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The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

SUMMARY Human skin from forehead, cheek and axilla was incubated in vitro with [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]DHA sulphate, [7α-3H]-androstenedione and [7α-3H]testosterone. The following enzyme activities were detected: 3β-hydroxysteroid dehydrogenase Δ4-5 isomerase, 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 5α-reductase, 5β-reductase, sulphotransferase, sulphatase, steroid hydroxylase. 5α-Reduced steroids were the major metabolites. All four substrates were converted to 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol. In axillary skin, conversion of 17-oxosteroids to 17β-hydroxysteroids was favoured, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol being major metabolites. In facial skin, formation of 17-oxosteroids predominated with little accumulation of 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol. 5α-Androstane-3β,17β-diol was a metabolite of DHA, androstenedione and testosterone but was found in lower amounts than 5α-androstane-3α,17β-diol. Similarly conversions to epiandrosterone were much lower than to androsterone in all the skin specimens. It was concluded that the differences in accumulation of 5α-dihydrotestosterone were determined by the differences in 17β-oxidoreduction rather than differences in 5α-reductase, the activity of which was high in all skin specimens.

SUMMARY The metabolism of [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]-androstenedione and [7α-3H]testosterone was studied in the ventral sebaceous gland patch of the Mongolian gerbil in vitro. The main enzyme activities found were 17β-hydroxysteroid dehydrogenase, 5α-reductase, 17α-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, sulphotransferase and hydroxylase. The active androgen 5α-dihydrotestosterone was formed in appreciable amounts from both testosterone and androstenedione. The 17β-hydroxysteroid dehydrogenase actively formed both 17-oxo and 17β-hydroxysteroids in this tissue. The conversion of DHA to C4–5 unsaturated steroids and 5α-steroids was not observed presumably due to a lack of 3β-hydroxysteroid dehydrogenase Δ4–5isomerase. The metabolism was compared with that in human and rat skin and its significance discussed.

The purified multifunctional enzyme, 3β-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3β-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.

The mechanism of the isomerization of androst-5-ene 3,17-dione by the isomerase of bovine adrenals has been reinvestigated using the methodology previously developed for the study of the bacterial enzyme of Pseudomonas testosteroni. However, owing to the lower activity of the mammalian enzyme, competitive non-enzymic reaction cannot be neglected. It has been shown that even in the absence of spontaneous isomerization, epimerization and exchange of the label on C4 takes place in the buffer. This prevents any quantitative discussion of the course of the reaction. It is however possible to conclude that the mechanism we have proposed for the bacterial enzyme that is, besides the classical 4 beta leads to 6 beta transfer and exchange with the medium, a competitive abstraction of the 4 alpha proton, accounts for the data obtained with the mammalian microsomes.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.

Through the treatment of rat testicular microsomes with sodium cholate, 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3β -hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: 1. With this purification procedure, the 3β-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. 2. For 3-oxo-4-ene-steroid formation from 3β-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3β-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. 3. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. 4. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. 5. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

SUMMARY Shoulder skin from young men with and without acne was incubated in vitro in Krebs Improved Ringer I medium with 7α-3Hdehydroepiandrosterone,7α-3Handrostenedione and 7α-3Htestosterone. There were no marked differences in steroid metabolism between the acne and control groups but skin from acne patients did form rather more 5α-dihydrotestosterone from testosterone than did skin from control subjects. Skin from the shoulder (acne area) of acne and control subjects metabolized dehydroepiandrosterone more rapidly than did skin from the thigh (non-acne area); this appeared to be due to higher activity of 3β-hydroxysteroid dehydrogenase Δ4-5 isomerase in shoulder than thigh skin. A comparison of steroid metabolism in skin incubated in Krebs Ringer medium with that in cryostat sections of skin incubated in a tris-buffered medium showed that the method of incubation could affect the quantitative pattern of steroid metabolism in vitro.

