F(2) isoprostanes, potential specific markers of oxidative damage in human retina

F(2) isoprostanes (F(2)-IPs) are a series of prostaglandin-F(2)(PGF(2))-like compounds produced during peroxidation of arachidonic acid (AA) by a mechanism independent of the cyclo-oxygenase. Of these, 8-epi-PGF(2alpha) has proved a reliable marker of oxidative stress in studies both in vitro and in vivo. Human retinas (n = 28) were obtained from healthy donors (age 15-59 years) and analysed for the content of free (non-esterified) as well as total (sum of free and esterified) F(2)-IPs. Retina tissue was homogenised, total lipids were partitioned with ethyl acetate and isoprostanes were isolated by chromatography on an NH(2) cartridge. After formation of pentafluorobenzyl ester and trimethylsilyl derivatives, F(2)-IPs were analysed by negative ion-chemical ionisation mass spectrometry using tetradeuterated PGF(2alpha) as the internal standard. Analysis of retina samples revealed the presence of measurable amounts of esterified 8-epi-PGF(2alpha) (mean: 81.71 microg/g AA; range: 3.29-187.89 microg/g AA) but not free F(2)-IPs. There was no correlation between age and content of 8-epi-PGF(2alpha). Detection of esterified F(2)-IPs in human retina provides the first direct evidence for free-radical-catalysed peroxidation of AA in retinal tissue. Analysis of F(2)-IPs should provide new opportunities for studying the implications of oxidative injury in various diseases of retina, including diabetic retinopathy and age-related macular degeneration.