Membrane changes associated with lysis of red blood cells by hypochlorous acid.
This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidenced by ektacytometry, and an increase in K(+)-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, but complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.
Vissers
M C
MC
Pathology Department, Christchurch School of Medicine, New Zealand.
Stern
A
A
Kuypers
F
F
van den Berg
J
J
Winterbourn
C C
CC
D003958:Diamide; D004591:Electrophoresis, Polyacrylamide Gel; D004907:Erythrocyte Deformability / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D004912:Erythrocytes / Q000187:drug effects*; D005033:Ethylmaleimide; D016166:Free Radical Scavengers; D006461:Hemolysis; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood* / Q000187:drug effects / Q000302:isolation & purification; D008715:Methionine / Q000494:pharmacology; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D011188:Potassium / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000097:blood; D013654:Taurine / Q000494:pharmacology; D013997:Time Factors
D016166:Free Radical Scavengers; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D013654:Taurine; D006997:Hypochlorous Acid; D008715:Methionine; D005033:Ethylmaleimide; D011188:Potassium