Exposure of induced Pseudomonas testosteroni to osmotic shock resulted in the release of androgen and estrogen-binding proteins, 5-ene-3-ketosteroid isomerase and (3 and 17)β-hydroxysteroid dehydrogenase activities, and variable amounts of 3α-hydroxysteroid dehydrogenase, 1-ene-dehydrogenase and 4-ene-5α-dehydrogenase activities. When binding of C19 and C21 steroids to induced bacteria and to periplasmic proteins was examined, binding correlated with the ability of the steroids to support growth of this organism. Except for estradiol-17β, steroids incapable of supporting growth, such as aldosterone, cortisol, and corticosterone, were not bound. In the case of C19 steroids, the bound form of the steroid was androstanedione and/or androstenedione. On the other hand, progesterone and 17α-hydroxyprogesterone were bound in the form in which they were added. Displacement studies with unlabeled steroids indicated the presence of two steroid receptors, one which bound C19 and C21 steroids, and another which bound estra-diol-17β.

The steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase (3 beta-HSD) that convert delta 5-hydroxysteroids to delta 4-ketosteroids were measured in human follicular tissue collected during the follicular phase of the menstrual cycle. This study was done in order to determine whether 3 beta-HSD enzyme activity fluctuated during the follicular phase of the menstrual cycle and, if so, whether these changes were reflected by the concentration of steroids in the follicular fluid. A microsomal fraction was prepared from each of 14 follicular-phase follicles, and 3 beta-HSD enzyme activity was estimated by the amount of androstenedione synthesized in the presence of excess substrate (dehydroepiandrosterone) and cofactor (nicotinamide adenine dinucleotide). Androstenedione, dehydroepiandrosterone, and progesterone were measured in the aspirated follicular fluid of each follicle. 3 beta-HSD enzyme activity was undetectable in the smallest (3 to 5 mm) follicles, increased in 5 to 6 mm follicles to 363.2 +/- 60 pmol of androstenedione per milligram per hour, and increased still further in 9 to 11 mm follicles to 5,000 +/- 200 pmol of androstenedione per milligram per hour. The increase in 3 beta-HSD enzyme activity was accompanied by an increase in the concentration of androstenedione in the follicular fluid as well as of progesterone in larger follicles. These data indicate that 3 beta-HSD activity increases during the follicular phase of the menstrual cycle. It is suggested that the primary product of the increased enzyme activity is androstenedione. Since androstenedione is the principal C19 steroid produced by the ovulatory follicle and serves as a substrate for estradiol production, this increase in 3 beta-HSD activity may be important for the associated changes in the late follicular phase that lead to ovulation.

Radioactive progesterone, 17α-hydroxypregnenolone, androstenedione and testosterone were incubated with midterm human placenta and fetal liver. After isolation of the radioactive metabolites, purification and identification was achieved by paper and thin-layer chromatography and crystallization to constant specific activity. The analysis of the metabolites formed during incubation revealed the presence in fetal liver and midterm placenta of all enzyme systems necessary for the biosynthesis of estrone, 17β-estradiol and estriol from C21- and C19- steroids. Evidence for the presence of the following enzymes could be found: Δ5-isomerase, 3β- hydroxy dehydrogenase, 16α-hydroxylase, 17α-hydroxylase and C17,2o-desmolase. The in vitro enzymatic activities varied quantitatively. Under the in vitro incubation conditions used, progesterone was not significantly metabolized by placenta or liver. 17α-Hydroxypregnenolone was oxidized and isomerized in high yield to 17α-hydroxyprogesterone by midterm placenta;...

To assess comparative 3β-hydroxysteroid dehydrogenase-isomerase and 17β-steroid dehydrogenase activities in human fetal testis and adrenal, equal weights of adrenal and testicular tissue from the same fetuses were incubated with dehydroepiandrosterone or pregnenolone, under identical conditions. Tissues from five fetuses were investigated. The following products were isolated and quantitated: dehydroepiandrosterone, androstenediol, androstenedione and testosterone. The 3β-hydroxysteroid dehydrogenase-isomerase activity, expressed as pmoles found in androstenedione plus testosterone per mg protein incubated, was 3·5–138 times higher in testicular than in adrenal tissue. 17β-Steroid dehydrogenase activity expressed as pmoles found in testosterone plus androstenediol per mg protein incubated was 73–313 in testicular tissue compared to 0–2.64 in adrenal tissue. When the same activities were expressed in terms of activity per total adrenal and testicular tissue per fetus, the 17β-steroid dehydrogenase activity was still considerably higher in testes than in adrenals, while the 3β-hydroxysteroid dehydrogenase-isomerase activity was higher in the testes of the three smaller fetuses investigated. These data provide additional evidence that the human fetal testis is geared to the formation of testosterone which is essential for genital development in the male fetus.

