Divergence in urinary 8-iso-PGF(2alpha) (iPF(2alpha)-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(2alpha). Possible methodological, mechanistic and clinical implications.
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(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF(2alpha) (i.e. 15(S)-8-iso-PGF(2alpha)) has been shown by others to be highly selective and specific for this 8-iso-PGF(2alpha) isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF(2alpha) 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(2alpha). 8-iso-PGF(2alpha) was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF(2alpha) 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(2alpha) at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF(2alpha) is heterogenous, with 15(S)-8-iso-PGF(2alpha) contributing by approximately 50%. PGF(2alpha) and other 8-iso-PGF(2alpha) isomers including 15(R)-8-iso-PGF(2alpha) are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF(2alpha). We assume that ent-15(S)-8-iso-PGF(2alpha) is also contributing by approximately 50% to urinary 8-iso-PGF(2alpha). This finding may have methodological, mechanistic and clinical implications.
Tsikas
Dimitrios
D
Institute of Clinical Pharmacology, Hannover Medical School, D-30625 Hannover, Germany. tsikas.dimitros@mh-hannover.de
Schwedhelm
Edzard
E
Suchy
Maria Theresia
MT
Niemann
Jonas
J
Gutzki
Frank Mathias
FM
Erpenbeck
Veit J
VJ
Hohlfeld
Jens M
JM
Surdacki
Andrzej
A
Frölich
Jürgen C
JC
D000328:Adult; D016022:Case-Control Studies; D002846:Chromatography, Affinity / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D015237:Dinoprost / Q000031:analogs & derivatives*; D028441:F2-Isoprostanes / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006973:Hypertension / Q000652:urine; D008055:Lipids / Q000097:blood; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012907:Smoking / Q000652:urine
D028441:F2-Isoprostanes; D008055:Lipids; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost