Age-associated perturbations in glutathione synthesis in mouse liver

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Abstract
The nature of the mechanisms underlying the age-related decline in glutathione (GSH) synthetic capacity is at present unclear. Steady-state kinetic parameters of mouse liver GCL (glutamate–cysteine ligase), the rate-limiting enzyme in GSH synthesis, and levels of hepatic GSH synthesis precursors from the trans-sulfuration pathway, such as homocysteine, cystathionine and cysteine, were compared between young and old C57BL/6 mice (6- and 24-month-old respectively). There were no agerelated differences in GCL Vmax, but the apparent Km for its substrates, cysteine and glutamate, was higher in the old mice compared with the young mice (∼800 compared with ∼300 μM, and ∼710 compared with 450 μM, P<0.05 for cysteine and glutamate in young and old mice respectively). Amounts of cysteine, cystathionine and Cys-Gly increased with age by 91, 24 and 28% respectively. Glutathione (GSH) levels remained unchanged with age, whereas GSSG content showed an 84% increase, suggesting a significant pro-oxidizing shift in the 2GSH/GSSG ratio. The amount of the toxic trans-sulfuration/glutathione biosynthetic pathway intermediate, homocysteine, was 154% higher (P<0.005) in the liver of old mice compared with young mice. The conversion of homocysteine into cystathionine, a rate-limiting step in trans-sulfuration catalysed by cystathionine β-synthase, was comparatively less efficient in the old mice, as indicated by cystathionine/homocysteine ratios. Incubation of tissue homogenates with physiological concentrations of homocysteine caused an up to 4.4-fold increase in the apparent Km of GCL for its glutamate substrate, but had no effect on Vmax. The results suggest that perturbation of the catalytic efficiency of GCL and accumulation of homocysteine from the trans-sulfuration pathway may adversely affect de novo GSH synthesis during aging.