Cytochrome P450 profiles in rat and their prediction from UPLC-MS metabolic profiles
Author(s)
Masson, Perrine Anne Sophie
Type
Thesis
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.
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.
Date Issued
2011
Date Awarded
2012-02
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Want, Elizabeth
Nicholson, Jeremy
Sponsor
Servier Laboratories Ltd.
Publisher Department
Surgery and Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)