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Background: Endogenous reactive oxygen species appear to contribute to aging and cancer and dietary antioxidants, present in fruit and vegetables, counteract these effects. Objective: The objective was to examine the association between intracellular glutathione, ascorbate (vitamin C), and [Alpha]-tocopherol (vitamin E) in human lymphocytes. Design: The study group consisted of 240 healthy nonsmoking volunteers with an approximately equal number of male and female subjects subdivided into 3 age groups: 18-39, 40-59, and [is greater than or equal to] 60 y). Glutathione, glutathione disulfide, ascorbate, and [Alpha]-tocopherol were measured in lymphocytes by HPLC. Results: The average concentration of antioxidants in lymphocytes was 27 [+ or -] 8 nmol/mg protein for glutathione, 21 [+ or -] 8 nmol/mg protein for ascorbate, and 0.4 [+ or -] 0.2 nmol/mg protein for [Alpha]-tocopherol. There was a strong positive correlation between glutathione and ascorbate (r = 0.62, P [is less than] 0.001). No correlation was observed for glutathione and ascorbate with [Alpha]-tocopherol. The concentration of glutathione in lymphocytes was inversely correlated with age (r = - 0.19, P [is less than] 0.01), as was that of ascorbate (r = - 0.22, P [is less than] 0.01), with 10-20% lower values in elderly than in young and elderly subjects. The concentrations of glutathione in lymphocytes were as much as 25% higher and those of ascorbate were as much as 38% higher during the summer than during the winter. The seasonal variation of ascorbate in lymphocytes was described by a linear function for age and a periodic sine function for season. Conclusion: Glutathione and ascorbate are directly correlated in human lymphocytes. Am J Clin Nutr 2000;71:1194-200. KEY WORDS Vitamin C, vitamin E, glutathione, lymphocytes, ascorbic acid, antioxidants, micronutrients, aging, elderly, cancer, reactive oxygen species, season