Amet Y, Berthou F, Baird S, Dreano Y, Bail J P, Menez J F
Equipe d'Accueil DGRT EA 948, Laboratoire de Biochimie-Nutrition Faculté de Médecine, Brest, France.
Biochem Pharmacol. 1995 Nov 27;50(11):1775-82. doi: 10.1016/0006-2952(95)02040-3.
The (omega-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes. The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot (r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation (r = 0.73), 4-nitrophenol hydroxylation (r = 0.84), N-nitrosodimethylamine demethylation (r = 0.79) and n-butanol oxidation (r = 0.73). The (omega-1)-hydroxylation of lauric acid was inhibited by ethanol (Ki = 3.5 mM), acetone (IC50 = 10 mM) dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol Ki on the (omega-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (omega-1)-hydroxylation of lauric acid in human liver microsomes, and omega-hydroxylation is mediated by another enzyme. Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (omega-1)-hydroxylation of lauric acid.
月桂酸（11-羟基月桂酸）是脂肪酸的一种模型底物，其（ω-1）-羟基化反应先前已证明是由大鼠肝微粒体中的CYP2E1介导的。本研究检测了29个人肝微粒体中肝CYP2E1含量和11-羟基月桂酸的变化。11-羟基月桂酸活性与通过免疫印迹定量的CYP2E1含量密切相关（r = 0.75），也与已知由CYP2E1介导的四种单加氧酶活性密切相关：氯唑沙宗-6-羟基化反应（r = 0.73）、4-硝基苯酚羟基化反应（r = 0.84）、N-亚硝基二甲胺去甲基化反应（r = 0.79）和正丁醇氧化反应（r = 0.73）。月桂酸的（ω-1）-羟基化反应受到乙醇（Ki = 3.5 mM）、丙酮（IC50 = 10 mM）、二甲基亚砜、氯唑沙宗（CYP2E1的竞争性抑制剂）、二乙基二硫代氨基甲酸盐和二烯丙基硫醚（均为CYP2E1的选择性基于机制的失活剂）的抑制。乙醇对月桂酸（ω-1）-羟基化反应的Ki值较低，表明低水平的酒精可能会改变肝脏中的脂肪酸代谢。分别作为CYP1A和CYP3A4自杀底物的呋拉茶碱和孕二烯酮，并未改变月桂酸的11-羟基化反应。针对大鼠CYP2E1的多克隆抗体抑制了11-羟基月桂酸的形成，但不影响12-羟基月桂酸的活性。综上所述，这些结果表明CYP2E1参与了人肝微粒体中月桂酸的（ω-1）-羟基化反应，而ω-羟基化反应是由另一种酶介导的。最后，使用经基因工程改造用于表达9种人P450的酵母和哺乳动物细胞表明，CYP2E1是参与月桂酸（ω-1）-羟基化反应的唯一一种酶。
