Open AccessEnergy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress
Author(s) -
Tavazzi Barbara,
Di Pierro Donato,
Amorini Angela Maria,
Fazzina Giovanna,
Tuttobene Michele,
Giardina Bruno,
Lazzarino Giuseppe
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01042.x
Subject(s) - lipid peroxidation , malondialdehyde , hypoxanthine , chemistry , oxidative stress , biochemistry , adenine nucleotide , xanthine oxidase , xanthine , oxidative phosphorylation , uric acid , metabolism , intracellular , nucleotide , enzyme , gene
To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5–10 m m ) of hydrogen peroxide for 1 h at 37 °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 m m H 2 O 2 ), oxidative stress produced, starting from 1 m m H 2 O 2 , 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 H 2 O 2 concentration (4 m m ). 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 H 2 O 2 used; this enzymatic activity increased with increasing H 2 O 2 in the medium, and reached its maximum at 4 m m H 2 O 2 ‐treated erythrocytes (10‐fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H 2 O 2 , being detectable at the lowest H 2 O 2 concentration and increasing without appreciable haemolysis up to 4 m m H 2 O 2 . 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.