Peroxidation of erythrocytes: FTIR spectroscopy studies of extracted lipids, isolated membranes, and intact cells

Abstract

AbstractFourier transform infrared (FTIR) spectroscopy was used to directly monitor peroxidative damage to membrane phospholipid acyl chains in erythrocyte membranes. Samples were suspended in a mixed 2H2O/H2O buffer system, thereby producing a “spectral window” in the C‐H stretching region of the infrared spectrum. A decrease in the number of acyl chain CC bonds upon erythrocyte peroxidation was quantitated directly from the spectra of isolated membranes. Second‐derivative spectroscopy permitted the conformationally sensitive membrane acyl chain methylene stretching modes to be separated from the protein (mostly hemoglobin) vibrations that dominate the spectra of intact cells. The sensitivity of these modes in erythrocytes was then determined in a series of thermotropic experiments. The effect of peroxidation upon the membrane acyl chain conformational order was monitored in isolated membranes and intact cells. No change in conformational order was detected upon peroxidation in intact cell and ghost spectra. In contrast, experiments with pure unsaturated phospholipids demonstrated that decreasing the CC bond population results in increased conformational order. The finding of identical results for peroxidation versus control samples in isolated membranes (“ghosts”) validates the results observed for intact cells. FTIR technology permits the direct monitoring of conformational order in the acyl chains of intact cells. © 1995 John Wiley & Sons, Inc.