Testicular glands of 12-month-old male chicken were homogenized and fractionated by differential centrifugation. After subcellular fractions were incubated with several radioactive steroid precursors, the metabolic products were identified on the basis of the mobilities on the thin-layer chromatogram, chemical reactions, and recrystal-lization with authentic preparations to constant specific activities. Enzyme activities related to androgen formation from pregnenolone were found to exist mainly in the microsomal fraction (10,000–105,000g precipitate). Activities of 20α- and 20β-hydroxysteroid dehydrogenases were exclusively in supernatant fluid at 105,000g. Pregnenolone was transformed to testosterone in the microsome fraction via the following two pathways: through progesterone, 17α-hydroxyprogesterone, and androstenedione; and also through 17α-hydroxypregnenolone, dehydroepiandrosterone, and androstenedione, depending on the cofactors. The biosynthetic pathways to testosterone in chicken testes were discussed in comparison with those of other vertebrates.

The small dysgenetic gonads which are induced in mice by neonatal thymeetomy are characterized by predominance of interstitial cells and absence of corpora lutea and follicles. In addition, the animals have stimulated uteri and masculinization of the salivary gland. Since these latter features suggested an abnormal pattern of steroid formation in vitro, ovarian steroid biosynthesis was studied. When 10 nmoles of 3H-3β hydroxy- pregn-S-en-20-one, 14C-progesterone or 14C-17-hydroxyprogesterone was incubated with 5 or 10 mg of ovarian homogenates, androstenedione and testosterone were synthesized by the dysgenetic ovaries, while much less of these C19- steroids was formed by control ovaries of normal mice. Enzyme activities related to steroid conversion were also estimated. In homogenates, the activity of 20α-hydroxysteroid dehydrogenase per g tissue was found to be about 15 times higher in the control ovaries than in the dysgenetic ovaries, while 3β-hydroxysteroid dehydrogenase activity per g tissue was sim...

Incubation of 4- 14 C-labeled pregnenolone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, and androstenedione with homogenates of head kidneys containing interrenal tissue of the rainbow trout ( Salmo gairdneri ) resulted in the production of 17α-hydroxypregnenolone, 11-deoxycortisol, and cortisol from pregnenolone; 11-deoxycortisol, cortisol, and androstenedione from 17α-hydroxyprogesterone; corticosterone and 18-hydroxy-11-deoxycorticosterone from 11-deoxycorticosterone; and testosterone and 11β-hydroxyandrostenedione from androstenedione. After incubation of progesterone with homogenates of the body kidney, 5α- and 5β-pregnanedione, 3α- and 3β-hydroxy-5α-pregnan-20-one, and 3α- and 3β-hydroxy-5β-pregnan-20-one were obtained as the metabolites of progesterone. When homogenates of corpuscles of Stannius of the rainbow trout were incubated with progesterone or 11-deoxycorticosterone, no corticoid could be detected as metabolite of the substrates, but 5α-pregnanedione and its 5β-isomer were obtained as the main products from progesterone. These results demonstrated the presence of 11β-hydroxylase, 17α-hydroxylase, 18-hydroxylase, 21-hydroxylase, 17β-hydroxysteroid dehydrogenase, Δ 5 -3 β -hydroxysteroid dehydrogenase, C 17 C 20 lyase, and Δ 5 -Δ 4 isomerase activities in the head kidney of the rainbow trout. On the other hand, pregnene-5α- and 5β-hydrogenase activities were predominant in the corpuscles of Stannins as well as in the body kidney of the rainbow trout, while 3α- and 3β-hydroxysteroid dehydrogenase activities were also observed in the body kidney of the fish.

3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (delta 5-3 beta-HSD) catalyzes an early step in the synthesis of testosterone from dehydroepiandrosterone (DHA). We compared enzyme activity in back skin biopsies with sebum excretion rate (SER) in 14 individuals. The rate of conversion of [7 alpha-3H]DHA into [3H]-4-androstene-3,17-dione was measured in cryostat sections of skin and compared with the sebaceous gland content of the same biopsies. Reaction rate was proportional to the volume of sebaceous gland tissue in the sections. Enzyme activity was absent from sections without histologically identifiable sebaceous gland tissue. This suggests that the delta 5-3 beta-HSD is localized in sebaceous glands. SER, measured by a modified photometric technique at the biopsy site, correlated highly with sebaceous gland volume and with the rate of conversion of DHA into androstenedione in the biopsy. For each biopsy, specific activity of delta 5-3 beta-HSD in sebaceous glands was calculated by dividing the rate of formation of [3H]-4-androstene-3,17-dione by sebaceous gland volume. Specific activity of delta 5-3 beta-HSD did not correlate significantly with SER, suggesting that variations in concentration of delta 5-3 beta-HSD in sebaceous glands probably do not underlie variations in sebaceous gland activity.

