A comparison of methods for the measurement of 8-isoPGF 2 α : a marker of oxidative stress

BACKGROUND: Oxidative stress describes the cellular damage caused by excess reactive oxygen species not adequately inactivated by antioxidants. Oxidative stress has been implicated in playing a role in many disorders. Lipid peroxidation end-products are employed as markers of oxidative stress, of which the isoprostane, 8-iso-PGF(2α), is widely used. 8-iso-PGF(2α) is measured in plasma or urine by gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectrometry (LC/MS), tandem-mass spectrometry or enzyme-linked immunosorbent assay (ELISA). However, discrepancies between the specificity of these methods means correlation is poor. METHODS: A tandem-mass spectrometric (LC/MS/MS) method, using immunoaffinity purification, for urinary 8-iso-PGF(2α) was developed and compared with two commercial ELISAs (A--Cayman Chemicals, B--Oxford Biomedical Research) in urine samples (n = 156). RESULTS: An LC/MS/MS method coupled to immunoaffinity purification was developed with satisfactory performance and comparison to ELISAs A and B. Spearman rank correlation demonstrated significant correlation between all methods (P = <0.0001); however, r² values ranged from 0.68 to 0.72. Bland-Altman plots revealed a proportional positive bias of ELISA B when compared with ELISA A and LC/MS/MS. Furthermore, the agreement between ELISA A and LC/MS/MS was poor. CONCLUSIONS: The poor agreement between methods for measurement of 8-iso-PGF(2α) highlights differences in selectivity. 8-iso-PGF(2α) is an isoprostane, a family of isomeric end-products of arachidonic acid peroxidation, which are produced by peroxidation or enzymatically. This makes avoiding cross-reactivity between 8-iso-PGF(2α) and related isomers challenging. When assessing oxidative stress studies, the selectivity of the methods used should be taken into account, particularly when comparing studies.