We have investigated the influence of vitamin C diet supplementation on the antioxidant response and nitrite levels in lymphocytes and erythrocytes during diving apnea.
Seven male professional apnea divers participated in a double blind crossover study. Divers were randomly assigned to either vitamin C supplemented or placebo groups. The subjects did not take any other supplements than the ones provided for this study.
One group was supplemented with vitamin C capsules (1 g per day) for 7 days while the other group took a placebo composed of lactose. The usual dietary habits of participants were assessed using a self-reported 7-days 24-h recall before the day of the study. Blood samples were taken under basal conditions, immediately after diving apnea for 4 h and after 1 h of recovery.
Catalase activity increased in erythrocytes (23%) and superoxide dismutase increased in lymphocytes (35%) during the recovery only in the placebo group. Lymphocyte ascorbate levels increased in the supplemented group after diving (85%) and maintained high at recovery. Plasma nitrite levels increased about twofold in both groups during the recovery. Erythrocyte nitrite levels increased after diving (50%) and about twofold during the recovery in the supplemented group. Nitrite levels and iNOS levels in lymphocytes were higher in the placebo group than in the supplemented during the recovery. Erythrocyte carbonyl derivates were unchanged in all situations.
Vitamin C supplementation influenced the antioxidant response and NO handling in erythrocytes and lymphocytes to the oxidative stress induced by hypoxia–reoxygenation.
Cellular hypoxia and reoxygenation are two essential elements of ischemia-reperfusion injury and they participate in a wide variety of human diseases such as circulatory shock, myocardial ischemia, stroke and organ transplantation (McCord, 1985; Li and Jackson, 2002). Hypoxia induces specific changes in enzyme activities, mitochondrial function, membrane transport and antioxidant defences, which predispose to reoxygenation injury (Li and Jackson, 2002). These changes include reduction of the mitochondrial respiratory chain, conversion of xanthine dehydogenase to xanthine oxidase, increased reactive species formation, and activation of signaling pathways (Murphy et al., 1984; Kayyali et al., 2001; Kokura et al., 2002). Overproduction of reactive species during hypoxia–reoxygenation induces oxidative stress (Tan et al., 1999; Schulz et al., 2000).
Many ‘in vitro’ studies have been carried out on hypoxia–reoxygenation; however, there are few ‘in vivo’ human studies about the effects of the hypoxic conditions on the cellular antioxidant defences (Vina et al., 1996; Vento et al., 2003). Exercise in chronic obstructive pulmonary disease (COPD) patients is a hypoxic situation because there is a lack of oxygen relative to the metabolic needs. Exercise in these patients causes glutathione oxidation, which can be partially prevented by oxygen therapy (Vina et al., 1996). In a previous study, we used exercise in diving apnea as a model of repetitive hypoxia–reoxygenation situation (Sureda et al., 2004a, 2004b). Exercise increases tissue oxygen demand, but a limited amount of oxygen is available in diving apnea, thus inducing tissue hypoxia. We evidenced that diving apnea induces oxidative stress and initiates neutrophil reactions resembling the acute phase immune response (APIR) (Sureda et al., 2004a). We also evidenced that chronic and repetitive episodes of diving apnea induce neutrophil adaptations in order to delay the oxidative burst response and enhance antioxidant levels (Sureda et al., 2004b). Several data show that antioxidant enzymes and low-molecular-weight reactive oxygen species (ROS) scavengers inhibit reoxygenation injury in cellular models (Horakova et al., 1997; Colantoni et al., 1998; Isowa et al., 2000). Studies with dietary vitamin C supplementation evidence an important role of vitamin C in the defence against oxidative stress induced by exercise and avoiding its negative effects on erythrocyte and lymphocyte integrity (Campbell et al., 1999).
Decompression sickness is often related with scuba diving as a result of wrong decompression habits (Hagberg and Ornhagen, 2003), and its incidence has been recently described in apnea divers (Batle, 2002) – also probably due to the wrong diving habits. Oxygen availability to the hypoxic tissue is mediated by the vasodilator effects of nitric oxide (NO) (Kelm and Schrader, 1990). An inactive nitric oxide synthase (NOS) has been immunodetected in erythrocytes (Kang et al., 2000); however, the erythrocyte generates NO from nitrite because hemoglobin has a dual capability to transform nitrite (Gladwin et al., 2004). Nitrite is reduced to NO by hemoglobin in hypoxic conditions and it is oxidized to nitrate in presence of oxygen. Nitric oxide generation along the physiological oxygen gradient suggests a role in vasodilatation responses to hypoxia (Datta et al., 2004; Gladwin et al., 2004). Peripherical vasodilatation could decrease the generation of gas bubbles in the main vessels and could thus contribute to decrease the pernicious effects of the decompressive sickness. The deoxygenation of NO by hemoglobin is parallel to the oxidation of hemoglobin to methemoglobin (Dou et al., 2002). The availability of molecules that reduce methemoglobin to hemoglobin as glutathione or ascorbate could be necessary to maintain the oxidative equilibrium in erythrocytes and facilitate the peripherical vasodilatation (Sharma et al., 2003). The administration of a NO donor before dive protects against bubble formation in rats probably changing the properties of the vascular endothelium (Wisloff et al., 2004).
