Measurement of Urinary F₂-Isoprostanes as Markers of in Vivo Lipid Peroxidation—A Comparison of Enzyme Immunoassay with Gas Chromatography/Mass Spectrometry

Abstract

This study aimed at comparing the two most commonly utilized methods for measuring urinary F₂-isoprostanes, currently considered one of the best available markers of in vivo lipid peroxidation. The F₂-isoprostanes were measured in 24-h urine samples from 14 male subjects using electron capture negative ionization gas chromatography–mass spectrometry (ECNI–GCMS) with 8-iso-PGF_2α-d₄ as an internal standard and compared with levels obtained using an enzyme immunoassay (EIA, 8-iso-PGF_2α kit, Cayman Chemical Co.). The methods were compared using Pearson correlation coefficients, and Bland–Altman plots were constructed for the difference in F₂-isoprostane against the average F₂-isoprostane measured by either method. Weighted least-products regression was used to determine fixed bias (where there is a consistent difference between the methods) and proportional bias (where one method gives values higher or lower than the other method by an amount proportional to the size of the measurement). The correlation between F₂-isoprostane levels obtained using EIA and GCMS methods, although significant, was poor (r = 0.628, P < 0.02). Comparison of the methods using the Bland–Altman analysis showed that there were wide limits of agreement between the two methods with only 28% of the values falling within the 95% confidence limits for the difference. The GCMS gave higher values with a mean difference of 298.1 pM (636.6, −40.2; P = 0.079), and a near significant linear association between the differences and the mean F₂-isoprostane level (r = −0.559, P = 0.05). Weighted least-product regression analysis confirmed the presence of both significant fixed and proportional bias with the EIA giving lower levels of F₂-isoprostanes at low concentrations and higher levels at higher concentrations. The cross-reactivity in the EIA of 8-iso-15(R)-PGF_2α and 9β-PGF_2α which coelute with the F₂-isoprostane peak measured by GCMS was very low, 0.2 and 0.1%, respectively. The proportional bias observed between the methods may in part be due to differences in the relative amounts of 8-iso-15(R)-PGF_2α, 9β-PGF_2α, and 8-iso-PGF_2α with increasing lipid peroxidation. This study shows that the measurements of F₂-isoprostanes by EIA and GCMS are not equivalent. Therefore, comparison of levels derived using a GCMS method which estimates concentration from a peak encompassing a number of F₂-isoprostane isomers, and levels derived from enzyme immunoassay measuring a specific isoprostane, may be inappropriate.