Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.
To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.
Tavazzi
B
B
Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Italy.
Di Pierro
D
D
Amorini
A M
AM
Fazzina
G
G
Tuttobene
M
M
Giardina
B
B
Lazzarino
G
G
D000659:AMP Deaminase / Q000097:blood; D000227:Adenine Nucleotides / Q000097:blood; D004734:Energy Metabolism / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000097:blood; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D066298:In Vitro Techniques; D007291:Inosine Monophosphate / Q000097:blood; D015227:Lipid Peroxidation / Q000187:drug effects; D008315:Malondialdehyde / Q000097:blood; D018384:Oxidative Stress / Q000187:drug effects
D000227:Adenine Nucleotides; D007291:Inosine Monophosphate; D008315:Malondialdehyde; D006160:Guanosine Triphosphate; D006861:Hydrogen Peroxide; D000659:AMP Deaminase