Direct Measurement by Single Photon Counting of Lipid Hydroperoxides in Human Plasma and Lipoproteins

Abstract

A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL–VLDL, escaping from extraction and chromatography, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical point which, in our hands, was limited and poorly reproducible. The sample is added to a reaction mixture containing luminol, hemin, and Triton X-100 in an alkaline buffer, the photon emission is recorded, and the data are processed using the monoexponential decay of the photon emission rate. The measurement is applied to (a) plasma passed through a “desalting” cartridge to eliminate the small water-soluble antioxidants which inhibit the chemiluminescent process or (b) apo-B-containing lipoproteins (LDL–VLDL) isolated by heparin–Sepharose affinity chromatography. The content of lipid hydroperoxides is calculated using an internal calibration with palmitoyllinoleoylphosphatidylcholine hydroperoxide. This procedure, based on a single photon counting technology, was adopted to produce reliable results using samples from which inhibitors of the photon emission process have not been completely eliminated. The specificity of the signal for lipid hydroperoxides was validated by its complete disappearance following incubation of the sample with glutathione and phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12), the sole enzyme specific for all classes of lipid hydroperoxides in lipoproteins. The interassay variability was <10%. The results indicated that the concentration of lipid hydroperoxides in the plasma of 20 healthy subjects was 353 ± 78 nM. In different subjects, LDL–VLDL accounted for 40–80% of the lipid hydroperoxides in plasma.