DIRECT MEASUREMENT BY SINGLE-PHOTON COUNTING OF LIPID HYDROPEROXIDES IN HUMAN PLASMA AND LIPOPROTEINS

A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL-VLDL, escaping from extraction and chromatograp hy, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical poin t 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 emiss ion rate. The measurement is applied to (a) plasma passed through a '' desalting'' cartridge to eliminate the small water-soluble antioxidant s which inhibit the chemiluminescent process or (b) apo-B-containing l ipoproteins (LDL-VLDL) isolated by heparin-Sepharose affinity chromato graphy. The content of lipid hydroperoxides is calculated using an int ernal calibration with palmitoyllinoleoylphosphatidylcholine hydropero xide. This procedure, based on a single photon counting technology, wa s adopted to produce reliable results using samples from which inhibit ors 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 hydrope roxides in lipoproteins. The interassay variability was <10%. The resu lts indicated that the concentration of lipid hydroperoxides in the pl asma of 20 healthy subjects was 353 +/- 78 nM. In different subjects, LDL-VLDL accounted for 40-80% of the lipid hydroperoxides in plasma. ( C) 1995 Academic Press, Inc.