Watson & Marrian (1955) detected the presence in extracts of the urine of pregnant women of a ketonic Kober chromogen which was more 'polar' than oestrone, and, on solvent-partition evidence, concluded that it was 16-oxo-oestradiol-17P (16-oxo-oestra-1:3:5-triene-3:17,B-diol). More recently Marrian, Watson & Panattoni (1957) concentrated this Kober chromogen (KC-5) and showed conclusively that the major component in it must have been either 16m-hydroxyoestrone (17-oxo-oestra-1:3:5-triene-3:160-diol) or 16p-hydroxyoestrone (17-oxo-oestra-1 :3:5-triene-3: 16,B-diol), and not 16-oxo-oestradiol-17,. On biogenetic grounds the view was favoured that the major component in KC-5 was 16m-hydroxyoestrone rather than 16phydroxyoestrone, but since reduction with sodium borohydride yielded a mixture of oestriol (oestra-
In the course of column partition-chromatographic analysis of ketonic-phenolic fractions obtained from ether extracts of enzymically hydrolysed pregnancy urine, Kober-chromogenic material slightly more 'polar' in its chromatographic behaviour than 16oc-hydroxyoestrone (17-oxooestra-1:3:5-triene-3:16oa-diol) was regularly detected. The two possibilities were at first considered that this material might have arisen by 'leakage' of the chromatographically similar 16-epioestriol (oestra-1:3:5-triene-3:16/3: 17/3-triol) into the ketonic fractions or by 'tailing' of the 16oc-hydroxyoestrone fraction on the columns. However, when a concentrate of this Kober-chromogenic material was subjected to a second Girard separation and the resulting ketonic fraction rechromatographed in the same system nearly 60% of the starting material was recovered. This finding disposed of these two possibilities and it became evident that a hitherto unknown ketonic Kober chromogen (KC-6) had been detected. Subsequently it was found that 'KC-6' was in fact a mixture of two different Kober chromogens; and in the present paper the isolation and complete characterization of one of these (KC-6A) is described. Brief preliminary accounts of the main findings reported here have been published (Loke, Watson & Marrian, 1957; Loke, Marrian, Johnson, Meyer & Cameron, 1958). EXPERIMENTAL AND RESULTS Methods Hydrolysis and extraction of urine. The procedure used for preparing ketonic-phenolic fractions from enzymically hydrolysed urine was essentially similar to that described by Marrian, Watson & Panattoni (1957), but two modifications of this procedure were introduced: (i) as the result of a suggestion made to us by Dr J. R. K. Preedy (The London Hospital) Bradosol (5% solution of /-phenoxy
1. The possible involvement of 5-pregnene-3beta,20beta-diol in 16-unsaturated C(19) steroid biosynthesis has been investigated. 2. 5,16-Androstadien-3beta-ol (andien-beta) formation from [4-(14)C]pregnenolone (3beta-hydroxy-5-pregnen-20-one), 5-pregnene-3beta,20alpha-diol and 5-pregnene-3beta,20beta-diol was studied in homogenates of boar testis and the mean yields obtained were 25.6, 2.7 and 16.0% respectively. 3. Short-term kinetic studies with pregnenolone and 5-pregnene-3beta,20beta-diol separately and together suggested that the latter compound might be an intermediate in the biosynthesis of andien-beta. 4. In agreement with this interpretation, radioactive 5-pregnene-3beta,20beta-diol has been isolated during andien-beta biosynthesis from [4-(14)C]pregnenolone in the presence of NADPH, more radioactivity being trapped under limiting conditions of andien-beta formation with NADH present as cofactor. 5. Further, 5-pregnene-3beta,20beta-diol and andien-beta have been shown to inhibit the formation of the 16-unsaturated C(19) steroid from [4-(14)C]pregnenolone, the yield of radioactive 5-pregnene-3beta,20beta-diol increasing in the presence of added unlabelled andien-beta. 6. It is concluded that there may be two pathways leading to 16-unsaturated C(19) steroid formation from pregnenolone, one of these involving 5-pregnene-3beta,20beta-diol as an intermediate. Possible mechanisms are presented and discussed.
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