the pediatric population. OBJECTIVE: To examine whether circulating glucocorticoid metabolites and their ratios reflecting the activities of metabolic enzyme are associated with obesity and body composition in girls. METHODS: A total of 227 girls aged 7-13 years (131 control, 45 overweight, 51 obese) were enrolled in this study. Serum concentrations of glucocorticoids (11-deoxycortisol, cortisol, tetrahydrocortisol [THF], allo-THF, allo-dihydrocortisol [allo-DHF], and cortisone) were evaluated by gas chromatography-mass spectrometry. Enzyme activities corresponding to the ratios of cortisol and cortisone to their respective precursors and metabolites were also assessed. RESULTS: Serum levels of allo-THF were significantly higher in obese girls compared with those in overweight and control girls (P = 0.018); however, concentrations of other cortisol metabolites were not significantly different between the groups studied. According to the severity of obesity, increasing trends in the metabolic ratios reflecting the activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) [(cortisol + allo-DHF + allo-THF + THF)/cortisone], relative 5α/5ß-reductase [allo-THF/THF] activity, and 3α-HSD [allo-THF/allo-DHF] activity, were noted (P-for-trend <0.05). Body fat percentage and waist-to-height ratio positively correlated with the activities of 11ß-HSD1 and 3α-HSD (P < 0.05). Following covariate control, girls with central obesity demonstrated significantly higher metabolic ratios reflecting 11ß-HSD1, relative 5α/5ß-reductase, and 3α-HSD activities (P < 0.05). CONCLUSIONS: We found an altered glucocorticoid metabolism suggesting increased production of cortisol by 11ß-HSD1 and increased metabolic clearance of cortisol catalyzed by 3α-HSD in girls with central obesity.

Clinical evaluation of non-surgical sterilization of male cats with single intra-testicular injection of calcium chloride.

BACKGROUND: Calcium chloride solution is an established injectable sterilant in dogs and other mammals. With cat populations a continuing problem, we sought to explore its first use in cats. Six cats per group were injected with 5%, 10% or 20% calcium chloride dihydrate in saline solution with lignocaine hydrochloride, a local anaesthetic. RESULTS: At the 60th day post-injection, cat testes were collected and showed complete testicular necrosis and replacement by fibrous tissue; very low sperm counts; and reduction of serum testosterone by at least 70% in 20% dose. Androgenic enzyme activities and their expressions were also reduced in all the treated groups along with intra-testicular testosterone concentration was also low. Increased testicular lipid peroxidation, with reduced antioxidants and mitochondrial membrane potential, were evident following calcium chloride treatments. However, there were no apparent changes in serum concentrations of cortisol, fasting blood sugar level, blood urea nitrogen, packed cell volume, or total serum protein following calcium chloride injection, suggesting that this method of sterilization is not associated with any general stress response. CONCLUSION: Calcium chloride solution demonstrates potential for androgenesis-eliminating nonsurgical sterilization of male cats in addition to its proven efficacy in dogs and other mammals.

New Insight in Loss of Gut Barrier during Major Non-Abdominal Surgery.

BACKGROUND: Gut barrier loss has been implicated as a critical event in the occurrence of postoperative complications. We aimed to study the development of gut barrier loss in patients undergoing major non-abdominal surgery. METHODOLOGY/PRINCIPAL FINDINGS: Twenty consecutive children undergoing spinal fusion surgery were included. This kind of surgery is characterized by long operation time, significant blood loss, prolonged systemic hypotension, without directly leading to compromise of the intestines by intestinal manipulation or use of extracorporeal circulation. Blood was collected preoperatively, every two hours during surgery and 2, 4, 15 and 24 hours postoperatively. Gut mucosal barrier was assessed by plasma markers for enterocyte damage (I-FABP, I-BABP) and urinary presence of tight junction protein claudin-3. Intestinal mucosal perfusion was measured by gastric tonometry (P(r)CO2, P(r-a)CO2-gap). Plasma concentration of I-FABP, I-BABP and urinary expression of claudin-3 increased rapidly and significantly after the onset of surgery in most children. Postoperatively, all markers decreased promptly towards baseline values together with normalisation of MAP. Plasma levels of I-FABP, I-BABP were significantly negatively correlated with MAP at (1/2) hour before blood sampling (-0.726 (p<0.001), -0.483 (P<0.001), respectively). Furthermore, circulating I-FABP correlated with gastric mucosal P(r)CO2, P(r-a)CO2-gap measured at the same time points (0.553 (p = 0.040), 0.585 (p = 0.028), respectively). CONCLUSIONS/SIGNIFICANCE: This study shows the development of gut barrier loss in children undergoing major non-abdominal surgery, which is related to preceding hypotension and mesenterial hypoperfusion. These data shed new light on the potential role of peroperative circulatory perturbation and intestinal barrier loss.

