Improved quantification of 8-epi-prostaglandin F2 alpha and F2-isoprostanes by gas chromatography/triple-stage quadrupole mass spectrometry: partial cyclooxygenase-dependent formation of 8-epi-prostaglandin F2 alpha in humans.
F2-isoprostanes are considered to be novel markers of lipid peroxidation. To study the in vivo formation of F2-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 F2-isoprostanes) and high-performance liquid chromatographic (HPLC) purification (prostaglandin F2 alpha (PGF2 alpha) and 8-epi-PGF2 alpha). Following the addition of isotopically labeled prostaglandins to urine, the sample was acidified and applied to a C18 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 F2-isoprostanes was determined by GC/MS/MS. For the determination of PGE2 alpha and 8-epi-PGF2 alpha prior to trimethylsilylation an additional HPLC step was performed and the fractions containing PGF2 alpha and 8-epi-PGF2 alpha were analyzed by GC/MS/MS. Using this technique, 8-epi-PGF2 alpha 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 PGE2 synthesis. F2-isoprostanes represented the main arachidonic acid metabolites in these groups and 8-epi-PGF2 alpha 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 F2-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 F2-isoprostanes and 8-epi-PGF2 alpha. However, the suppression of F2-isoprostanes and 8-epi-PGF2 alpha excretion rates was less pronounced in comparison with the classical prostanoids. An improved and reliable method for the determination of F2-isoprostanes and especially 8-epi-PGF2 alpha has been developed. The data obtained on human urine samples indicates a contribution of the cyclooxygenase pathway to the formation of isoprostanes.
Schweer
H
H
Children's Hospital, Philipps University Marburg, Germany.
Watzer
B
B
Seyberth
H W
HW
Nüsing
R M
RM
D000293:Adolescent; D000328:Adult; D002648:Child; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D016861:Cyclooxygenase Inhibitors / Q000627:therapeutic use; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000652:urine; D004591:Electrophoresis, Polyacrylamide Gel; D028441:F2-Isoprostanes; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007202:Indicators and Reagents; D007213:Indomethacin / Q000627:therapeutic use; D008297:Male; D008661:Metabolism, Inborn Errors / Q000188:drug therapy / Q000652:urine; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012016:Reference Values
D016861:Cyclooxygenase Inhibitors; D028441:F2-Isoprostanes; D007202:Indicators and Reagents; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin