Experimental Studies on the Steroid Aromatization

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Estrogen has been well known for many years, but the entire route of its biosynthesis has not yet been sufficiently understood. It has been recently reported by Ryan that a conversion experiment was made to obtain Estrogens from some steroids in vitro by using placental aromatizing enzyme system.The principal target was the possibility of conversion of 19-Norsteroids to Estrogen. These 19-Norsteroids are of great interest as a potent progestin.Quantitative measurement of Estrogens was performed by a modified Engel's method. The ex-traction from the tissue was done by acetone. Additional washing with toluene proved more effective in cleaning out the substrate as well as in removing the impurities.Stimmel's almina column chromatography method was perfomed for the purified fractionation of EstrogenHydroquinone Kober reaction also was used for colorimetric determinaton, according to Brown's mdthod.It was learned empirically during the course of such experiments that the quality of sulfuric acid could bring about a diversity in results.Identification of steroids was made by the use of paper chromatography and its hydroquin one-Kober chromogen spectrum.The optimal pH of the reaction was about 7.0, and the optimal enzyme concentration was prepared by homogenizing the placental tissue with its 1/3 volume 0.25 Mol sucrose solution, containing 0.04 Mol nicotinamide and 0.05 Mol phosphate buffer ; the maximal amount of Estrogens was obtained after 2 hours incubation with 500/2g of substrate.Calculation of the Estorogen amounts was made according to the following equation ;CE- (S2-B2) - (So-Bo) where abbreviations CE stand for Estrogen corrected, S with 500μg of substrate, B blank without substrate, 0 and 2 indicating 0 and 2 hours of incubation period, respectively.The interconversion from Estradiol to Estrone was only demonstrable and no other interconversions were observed. Comparatively large amounts of conversion to Estrogen was observed from Androgens such as Testosterone, Δ4-Androstenedine, Dehydro-epiandrosterone, whereby the conversion ratio was almost identical in every sample. The conversion of 19-Norsteroid to Estrogen occured only from 19-Nortestosterone and not from its derivatives, including Ethinyl-nortestosterone and Methyl-nor-testosterone.Conversion ratio in the case of 19-Nortestosterone was calculated to be about 20% of that of Testosterone. Further experiments were undertaken with Ethinyl-estradiol as the substrate, and we observed no appreciable conversion to Estrogen. Likewise, conversion of Testosterone derivatives to Estrogen was not demonstrable.Of the tentative schema of conversion process from 19-Nortestosterone derivatives to Estrogen, many experiments were done, but only one route from 19-Nortestosterone to Estrone (or Estradiol) was observed and no other routes could be demonstrated.<BE> From these results, it was concluded, that the side chain of 17-position blocked the aromatization in some way. If conversion of 19-Nortestosterone derivatives to Estrogen occurs in vivo, some mechanisms other than that found in the case of Androgen in vitro seem to play a role in the conversion.