Increased collecting duct urea transporter expression in Dahl salt-sensitive rats.

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11beta-HSD2 abundance in DS rats. Consistent with the view that an elevated 11beta-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11beta-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

Effect of intensive exercise-induced testicular gametogenic and steroidogenic disorders in mature male Wistar strain rats: a correlative approach to oxidative stress.

AIMS: In order to investigate the effects of intensive exercise on reproductive dysfunctions in relation to oxidative stress, a total of 12 male rats (age: 3 months, weight: 127 +/- 2.86 g) were randomly divided into: (1) control group (CG, n = 6) and (2) experimental group (Exp. G, n = 6). METHODS: An exercise protocol of 3 h swimming day(-1), 5 days week(-1) was followed for 4 weeks in Exp. G, with no exercise in CG. All the animals were killed; blood, testes and the accessory sex organs were collected for estimation of different parameters. RESULTS: A significant diminution (P < 0.001) was noted in testicular Delta5, 3beta-hydroxy-steroid dehydrogenase (Delta5, 3beta-HSD), 17beta-hydroxy steroid dehydrogenase (17beta-HSD); plasma levels of testosterone, luteinizing hormone (LH); preleptotine spermatocytes (pLSc), midpachytene spermatocytes (mPSc) and stage 7 spermatids (7Sd); with no significant alteration in follicle stimulating hormone (FSH) and spermatogoia A (Asg) after intensive exercise. A significant elevation (P < 0.001) in malondialdehyde (MDA) and conjugated dienes (CD) along with significant reduction (P < 0.001) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) and peroxidase were found in testes of Exp. G. Moreover, the somatic index of testes and accessory sex organs were also decreased significantly (P < 0.001) after exercise. High correlations have been found in 17 beta-HSD with CAT (r = 0.90, P < 0.05) and peroxidase (r = 0.83, P < 0.05), epididymal somatic index with CD (r = -0.91; P < 0.05) and GSH (r = 0.84, P < 0.05). CONCLUSION: The present study focused an chronic intensive exercise-induced oxidative stress that may cause dysfunctions in male reproductive system including steroidogenesis and spermatogenesis.

Sexually dimorphic effects of maternal alcohol intake and adrenalectomy on left ventricular hypertrophy in rat offspring.

In humans, low birth weight and increased placental weight can be associated with cardiovascular disease in adulthood. Low birth weight and increased placental size are known to occur after fetal alcohol exposure or prenatal glucocorticoid administration. Thus the effects of removing the alcohol-induced increase in maternal corticosterone by maternal adrenalectomy on predictors of cardiovascular disease in adulthood were examined in rats. Alcohol exposure of dams during the last 2 wk of gestation resulted in significantly decreased fetal weight and increased placental weight on gestational day 21. Adult female, but not male, offspring of alcohol-consuming mothers exhibited left ventricular hypertrophy. Placental 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD-2) mRNA levels, measured by Northern blot, were decreased in females but not males. Adrenalectomy of alcohol-consuming dams reversed the increase in placental weight and the decrease in female placental 11beta-HSD-2 expression and eliminated the left ventricular hypertrophy of adult female offspring. These data suggest that alcohol-induced changes in placental 11beta-HSD-2 mRNA levels and left ventricular weight are coupled in female offspring only and depend on maternal adrenal status.

