Cytochrome P450 profiles in rat and their prediction from UPLC-MS metabolic profiles

Abstract

Cytochrome P450 enzymes (CYPs) are of great interest to pharmaceutical industry due to their major role in drug metabolism and drug-drug interactions in man. As CYPs also act on endogenous substrates and are regulated by nuclear receptors involved in biochemical pathways, their profiles are likely to affect endogenous metabolic profiles. The work in this thesis aimed to establish baseline rat liver CYP levels and assess relationships between CYP profiles and corresponding endogenous metabolic profiles, before introducing CYP inducers in subsequent projects to identify early biomarkers of CYP induction. An efficient and reproducible protocol was first developed and optimised for liver untargeted metabolic profiling by ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Comparison of technical and biological variation for this liver protocol then demonstrated that sample preparation and UPLC-MS variability was mostly small compared to inter-animal variability. Subsequently, an in vivo study was designed including 20 male and 20 female Wistar rats. Liver mRNA of 81 CYPs was quantified, of which 23 exhibited significant gender differences with a gender ratio > 2. Clear gender differences were also observed in serum, urine and liver UPLC-MS metabolic profiles, e.g for metabolites belonging to steroid, triglyceride and phospholipid families. Multivariate models constructed to investigate relationships between CYP and metabolic profiles mainly highlighted the latent variable “gender”. Separate investigations for each gender yielded good prediction of CYP mRNA profiles from male liver aqueous metabolic profiles and female urine metabolic profiles. For prediction of CYP activities, the best models were obtained from serum metabolic profiles. In conclusion, this work improved our knowledge of rat basal CYP and metabolic profiles, and provides a strong basis for subsequent studies on CYP inducers. Importantly, strategies developed for UPLC-MS sample preparation, study design and data pre-processing/analysis are now routinely employed in our laboratory and have applications in many metabolic profiling studies.