EFFECT OF OXYGEN-TENSION ON THE GENERATION OF F-2-ISOPROSTANES AND MALONDIALDEHYDE IN PEROXIDIZING RAT-LIVER MICROSOMES

Although numerous methods have been developed for the detection of lipid peroxidation, it is generally recognized that most of these lack specificity and/or sensitivity, particularly when applied to in vivo situations. We have reported recently that a series of prostaglandin F-2-like compounds, termed F-2-isoprostanes, are formed in vivo from the free radical catalyzed peroxidation of arachidonic acid and appear to be a useful marker of oxidant stress. Because the formation of other products of lipid peroxidation, such as alkanes and malondialdehyde (MDA), are affected by oxygen tension, which may influence their usefulness as markers of oxidant stress, we carried out a systematic study of the generation of F-2-isoprostanes at various oxygen concentrations and compared these changes with the generation of MDA. The disappearance of the F-2-isoprostane precursor, arachidonic acid, was used as a reference measure. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N-2 and various concentrations of O-2 up to 100%. F-2-isoprostanes were quantified by mass spectrometry and MDA by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Both MDA and F-2-isoprostane formation increased in a time-dependent manner up to 15 min. Their formation correlated with a loss of polyunsaturated fatty acid and with an increase in O-2 tension up to 21% O-2 At oxygen tensions above 21%, MDA generation continued to increase, while F-2-isoprostane generation and arachidonic acid loss did not. Levels of MDA and F-2-isoprostanes increased a maximum of 65 and 9.4 times baseline values, respectively. These studies, therefore, define factors that influence the formation of F-2-isoprostanes in an in vitro model of lipid peroxidation. Further, they demonstrate that higher O-2 tensions do not block formation of F-2-isoprostanes and validate their usefulness for assessing lipid peroxidation under high, as well as low, oxygen tension.