Increased diurnal plasma concentrations of cortisone in depressed patients.

The enzyme 11-beta-hydroxysteroid dehydrogenase (11-beta-HSD) regulates glucocorticoid activity by converting cortisol into cortisone and vice versa. Frequent signs of major depression are elevated concentrations of circulating cortisol and ACTH. However, no information is available about the activity of 11-beta-HSD in this disorder. Therefore, we compared diurnal plasma concentrations of cortisol and cortisone and their ratios, reflecting 11-beta-HSD activity, in 25 severely depressed patients (Hamilton Depression Scale, 29 +/- 6; 14 men, 11 women, age 22-77 yr; mean, 47 +/- 16) and 30 control persons (20 men, 10 women age 23-85 yr; mean, 51 +/- 19). Cortisol and cortisone were measured at 0900 h, 1100 h, 1300 h, 2000 h, 2200 h, 0100 h, 0300 h, and 0700 h with specific RIAs after extraction. Both cortisol and cortisone concentrations were significantly increased in patients compared with controls (cortisol, 251.7 +/- 113.1 vs. 160 +/- 96.6 nmol/L; cortisone, 32.8 +/- 10.9 vs. 21.9 +/- 10.9 nmol/L). The calculated ratios of cortisol to cortisone were similar in controls and patients. Similar to cortisol, the circadian variation of cortisone was flattened in patients with the ratio of maximal cortisone to minimal cortisone being 1.9-fold higher in controls than in patients. There was no gender-specific difference in cortisone values neither in patients nor in controls. We conclude that in major depression increased cortisol is not due, at least partly, to an altered 11-beta-HSD activity or to a decrease in cortisone.

Effects of cholic acid on blood pressure and production of vascular aldosterone and corticosterone.

The aims of this study were to search for the role of cholic acid in the regulation blood pressure of humans and rats and to investigate the effects of cholic acid on the production of vascular aldosterone and corticosterone in rats. Levels of serum total bile acids were measured by an enzymic spectrophotometeric method in normal controls, patients with essential hypertension, and in Wistar and spontaneously hypertensive rats. Levels in essential hypertension (7.3+/-3.4 micromol/l, n = 88) were higher than those of normal subjects (4.9+/-3.3 micromol/l, n = 86), and levels in SHR (13.9+/-3.8 micromol/l, n = 11) were slightly increased, but not significantly different from Wistar rats (10.4+/-5.1 micromol/l, n = 12). Male Wistar rats received cholic acid 80 mg/kg/day, orally, for 30 days, and blood pressure was monitored by a pressure transducer. Systolic blood pressure increased in Wistar rats treated with cholic acid compared to control rats. Mesenteric artery perfusion ex vivo was performed, and pressor responses to norepinephrine were determined in Wistar rats. The pressor responses to norepinephrine in mesenteric arteries treated with cholic acid were significantly increased. The perfusate from the mesenteric arteries was collected and applied to a Sep-Pak C 18 cartridge column for reverse phase high performance liquid chromatography, and levels of both aldosterone and corticosterone were determined by radioimmunoassay. Levels of aldosterone were decreased but those of corticosterone increased in the perfusate from arteries treated with cholic acid. Reverse transcriptase polymerase chain reaction showed that cholic acid inhibited the expression of 11beta-HSD2 and CYP11B2 mRNA in mesenteric arteries. These results reveal that cholic acid is able to induce hypertension and provide evidence that cholic acid inhibits the transcription of both 11beta-HSD2 and CYP11B2 in vasculature, leading to lower aldosterone and higher corticosterone production in vessels and increased vasoconstrictor responses to norepinephrine.

In vivo evidence that human adrenal glands possess 11 beta-hydroxysteroid dehydrogenase activity.

