Sherratt A J, Damani L A
Department of Pharmacy, University of Manchester, London, UK.
Drug Metab Dispos. 1989 Jan-Feb;17(1):20-5.
Sensitive and specific chromatographic assays for the measurement of flavin-containing monooxygenase (N-oxygenase and S-oxygenase activities) and aldehyde oxidase activities in rat hepatocyte primary cultures were developed. Conditions for the measurement of enzymatic activities in rat liver cell cultures were first optimized using freshly isolated cell suspensions. Activities of the cytochrome P-450/P-448 isozymes in rat hepatocytes maintained in primary culture [assessed by the O-deethylation of 7-ethoxycoumarin (P-450/P-448) and the N-demethylation of N,N-dimethylaniline (P-450)] rapidly declined to 25% of the initial levels by 48 hr in culture. The flavin-containing monooxygenase system was considerably more stable in cell culture. Flavin N-oxygenase activity (assessed by the N-oxidation of N,N-dimethylaniline) declined slightly (10-15%) and remained almost constant over the 48-hr culture period, whereas S-oxygenase activity (assessed by the S-oxygenation of tetrahydrothiophen) gradually declined and stabilized at approximately 65% of its initial activity at 48 hr in culture. Aldehyde oxidase activity (assessed by the 1-hydroxylation of phthalazine) declined to approximately 20% of the initial value by 48 hr in culture. The differential stability of the microsomal and cytosolic drug oxidases in rat hepatocytes in primary culture demonstrates some of the limitations of this model for metabolic studies.
已开发出灵敏且特异的色谱分析方法，用于测定大鼠原代肝细胞培养物中含黄素单加氧酶（N-加氧酶和S-加氧酶活性）及醛氧化酶活性。首先使用新鲜分离的细胞悬液优化大鼠肝细胞培养物中酶活性的测定条件。原代培养的大鼠肝细胞中细胞色素P-450/P-448同工酶的活性[通过7-乙氧基香豆素的O-脱乙基作用（P-450/P-448）和N,N-二甲基苯胺的N-脱甲基作用（P-450）进行评估]在培养48小时后迅速降至初始水平的25%。含黄素单加氧酶系统在细胞培养中稳定性更高。黄素N-加氧酶活性（通过N,N-二甲基苯胺的N-氧化进行评估）略有下降（10%-15%），并在48小时的培养期内几乎保持恒定，而S-加氧酶活性（通过四氢噻吩的S-氧化进行评估）逐渐下降，并在培养48小时时稳定在其初始活性的约65%。醛氧化酶活性（通过酞嗪的1-羟基化进行评估）在培养48小时后降至初始值的约20%。原代培养的大鼠肝细胞中微粒体和胞质药物氧化酶的不同稳定性表明了该模型在代谢研究中的一些局限性。
