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Quantitative assays for esterified oxylipins generated by immune cells.

ABSTRACT: Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins--in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids--attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC-tandem mass spectrometry (LC/MS/MS) in multiple reaction-monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ?2 d. Following this, extraction of 30 samples takes ?3 h, with LC/MS/MS run time of 50 min per sample.

SUBMITTER: Morgan AH

PROVIDER: S-EPMC3678246 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Quantitative assays for esterified oxylipins generated by immune cells.

Morgan Alwena H AH Hammond Victoria J VJ Morgan Lloyd L Thomas Christopher P CP Tallman Keri A KA Garcia-Diaz Yoel R YR McGuigan Christopher C Serpi Michaela M Porter Ned A NA Murphy Robert C RC O'Donnell Valerie B VB

Nature protocols 20101110 12

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins--in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids--attached to either phosph ...[more]

PMID: 21127486

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Project description:INTRODUCTION:Oxylipins are bioactive oxidation products derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs) in the linoleic acid and α-linolenic desaturation pathways. PURPOSE:This study determined if carbohydrate intake during prolonged and intensive cycling countered post-exercise increases in n-6 and n-3 PUFA-derived oxylipins. METHODS:The research design utilized a randomized, crossover, counterbalanced approach with cyclists (N = 20, overnight fasted state, 7:00 am start) who engaged in four 75-km time trials while ingesting two types of bananas (Cavendish, Mini-yellow), a 6% sugar beverage, and water only. Carbohydrate intake was set at 0.2 g/kg every 15 minutes, and blood samples were collected pre-exercise and 0 h-, 0.75 h-,1.5 h-, 3 h-, 4.5 h-, 21 h-, 45 h-post-exercise. Oxylipins were measured with a targeted liquid chromatography-multiple reaction monitoring mass spectrometric method. RESULTS:Significant time effects and substantial fold-increases (immediately post-exercise/pre-exercise) were measured for plasma levels of arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and 43 of 45 oxylipins. Significant interaction effects (4 trials x 8 time points) were found for plasma ARA (P<0.001) and DHA (P<0.001), but not EPA (P = 0.255), with higher post-exercise values found in the water trial compared to the carbohydrate trials. Significant interaction effects were also measured for 12 of 45 oxylipins. The data supported a strong exercise-induced increase in plasma levels of these oxylipins during the water trial, with carbohydrate ingestion (both bananas types and the sugar beverage) attenuating oxylipin increases, especially those (9 of 12) generated from the cytochrome P-450 (CYP) enzyme system. These trials differences were especially apparent within the first three hours of recovery from the 75-km cycling bout. CONCLUSIONS:Prolonged and intensive exercise evoked a transient but robust increase in plasma levels of oxylipins, with a significant attenuation effect linked to acute carbohydrate ingestion for 28% of these, especially those generated through the CYP enzyme system. TRIAL REGISTRATION:ClinicalTrials.gov, U.S. National Institutes of Health, NCT02994628.

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Quantitative assays for esterified oxylipins generated by immune cells.

Publications

Quantitative assays for esterified oxylipins generated by immune cells.

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