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Intracellular antioxidants, glutathione and ascorbate, and two molecular markers of oxidative DNA damage, 5-hydroxy-2'-deoxycytidine (5-OH-dCyd) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo), were measured in lymphocytes from 105 healthy volunteers. The analysis of 5-OH-dCyd and 8-oxo-dGuo was carried out by HPLC with electrochemical detection such that both compounds were detected on the same chromatography run. There was no significant difference in oxidative DNA damage when the extraction of DNA from cells using phenol was carried out under anaerobic conditions or in the presence of metal ion chelators. This indicates that auto-oxidation of DNA during sample preparation was minimal. Using the above methods, the average level of oxidative DNA damage in lymphocytes was 2.9 +/- 1.4 for 5-OH-dCyd and 4.5 +/- 1.8 for 8-oxo-dGuo lesions per 10(6) dGuo (n = 105). It is unlikely that artifactual oxidation contributed to the observed damage because the level of 5-OH-dCyd was comparable with that of 8-oxo-dGuo in lymphocyte DNA, whereas 8-oxo-dGuo outnumbers 5-OH-dCyd by a ratio of >5:1 when DNA is exposed to various oxidants, including ionizing radiation or Fenton reagents. Rather, the nearly equal levels of 5-OH-dCyd and 8-oxo-dGuo in cellular DNA implies that 8-oxo-dGuo may be more efficiently removed by DNA repair. Finally, and most importantly, the correlation of our endpoints revealed that the naturally occurring level of intracellular antioxidants was negatively correlated to the level of oxidative DNA damage with the strongest correlation observed for glutathione and 8-oxo-dGuo (r = -0.36; P < 0.001). These results strongly suggest that intracellular glutathione and ascorbate protect human lymphocytes against oxidative DNA damage.

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 ␣-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 ≥ 60 y). Glutathione, glutathione disulfide, ascorbate, and ␣-tocopherol were measured in lymphocytes by HPLC. Results: The average concentration of antioxidants in lymphocytes was 27 ± 8 nmol/mg protein for glutathione, 21 ± 8 nmol/mg protein for ascorbate, and 0.4 ± 0.2 nmol/mg protein for ␣-tocopherol. There was a strong positive correlation between glutathione and ascorbate (r = 0.62, P < 0.001). No correlation was observed for glutathione and ascorbate with ␣-tocopherol. The concentration of glutathione in lymphocytes was inversely correlated with age (r = Ϫ0.19, P < 0.01), as was that of ascorbate (r = Ϫ0.22, P < 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.

Vitamin C supplements increase glutathione in human lymphocytes.

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