Effect of Fish Oil on Levels of R- and S-Enantiomers of 5-, 12-, and 15-Hydroxyeicosatetraenoic Acids in Mouse Colonic Mucosa

Abstract

The balance of putative pro- and antiinflammatory lipoxygenase (LOX)-derived S-hydroxyeicosatetraenoic acids (S-HETEs) in colon mucosa is a potential target for modulating colon cancer risk and progression. The biological effects of S-HETEs and R-hydroxyeicosatetraenoic acids (produced by distinct pathways) may differ, but levels of these compounds in the colon are unknown. The objective of this study was to develop chiral methods to characterize hydroxyeicosatetraenoic (HETE) enantiomers in colonic mucosa and evaluate the effects of fish oil on HETE formation. C57BL/6 mice (COX-1 null, COX-2 null, wild-type) were fed a diet supplemented with either olive oil or menhaden oil for 11 wk, and R-/S-HETEs in colonic mucosa were quantified by chiral LC-MS/MS. The R-enantiomer comprised 60–72% of 5-HETE, 18–58% of 15-HETE, and 1–16% of 12-HETE in colonic mucosa, suggesting that non-LOX sources contribute to HETE profiles. Fish oil reduced levels of both R- and S-HETEs, and increased the preponderance of the R-enantiomers (particularly 12- and 15-HETEs). There was apparent shunting of arachidonic acid to 12-/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo and shows large effects of fish oil in the normal colon.

ACKNOWLEDGMENTS

We thank Dr. Robert Langenbach for providing the transgenic animals (originally obtained from Taconic) and Dr. William Lands for advice on formulation of the rodent diets. Patrick Brown assisted with statistical data analysis, and Dr. Kathleen Noon assisted with chiral chromatographic LC-MS-MS methods.

This research was supported by the Michigan Institute for Clinical & Health Research (grant GM 48864), the University of Michigan Comprehensive Cancer Center (grant P30-CA46592), NCI grant RO1 CA120381, NIH grant R01 GM68848, and the Kutsche Memorial Endowment in Internal Medicine at the University of Michigan Medical School. A. Neilson was supported by the NCI T32 Cancer Biology Training Program at the University of Michigan (grant 5T32CA009676-18).

Andrew P. Neilson is now with the Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.

Notes

^†In chiral LC, the R- and S-enantiomers of each HETE are resolved and elute as 2 distinct peaks. Both peaks for each HETE were quantified relative to the respective S-HETE-d₈ internal standard.

^†Values represent mean ± SEM.

^‡Means that differ significantly within each column are denoted as follows: significantly different than oleate diet for mice with the same genotype at ^a P< 0.05, ^b P< 0.01, ^c P< 0.001. No differences were detected between genotypes within the same diet.

^ζThe % of highly unsaturated fatty acids (≥20 carbons and ≥3 double bonds) that are ω3.

^†Values represent mean ± SEM.

^‡Within each column, statistically significant differences (P < 0.05) between means are indicated by superscripts: ^aSignificantly different from wild-type mice fed the same diet. ^bSignificantly different from COX-1 null mice fed the same diet, ^cSignificantly different from mice fed the oleate diet in the same genotype.