Chen C H, Fung P T, Hecht S S
Cancer Res. 1979 Dec;39(12):5057-62.
A high-pressure liquid chromatographic assay was developed for microsomal alpha-hydroxylation (2'-hydroxylation and 5'-hydroxylation) of N'-nitrosonornicotine. N'-Nitrosonornicotine was incubated with rat liver microsomes and a reduced nicotinamide adenine dinucleotide phosphate-generating system at 37 degrees. After addition of 2,4-dinitrophenylhydrazine reagent, the mixtures were analyzed by reverse-phase high-pressure liquid chromatography. The 2,4-dinitrophenylhydrazones of 4-hydroxyl-1-(3-pyridyl)-1-butanone and 4-hydroxy-1-(3-pyridyl)-butanal, which are the products of 2'-hydroxylation and 5'-hydroxylation, were quantified by ultraviolet light detection at 254 nm. Km's for 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine by liver microsomes from Aroclor-treated male F-344 rats were 1.81 and 1.96 mM, while Vmax's were 0.53 and 1.05 nmol/min/mg protein, respectively. Aroclor pretreatment of rats resulted in a 20-fold induction of 2'-hydroxylation, but only a 1.9-fold induction of 5'-hydroxylation. The deuterium isotope effect for alpha-hydroxylation of N'-nitrosonornicotine was determined by comparing the rates of 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine, N'-[2',5',5'-D]nitrosonornicotine, N'-[2'-D]nitrosonornicotine, and N'-[5',5'-D]nitrosonornicotine. The deuterium isotope effect (Vmax H/Vmax D) was 2.4 to 2.7 for 5'-hydroxylation and 1.2 for 2'-hydroxylation.
开发了一种用于检测N'-亚硝基去甲烟碱微粒体α-羟基化作用（2'-羟基化和5'-羟基化）的高压液相色谱分析方法。将N'-亚硝基去甲烟碱与大鼠肝脏微粒体及一个生成还原型烟酰胺腺嘌呤二核苷酸磷酸的系统在37℃下孵育。加入2,4-二硝基苯肼试剂后，通过反相高压液相色谱对混合物进行分析。通过在254nm处进行紫外光检测，对2'-羟基化和5'-羟基化产物4-羟基-1-(3-吡啶基)-1-丁酮和4-羟基-1-(3-吡啶基)-丁醛的2,4-二硝基苯腙进行定量。经多氯联苯混合物处理的雄性F-344大鼠肝脏微粒体对N'-亚硝基去甲烟碱进行2'-羟基化和5'-羟基化的米氏常数（Km）分别为1.81和1.96mM，而最大反应速率（Vmax）分别为0.53和1.05nmol/min/mg蛋白质。大鼠经多氯联苯混合物预处理后，2'-羟基化作用诱导了20倍，但5'-羟基化作用仅诱导了1.9倍。通过比较N'-亚硝基去甲烟碱、N'-[2',5',5'-D]亚硝基去甲烟碱、N'-[2'-D]亚硝基去甲烟碱和N'-[5',5'-D]亚硝基去甲烟碱的2'-羟基化和5'-羟基化速率，确定了N'-亚硝基去甲烟碱α-羟基化作用的氘同位素效应。5'-羟基化的氘同位素效应（Vmax H/Vmax D）为2.4至2.7，2'-羟基化的为1.2。
