CONTINUOUS MONITORING OF INTERMEDIATES AND FINAL PRODUCTS OF OXIDATION OF LOW-DENSITY-LIPOPROTEIN BY MEANS OF UV-SPECTROSCOPY

The most widely used routine technique for determination of LDL 'oxidi zability' is the continuous monitoring of the absorption at 234 nm in the UV spectrum of LDL, following the addition of an oxidation promoto r such as copper ions. This absorption is commonly attributed to the c onjugated dienes formed upon oxidation as the major intermediate, name ly the hydroperoxides of polyunsaturated fatty acids, mostly linoleate hydroperoxides. These, however, are not the only products of oxidatio n that absorb light at 234 nm. Other products, particularly 7-ketochol esterol, also absorb light at the same wavelength. Furthermore, enals and dienals also absorb in the wavelength range of 210-300 nm. The aim of the present work was to develop a simple spectroscopic method for more detailed investigation of the kinetics of lipoprotein oxidation. The method is based on continuous measurement of the UV spectrum in th e wavelength range of 210-300 nm and subsequent decomposition of the s pectra into four absorption bands due to hydroperoxides, 7-ketocholest erol, dienals and enals. The sixth derivatives of the spectra, recorde d during the first seven hours of copper-induced oxidation of LDL were used to monitor the growth and subsequent decay of the hydroperoxides . The resultant time course, in conjunction with difference spectra ob tained after the concentration of these intermediates decay to zero, e nabled us to determine the spectra of the other oxidation products and , by that, to evaluate their time dependencies. Based on these results , we present a series of four simple equations that can be used to eva luate the concentrations of the individual products of LDL oxidation f rom UV absorption measurements of the mixtures at merely four differen t wavelengths. The resultant time dependencies of the accumulation of four major products of lipid oxidation are consistent with published d ata obtained through separation and chemical analysis. This simple met hod can be used for more meaningful routine kinetic measurements of li pids oxidation.