Homocysteine Production and membrane oxidative stress in erythrocytes

Background: Homocysteine (Hcy) seems to induce oxidative stress (H2O2 production). Hcy synthesis occurs in erythrocytes (RBC).Aims: To assess if Hcy production in RBC may contribute to membrane lipid peroxidation.Methods: 15 RBC samples from patients with normal plasma Hcy (7.8 ± 3.1 mol/L) (controls) and 15 from megaloblastic anemia patients, [with higher Hcy plasma levels (19.1 ± 6.3 mol/L , p<0.01 with respect to controls) and RBC folate and serum B12 levels significantly lower (145.6 ± 34.1 vs. 272.6 ± 61.3 g/L, p<0.01, and 152 ± 65.5vs.582.6±195.6 pmol/ml,p<0.01, respectively)] were incubated both at 4°C and at 37° C in a RPMI folate and B12-free medium, containing methionine 50 mol. In each sample supernatant we measured at 4, 8 and 24 h Hcy, malondialdehyde (MDA) (HPLC) (marker of lipid peroxidation) and LDH levels (marker of early membrane damage) Results: In 37°C incubation samples Hcy production resulted higher in samples from megaloblastic anemia, whereas a slight Hcy increase was observed during incubation at 4°C in both groups (see figure). Hcy production (incubation at 37°C) seems associated to an higher lipid peroxidation and membrane functional derangement (see table) and a significant correlation ( 4 h : r=.668, p<0.01; 8h : r=.774, p<0.01; 24h : r= .556, p<0.01) was found between RBC Hcy production and MDA levels in the supernatant from samples at 37° Conclusions: Hcy production by RBC contribute to oxidative stress of membrane lipids. Lipoperoxidation (higher MDA levels in the medium) and membrane damage (LDH levels) in vitamin-deficient RBC may account for higher frequency of haemolysis in patients with megaloblastic anemia.