HEPSYS-II METABOLOMICS PROFILING OF AN IN VITRO MODEL OF CELLULAR STEATOSIS: DICARBOXYLIC ACIDS AND EICOSANOIDS AS MARKERS OF ENHANCED LIPID PEROXIDATION AND FATTY ACID OXIDATION DYSREGULATION

K. A. Lockman, Philipp Treskes, Leonard Nelson, Karl Burgess, Patricia Lee, Steven D Morley, Peter Hayes, John Plevris

Research output: Contribution to conferencePosterpeer-review

Abstract

Introduction: Oxidative stress is one of the hallmarks of nonalcoholic fatty liver disease (NAFLD).
Reactive oxygen species (ROS) can activate prooxidant cascades, including lipid peroxidation species.
Lipid peroxidation products such as eicosanoids and dicarboxylic acids can potentially promote fatty acid
(FA) dysregulation. We have previously shown that in vitro cellular steatosis model using C3A cells treated
with energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N), recapitulates the
sequence of events in dietary-induced NAFLD; enhanced acute respiration and ROS formation resulted
in mitochondrial impairment. In contrast, treatment with oleate led to relatively low ROS and preserved
mitochondrial function. Using metabolomics approach, we aimed to explore, in vitro, the relationship
between lipid peroxidation and perturbed FA oxidation.
Methods: C3A cells were treated with either LPON or oleate for 72 hours. Metabolites were extracted from
the cell pellet using chloroform/methanol/water (1:3:1). LC separation was performed using hydrophilic
interaction chromatography with a ZIC–HILIC. Mass spectrometry was performed using Orbitrap
Exactive with HESI 2 probe. Raw LC/MS data were processed with XCMS Centwave for peak picking
and mzMatch for alignment and annotation of related peaks. Metabolite identification was performed by
matching masses and retention times to the database, with a mass accuracy window of 3, and RT window
of 35% (by in-house VBA :
Results: Ten out of 13 metabolites, corresponding to eicosanoids, were increased by LPON treatment
when compared with oleate or untreated cells. Importantly, F2-isoprostane, an established marker
of oxidative stress, was increased by LPON, but not with oleate. Similarly, the concentrations of all
9 medium and 4 long chain dicarboxylic acids were higher with LPON than oleate. Enhanced lipid
peroxidation with LPON was paralleled by increased levels of all acyl glycine species (N-nonanoylglycine,
N-undecanoylglycine, glutarylglycine and isobutyrylglycine) and even-chain acyl carnitine species
(butanoylcarnitine, hexanoylcarnitine and octanoylcarnitine) - markers of defective beta-oxidation.
Conclusions: Our data suggest that oxidative stress increases formation of both eicosanoids and
dicarboxylic acids, suggesting that the mitochondrial beta-oxidation pathway is overwhelmed, whilst
minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation), are likely to be activated.
Original languageEnglish
Publication statusPublished - Feb 2013
EventlEASL Monothematic Conference: Systems Biology of the Liver: Systems Biology and Clinics Face-à-Face - Luxemburg, Luxembourg
Duration: 21 Feb 201323 Feb 2013

Conference

ConferencelEASL Monothematic Conference: Systems Biology of the Liver: Systems Biology and Clinics Face-à-Face
Country/TerritoryLuxembourg
CityLuxemburg
Period21/02/1323/02/13

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