GLUTATHIONE .3. BIOLOGICAL ASPECTS OF AZOESTER PROCEDURE FOR OXIDATION WITHIN NORMAL HUMAN ERYTHROCYTE

1. 1. The age of human erythrocytes plays no significant role in affecting (a) the GSH content of the cells, (b) the ease of azoester oxidation of the GSH to the disulfide or (c) the ease of intracellular regeneration of GSH from GSSG. 2. 2. The chemical state of the hemoglobin (oxy-, deoxy-, carbon monoxide or met-) within the human erythrocyte has no influence on the ease of oxidation of GSH by azoester or on the subsequent regeneration of GSH from GSSG. 3. 3. Hemoglobin SH groups are unaffected by quantities of azoester sufficient to oxidize all the GSH to GSSG. 4. 4. O2 is not required for oxidation of GSH. The implication is clear that H2O2 is not a necessary intermediate for drug-induced damage of cells. 5. 5. Excess azoester, which generates reactive free radicals, has the following effects: (a) The quantity of GSH regenerated from GSSG falls from 90% of the theoretical to about 60% of that expected when a 4-fold excess of azoester is used. Further increases in the excess of azoester used do not diminish the quantity of GSH regenerated, implicating a limited number of intracellular sites for the disappearance of GSH. These sites are called oxidant target sites". (b) Methemoglobin formation rises from 3-6% under the usual conditions for azoester oxidation of GSH to 15-20% when a large excess (5-fold) of reagent is utilized. (c) Heinz body formation becomes evident at large (above 4-fold) excesses of azoester. © 1969."