Measurement of urinary F-2,-isoprostanes as markers of in vivo lipid peroxidation - A comparison of enzyme immunoassay with gas chromatography/mass spectrometry

This study aimed at comparing the two most commonly utilized methods for me asuring urinary F-2-isoprostanes, currently considered one of the best avai lable markers of in vivo lipid peroxidation. The F-2-isoprostanes were meas ured in 24-h urine samples from 14 male subjects using electron capture neg ative ionization gas chromatography-mass spectrometry (ECNI-GCMS) with 8-is o-PGF(2 alpha)-d(4) as an internal standard and compared with levels obtain ed using an enzyme immunoassay (EIA, 8-iso-PGF(2 alpha) kit, Cayman Chemica l Co.). The methods were compared using Pearson correlation coefficients, a nd Bland-Altman plots were constructed for the difference in F-2-isoprostan e against the average F-2-isoprostane measured by either method. Weighted l east-products regression was used to determine fixed bias (where there is a consistent difference between the methods) and proportional bias (where on e method gives values higher or lower than the other method by an amount pr oportional to the size of the measurement). The correlation between F-2-iso prostane levels obtained using EIA and GCMS methods, although significant, was poor (r = 0.628, P < 0.02). Comparison of the methods using the Bland-A ltman analysis showed that there were wide limits of agreement between the two methods with only 28% of the values falling within the 95% confidence l imits for the difference. The GCMS gave higher values with a mean differenc e of 298.1 pM (636.6, -40.2; P = 0.079), and a near significant linear asso ciation between the differences and the mean F-2-isoprostane level (r = -0. 559, P = 0.05). Weighted least-product regression analysis confirmed the pr esence of both significant fixed and proportional bias with the EIA giving lower levels of F-2-isoprostanes at low concentrations and higher levels at higher concentrations. The cross-reactivity in the EIA of 8-iso-15(R)-PGF( 2 alpha) and 9 beta-PGF(2 alpha) which coelute with the F-2-isoprostane pea k measured by GCMS was very low, 0.2 and 0.1%, respectively. The proportion al bias observed between the methods may in part be due to differences in t he relative amounts of 8-iso-15(R)-PGF(2 alpha), 9 beta-PGF(2 alpha), and 8 -iso-PGF(2 alpha) with increasing lipid peroxidation. This study shows that the measurements of F-2-isoprostanes by EIA and GCMS are not equivalent. T herefore, comparison of levels derived using a GCMS method which estimates concentration from a peak encompassing a number of F-2-isoprostane isomers, and levels derived from enzyme immunoassay measuring a specific isoprostan e, may be inappropriate. (C) 1999 Academic Press.