Quantification of 15‐F_2t‐isoprostane in human plasma and urine: results from enzyme‐linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared

Abstract

Quantification of F(2)-isoprostanes is considered a reliable index of the oxidative stress status in vivo. Several immunoassays and chromatography/mass spectrometry-based assays are available for 15-F(2t)-isoprostane quantification. However, it remains unclear if results of immunoassays using different assays can be compared with those of liquid chromatography/mass spectrometry (LC/MS) assays. Previous studies comparing enzyme-linked immunosorbent assay (ELISA) and more specific gas chromatography/mass spectrometry assays have already indicated that ELISAs may overestimate 15-F(2t)-isoprostane concentrations in human plasma. Concentrations of 15-F(2t)-isoprostane in 25 human plasma and urine samples were measured by three commercially available ELISA assays (Assay Designs, Cayman Chemical and Oxford Biomedical Research) and compared with the concentrations measured with a validated, semi-automated high-throughput HPLC tandem mass spectrometry assay (LC/LC-MS/MS). All three ELISAs measured substantially higher 15-F(2t)-isoprostane concentrations (2.1-182.2-fold higher in plasma; 0.4-61.9-fold higher in urine) than LC/LC-MS/MS. Utilization of solid-phase extraction (SPE) columns, especially isoprostane affinity purification columns, brought ELISA isoprostane urine concentrations closer to the LC/LC-MS/MS results. However, SPE did not have much of an effect on ELISA plasma concentrations which remained significantly higher than corresponding LC/LC-MS/MS results. A poor correlation not only between LC/LC-MS/MS and immunoassay results, but also among the immunoassays was found. Especially in plasma, ELISAs grossly overestimate 15-F(2t)-isoprostane concentrations and are not comparable with each other or with LC/LC-MS/MS. It is most disturbing that a sample with relatively high concentrations measured with one ELISA may show low concentrations with another ELISA, and vice versa, potentially affecting the conclusions drawn from such data. The use of specific mass spectrometry-based assays seems advisable.

Endothelial dysfunction and oxidative stress in polycystic kidney disease

“…Altered levels of these oxidative stress markers were apparent in early ADPKD subjects and did not appear to change with worsening kidney function or hypertension (35). In our study, we confirmed markedly increased serum concentrations of the stable oxidative stress markers 8-isoprostane and PGF 2␣ (15,25,37,46) in ADPKD patients with preserved eGFR compared with healthy individuals. Both markers were positively associated with TKV/BSA, PGF 2␣ also with eGFR.…”

“…Prostaglandins. Protaglandins were extracted from 400 l plasma following protein precipitation with 400 l methanol/0.2 M ZnSO 4 (70/30 vol/vol) as previously described (25). Automated inline extraction was used for sample analysis.…”

Passive smoking reduces and vitamin C increases exercise-induced oxidative stress: Does this make passive smoking an anti-oxidant and vitamin C a pro-oxidant stimulus?

“…There are some reservations regarding the validity and reliability of F 2 -isoprostane ELISA kits [13]. Nevertheless, it has been shown that the kit employed in the present study for F 2 -isoprostanes resulted in less variability among the ELISAs and reduced the positive analytical bias between the ELISA and LC/LC-MS/MS results in urine samples [14]. Plasma protein carbonyls and erythrocyte glutathione were determined spectrophotometrically, as previously described [10].…”

Isoprostane as a promising prognostic biomarker in pulmonary arterial hypertension: preanalytical and analytical viewpoints

“…Did Zhang et al quantify free or total Pl-15-F2t-isoP concentrations? Thirdly, the ELISA kit booklet (Cayman Chemical, Ann Arbor, USA, [5]) mentions that the analysis of plasma without purification may lead to inconsistent results and recommends an immunoaffinity or solid phase extraction (IAE or SPE) before quantification, as elsewhere done [2,6]. Indeed, this purification pre-step allows the collection of most isoprostane, while removing major interferential molecules, thus providing quantification with a very low limit of detection (LOD) and high specificity [7].…”