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105. Metabolism of estrone-14C by the rhesus monkey kidney under in vitro conditions
Abstracts data fitted a 3-~mpartment model for the tritium migration, which allowed computer-simulation of the partial processes. The portion of methoxy-3H-ME demethylated to EE amountsto 53.7 + 5.0% (SD) corresponding to clinical observations of ME being half as potent in its estrogenic action as EE. (2) In rats 2-hydroxylation was the main metabolic pathway for EE, exceeding even the 2-hydroxylation of E,. Recent studies confirmed this finding also for humans: EE and E, were incubated with human liver microsomes using an NADPH-regenerating system. The extent of 2-hydroxylation was measured by replacement of tritium from that position. It was found that the 2-hydroxylation of EE proceeded about 3 times as fast as that of E,. This is thought to be due to the hindering of hydroxylation of EE at C-16 by its 17ct-alkyl substituent, and to the competitive nature of 16a- and 2-hydroxylation of estrogens.
105. Metabolism of estrone-‘4C by tbe rhesus monkey kidney under in vitro conditions BRECKWOLDT, M., TRAUTWEIN, A. and LISBOA,B. P., Departments of Obstetrics and Gynaecology of the Universities of Freiburg i.Br. and Hamburg-FRG The present study investigates the metabolizing capacity of the Rhesus monkey kidney of estrone under in vitro conditions. Purified i4C-estrone (1 ug) was incubated with 5 g of kidney slices, freshly obtained from a female Rhesus monkev in a KrebsRinaer bicarbonate buffer (~H7.0) in the p&ence of NADPH-at 37°C for 60 min. The extr&ts were purified on amberhte XAD-2 and subjected to columnchromatography on Sephadex LH 20 using various solvent systems. Isolation and identification of the metabolites was performed by thin-layer-, column- and radio gas-chromatography. 29.3% of the radioactivity recovered from the incubation corresponded to unmetabolized estrone, while 58.29; was identified by radio gas-chromatography as estradiol-17/I and 4.5% were polar C,,O,-metabolites; 4.9% of the recovered radioactivity had the same chromatographic behaviour as estrone conjugates. The present data indicate that the kidney of the Rhesus monkey is highly active in converting estrone to estradiol-17/I and in the formation of conjugates. 106. Pe~usion of the Rhesus monkey ovary witb Z,4,6,7-3H-
e&one in viva WORTMANN,W., WORTMANN,B., SCHENKER,J. and TOUCHSTONE, J. C., Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, U.S.A. and Universitlts-Frauenklinik Mainz, West Germany The interconversion and conjugation of estrogens in 5 rhesus monkey ovaries in viva was studied for the first time with 2,4,6,7-3H-estrone(3H-E,). To minimize disturbances of the physiological conditions a new method was used. 3H-E, was administered by phonophoresis directly to the ovary with an energy of 0,5 Watt/cm2 and a frequency of 1 MHz. 79% of the applied radioactivity was collected from the ovarian veins over a period of 30-75 min. The venous ovarian blood collected was analyzed for free and conjugated steroids. The analysis included separation by thin layer chro~tography, paper chromatography and derivative formation. Specific activities were determined. Between 2.7% and 4.8% of the recovered radioactivity was found in the conjugated fraction. More radioactivity was counted in the sulfate fraction as
321
compared to the glucuronide fraction. Estrone was the main steroid recovered. Conversion to estradiol was not extensive. Estriol was found in very small amounts. (Supported by The Ford Foundation and Deutsche Forschungsgemeinschaft.)