Blood samples were collected intraoperatorily from inferior vena cava (VC) and adrenal vein (AV) of 8 male and 9 female consenting adult patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for kidney cancer, and steroid-hormone concentrations were assayed by quantitative HPLC. Hormonal concentrations were significantly higher in AV than in VC (systemic) blood and did not display significant differences between males and females. Higher levels not only of the main glucocorticoids cortisol and corticosterone, but also of their inactive oxidized forms corticosterone and 11-dehydrocorticosterone (DH-B), respectively, were detected in AV than in VC blood. Highly significant inverse correlations between cortisol and cortisone, and corticosterone and DH-B concentrations were observed in AV, but not in VC blood. Moreover, in AV blood the concentration of the main cortisol precursor 11-deoxycortisol correlated inversely with those of both cortisone and DH-B. Taken together, these findings are in keeping with previous in vitro evidence that human adrenal glands possess 11beta-hydroxysteroid dehydrogenase activity, which is engaged in the inactivation of newly formed glucocorticoids and is probably negatively regulated by the local concentrations of non-11beta-hydroxylated steroid-hormone precursors.

[Defects of adrenal steroidogenesis in patients with hirsutism]. / Defectos de la esteroidogénesis adrenal en pacientes con hirsutismo.

BACKGROUND: To determine the frequency and the type of adrenal steroidogenic abnormalities in hirsute women. SUBJECTS AND METHODS: ACTH test was performed during follicular phase in 127 hirsute and 40 normal (control) women. Before ACTH injection we measured in serum by RIA: 17-OH-pregnenolone (17-OH-P5), 17-OH-progesterone (17-OH-P4), androstenedione (AN), cortisol (CT), 11-deoxycortisol (DCT), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), total (TT) and free (FT) testosterone, oestradiol (E2), progesterone (PR), androstenediol glucuronide (AG), LH, FSH and prolactin. After 60 min of ACTH injection 17-OH-P5, 17-OH-P4, AN, DHEA, CT and DCT were measured. Net increment of stimulated steroids and the ratios 17-OH-P5/17-OH-P4, DHEA/AN, 17-OH-P4/CT, 17-OH-P5/CT and DCT/CT were calculated. Pelvic ultrasonographic exploration was done when irregular menses were reported. RESULTS: Up to 31% of the patients presented enzymatic defects in adrenal steroidogenesis. Diagnostic criteria for enzyme defects were established. Late-onset 21-hydroxylase deficiency was diagnosed in 6 (4.5%) patients, HLA typing of these patients demonstrated that 4 out of 6 had B14-DR1. Sixteen women (12.6%) displayed a 17-OH-P4 response and the net increment 2 SD above the normal mean concentration, which are diagnostic criteria for late-onset 21-hydroxylase deficiency carriers. We diagnosed a 3 beta-hydroxysteroid dehydrogenase defect when 17-OH-P5 and DHEA responses, their net increment and the 17-OH-P5/17-OH-P4 and 17-OH-P5/CT ratios were 2 SD above the normal mean after ACTH: 14 women were diagnosed. 11 beta-hydroxylase deficiency diagnosis was made when DCT response, its net increment and the DCT/CT ratio after ACTH were 2 SD above the normal mean: 7 women were detected. Associated biosynthetic defects were described. CONCLUSIONS: One third of our patients with hirsutism presented anomalous response to ACTH, consistent with enzymatic abnormalities in adrenal steroidogenesis.

11 beta-Hydroxysteroid dehydrogenase deficit: a rare cause of arterial Hypertension. Diagnosis and therapeutic approach in two young brothers.

