Integrated Comparison of Drug-Related and Drug-Induced Ultra Performance Liquid Chromatography/Mass Spectrometry Metabonomic Profiles Using Human Hepatocyte Cultures

Abstract

The biochemical variations induced in human primary hepatocyte cultures by reference activators of xenoreceptor CAR (NR1I3) and PXR (NR1I2), i.e., rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO), were investigated using a global metabonomics approach. Cultured human hepatocytes were treated with the three drugs before analysis of intracellular and extracellular media by ultra performance liquid chromatography/time-of-flight-mass spectrometry (UPLC/TOF-MS) technique, in order to list endogenous compounds potentially related to a PXR or CAR induction mechanism and to identify drug metabolites related to each treatment. The emphasis was put on the quality of the analytical data (dilution/filtration strategy before data processing) and on the appropriate pattern recognition techniques. In cellular media, the most significant variations seen in the data are not related to the treatments but to the source of hepatocytes, illustrating the importance of the genetic and/or environmental background in human liver experiments. However when applying classical multivariate statistical approaches (principal component analysis (PCA) and orthogonal partial least squares (O-PLS)), the statistical weight due to drug metabolites, present only in the treated groups, hinders the interpretation because of their predominance compared to most of the changes seen in endogenous metabolites. A new statistical approach, called shared and unique structure (SUS) plot, enabling the comparison of different treatments having the same control has been applied, allowing separation of clearly exogenous variables (drug metabolites) from endogenous biomarkers. Endogenous variables (either up- or down-regulated) have been attributed specifically to the impact of rifampicin (PXR ligand), CITCO (CAR ligand), and phenobarbital (CAR and PXR activator) on the biological regulation pathways of the hepatocytes. This global approach coupled to a statistical pretreatment of the data, enabling the separate capture of both drug related and drug induced biomarkers, represents a powerful technique for future mechanistic studies using cellular tools.

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

“…21 These artifactual signals can be highlighted and removed as their intensities do not correlate with the dilution factor. 21,22 The linearity of the signal response was investigated to search for such signals and to ensure that the variations in ion abundances observed in the course of metabolomic studies can be detected despite the occurrence of ion suppression effects. 23 Among 3000 signals that could be detected in the positive and negative ion modes, more than 1700, including all the identified metabolites, exhibited coefficient correlation more than 0.7 and were thus found to be analytically relevant ( Figure 1C).…”

Current trends and challenges in sample preparation for global metabolomics using liquid chromatography–mass spectrometry

“…For analysis of extracellular metabolites, the possibility of inadvertent leakage of intracellular metabolites must be examined during method development and evaluation, whereas for analysis of intracellular species effective methods of disruption of the cell membrane are necessary. Extracellular metabolites are usually analyzed after a simple centrifugation step, with no further sample treatment performed, or after simple dilution with water [90]. The procedures for intracellular metabolite profiling are more involved and generally include sampling; metabolism quenching; washing; and cell lysis and metabolite extraction from cell pellet.…”

“…However, the explanations of the differences between observed NAD recovery in the two studies are less clear. Croixmarie et al omit the quenching step in analysis of hepatocyte intracellular metabolites, and the effect of this is not evaluated [90].…”

A Perceptive of Mass Spectrometry in Clinical Chemistry: Hyphenation for Benefit of Mankind

“…There are a large number of procedures like protein precipitation, liquid-liquid extraction, immunoaffinity purification, dilution and solid phase extraction. However in practice, it is usually a combination of all these procedures that are used [6][7][8][9][10][11][12]. This is because the sample matrix decides the procedure adopted.…”