A sensitive chemiluminescence method to measure the lipoxygenase catalyzedoxygenation of complex substrates

Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenas es (15-LOX) may be involved. While studying the in vitro kinetics of the 15 -LOX/LDL interaction, we found that the conventional spectrophotometric ass ays failed in the range of substrate saturation owing to the high optical d ensity of concentrated LDL solutions. Therefore, we developed a much more s ensitive assay system which was based on peroxide induced isoluminol enhanc ed chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric metho d the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid ( K-m = 3.7 mu M, k(cat) = 17 s(-1)) were determined and we observed a good a greement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LD L oxidation (K-m = 0.64 mu M, k(cat) = 0.15 s(-1)) are quite different from those of free fatty acid oxygenation and that the cholesterol eaters are p referentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion d oes not reduce the rate of LDL oxidation and analysis of the structure of t he oxygenation products suggests that the majority of the products were for med via direct LOX catalyzed oxidation of LDL ester lipids. The luminometri c method described here is not restricted to the measurement of LOX catalyz ed LDL oxidation, but may also be used to determine kinetic constants for t he oxidation of other complex substrates such as biomembranes or liposomes. (C) 1999 Elsevier Science B.V. All rights reserved.