Measurement of Urinary F₂-Isoprostanes as Markers of in Vivo Lipid Peroxidation: A Comparison of Enzyme Immunoassays with Gas Chromatography–Mass Spectrometry in Domestic Animal Species

Abstract

F(2)-isoprostanes are useful markers for assessing oxidant injury; however, the validity of measuring urinary 15-F(2t)-isoprostane concentration by enzyme-linked immunosorbent assay (ELISA) has not been evaluated in veterinary species. The current study assesses the agreement between 2 commercially available urinary isoprostane kits and gas chromatography and negative ion chemical ionization-mass spectrometry (GC/NICI-MS). The results indicate that only feline urinary isoprostane measurement by glucuronidase (GL)-ELISA has acceptable agreement with GC/NICI-MS. Urinary isoprostane concentration was highly variable in critically ill animals, but there were too many variations between healthy and critically ill animals to draw meaningful conclusions. Currently, GC/NICI-MS is the only method that can be recommended for the assessment of urinary isoprostanes in dogs, cattle, and horses. Feline urinary isoprostanes can be assessed by GL-ELISA, but caution is still warranted when comparing data from manuscripts using different methods given the relatively low Spearman rank correlation coefficient. Future studies may require large sample sizes or focused inclusion criteria to account for variability in isoprostane concentration.

Urinary F2‐Isoprostanes in Cats with International Renal Interest Society Stage 1–4 Chronic Kidney Disease

“…We used an immunoassay for IsoP measurements, rather than 1 of several mass spectrometry (MS) methods that are considered gold standards,41 but MS methods are more expensive and labor‐intensive,41 and suboptimal for veterinary use. In a previous study in 20 cats, the same Cayman ELISA (with slightly different preparative steps) correlated significantly with gas chromatography/negative ion chemical ionization‐MS, but the correlation was somewhat weak ( r = 0.678), with the ELISA measuring lower concentrations than MS (fixed bias), and sometimes missing low concentrations that were detectable by MS (proportional bias) 42. In our study, we did not have the same sensitivity problem and were able to detect urinary IsoPs in all cats.…”

Lipid Peroxidation and Antioxidants in Arterial Hypertension

“…Second, they provide a noninvasive route for systemic oxidative stress evaluation. Although they can also be locally produced in the kidney, many studies have demonstrated that urinary F2-isoprostanes are mainly derived from free F2-isoprostanes filtered from the circulation [97,149,151,152]. Only hydrolyzed isoprostanes are excreted into the urine whereas blood plasma samples contain both free and esterified isoprostanes.…”

“…Furthermore, F2-isoprostanes have been shown to exert potent vasoconstrictor effects on animal and human vessels, suggesting a pathogenic role in cardiovascular diseases and have been extensively used as markers of lipid peroxidation in human diseases [74,75,148]. Their high stability and presence in measurable concentrations in many biological tissues and fluids, under physiological and pathological conditions, has also allowed the establishment of reference intervals and the comparison or monitoring of disease states [97,149,150]. Urine specimens are particularly suited for F2-isoprostanes measurements.…”

“…Urine specimens are particularly suited for F2-isoprostanes measurements. First, the ex vivo formation of F2 isoprostanes is minimized in these samples due to the low urinary lipid content, avoiding the need for time-sensitive sample processing [97,149,151,152]. Second, they provide a noninvasive route for systemic oxidative stress evaluation.…”

Automation of Methods for Determination of Lipid Peroxidation

“…For the monitoring of lipid peroxidation, spectrophotometric [30,31], chromatographic [32] and immunochemical [33] methods can be used. The analysis itself may be based on the analysis of the primary products of lipid peroxidation as conjugated dienes [34] and lipid hydroperoxides [35], or secondary products, such as malondialdehyde [36], alkanes [37] or isoprostanes [32,[38][39][40]. Chromatographic methods represent the special group of methods, which are mostly based on the decrease of unsaturated fatty acids' concentration [41].…”