Triphenylphosphine assay for nanomolar detection of lipid hydroperoxides

Abstract

As the first stable product of lipid oxidation, hydroperoxides have often been the most common marker of oxidation progress. Their importance in the integrated alternated reaction scheme for lipid oxidation merits the need for a sensitive assay. The gold standard assay of hydroperoxide content -- iodometric titration with thiosulfate – has low sensitivity for the amount of sample required, tedious handling, and no possibility for high throughput. More sensitive methods such as xylenol orange and ferric thiocyanate colorimetric assays have debatable stoichiometry and specificity, limited range of quantification, and low stability. Triphenylphosphine (TPP), used for decades to detect trace levels of hydroperoxides in solvents, offers excellent promise as an alternative reagent for specific quantitation of lipid hydroperoxides with greater sensitivity than previous methods. TPP reaction with hydroperoxides yields the optically active triphenylphosphine oxide (TPPO) which can be separated from other reactants by HPLC to create an assay with greater sensitivity, specificity, ease of handling, stability, confirmation of reaction completion, and possibility of high-throughput analyses using autosamplers. A previous study using standards verified quantitation of nanomolar hydroperoxides but did not optimize applicability to lipid hydroperoxides. This thesis extended TPP method development to optimize reaction specifically with lipid hydroperoxides (LOOH). A gradient program that separated TPP, TPPO, LOOH, and product hydroxylipids on a pentafluorophenyl column provided a means of following reaction completion with samples of unknown LOOH content and allowed determination of TPP excess required for complete reaction. Reversed-phase HPLC proved suitable for quantitating hydroperoxides of fatty acid methyl esters, but quantitating hydroperoxides of triacylglycerols required adapting the method to normal-phase HPLC due to their hydrophobicity. Standard curves of TPPO were generated for quantitating lipid hydroperoxides by both reversed-phase and normal phase HPLC. Response curves for TPP reaction with oxidized lipids of various hydroperoxide concentrations (predicted by iodometric titration) were co-linear with the TPPO standard curve, showing that the TPP-lipid hydroperoxide reaction is stoichiometrically 1:1. With reversed-phase HPLC, limits of detection and quantification were 2 pmol and 6.03 pmol TPPO injected (LOOH reacted), respectively. With normal-phase HPLC, limits of detection and quantification were 0.19 pmol and 0.57 pmol TPPO injected (LOOH reacted), respectively.M.S.Includes bibliographical reference