Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive ‘single photon counting’ luminometer

Abstract

The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present in the sample (as in the case of lipoproteins). Thus, by using an internal calibration with a phospholipid hydroperoxide, the evaluation of the lipid hydroperoxide content in whole, native lipoproteins was possible without previous extraction and chromatographic separation. Data obtained from plasma lipoproteins isolated by different procedures suggest that lipid hydroperoxide content increases during ultracentrifugation