OxyFile #90

TI:  Effect of Hydrogen Peroxide Exposure on Normal Human 
     Erythrocyte Deformability, Morphology, Surface 
     Characteristics, and Spectrin-Hemoglobin Cross-linking.

DT:  November 1985

AU:  L.M. Snyder, N.L. Fortier, J. Trainor, J. Jacobs, L. Leb,
     B. Lubin, D. Chiu, S. Shohet and N. Mohandas

SO:  J. Clin. Invest, Vol. 76, November 1985, 1971-1977

AB:  To further define the conditions for forming spectrin-
     hemoglobin cross-linking in human erythrocyte membranes and 
     to examine its possible effects on membrane function, we 
     incubated normal human erythrocytes for up to 3 h in 
     concentrations of H2O2, varying from 45 to 180 uM, in a 
     azide phosphate buffer, pH 7.4.  The chemical changes 
     observed indicated that methemoglobin formation occurred 
     early and at a low concentration (45 uM).  Morphologic 
     changes characterized by increased echinocyte formation 
     occurred in a dose-dependent fashion.  In addition, 
     decreased cell deformability commensurate with increased 
     membrane rigidity was found.  Finally, an increase in cell 
     recognition as determined by monocyte phagocytosis and 
     adherence in vitro, as well as decreased phosphatidylcholine 
     accessibility to bee venom phospholipase A2, was found in 
     H2O2 treated erythrocytes compared with controls.  Both of 
     these latter changes were closely correlated with the extent 
     of spectrin-hemoglobin cross-linking.

     In addition to these protein-mediated interactions, lipid 
     peroxidation also occurred after H2O2 exposure, as shown by 
     generation of fluorescent amino propene derivatives.  The 
     addition of the antioxidant, butylated hydroxytoluene, 
     decreased the fluorescent derivatives, but did not prevent 
     the effects on membrane function.  This suggests that lipid 
     peroxidation, though present, was not necessary for the 
     membrane changes found.  In contrast, spectrin-hemoglobin 
     aggregation and the alterations in membrane function were 
     completely prevented by prior exposure of the erythrocytes 
     to carbon monoxide.