Regeneration of Reduced Glutathione in Erythrocytes: Stoichiometric and Temporal Relationship to Hexose Monophosphate Shunt Activity

The stoichiometric and temporal relationships between glutathione reduction and hexose monophosphate shunt (HMPS) activity in normal red blood cells were investigated using azoester to oxidize reduced glutathione (GSH) and an ionization chamber-electrometer apparatus to measure continuously the ¹⁴CO₂ derived from ¹⁴C-glucose. Under air, azoester produced rapid oxidation of GSH followed by rapid regeneration. The HMPS response was delayed and occurred after the period of maximal GSH regeneration. Due to the generation of hydrogen peroxide by azoester, cumulative shunt activity was far in excess of that theoretically required to regenerate GSH oxidized directly by azoester. Under carbon monoxide, no hydrogen peroxide was generated by azoester, and a stoichiometric relationship existed between GSH regeneration and HMPS activity. Again, however, the response of the HMPS was temporally dissociated from GSH regeneration. These findings demonstrate that under carefully controlled conditions there is a stoichiometric relationship between the regeneration of GSH and CO₂ production by the HMPS but that this stoichiometry is not the result of a "direct linkage" of the two reactions.