Session: Session 3

Diet, lipid peroxidation and colorectal cancer: Targeted UHPLC-ESI-MS/MS analysis of hydroxyalkenal biomarkers in rat serum subjected to different diets.

Romain VUILLAUME^1,2, Ndeye DIENG^1,2, Loic MERVANT^1,2, Charline BUISSON¹, Françoise GUÉRAUD¹, Fabrice PIERRE ¹, Laurent DEBRAUWER^1,2, Sylvie CHEVOLLEAU^1,2

¹Toxalim (Research Center in Food Toxicology), INRAE, UMR1331, ENVT, INP-Purpan, Université de Toulouse, Toulouse, France
²Metatoul-Axiom Platform, UMR 1331 Toxalim, INRAE, MetaboHUB-MetaToul, National Infrastructure of Metabolomics and Fluxomics, Toulouse, France

Introduction

Diets rich in red meat are known to be associated with a high risk of colorectal cancer. A major hypothesis involves dietary heme iron catalyzing lipid peroxidation inducing the formation of hydroxyl alkenals from polyunsaturated fatty acids. Several aldehydes, in particular 4-hydroxy-2(E)-nonenal (HNE) and 4-hydroxy-2(E)-hexenal (HNE), are known for their reactivity, cytotoxic and genotoxic activities against colon epithelial cells. Quantifying these representative hydroxyl alkenals in different diets, during digestion and in biofluids is highly instructive in a toxicological context, enabling correlation with other biological and biochemical markers of colorectal cancer promotion.

In previous works, we have developed, validated and published a methodology combining isotope-labeling derivatization of these unstable aldehydes and LC-MS/MS analysis for the quantification of HNE and HHE in faeces in accordance with EMEA guidelines (1).

Methodology

The methodology was transposed to rat serum matrix in order to assess the circulation of these compounds in the blood. Serum samples were derivatized in situ with Bromo benzene hydroxylamine (BBHA) in the presence of deuterated internal standards (HNE-D11/HHE-D5), extracted by solid-phase extraction, then analyzed by UHPLC-ESI-MS/MS on a C18 column, in MRM mode on an ALTIS + triple quadrupole instrument (ThermoScientific).

Results and conclusion

After optimization, the entire method, validated in terms of sensitivity, linearity, memory effect, repeatability, trueness and accuracy, was applied to the analysis of serum samples obtained from rats fed different diets containing red or white meat, lipids rich in omega-6 (HNE precursors) or omega-3 (HHE precursors), and antioxidants. Results show specific modulation of the formation of these lipid peroxidation products depending on the diet composition.

(1) S. Chevolleau et al. (2018) J. Chrom. B, 1083, 171.