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Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Published on: November 17, 2011
Characterization of lipoxygenase oxidation products by high-performance liquid chromatography with electron
A Nuñez1, T A Foglia, G J Piazza
1USDA, ARS, ERRC, Wyndmoor, Pennsylvania 19038, USA. anunez@arserrc.gov
Abstract:
Lipoxygenase (LOX) is an enzyme that oxygenates polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. For example, LOX found in plants produce the corresponding 13- and 9-hydroperoxide derivatives of linoleic acid (13-HPOD and 9-HPOD). Identification of the HPOD products is usually accomplished by using gas chromatography with mass spectrometric (MS) detection, which requires extensive derivatization of the thermally unstable hydroperoxy group. Here we report a high-performance liquid chromatographic method in combination with electron impact (EI)-MS detection that separates and characterizes the HPOD isomers generated by soybean LOX type I oxygenation of linoleic (LA) and linolenic acids as well as HPOD products produced by photosensitized oxidation of LA. The method does not required derivatization of the hydroxyperoxide group, and location of its position can be determined by the EI-MS fragmentation pattern. The method has been used for the analysis of HPOD produced by action of partially purified LOX from the micro-alga Chlorella pyrenoidosa on LA. The study suggests the presence of two LOX isozymes in the micro-alga that oxygenate LA to its 13-HPOD and 9-HPOD derivatives. Moreover, the 9-LOX isozyme under anaerobic conditions cleaves 13-HPOD to 13-oxo-tridecadienoic acid and pentane but does not cleave 9-HPOD.
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