Determination Of Cholesterol 7α-hydroxylase (CYP7A1) Activity By Reverse Phase HPLC

Background:Bile acids are synthesized in the liver hepatocyte from free cholesterol. The initial and rate-limiting step in the biosynthesis is the 7a-hydroxylation of cholesterol, which is catalyzed by cholesterol 7a-hydroxylase, a cytochrome P450-dependent enzyme. The activity of cholesterol 7a-hydroxylase has been reported to be regulated by negative feedback by bile acids returning to the liver via the portal vein, hormones, drugs, and alterations of the cholesterol substrate pool in the liver. For example, this enzyme has been reported to be regulated by phosphorylation-dephosphorylation control, cytosolic proteins, biliary drainage, bile acid feeding, and glucocorticoid hormones.Three different assay techniques have been developed for measuring cholesterol 7a-hydroxylase activity. These include an isotope incorporation method using thin-layer chromatography to separate radiolabeled cholesterol from 7a-hydroxylase, a GLC-MS procedure, and more recently a HPLC-spectrophotometric method which quantitates the amount of 7a-hydroxycholesterol formed after enzymatic conversion of 7a-hydroxy-4-cholesten-3-one(HCO) using cholesterol oxidase. This method used normal-phase HPLC to separate and quantitate the amount of enzyme product formed. The latter two methods rely on endogenous cholesterol as the substrate for cholesterol 7a-hydroxylase. In the present, it has reported two modifications in the HPLC-spectrophotometric method which should improve this assay procedure and make it more readily usable. These two modifications include the use of an internal steroid recovery standard(7β-hydroxycholesterol) and the use of reverse-phase, rather than normal-phase, HPLC to separate and quantitate enzyme product.Objective:The methodology setup for detecting CYP7A1 activity in rat hepatic microsomes. In this study, we adopted the reverse-phase HPLC to separate and quantiate the enzymatic product of 7α-hydroxy-4-cholesten-3-one.Methods:Residues were analyzed by C-18 reverse-phase HPLC using an Agilent XDB C18 (4.6×250mm,5μm) column equilibrated with 95% acetonitrile and 5% methanol(v/v), at a flow rate of 0.80 ml/min, VWD=240nm. The rat hepatic microsomes were incubated with an internal steroid recovery standard(7β-hydroxycholesterol), and then the enzymatic product of 7a-hydroxy-4-cholesten-3-one was assayed by reverse phase HPLC.Results:1. The standard curve:Y=1.31362786X+0.0268326, R2=0.99603.2. The retention time of 7a-hydroxy-4-cholesten-3-one was 13.6min.3. The activity of cholesterol 7a-hydroxylase was 1.214±1.153 nmol/h/mg protein.Conclusions:The reverse phase HPLC can detect the activity of cholesterol 7a-hydroxylase in rat hepatic microsomes accurately.