Divergence in urinary 8- iso-PGF 2α (iPF 2α-III, 15-F 2t-IsoP) levels from gas chromatography–tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8- iso-PGF 2α: Possible methodological, mechanistic and clinical implications

Abstract

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F 2-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC–tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8- iso-PGF 2α (iPF 2α-III, 15-F 2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8- iso-PGF 2α (i.e. 15( S)-8- iso-PGF 2α) has been shown by others to be highly selective and specific for this 8- iso-PGF 2α isomer when quantified by GC–MS. In the present study we established IAC for urinary 8- iso-PGF 2α for subsequent quantification by GC–tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15( S)-8- iso-PGF 2α. 8- iso-PGF 2α was measured in urine of 10 young healthy humans by both methods. 8- iso-PGF 2α was determined to be 291±102 pg/mg creatinine by method A and 141±41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8- iso-PGF 2α at 128±55 pg/mg creatinine. This finding suggests that urinary 8- iso-PGF 2α is heterogenous, with 15( S)-8- iso-PGF 2α contributing by ∼50%. PGF 2α and other 8- iso-PGF 2α isomers including 15( R)-8- iso-PGF 2α are not IAC-immunoreactive and are chromatographically separated from 15( S)-8- iso-PGF 2α. We assume that ent-15( S)-8- iso-PGF 2α is also contributing by ∼50% to urinary 8- iso-PGF 2α. This finding may have methodological, mechanistic and clinical implications.