We report the clinical history and results of endocrine investigations in two brothers born to consanguineous parents, who presented with hypokalemia and arterial hypertension when they were aged 2 and 6 years. The hormonal serum assay results, including extremely low values for aldosterone and plasma renin activity, favored the existence of apparent mineralocorticoid excess. A diagnosis of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) deficiency was made, based on assays of the hydrogenated urinary metabolites of cortisol and cortisone, as well as of corticosterone and dehydrocorticosterone. Indeed we found a very low rate of urinary elimination of cortisone metabolites: tetrahydrogenated cortisone was reduced to between 0.10 and 30 mumol/24 h, which is 15-100 times lower than the normal rate; hexahydrogenated cortolones alpha and beta were found to be 7- to 20-fold lower than normal levels; and the 11-keto-17-ketosteroid derivatives of cortisone were also reduced. Urinary elimination of the cortisol-reduced metabolites 5 beta- and 5 alpha-tetrahydrogenated cortisol were slightly reduced or normal. These results argue in favor of a deficit in the enzyme 11 beta-HSD, which oxidizes cortisol into cortisone. A moderate defect in the conversion of cortisol into 5 beta-THF compared to normal conversion into 5 alpha-THF was also found. With respect to corticosterone metabolism, we demonstrated the presence of a defect in the oxidation of that steroid into dehydrocorticosterone, also due to the deficit in 11 beta-HSD. Arterial hypertension and hypokalemia were corrected by treatment with dexamethasone, concomitantly with correction of the low aldosterone and plasma renin activity levels. On the other hand, during this treatment, urinary concentrations of the metabolites of cortisol, cortisone and corticosterone were only moderately affected.

11 beta-Hydroxysteroid dehydrogenase type I enzyme in the hearts of normotensive and spontaneously hypertensive rats.

1. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to their inactive 11-keto metabolites. The ubiquitous expression of the NADP-dependent isoform (11 beta HSD1) suggest an important role in modulating glucocorticoid action, but little is known about 11 beta HSD1 gene expression and enzymatic activity in the rat heart. 2. In the present study rat cardiac 11 beta HSD1 activity and ontogeny of gene expression have been characterized. The addition of NADP, but not NAD, to heart homogenates resulted in significant increases in the metabolism of both corticosterone and cortisol, with the former substrate displaying far greater metabolism. Both 11 beta HSD1 gene expression and enzyme activity increased in parallel from low levels at 1 week of age to maximal levels at 8 weeks, with no further change by 16 weeks of age. 3. We also compared the activity of 11 beta HSD1 in the hearts of male and female spontaneously hypertensive rats (SHR) with normotensive Wistar-Kyoto (WKY) controls. Enzyme activity in the pooled atria of female SHR was significantly higher than in male SHR atria (7.6 +/- 0.6% conversion of corticosterone vs 4.5 +/- 0.5%; P < 0.05). The left ventricles of female WKY rats contained significantly less 11 beta HSD activity than either male WKY rats or female SHR (8.6 +/- 0.8% conversion vs 17 +/- 1.4 and 13.6 +/- 0.5%, respectively; P < 0.05). In the right ventricle, female WKY rats also had significantly less enzyme activity than either female SHR or male WKY rats (4.9 +/- 0.7 vs 10.0 +/- 1.7 and 10.2 +/- 1.4%; P < 0.05). 4. These results clearly show that the rat heart contains significant amounts of the 11 beta HSD1 enzyme and that this activity is sexually dimorphic. Furthermore, significant differences were observed between a normotensive and hypertensive strain of rat. The relevance of these observations to the aetiology and maintenance of hypertension remains to be explored.

11 beta-Hydroxysteroid dehydrogenase and its inhibitors in hypertensive pregnancy.

Preeclampsia is accompanied by amplification of the sodium retention that is a feature of normal pregnancy. Recent evidence suggests that mineralocorticoid receptor activation is increased in preeclampsia, but classic mineralocorticoids (aldosterone, 11-deoxycorticosterone) are not present in excess. Cortisol can act as a mineralocorticoid receptor agonist only when its renal inactivation to cortisone by 11 beta-hydroxy-steroid dehydrogenase is impaired, for example, in congenital enzyme deficiency and after administration of exogenous inhibitors (eg, licorice). Endogenous inhibitors of this enzyme have been detected in human urine and are increased in pregnancy. To establish whether cortisol causes mineralocorticoid excess in hypertensive pregnancy and whether endogenous inhibitors of 11 beta-hydroxysteroid dehydrogenase are responsible, we studied 25 hypertensive pregnant patients (13 with preeclampsia and 12 with gestational hypertension), 16 normotensive pregnant subjects, and 13 nonpregnant control subjects. Concentrations of plasma renin and aldosterone were increased in pregnancy, but less so in hypertensive pregnancy. Plasma potassium and urinary electrolytes were not different between the groups. Plasma cortisol was increased in pregnancy but not different in hypertensive pregnancy, and urinary cortisol, plasma and urinary cortisone, and urinary tetrahydrocortisol and tetrahydrocortisone were not different between the groups. Endogenous inhibitors of 11 beta-hydroxysteroid dehydrogenase were more active in urine from pregnant women but were not increased further in hypertensive pregnancy. There were no differences in these parameters between patients with preeclampsia and gestational hypertension. We conclude that deficient inactivation of cortisol to cortisone does not contribute to the sodium retention of normotensive or hypertensive pregnancy and that endogenous inhibitors of 11 beta-hydroxysteroid dehydrogenase have no evident pathophysiological significance in pregnancy.

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone.

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood.

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.

Dual activity at an enzyme active site: 3 beta,20 alpha-hydroxysteroid oxidoreductase from fetal blood.

An enzyme exhibiting both 3 beta and 20 alpha steroid reductase activities from calf fetal red blood cells was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3 beta,20 alpha-Hydroxysteroid oxidoreductase (3 beta,20 alpha-HSD) was found to be a single-stranded polypeptide with a molecular weight of 55 000 +/- 1 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-100 chromatography. The amino acid composition of 3 beta,20 alpha-HSD was obtained. 17 beta-Hydroxy-5 alpha-androstan-3-one and progesterone were substrates for the enzyme's 3 beta and 20 alpha reductase activities, respectively, which required NADPH for both 3 beta [Km = 9.4 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] and 20 alpha [Km = 2.5 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] reductase activities. 17 beta-Hydroxy-5 alpha-androstan-3-one competitively inhibited (Ki = 35 microM) 20 alpha reduction of progesterone. Incubating 3 beta,20 alpha-HSD with 19-nortestosterone 17-bromoacetate at pH 7.0 and 25 degrees C caused simultaneous, time-dependent, and irreversible losses of 3 beta and 20 alpha activities by a first-order kinetic process. Similar incubations with either of the 3 beta or 20 alpha substrates present at concentrations equal to their respective Km values practically doubled the time required for loss of 3 beta and 20 alpha enzyme activities. These data lead us to conclude that the active site of 3 beta,20 alpha-HSD contains 3 beta and 20 alpha dual activity.

20 alpha-Hydroxysteroid oxidoreductase activity and 20 alpha-dihydroprogesterone concentration in human placenta before and after parturition.

The objective of this investigation was to determine if decreased biosynthesis or increased catabolism of progesterone (P) during labor was responsible for the decreased concentration of hormone observed in the human placenta after labor and vaginal delivery. No significant difference was found in P biosynthesis by placental tissues examined before and after labor as evidenced by a similar activity of 3 beta-hydroxysteroid dehydrogenase: delta 5-isomerase. In contrast, there was a marked increase in P catabolism during labor, as shown by a significant (p less than 0.05) change in placental 20 alpha-hydroxysteroid oxidoreductase activity which increased from 835.5 +/- 103 (mean +/- SEM) pmoles of 20 alpha-dihydroprogesterone (20 alpha-DHP) generated per milligram of protein per hour before labor to 1,160.4 +/- 101 pmoles/mg/hr after labor. A similar significant (p less than 0.02) increase in enzyme activity was noticed in parallel assays of the reverse (20 alpha-DHP leads to P) reaction. As a consequence of increased P catabolism, placentas after labor had a 20 alpha-DHP concentration of 63.7 +/- 9.5 (mean +/- SEM) ng/mg protein, a value which was significantly (p less than 0.05) larger than that found before labor (37.8 +/- 8.3 ng/mg protein). These changes resulted in a modification of the placental tissue P/20 alpha-DHP ratio which decreased from 2/1 before to 1/1 after labor. The results indicate that the catabolism of P to 20 alpha-DHP increases significantly during human parturition. This phenomenon may be of importance in the mechanism of initiation and continuation of labor.