White I N
Chem Biol Interact. 1980 Jan;29(1):103-15. doi: 10.1016/0009-2797(80)90090-3.
A method has been developed to separate norethindrone and norethindrone-4 beta,5 beta-epoxide by high performance liquid chromatography using isocratic solvent systems with either ODS-reverse phase or conventional silica gel columns. Using these techniques it was found that norethindrone epoxide, prepared chemically, was stable in aqueous buffer (pH 7.4) at 37 degrees C for at least 1 h. Under similar conditions, in the presence of a 10-fold molar excess of cysteine or glutathione, the half life for norethindrone epoxide was 15 and 32 min respectively. In 0.01 M perchloric acid at 37 degrees C the t 1/2 of norethindrone epoxide was 17 min. Norethindrone epoxide was rapidly degraded by rat liver microsomal epoxide hydratase to give a metabolite having properties consistant with it being norethindrone-4,5-dihydrodiol. Epoxide hydratase activities were stimulated about three fold by pretreating rats with phenobarbitone. The pH optimum for this reaction was pH 7.4. Conversion of norethindrone epoxide to norethindrone-dihydrodiol was inhibited by the epoxide hydratase inhibitor 1,2-epoxytrichloropropane. Although norethindrone was extensively metabolised in the presence of NADPH and rat liver microsomes, no conversion to norethindrone-4 beta,5 beta-epoxide could be demonstrated, either in the presence or absence of epoxytrichloropropane in the reaction mixture. If norethindrone epoxide was produced under these conditions it was suggested that it either reacted with microsomal proteins at or close to the site or production or was further metabolised. Norethindrone-4 beta,5 beta-epoxide did not cause any loss of cytochrome P-450 when incubated with rat liver microsomes in the absence of NADPH. Only in the presence of NADPH did further metabolism of norethindrone epoxide occur leading to the formation of active metabolites capable of breaking down cytochrome P-450. The initial rate of loss of cytochrome P-450 under these conditions was greater with norethindrone than with norethindrone epoxide as the substrate.
已开发出一种通过高效液相色谱法分离炔诺酮和炔诺酮 - 4β,5β - 环氧化物的方法，该方法使用等度溶剂系统，采用ODS反相柱或常规硅胶柱。使用这些技术发现，化学制备的炔诺酮环氧化物在37℃的水性缓冲液（pH 7.4）中至少稳定1小时。在类似条件下，在半胱氨酸或谷胱甘肽摩尔过量10倍的情况下，炔诺酮环氧化物的半衰期分别为15分钟和32分钟。在37℃的0.01 M高氯酸中，炔诺酮环氧化物的t1/2为17分钟。炔诺酮环氧化物被大鼠肝微粒体环氧化物水解酶迅速降解，产生一种代谢产物，其性质与炔诺酮 - 4,5 - 二氢二醇一致。用苯巴比妥预处理大鼠可使环氧化物水解酶活性提高约三倍。该反应的最适pH为pH 7.4。环氧化物水解酶抑制剂1,2 - 环氧三氯丙烷可抑制炔诺酮环氧化物向炔诺酮 - 二氢二醇的转化。尽管炔诺酮在NADPH和大鼠肝微粒体存在下会广泛代谢，但在反应混合物中无论是否存在环氧三氯丙烷，均未证明其转化为炔诺酮 - 4β,5β - 环氧化物。如果在这些条件下产生了炔诺酮环氧化物，则表明它要么在产生部位或其附近与微粒体蛋白反应，要么进一步代谢。在没有NADPH的情况下，炔诺酮 - 4β,5β - 环氧化物与大鼠肝微粒体孵育时不会导致细胞色素P - 450的任何损失。只有在NADPH存在下，炔诺酮环氧化物才会进一步代谢，导致形成能够分解细胞色素P - 450的活性代谢产物。在这些条件下，以炔诺酮为底物时细胞色素P - 450的初始损失速率比以炔诺酮环氧化物为底物时更大。
