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Improved quantification of 8‐ epi ‐prostaglandin F 2α and F 2 ‐isoprostanes by gas chromatography/triple‐stage quadrupole mass spectrometry: partial cyclooxygenase‐dependent formation of 8‐ epi ‐prostaglandin F 2α in humans
Author(s) -
Schweer Horst,
Watzer Bernhard,
Seyberth Hannsjörg W.,
Nüsing Rolf M.
Publication year - 1997
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199712)32:12<1362::aid-jms606>3.0.co;2-n
Subject(s) - isoprostanes , chemistry , isoprostane , chromatography , isotope dilution , prostaglandin , urine , gas chromatography–mass spectrometry , arachidonic acid , mass spectrometry , cyclooxygenase , lipid peroxidation , biochemistry , oxidative stress , enzyme
F 2 ‐isoprostanes are considered to be novel markers of lipid peroxidation. To study the in vivo formation of F 2 ‐isoprostanes, an improved method was developed for isotope dilution assays involving gas chromatography/triple‐stage quadrupole mass spectrometry (GC/MS/MS) including thin‐layer chromatography (TLC) (sum of all F 2 ‐isoprostanes) and high‐performance liquid chromatographic (HPLC) purification (prostaglandin F 2α (PGF 2α ) and 8‐ epi ‐PGF 2α ). Following the addition of isotopically labeled prostaglandins to urine, the sample was acidified and applied to a C 18 cartridge. After elution, prostaglandins were derivatized to pentafluorobenzyl esters and subjected to TLC. A broad zone was scratched off, isoprostanes were eluted and after formation of their trimethylsilyl ether derivatives the sum of F 2 ‐isoprostanes was determined by GC/MS/MS. For the determination of PGF 2α and 8‐ epi ‐PGF 2α prior to trimethylsilylation an additional HPLC step was performed and the fractions containing PGF 2α and 8‐ epi ‐PGF 2α were analyzed by GC/MS/MS. Using this technique, 8‐ epi ‐PGF 2α concentrations in urine samples as low as 5 pg ml ‐1 could be determined with high accuracy. The excretion rates of isoprostanes were studied in comparison with the classical prostaglandins in three different groups: healthy adults, healthy children and children with hyper‐PGE syndrome (HPS), a pathological situation associated with a stimulated PGE 2 synthesis. F 2 ‐isoprostanes represented the main arachidonic acid metabolites in these groups and 8‐ epi ‐PGF 2α excretion was comparable in its amount to the classical prostanoids. To delineate the cyclooxygenase‐catalyzed contribution, the influence of indomethacin, an inhibitor of cyclooxygenases, on F 2 ‐isoprostane formation in healthy adults and in HPS children was analyzed. Significantly decreased excretion rates were observed 2 days after indomethacin administration for all prostanoids, including F 2 ‐isoprostanes and 8‐ epi ‐PGF 2α . However, the suppression of F 2 ‐isoprostanes and 8‐ epi ‐PGF 2α excretion rates was less pronounced in comparison with the classical prostanoids. An improved and reliable method for the determination of F 2 ‐isoprostanes and especially 8‐ epi ‐PGF 2α has been developed. The data obtained on human urine samples indicates a contribution of the cyclooxygenase pathway to the formation of isoprostanes. © 1997 John Wiley & Sons, Ltd.