Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle

Abstract

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant-induced expression of protective enzymes and HSP content. Lymphocytes from non-supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day-1 for 8 weeks was found to increase the serum vitamin C concentration by ~50 %. Lymphocytes from vitamin C-supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non-supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C-supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).

Figure 1

Lymphocyte superoxide dismutase (SOD) activity following exposure to 15 μm H2O2 (A) and 25 μm H₂O₂ (B), and catalase activity following exposure to 15 μm H₂O₂ (C) and 25 μm H₂O₂ (D) in subjects prior to (open bars), and following 8 weeks of supplementation with 500 mg day⁻¹ vitamin C (grey bars). Data are presented as means ±s.e.m. ^* Values significantly different from pre-stimulation, non-supplemented values.

Figure 3

Lymphocyte HSP70 content following exposure to 15 μm (A) and 25 μm H₂O₂ (B) in volunteer subjects prior to (open bars), and after 8 weeks supplementation with 500 mg day⁻¹ vitamin C (grey bars). Data are presented as means ±s.e.m. ^* Values significantly different from pre-stimulation, non-supplemented values; † value significantly different from data from non-supplemented subjects at the same time post-H₂O₂. C, example Western blots are also shown for pre-supplementation (lanes 1–4) and post-supplementation (lanes 5–8) samples.

Figure 2

Lymphocyte HSP60 content following exposure to 15 μm (A) and 25 μm H₂O₂ (B) in volunteer subjects prior to (open bars), and after 8 weeks supplementation with 500 mg day⁻¹ vitamin C (grey bars). Data are presented as means ±s.e.m. ^* Values significantly different from pre-stimulation, non-supplemented values. C, example Western blots are also shown for pre-supplementation (lanes 1–4) and post-supplementation (lanes 5–8) samples.

Figure 4

Relative contents of HSP70 (A) and HSP60 (B) in the vastus lateralis muscles of a group of volunteer subjects prior to, and at 2 days following, 45 min of exercise on a cycle ergometer. The effect of supplementation for 8 weeks with 500 mg day⁻¹ vitamin C is also shown (grey bars). Data are presented as means ±s.e.m. ^* Values significantly different from pre-stimulation, non-supplemented values. C, example Western blots are shown for pre-supplementation (lanes 1 and 2) and post-supplementation samples (lanes 3 and 4).