Glutamine and α‐ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α‐ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α‐ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)−1·min−1, respectively) compared with 0.52 ± 0.09 μmol·(L RBC)−1·min−1 for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)−1·min−1, while the initial rate for 0.8 mmα‐ketoglutarate was 0.97 μmol·(L RBC)−1·min−1. However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α‐ketoglutarate (0.31 and 0.25 μmol·(L RBC)−1·min−1, respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α‐ketoglutarate and/or glutamine. Database The mathematical models described here have been submitted to the Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/Whillier/index.html free of charge. We investigated the contributions of glutamine and α‐ketoglutarate/alanine as substrates for glutamate in glutathione synthesis. When extracellular glutamine or α‐ketoglutarate/alanine were at 10 mm, erythrocyte glutathione synthesis was faster with α‐ketoglutarate/alanine. Mathematical modelling of glutathione metabolism demonstrated that for erythrocytes in plasma glutamate from both glutamine and α‐ketoglutarate/alanine was required to maintain a normal rate of glutathione production.