107. Participation of glucuronidationin selective O-methylation of catechol estrogen in the rat NAMBARA,T. and HONMA,S., Pharmaceutical Institute, Tohoku University, Sendai, Japan In view of the accumulating evidence of the importance of the steroid conjugates, studies of the mechanisms involved in the O-methylat~on of catechol estrogen in the rat have been undertaken. First, the isolation and characterization ofthe metabolites excreted in rat bile after administration of estrone (E,) were carried out. Of the biliary metabolites 2-OHE, 2glucuronide (G) was the principal conjugate. Formation of the isomeric catechol monomethyl (Me) ethers with rat liver 15OOng supernatant and S-adenosylL-methionine-Me3H (or I‘%) was examined employing E,. 2-OHE,, 2-OHE,2-G, and 2-OHE,3-G as substrate. In consequence it was found that 0-methylation of 2-OHE,2-G was directed preferentially to the unconjugated phenol. The structure of the 2-OHE,2-G 3-Me ether formed was definitely characterized by transforming it to the acetatemethyl ester in the usual manner, followed by reverse isotope dilution anaIysis. The results of the 0-methyiation of 2-OHE,2-G in the living animal were subs~ntially compatible with those of the in vitro study. These findings suggest that glu~uronidation at C-2 participates in the selective 0-methylation, resulting in the formation of the catechol estrogen 3-Me ether.
108. New metabolites of l’la-ethynylestradiol WALL, M. E., JEFFCOAT,A. R. and SCHIAPPA,G. J., Chemistry and Life Sciences Division, Research Triangle Institute, Research Triangle Park, N. C. 27709 U.S.A. Radiolabelled ethynylestradiol (EE) was incubated with microsomal preparations (pellet, 105,#0 g) from beagle and rat liver in the presence of NADPH. After extraction and preliminary purification combined gas-liquid chromatographic-mass spectral analysis of the beagle liver metabolites showed the presence of 17a-D-homoestrone and a hydroxylated ethynylestradiol. From the mass spectral fragmentation pattern of the latter species, the enzymatic hydroxylation must have occurred in either Ring B or C. The major metabolite, however, from this incubation is a compound of molecular weight 590 (mol wt EE = 296). Mass spectral fragmentation shows the involvement of two molecules of EE, and therefore the metabolite must be a dehydro-dimer of this steroid. n.m.r. analysis of this compound indicates a covalent attachment between Ring A of one EE molecule and the phenolic oxygen of the second. That this highly unusual metabolite is not an experimental artifact was established by the fact that it is not found when the enzymes are denatured by heating. This dehy~o-dimer is also the major in vitro EE metabolite from rat liver. Estradiol also yields a similar dehydro-dimer metabolite. (These studies were supported by Contract PH-43-NIGMS-65-1057 with the National Institute of General Medical Sciences).
105. Metabolism of estrone-‘4C by tbe rhesus monkey kidney under in vitro conditions BRECKWOLDT, M., TRAUTWEIN, A. and LISBOA,B. P., Departments of Obstetrics and Gynaecology of the Universities of Freiburg i.Br. and Hamburg-FRG The present study investigates the metabolizing capacity of the Rhesus monkey kidney of estrone under in vitro conditions. Purified i4C-estrone (1 ug) was incubated with 5 g of kidney slices, freshly obtained from a female Rhesus monkev in a KrebsRinaer bicarbonate buffer (~H7.0) in the p&ence of NADPH-at 37°C for 60 min. The extr&ts were purified on amberhte XAD-2 and subjected to columnchromatography on Sephadex LH 20 using various solvent systems. Isolation and identification of the metabolites was performed by thin-layer-, column- and radio gas-chromatography. 29.3% of the radioactivity recovered from the incubation corresponded to unmetabolized estrone, while 58.29; was identified by radio gas-chromatography as estradiol-17/I and 4.5% were polar C,,O,-metabolites; 4.9% of the recovered radioactivity had the same chromatographic behaviour as estrone conjugates. The present data indicate that the kidney of the Rhesus monkey is highly active in converting estrone to estradiol-17/I and in the formation of conjugates. 106. Pe~usion of the Rhesus monkey ovary witb Z,4,6,7-3H-
e&one in viva WORTMANN,W., WORTMANN,B., SCHENKER,J. and TOUCHSTONE, J. C., Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, U.S.A. and Universitlts-Frauenklinik Mainz, West Germany The interconversion and conjugation of estrogens in 5 rhesus monkey ovaries in viva was studied for the first time with 2,4,6,7-3H-estrone(3H-E,). To minimize disturbances of the physiological conditions a new method was used. 3H-E, was administered by phonophoresis directly to the ovary with an energy of 0,5 Watt/cm2 and a frequency of 1 MHz. 79% of the applied radioactivity was collected from the ovarian veins over a period of 30-75 min. The venous ovarian blood collected was analyzed for free and conjugated steroids. The analysis included separation by thin layer chro~tography, paper chromatography and derivative formation. Specific activities were determined. Between 2.7% and 4.8% of the recovered radioactivity was found in the conjugated fraction. More radioactivity was counted in the sulfate fraction as
321
compared to the glucuronide fraction. Estrone was the main steroid recovered. Conversion to estradiol was not extensive. Estriol was found in very small amounts. (Supported by The Ford Foundation and Deutsche Forschungsgemeinschaft.)
