Abstract
In this study, a simple and reliable high performance liquid chromatographic method with UV detection was developed and validated for rapid determination of coumarin hydroxylase activity in rat hepatic microsomes. The chromatographic separation was achieved using Zorbax Eclipse XDB C18 column (150×4.6 mm, 5 μm), which was kept at 40°C. The isocratic mobile phase consisted of methanol and 1% glacial acetic acid mixture (35:65, v/v) with a flow rate of 0.6 ml/min. The effluent was monitored at 320 nm using photodiode array detector (PDA). 6-hydroxychlorzoxazone (6-OH CZX) was used as internal standard. The method exhibited good linearity (R2 >0.999) for both coumarin (COUM) and its 7-hydroxy metabolite (7-OH COUM) over the assayed concentration range (0.025-5.0 μM) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were less than 15%). The detection limits were 0.001 and 0.005 μM for coumarin and 7-hydroxycoumarin, respectively. This method was also successfully applied for studying the effect of three phytochemicals on hepatic CYP2A6 activity in rats.
Highlights
Cytochrome P450 enzymes consist of a superfamily of enzymes and plays a pivotal role in the metabolism of xenobiotics and synthesis of endogenous substrates (Schneider and Clark, 2013)
As studying the effect of herbal remedies on the activity of drug metabolizing enzymes is a routine experiment in our laboratory, development of simple, cost-effective, accurate, fast and sensitive HPLC methods is a key
We described in this article a new UV-based HPLC assay for COUM and its 7-OH metabolite as a marker of CYP2A6
Summary
Cytochrome P450 enzymes consist of a superfamily of enzymes and plays a pivotal role in the metabolism of xenobiotics and synthesis of endogenous substrates (Schneider and Clark, 2013). The CYP2A6 enzyme is a primary member of the CYP subfamily and is predominantly expressed in the human liver (Oscarson, 2001). CYP2A6 is involved in the metabolism of several drugs including phenobarbital, dexamethasone and rifampin (Le et al, 2003). CYP2A6 metabolizes or bioactivate many environmental toxins including coumarin, aflatoxin B, nicotine and N-nitrosodiethylamine and tobaccospecific nitrosamines (Oscarson, 2001). It is pertinent to study CYP2A6 metabolism in order to evaluate its clinical and toxicological significance.
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