Erythrocyte Peroxidation: Quantitation by Fourier Transform Infrared Spectroscopy

We used Fourier transform infrared spectroscopy (FT-IR) to quantitatively monitor peroxidative damage in model phospholipids and in human erythrocyte membranes. The 3012-cm−1 band arising from olefinic C-H stretching modes diminished in intensity during membrane peroxidation as a result of loss of unsaturation in the phospholipid acyl chains. A precise (correlation coefficient = 0.99) linear relationship between the number of CC bonds in the chains and the fraction of the total C-H stretching contour occupied by the olefinic C-H stretch was established for model phospholipids. Intact human erythrocytes and isolated ("ghost") membranes were also peroxidized and the loss of CC bonds was demonstrated by FT-IR. GC studies of erythrocyte membranes confirmed that the primary site of peroxidation occurred at phospholipids containing arachidonate acyl chains. Finally, the formation of a new feature at 1260 cm−1 in the FT-IR spectrum was observed in both model compounds and erythrocyte membranes undergoing peroxidative damage. The feature corresponds to a C-O stretching and/or O-H inplane bending vibration and is suggestive of the formation of these functional groups during peroxidation. FT-IR provides an accurate means for monitoring the effects of peroxidative damage in human erythrocytes.