Measurement of Lipid Hydroperoxides in Normal Human Blood Plasma Using HPLC-Chemiluminescence Linked to a Diode Array Detector for Measuring Conjugated Dienes

A modification of a method using high-performance liquid chromatography (HPLC) with chemilumi- nescence (CL) detection for the measurement of lipid hydroperoxides (LOOH) in human blood plasma has been developed. The system involves separation of different classes of LOOH using reverse-phase HPLC, and post-column detection of CL produced by isoluminol oxidation during the reaction of LOOH with microperoxidase. Complete ultra-violet absorption spectra are collected with an in-line diode-array detector and used to confirm a positive CL response due to LOOH, or other compounds, by the presence or absence, respectively, of the LOOH conjugated diene chromophore. We have used the method to investigate the stability of exogenous 15(S)-HPETE (a hydroperoxide of eicosatetraenoic acid) and conjugated dienes (of both 15(S)-HPETE and its reduced metabolite, 15(S)- HETE) in human plasma stored at various temperatures. A large and rapid loss of the hydroperoxide occurred in plasma incubated at 0°C or 27°C, whereas only a small reduction in the level of conjugated dienes was found. 15(S)-HPETE in PBS was stable under the same conditions, and zero time recovery of the hydroperoxide from denatured plasma and from buffer containing albumin was identical to that of fresh plasma. Our data suggest that the observed temperature-dependent loss of exogenous hydroperoxide from fresh plasma results from a combination of enzymatic degradation to the hydroxy derivative and binding to plasma albumin. 15(S)-HPETE was found to be stable in plasma stored at - 70°C for up to 2 weeks and in liquid nitrogen for 3 months in the presence of the antioxidants butylated hydroxytoluene (BHT) and desferal, with no significant loss of conjugated dienes.