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 'oxidizability' is the continuous monitoring of the absorption at 234 nm in the UV spectrum of LDL, following the addition of an oxidation promotor such as copper ions. This absorption is commonly attributed to the conjugated dienes formed upon oxidation as the major intermediate, namely the hydroperoxides of polyunsaturated fatty acids, mostly linoleate hydroperoxides. These, however, are not the only products of oxidation that absorb light at 234 nm. Other products, particularly 7-ketocholesterol, 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 the wavelength range of 210-300 nm and subsequent decomposition of the spectra into four absorption bands due to hydroperoxides, 7-ketocholesterol, dienals and enals. The sixth derivatives of the spectra, recorded 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 obtained after the concentration of these intermediates decay to zero, enabled 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 evaluate the concentrations of the individual products of LDL oxidation from UV absorption measurements of the mixtures ar merely four different wavelengths. The resultant time dependencies of the accumulation of four major products of lipid oxidation are consistent with published data obtained through separation and chemical analysis. This simple method can be used for more meaningful routine kinetic measurements of lipids oxidation.