[43] Measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function

This chapter discusses the measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function. The cytochrome P450s play active roles in the intermediary metabolism of several physiologically important endogenous substrates such as steroid hormones, bile acids, fatty acids, and prostaglandins to name but a few. The P450s participate in the oxidation of many xenobiotics leading, in some instances, to highly reactive products. The xenobiotic-metabolizing P450s have broad and overlapping substrate specificities. Throughout the years, a major obstacle in the characterization of P450 isozymes has been the lack of specific enzymatic assays to distinguish the individual P450 isozymes. It is etablished that the regio- and stereospecific hydroxylation of the steroid nucleus is a sensitive fingerprint for the identification of a specific type of cytochrome P450. This is of particular use in the characterization of mixed populations of P450s, such as exist in the endoplasmic reticulum of liver.