Major urinary metabolites of 6-keto-prostaglandin F2α in mice

Abstract

National Institutes of Health has taken the position that the evidence is inconclusive .The omega-3/omega-6 fatty acid "tone" of experimental animals or of humans ingesting nonprescription fi sh oil tablets or prescription supplements (Lovaza®) can be determined by measuring the ratio of omega-3 to omega-6 fatty acids in blood or in red blood cell membranes. Commercial kits are available for these relatively simple blood tests that involve quantifying fatty acid levels in plasma ( 4 ) or fatty acyl compositions of red blood cell lipids ( 5 ). Unfortunately, there is no validated method for estimating tissue levels of omega-3 versus omega-6 fatty acids noninvasively, such as by measuring appropriate metabolites in the urine.Prostaglandins (PG) can be formed from either arachidonic acid (AA), an omega-6 fatty acid, or from EPA, an omega-3 fatty acid, through the sequential actions of a phospholipase A 2 , cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2), and a PG synthase ( 6-10 ). Methods are available for measuring 2-series urinary PG metabolites formed from AA, including the major PGE 2 metabolite (PGEM) tetranor ( 11 ), two dinor metabolites of prostacyclin ( 12 ) and PGD 2 ( 13 ), and thromboxane B 2 metabolites ( 14 ). We reasoned that quantifi cation of a major urinary 3-series PG metabolite(s) of EPA in conjunction with measuring an appropriate 2-series PG metabolite might prove useful in estimating the AA/EPA ratio in tissue phospholipid precursors of PGs.Previous examination of the major urinary metabolites of PGE 3 performed in rats indicated that one of the metabolites is the tetranor derivative of PGE 3 formed via two steps of Abstract Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefi ts. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fi sh oil omega-3 fatty acid that becomes elevated in tissues following fi sh oil consumption. Quantifi cation of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium-and deuterium-labeled 6-keto-PGF 2 ␣ and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF 2 ␣ urinary metabolites included dinor-6-keto-PGF 2 ␣ ( ‫ف‬ 10%) and dinor-13, 14-dihydro-6,15-diketo-PGF 1 ␣ ( ‫ف‬ 10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI 3 by prostacyclin synthase and, fi nally, nonenzymatic hydrolysis to 6-keto-PGF 2 ␣ . The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases. -Kuklev,

Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis

“…[1-14 C]AA (1.85 GBq/ mmol) was from American Radiolabeled Chemicals. [5,8,11,14, 17-3 H]Eicosapentaenoic acid was prepared as described previously (18). Decyl maltoside (C 10 M), n-octyl ␤-D-glucopyranoside, and C 10 E 6 were purchased from Anatrace (Maumee, OH).…”

Biomarkers for Personalizing Omega-3 Fatty Acid Dosing

“…The internal standard d5-EPA was synthesized as described in a recent report (25). This material was used to synthesize d5-PGE 3 using human recombinant COX-2 as described previously (16), making a 800 μg/ml solution in 50 mM PBS.…”

“…The d5-PGE 3 and d5-EPA used in the assays were synthesized in our laboratory (25). Other eicosanoid standards were obtained from Cayman Chemical (Ann Arbor, MI).…”

Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway

“…Moreover, we had no access to potentially highly important local changes such as the formation of 15d-PGJ3 in adipose tissue ( 61 ). A further limitation may come from the use of currently available surrogate parameters (stable prostaglandin degradation products) that only partially refl ect the actual utilization of EPA by COX enzymes ( 62 ). However, based on the properties of the COXs, the capacity of COX-1 to metabolize EPA may be limited by the availability of endogenous hydroperoxides, and COX-2 preferentially oxygenates AA when EPA is simultaneously present ( 60 ).…”