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Urinary F2‐isoprostane metabolite analysis: a step closer to obtaining a reliable measure of oxidative stress?

Oxidative stress, the consequence of an unequal balance between reactive oxygen species (ROS) production and antioxidant defences has been implicated in a wide range of diseases, including those of the respiratory system. In its most severe form, redox imbalance can result in cell death following widespread macromolecule oxidation while more subtle redox changes appear to play a role in modulating a range of signal transduction pathways [1]. All molecules are potential targets for ROS, but because of their ubiquitous distribution within cell membranes and their propensity to contain double bonds, unsaturated lipids are often targeted. Indeed, investigators determining the impact of the oxidation of cell lipids drove the massive growth in free radical biology during the 1960s and 1970s. Unfortunately, most of the early methods used to assess lipid oxidation were non-specific, inaccurate and gave variable results in different laboratories. As a result of these limitations, their application in clinical studies provided little major advancement in knowledge. During the 1980s and 1990s methodological advances gave rise to more accurate ways to assess the extent of lipid peroxidation occurring in vivo. These included the measurement of malondialdehyde and lipid hydroperoxide content of biological samples by hplc [2]. In 1990, Morrow, Roberts and co-workers provided a further advance by reporting the discovery of prostaglandin F2 -like compounds, the F2 -isoprostanes [3]. Isoprostanes are formed by direct ROS attack of arachidonic acid, an unsaturated fatty acid component of cell membranes and low density lipoprotein (LDL). Unlike prostaglandins, enzymatic products of this fatty acid, isoprostanes are formed initially in situ in cell membranes from which they are subsequently cleaved by phospholipase(s). Although up to 64 different isoprostanes can arise from the oxidation of arachidonic acid, one of the most stable products is 8-iso-prostaglandin F2a (8-iso-PGF2a). As a consequence it is this isoprostane which has received most attention in human studies. Perhaps as the field was in such need of a reliable and meaningful measure of in vivo lipid peroxidation, 8-isoPGF2a was rapidly adopted by many as a `gold standard' marker. Although a little premature in this respect, determining 8-iso-PGF2a concentrations does have several advantages over many existing methods used to access oxidative stress. For example, 8-iso-PGF2a is both a specific and stable product of lipid peroxidation. Moreover, the quantities of 8-iso-PGF2a increase markedly in a number of animal models of oxidant injury and this can be modified by antioxidant status [3,4]. In humans, most 8-iso-PGF2a analyses have been undertaken using blood and urine samples. Measurements in blood provide an index of isoprostane formation over a relatively short period as their half-life in this compartment is only 20 min [5]. Moreover, because blood contains substantial quantities of arachidonic acid, the substrate for 8-iso-PGF2a, there is always the potential of artificial generation of 8-iso-PGF2a through autoxidation, if the samples are not processed and stored properly. Measurements of 8-iso-PGF2a in urine partially circumvent this problem but worries about potential contributions of local formation of isoprostanes in the kidney also exist [6]. As the urinary excretion of prostaglandin metabolites was the most reliable measure of total endogenous prostanoid production, efforts have been directed at developing a technique to measure metabolites of 8-iso-PGF2a in urine. Following such an approach, Roberts, Morrow and coworkers identified a single predominant metabolite of 8iso-PGF2a in urine [7]. The metabolite, identified by mass spectrometer analysis as 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M) is present at baseline concentrations of 0.39 ^ 0.18 ng/mg creatinine. Moreover, this level is increased 2.5-fold in patients with polygenic hypercholesterolemia and can be suppressed 54% following 8 weeks treatment with a combination of vitamin E, C and b-carotene [8]. Although now widely utilized in a number of fields, isoprostane analysis has only recently been undertaken in respiratory research. In the last 2 years studies by Barnes and colleagues have demonstrated that 8-iso-PGF2a concentrations are increased in breath condensate of patients with asthma [9], COPD and cystic fibrosis [10]. Although encouraging, these data obtained with use of a commercial ELISA kit for 8-iso-PGF2a need confirmation, as urinary analysis of 8-iso-PGF2a by ELISA and mass spectrometry have revealed considerable inconsistencies [11]. Even so while we wait for confirmation, such findings help support long-held suspicions of many investigators that oxidative