9-oxo-10-12-octadecadienoic-acid and 9-hydroxy-10-12-octadecadienoic-acid

Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-​octadecadienoic acid (HODE) and 9- and 13-oxo-​octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Topics: Animals; Chromatography, Liquid; Limit of Detection; Linoleic Acids; Linoleic Acids, Conjugated; Linolenic Acids; Rats; Reproducibility of Results; Tandem Mass Spectrometry

Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid, (10E,12Z,15Z)-9-oxo-10,12,15-octadecatrienoic acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and (10E,12Z)-9-oxo-10,12-octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co-chromatography with authentic standards on various types of HPLC columns including chiral-phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy-polyenoic fatty acids. Air-drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids. The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.

Topics: Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Esterification; Linoleic Acids; Linoleic Acids, Conjugated; Linolenic Acids; Mass Spectrometry; Plants