Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions.
Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions.
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使用猪肝微粒体组分生物合成 [3H]7 α-羟基-、7-β-羟基-和 7-氧代-脱氢表雄酮。
DOI：
10.1016/j.ab.2004.06.003
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Prough,RussellA
中科院分区：
文献类型：
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作者：
Robinzon,Boaz;Miller,KristyKMichael;Prough,RussellA
由于缺乏放射性标记的代谢物，目前对脱氢表雄酮（DHEA）的研究受到限制。我们利用猪肝微粒体 (PLM) 级分从 50μM [1,2,6,7-3H]DHEA（比放射性 60–80mCi/mmol)。使用乙酸乙酯：己烷：冰醋酸（18：8：3 v：v：v）作为流动相，通过制备薄层色谱（TLC）分离代谢物，用乙酸乙酯萃取，并在氮气流下干燥。通过 TLC 和气相色谱-质谱分析发现代谢物是纯的。在仅用 1mM NADPH 启动的还原型烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 再生系统存在的情况下，1mg/ml PLM 产生 7α-OH-DHEA 和少量 7-氧代-DHEA（分别为 68 和 14nmol/2h/2ml；82% 转化率），而在 1mM NADPH 和 1mM 氧化的存在下 烟酰胺腺嘌呤二核苷酸磷酸 (NADP+)，形成比 7α-OH-DHEA 更多的 7-氧代-DHEA（分别为 58 和 11nmol/2ml/120 分钟；69% 转化率）。当 NADPH 和 NADP+ 使用较长的反应时间时，会产生 7α-OH-DHEA、7β-OH-DHEA 和 7-氧代-DHEA 的混合物（分别为 19,14 和 35nmol/180min/2ml；转化率 62%）。使用猪肝微粒体，可以制备稳定、纯的 DHEA 代谢物，浓度为 10mM，放射性水平 >0.5mCi/mmol，用于生化研究。
Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [3H]-labeled 7α-hydroxy-DHEA (7α-OH-DHEA), 7β-hydroxy-DHEA (7β-OH-DHEA), and 7-oxo-DHEA substrates from 50μM [1,2,6,7-3H]DHEA (specific radioactivity 60–80mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography–mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1mM NADPH alone, 1mg/ml PLM produced 7α-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14nmol/2h/2ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1mM oxidized nicotinamide adenine dinucleotide phosphate (NADP+), more 7-oxo-DHEA than 7α-OH-DHEA (58 and 11nmol/2ml/120min, respectively; 69% conversion) was formed . When longer reaction times were used with NADPH and NADP+, a mixture of 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35nmol/180min/2ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5mCi/mmol levels of radioactivity for biochemical studies.