Formation and metabolism of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norandrogen produced by porcine granulosa cells from C19 aromatizable androgens.

The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.

Biosynthesis of 16 alpha, 17 alpha-epoxy-oestratrienol in rat liver microsomes.

After incubation of 1,3,5(10),16-oestratetraenol, a 16-dehydrosteroid, with rat liver microsomes, 16 alpha, 17 alpha-epoxy-oestratrienol was isolated as metabolite. The compound was detected by the use of mass fragmentography after purification of the incubation extract by thin-layer chromatography. Since the epoxide is rapidly hydrolysed by a hepatic epoxide hydratase, only very small concentrations of this metabolite were present in the incubation extract. When styrene oxide was added to the incubation mixture as inhibitor of the epoxide hydratase, the yield of the steroid epoxide increased considerably. Final identification of the oestrogen epoxide was performed by recording mass spectra and by comparison with authentic reference material.

Estrogen biosynthesis and 1beta-hydroxylation using C19 and 19-nor steroid precursors.

(1) In order to study the relationship between aromatization (estrogen biosynthesis) and 1beta-hydroxylation, the effects of a variety of factors on these processes were evaluated. (2) Using the C18 substrate, 4-estrene-3,17-dione, it was found that carbon monoxide, SU-4885, amphenone B, potassium cyanide, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione inhibited the above transformations significantly and to varying degrees. However, within a given experiment the inhibition of each process was similar. (3) SKF-525A did not inhibit either transformation. In addition, phosphate, Tris and barbital buffers, as well as pH changes from 6.9 to 7.7, had no stimulatory or inhibitory effect on the production of estrogen and 1beta-hydroxy compounds. (4) In contrast, several inhibitors affected the aromatization of C19 and C18 steroids differently. These include carbon monoxide, SU-4885 and amphenone B. (5) When a mixture of 4-[7beta-3Hi1estrene-3,17-dione and 19-[4-14C]nortestosterone were incubated together the former was preferentially converted to estrogen. This preference for the 17-keto steroidal form mimics results observed for C19 substrates. (6) We conclude that while estrogen biosynthesis and 1beta-hydroxylation appear to be mediated by the same enzyme system, the same conclusion cannot be drawn for the aromatization of C19 and C18 substrates.

PIP: Estrogen biosynthesis and lbeta-hydroxylation using carbon-19 and 19-nor steroid precursors were evaluated. 4-estrene-3, 17-dione was used to find that carbon monoxide, SU-4885, amphenone B, potassium cyanide, 4-androstene-3,17-dione, and 1,4-androstadiene-3,17-dione inhibited the above transformations to varying degrees. SKF-525A did not inhibit either transformation, and phosphate, Tris and barbital buffers as well as pH changes from 6.9 to 7.7 had no effect on the production of estrogen and lbeta-hydroxy compounds. Carbon monoxide, SU-4885, and amphenone B affected the aromatization of carbon-19 and carbon-18 steroids differently. A mixture of 4 (7 beta-tritium) estrene-3,17-dione and 19-(4-carbon 14) nortestosterone incubated together resulted in the former preferentially converted to estrogen. It is concluded that while estrogen biosynthesis and lbeta-hydroxylation appear to be mediated by the same enzyme system, however, the same conclusion cannot be drawn for the aromatization of carbon-19 and carbon-18 substrates.

[Production of the methyl ether of 8,14-seco-delta,3,5,(10),9(11)-estratetraendiol-3,17beta-one-14 using a culture of Saccharomyces cerevisiae BKMU-488]. / Poluchenie metilovogo éfira 8,14-seco-del'tal, 3,5(10),9(11)-éstratetradiola-3,17beta-ona-14 s pomoshch'iu kul'tury Saccharomyces cerevisiae BKMU-488

The culture Saccharomyces cerevisiae BKMU-499 was selected from 100 strains of different Saccharomyces species to obtain an optically active methyl ester 8,14-seco-delta1,3,5(10),9(11)-estratetraendiol-3-17beta-on-14. Various conditions of the transformation were examined. The yield of this keto-alcohol depended on the following factors: level of aeration, method of steroid administration, physiological state of the culture, and composition of the cultivation medium. The optimal yeild of the optically active product was 84%.