inosinic-acid and Hemolysis

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.

Topics: Adenine Nucleotides; AMP Deaminase; Energy Metabolism; Erythrocytes; Guanosine Triphosphate; Hemolysis; Humans; Hydrogen Peroxide; In Vitro Techniques; Inosine Monophosphate; Lipid Peroxidation; Malondialdehyde; Oxidative Stress

Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen.

Topics: 5'-Nucleotidase; Adenosine Monophosphate; Coformycin; Erythrocyte Count; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Inosine Monophosphate; Nucleotidases; Phosphates; Phosphorylation; Reticulocytes