Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise

Abstract

There has been no investigation to determine if the widely used over-the-counter, water-soluble antioxidants vitamin C and N-acetyl-cysteine (NAC) could act as pro-oxidants in humans during inflammatory conditions. We induced an acute-phase inflammatory response by an eccentric arm muscle injury. The inflammation was characterized by edema, swelling, pain, and increases in plasma inflammatory indicators, myeloperoxidase and interleukin-6. Immediately following the injury, subjects consumed a placebo or vitamin C (12.5 mg/kg body weight) and NAC (10 mg/kg body weight) for 7 d. The resulting muscle injury caused increased levels of serum bleomycin-detectable iron and the amount of iron was higher in the vitamin C and NAC group. The concentrations of lactate dehydrogenase (LDH), creatine kinase (CK), and myoglobin were significantly elevated 2, 3, and 4 d postinjury and returned to baseline levels by day 7. In addition, LDH and CK activities were elevated to a greater extent in the vitamin C and NAC group. Levels of markers for oxidative stress (lipid hydroperoxides and 8-iso prostaglandin F_2α; 8-Iso-PGF_2α) and antioxidant enzyme activities were also elevated post-injury. The subjects receiving vitamin C and NAC had higher levels of lipid hydroperoxides and 8-Iso-PGF_2α 2 d after the exercise. This acute human inflammatory model strongly suggests that vitamin C and NAC supplementation immediately post-injury, transiently increases tissue damage and oxidative stress.

Introduction

It is well established by in vitro experiments that vitamin C is reactive with free iron and produces the ascorbate radical [1], [2], [3], while causing oxidative damage to biomolecules [1], [4], [5]. In humans, scientists have claimed increases in DNA damage in healthy humans supplemented with vitamin C and iron salts [6], as well as ascorbyl radical formation in subjects with sepsis following ascorbate loading [7]. Podmore et al. [8] supplemented subjects with 500 mg/d of ascorbate and showed a drop in the content of 8-hydroxyguanine in lymphocyte DNA, however, there was a concurrent increase in 8-hydroxyadenine, a mutagenic base that causes adenine → guanine transitions and adenine → cytosine transversions. Although not as well documented as vitamin C, several reports suggest a pro-oxidant or adverse effect from N-acetyl cysteine (NAC) in vitro and in vivo [9], [10], [11], [12]. However, other studies performed show that vitamin C and NAC are protective antioxidants that can prevent oxidative stress [1], [13], [14], [15], [16], [17], [18], [19]. Therefore, there is still debate on whether supplements such as vitamin C and NAC could act as pro-oxidants in vivo. Moreover, no human or animal investigations have determined if immediately after an acute inflammation—characterized by release of free iron from its normal sequestration sites—these water-soluble antioxidants could cause oxidative stress.

Under normal physiological circumstances, metals are bound to circulating proteins and are rendered redox-inactive [1], [20], [21]. However, levels of free metal ions, including iron, may be elevated during acute inflammation and sepsis, becoming redox-active [1], [22]. Inflammation stimulates polymorphonuclear leukocytes and macrophages that produce large amounts of superoxide (O₂^•−) and hydrogen peroxide (H₂O₂) [1], [24]. The detrimental effects of these species may be amplified in the presence of iron by the subsequent formation of other reactive intermediates, such as the hydroxyl radical (HO^•). The hydroxyl radical has been widely postulated to cause significant damage to several biomolecules in vivo. The relevance of the hydroxyl radical in biology has been questioned because of the requirement of redox-active catalytic iron [21]. However, Biemond et al. have shown iron release from ferritin during inflammation [22]. In addition, Gutteridge et al. [23] show release of iron from heomoglobin following treatment with hydrogen peroxide.

The reduction potentials of Fe⁺³ (−0.4 V) and vitamin C (−0.17 V) easily allow the formation of the ascorbate radical and Fe⁺² iron. Therefore, in vitro vitamin C can exert pro-oxidant effects by converting Fe⁺³ into Fe⁺², which reacts with H₂O₂ to generate HO^• [1], [25]. Iron-ascorbate mixtures have been shown to stimulate free radical damage to DNA, lipids, and proteins in vitro [21]. In vivo, iron [26] supplementation and ascorbate-copper supplementation [27] to rats have been reported to stimulate HO^• generation. In addition, mixtures of thiols with transition-metal ions can be cytotoxic because of reactions that produce O₂^•−, H₂O₂, HO^•, and sulphur-containing radicals such as RS^• and RSO^• [1], [28]. It is feasible that the release of iron and the presence of vitamin C and NAC during acute inflammation could lead to HO^•, ascorbate^•, and thiol radical generation.

Inflammation can be induced by acute exercise in unadapted subjects and can increase the levels of free iron [29]. Several types of exercise damage enzymes and lipid membranes, increase DNA damage, stimulate oxidative stress, and increase plasma markers of cell damage [30], [31], [32], [33], [34], [35], [36], [37]. Specifically, eccentric exercise leads to a condition characterized by severe inflammation and edema. Examples of eccentric exercises are downhill running and eccentric arm exercises, which have been shown to increase neutrophil migration into the skeletal muscle after such injury [33], [34], [35], [37]. In the present study, we used an eccentric exercise protocol to induce a severe injury to a human arm muscle. Severe inflammation, pain, and a decrease in range of motion characterize this injury. This condition provides an informative experimental human model for evaluation of compounds believed to exhibit antioxidant properties. Supplements increased lipid peroxidation products, superoxide dismutase, glutathione peroxidase, markers of muscle damage, and increased bleomycin-detectable iron in the serum above levels of subjects receiving a placebo. Therefore, intake of these two supplements immediately following acute inflammatory conditions may not be advisable.