3 beta-Hydroxysteroid dehydrogenase / delta 5-4 isomerase activity was demonstrated in the human amniotic epithelium from the first trimester of pregnancy. The evidence was based on the in vitro formation of [4-14C] testosterone and [4-14C] androstenedione from [4-14C] 5-androstenediol and [4-14CA1 DEHYDROEPIANDROSTERONE, RESPECTIVELY. The activity of the enzyme studied in age dependent, reaching a maximum in the 8th-9th week of pregnancy and decreasing to negligible values at the end of the second trimester of gestation.

In order to document testicular 17beta-reduction deficiency (17RD) and to search for additional metabolic aberrations possibly associated with this disorder, the metabolism of 14C-labeled pregnenolone (delta5P), 17-HYDROXYPROGESTERONE (17OHP), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T) and estrone (E1) was studied in testicular minces from a 46-year-old male pseudohermaphrodite (MPH) with highly elevated testicular A and minimal T secretion but normal extragonadal conversion of A to T. Testicular minces from a 20-year-old MPH with apparently normal testicular T biosynthesis served as a control. The results of this investigation show that the 17RD testes metabolized delta5P along delta5- and delta4- pathways but, in contrast to the control, converted more 17OHP, metabolizing it predominantly to A rather than T, failed to reduce DHEA to androst-5-ene-3beta,17beta-diol, metabolized DHEA very efficiently to A and produced little T, and converted only minimal quantities of A and E1 to their 17beta-reduced counterparts. 17beta-Reduction increased slightly but was far from being restored to control levels upon addition of NADH plus NADPH. However, oxidation of T to A by 17RD testicular minces, with and without additional NAD plus NADP, was comparable to that by the control. These results document 17RD for A, DHEA and E1 and suggest that the lack of elevated 17OHP and DHEA secretion by the 17RD testes was due to increased 17, 20-lyase and perhaps elevated 3beta-hydroxysteroid dehydrogenase and/or isomerase activity. The observation that 17beta-reduction was only slightly increased upon addition of NADH plus NADPH, but that oxidation of T to A was normal, is consistent with the assumption that more than one 17beta-hydroxysteroid dehydrogenase may be involved in testicular 17beta-reduction and/or 17-oxidation, and that the 17RD testes studied either lacked the enzyme which acts predominantly as 17beta-reductase or that the affinity of this 17beta-reductase for reduced cofactor(s) and/or substrates was abnormal.

The mechanism of the isomerisation of delta 5-3,17-androstenedione by the isomerase (3-oxosteroid delta 4-delta 5-isomerase, EC 5.3.3.1) of Pseudomonas testosteroni has been reinvestigated with delta 5-[4-beta-2H]androstenedione as substrate in H2O and delta 5-androstenedione in 2H2O. A precise localisation of the label in delta 4-androstenendione has revealed that the previously reported 4 beta leads to 6 beta deuterium transfer accounts for only a part of the reaction. Along with this process, removal of the 4 alpha proton is also occurring. This has already been observed with mammalian isomerases. Hence the assumed difference in mechanism between the bacterial and mammalian enzymes is very unlikely.

In human pregnancy, placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5 → 4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric M r = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (M r = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37°C for the oxidation of pregnenolone ( K m = 1.9 μM, V max = 32.6 nmol/min/mg) and dehydroepiandrosterone ( K m = 2.8μM, V max = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione ( K m = 9.7 μM, V max = 618.3 nmol/min/mg) and 5-androstene-3,17-dione ( K m = 23.7μM, V max = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5 → 4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Delta(4)-Delta(5)-isomerase with Delta(5(6))- and Delta(5(10))-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Delta(5(6))-substrates have markedly high k(cat.) values, whereas those of 3-oxo-Delta(5(10))-substrates are very low and their apparent K(m) values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k(-1)/k(+1) ratio and is shown to be very similar for the two classes of substrates (3-oxo-Delta(5(6))- and -Delta(5(10))-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis-Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k(+1) and k(-1) as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k(+1)) is limited only by diffusion and the apparent K(m) does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ;catalytic perfection'.

