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.

Figure 1

A: Structures of measured HETEs in colonic mucosa. Chiral carbons are indicated by *. B: Pathways of arachidonic acid metabolism leading to the formation of R- and S-HETEs. The enantiomeric composition of non S-LOX mediated pathways are unknown.

Figure 2

Selected reaction monitoring (SRM) chromatograms demonstrating the resolution of R- and S-HETEs in mouse colonic mucosa by chiral LC-MS/MS.

Figure 3

Levels of R- and S-HETEs in mouse colon mucosa. Error bars represent SEM. Brackets indicate significant differences between means (*P<0.05). Note the different scales for 12-S-HETE and 12-R-HETE.