Crosslinking of aminophospholipids in cellular membranes of lens by oxidative stress in vitro

We have previously demonstrated by TLC an additional phospholipid spot between phosphatidylethanolamine (PE) and phosphatidylserine (PS) in human cataract. This was further identified as a fluorescent Schiff-base conjugate resulting from crosslinking of reactive carbonyl groups of malondialdehyde (MDA) with the primary amino groups of membrane phospholipids. Evidence presented here shows that such an adduct could be formed in rabbit lens subjected to oxidative stress in vitro. TLC analysis of a lipid extract of a crude membrane fraction obtained from the lens homogenate incubated with 1 mM H2O2, tert-butyl hydroperoxide (TBHP) or MDA for 1-6 h at 25 degrees C, showed that the oxidants and MDA produced time-dependent crosslinking of aminophospholipids. Under identical conditions of incubation with TBHP or MDA, development of the Schiff-base lipid fluorochrome in lens with peak emission at 470 nm when excited at 360 nm also showed a time-dependent increase. The PE . MDA . PS produced in cellular membranes of the lenses cultured for 3 h in Krebs-Ringer medium was 151 nmol/mu mol PE, and addition of 1 mM H2O2, TBHP or MDA, increased it to 881, 610 and 375 nmol/mu mol PE, respectively. Adduct was also formed when authentic samples of PE and PS were reacted with pure MDA. From the results it is clear that oxidants viz., H2O2 and TBHP, or MDA were effective in promoting crosslinking of lens membrane aminophospholipids by Schiff-base conjugation of primary amino groups with the carbonyl groups of the aldehyde, a breakdown product of lipid peroxides.