Adas F, Berthou F, Picart D, Lozac'h P, Beaugé F, Amet Y
Laboratoire de Biochimie-Nutrition, Faculté de Médecine, Brest, France.
J Lipid Res. 1998 Jun;39(6):1210-9.
In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is currently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its omega and (omega-1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The omega/omega-1 ratio of oleic acid metabolites (1.22+/-0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10+/-0.02), while in human liver microsomes this ratio was 0.75+/-0.5 for lauric acid and 5.2+/-2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal (omega-1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the (omega-1)-hydroxylation of oleic acid. Furthermore, the omega-hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of (omega-1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min(-1), respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.
体外技术已被用于研究参与生理单不饱和脂肪酸油酸代谢的微粒体细胞色素P450的性质。与目前用作脂肪酸代谢模型底物的月桂酸一样，油酸的烷基链在其ω和（ω-1）碳原子上被羟基化。这些羟基化代谢物的身份通过气相色谱/质谱联用仪（GC/MS）和液相色谱/质谱联用仪（LC/MS）得以确定。发现油酸代谢物的ω/ω-1比值（1.22±0.01）与在大鼠肝微粒体中月桂酸的该比值（1.10±0.02）相似，而在人肝微粒体中，月桂酸的该比值为0.75±0.5，油酸的该比值为5.2±2.6。在用乙醇或氯贝丁酯分别处理大鼠后，乙醇是细胞色素P450 2E1的诱导剂，氯贝丁酯是细胞色素P450 4A的诱导剂，结果显示油酸的羟基化比月桂酸的羟基化更不易被诱导。采用了五种体外方法来鉴定负责油酸微粒体（ω-1）-羟基化的细胞色素P450同工酶：各种诱导剂对大鼠的影响、25个人肝微粒体中特定细胞色素P450催化活性之间的相关性研究、化学抑制、免疫抑制以及由cDNA表达的人细胞色素P450酶进行的代谢研究。从上述结果可以确定，细胞色素P450 2E1是参与油酸（ω-1）-羟基化的主要酶。此外，在人肝微粒体中，油酸的ω-羟基化主要由细胞色素P-450 4A酶催化。月桂酸和油酸的（ω-1）-羟基化的转换数分别从7.8降至1.5 min⁻¹，这表明十二烷烷基链能使与细胞色素P450 2E1的活性位点实现最佳结合。