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with KM values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with KI values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with KI values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with KI values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the KM for pregnenolone as a 3β-HSD substrate, had no effect on the KM for the isomerase substrate 5-pregnene-3, 20-dione. It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.

Nine human ovaries were perfused in vitro with [4-14C] progesterone and in addition one ovary with [7-3H] pregnenolone. From the perfusate unchanged progesterone and five different metabolites were isolated: 20α-dihydroprogesterone, 17α-hydroxyprogesterone, 16α-hydroxyprogesterone, 5α-pregnane-3,20-dione and 4-androstene-3,17-dione. In the ovarian tissue saturated pregnane derivatives were the main metabolites. When [3H] pregnenolone and [14C] progesterone were perfused simultaneously a stimulation of Δ4–5-isomerase and 3β-dehydrogenase activity by HCG was demonstrated.

Tissue homogenates of badger (Taxidea taxus) ovaries and corpora lutea have been incubated with 7α-3H-pregnenolone together with either 4-14C-androstenedione or 4-14C-dehydroepiandrosterone substrates. Ovarian tissue from nonpregnant animals metabolized androstenedione very little, and 95% was recovered unchanged after a 3-hr incubation. Kinetic studies showed that pregnenolone was extensively metabolized via 17α-hydroxypregnenolone and dehydroepiandrosterone to androstenedione and possibly small amounts of testosterone. No detectable 3H-progesterone accumulated in these incubations. After removal of the corpora lutea, ovaries of two pregnant badgers in the pre- and postimplantation stages showed metabolic patterns of 7α-3H-pregnenolone similar to that of ovarian tissue from the nonpregnant badgers. Luteal tissue from the postimplantation animal formed primarily progesterone, 17α-hydroxypregnenolone and androstenedione from 7α-3H-pregnenolone, and testosterone from 14C-androstenedione substrate in 3-hr incubations. Similar tissue from an animal in the preimplantation stage of pregnancy formed less than half the amount of 3H-progesterone and eight times the amount of 17α-hydroxypregnenolone formed by the corpus luteum tissue from the postimplantation ovary. Dehydroepiandrosterone-4-14C was 90% metabolized to androstenedione in the latter incubations. These results demonstrate the predominance of a Δ5-pathway of pregnenolone metabolism to dehydroepiandrosterone and androstenedione in whole ovarian tissue homogenates from nonpregnant badgers and in ovarian tissue from pregnant badgers after the removal of the corpora lutea. The presence of 3β-ol-dehydrogenase, Δ5-isomerase, 17α-hydroxylase, C17–20-desmolase, and 17β-hydroxysteroid dehydrogenase enzymatic activities has also been demonstrated in badger corpus luteum tissue. No major qualitative differences were observed in the steroid metabolic patterns of similar tissue from badgers in different reproductive states.

The clinical and endocrine features of a unique form of adrenal insufficiency secondary to an inherited deficiency of 3 beta-hydroxysteroid dehydrogenase-isomerase (3-HSD) were studied. The propositus was a 19-yr-old man with a history of repeated episodes of acute adrenal crisis. Family study disclosed that a 6-yr-old female sibling also was affected, and a third sibling had died during the course of an adrenal crisis. The diagnosis of adrenal insufficiency was established on the basis of extremely low serum cortisol levels and urinary 17-hydroxycorticosteroid excretion with concomitantly elevated serum ACTH levels and lack of cortisol response to ACTH administration. Impairment of C-21 steroid 3-HSD activity was strongly suggested by persistency elevated serum 17-hydroxypregnenolone to 17-hydroxyprogesterone and pregnenolone to progesterone ratios, their significant increase after ACTH administration, and their return to normal during cortisol therapy in both patients. Nevertheless, the serum dehydroepiandrosterone to androstenedione ratio, both basally and after ACTH and/or hCG stimulation, was normal. These findings coupled with the normal phenotypic development and onset of puberty in the two patients indicated intact C-19 steroid 3-HSD activity. The overall results indicate an inherited impairment of 3-HSD activity confined only to C-21 steroid substrates and, thus, suggest the existence of at least two 3-HSD isoenzymes under independent genetic regulation.