107. Participation of glucuronidationin selective O-methylation of catechol estrogen in the rat NAMBARA,T. and HONMA,S., Pharmaceutical Institute, Tohoku University, Sendai, Japan In view of the accumulating evidence of the importance of the steroid conjugates, studies of the mechanisms involved in the O-methylat~on of catechol estrogen in the rat have been undertaken. First, the isolation and characterization ofthe metabolites excreted in rat bile after administration of estrone (E,) were carried out. Of the biliary metabolites 2-OHE, 2glucuronide (G) was the principal conjugate. Formation of the isomeric catechol monomethyl (Me) ethers with rat liver 15OOng supernatant and S-adenosylL-methionine-Me3H (or I‘%) was examined employing E,. 2-OHE,, 2-OHE,2-G, and 2-OHE,3-G as substrate. In consequence it was found that 0-methylation of 2-OHE,2-G was directed preferentially to the unconjugated phenol. The structure of the 2-OHE,2-G 3-Me ether formed was definitely characterized by transforming it to the acetatemethyl ester in the usual manner, followed by reverse isotope dilution anaIysis. The results of the 0-methyiation of 2-OHE,2-G in the living animal were subs~ntially compatible with those of the in vitro study. These findings suggest that glu~uronidation at C-2 participates in the selective 0-methylation, resulting in the formation of the catechol estrogen 3-Me ether.
108. New metabolites of l’la-ethynylestradiol WALL, M. E., JEFFCOAT,A. R. and SCHIAPPA,G. J., Chemistry and Life Sciences Division, Research Triangle Institute, Research Triangle Park, N. C. 27709 U.S.A. Radiolabelled ethynylestradiol (EE) was incubated with microsomal preparations (pellet, 105,#0 g) from beagle and rat liver in the presence of NADPH. After extraction and preliminary purification combined gas-liquid chromatographic-mass spectral analysis of the beagle liver metabolites showed the presence of 17a-D-homoestrone and a hydroxylated ethynylestradiol. From the mass spectral fragmentation pattern of the latter species, the enzymatic hydroxylation must have occurred in either Ring B or C. The major metabolite, however, from this incubation is a compound of molecular weight 590 (mol wt EE = 296). Mass spectral fragmentation shows the involvement of two molecules of EE, and therefore the metabolite must be a dehydro-dimer of this steroid. n.m.r. analysis of this compound indicates a covalent attachment between Ring A of one EE molecule and the phenolic oxygen of the second. That this highly unusual metabolite is not an experimental artifact was established by the fact that it is not found when the enzymes are denatured by heating. This dehy~o-dimer is also the major in vitro EE metabolite from rat liver. Estradiol also yields a similar dehydro-dimer metabolite. (These studies were supported by Contract PH-43-NIGMS-65-1057 with the National Institute of General Medical Sciences).