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

Abstract

The balance of putative pro- and anti-inflammatory 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-HETEs (produced by distinct pathways) may differ, but levels of these compounds in colon are unknown. The objective of this study was to develop chiral methods to characterize 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 weeks, 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 to12/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo.

Biomarkers for Personalizing Omega-3 Fatty Acid Dosing

“…Previously, our group developed a chiral LC/MS-MS method to quantify HETEs and HODEs in the colonic tissue of rodents (27). The technology permits more efficient interrogation of eicosanoid products beyond COX products.…”

“…Other eicosanoid standards were obtained from Cayman Chemical (Ann Arbor, MI). The LC-MS/MS method for eicosanoid analysis was adapted from the literature as previously described (27, 28). For extraction of eicosanoids, 300 μL of the colonic homogenate was added to 12 × 75 mm glass tubes on ice, along with 1 N citric acid (20 μL), 30 mM disodium EDTA (20 μL), and deuterated internal standard.…”

“…The linear gradient program was as follows: 30–50% B (0–5 min), 50–100% B (5–24 min), 100% B (25–30 min), and return to 30% B (30–37 min). The effluent was introduced into a Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer by electrospray ionization (ESI) and detection of negative ions was as previously described (27). Eicosanoid concentrations in colonic tissue were normalized to protein concentrations.…”

The Anti-inflammatory Effect of Personalized Omega-3 Fatty Acid Dosing for Reducing Prostaglandin E2 in the Colonic Mucosa Is Attenuated in Obesity

“…Three frozen biopsies were pulverized in liquid nitrogen, and pooled homogenates were prepared in buffered saline containing indomethacin as described previously (20, 21). Deuterated internal standards were added before extraction and analysis of eicosanoids (20).…”

Reducing Proinflammatory States with the Mediterranean Diet

“…Fortunately, methods for detecting proinflammatory states have improved over the years. Eicosanoids can be profiled in tissues and urine using chromatographic methods coupled with mass spectral detection [15]. Multiplex immunoassays allow for simultaneous detection of proinflammatory and immunoregulatory cytokines in small sample volumes [16].…”