The repetitive episodes of hypoxia–reoxygenation associated to diving apnea could increase ROS production, inducing oxidative stress and altering the NO availability by cells (Elsner et al., 1998; Sureda et al., 2004a). The effects of the diet supplementation with vitamin C in the erythrocyte capability to generate NO after hypoxia–reoxygenation could contribute to explain the vasodilatador effects of ascorbate. The aim of this study was to determine the influence of vitamin C diet supplementation on the response of lymphocyte and erythrocyte antioxidant defences and nitrite levels to hypoxia–reoxygenation during diving apnea.
Materials and methods
Seven voluntary male subjects participated in this study. They were all professional apnea divers. Subjects were informed of the purpose of this study and the possible risks involved before giving their written consent to participate. The study protocol was in accordance with the Declaration of Helsinki and was approved by the local ethics committee (Ethical Committee of ‘Hospital Universitari Son Dureta’). Dietary intake of energy, macronutrients, minerals and vitamins of each participant were assessed using a 7-day 24-h recall before the day of the study (Sureda et al., 2004a). This study was a double blind crossover study. The sportsmen were randomly divided into two groups. One group was supplemented with vitamin C capsules (1 g per day) for 7 days, and the other group took a placebo (composed of lactose). The exercise was a 4-h period of diving apnea, which subjects the organism to successive episodes of hypoxia and reoxygenation. Divers did not take the supplement on the day of the dive, and did not drink during the diving session. Ten days after the first diving apnea session of washout period, we repeated the procedure, but changing the diet supplemented capsules, that is the first group supplemented with vitamin C, were now supplemented with placebo. During the diving session divers remained more than 1 h without breathing under hypoxic conditions. Each immersion consisted in 68.9±6.5 s consumed per immersion and 178±22 s of recovery on the surface.
In a similar diving apnea session with the same divers participating in the study, the pulses of the divers were measured using a Polar Electro S18 pulsometer, and the results were treated with ‘Polar precision performance software version 3’.
Erythrocyte and lymphocyte purification and quantification
Blood samples were taken from the antecubital vein with suitable vacutainers with EDTA as anticoagulant. Samples were taken the morning of the diving apnea after overnight fasting, immediately after diving, and after 1 h of recovery. These blood samples were used to purify erythrocytes and lymphocytes following an adaptation of the method described by Boyum (1964). Blood was centrifuged at 900 g, at 4°C for 30 min after carefully introducing on Ficoll in a proportion of 1.5:1. The lymphocyte layer was carefully removed. The plasma and the organic phase were discarded. The erythrocyte phase at the bottom was washed twice with 10 ml of PBS and was finally reconstituted with distilled water in the same volume as plasma. Then the erythrocyte resuspension was hemolyzed with distilled water (1:1). The erythrocyte phase contained both erythrocytes and neutrophils. The lymphocyte slurry was washed twice with isotonic PBS and centrifuged for 10 min at 1000 g and 4°C. Finally, the cellular precipitate of lymphocytes was lysed with distilled water.
Hematological parameters such as erythrocyte, leukocyte and lymphocyte numbers, hemoglobin concentration and others were determined in an automatic flow cytometer analyzer Technicon H2 (Bayer) VCS system.
We determined the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, determined both with H2O2 (GPx) and cumene hydroperoxide (GPer) as substrate, glutathione reductase (GRd) and arginase in erythrocytes and lymphocytes. All the activities were determined with a Shimadzu UV-2100 spectrophotometer at 37°C.
CAT activity was measured by the spectrophotometric method of Aebi (Aebi, 1984) based on the decomposition of H2O2. GRd activity was measured by a modification of the Goldberg and Spooner spectrophotometric method (Goldberg and Spooner, 1984). This assay required oxidized glutathione as the substrate. GPx activity was measured using an adaptation of the spectrophotometric method of Flohé and Gunzler (1984). This assay required H2O2 and NADPH as substrates and GRd as enzyme indicator. GPer activity was carried out as for the GPx, but the substrate was cumene hydroperoxide. Superoxide dismutase activity was measured by an adaptation of the method of McCord and Fridovich (1969). The xanthine/xanthine oxidase system was used to generate the superoxide anion. This anion produced the reduction of cytochrome c, which was monitored at 550 nm. Arginase activity was determined by measuring an increase in the concentrations of ornithine (Colombo and Konarska, 1984). In the presence of acetic acid ornithine reacts with ninhydrin with a maximal absorbance at 515 nm.
Ascorbate was determined in lymphocytes by an HPLC method with electrochemical detection (Dhariwal et al., 1990). Immediately after lymphocyte purification samples were deproteinised with 30% trichloroacetic acid containing 2 mM EDTA. After complete precipitation of the proteins, samples were centrifuged for 5 min at 15 000 g at 4°C. Supernatants were stored at −70°C. Appropriate volumes of deproteinised samples were injected. The mobile phase consisted of 0.05 M sodium phosphate, 0.05 M sodium acetate, 189 μ M dodecyltrimethylammonium chloride and 36.6 mM tetraoctylammonium bromide in 25:75 methanol:water, pH 4.8. The HPLC system was a Shimadzu with a Waters Inc. electrochemical detector and Nova Pak, C18, 3.9 × 150 mm column. The potential of the chromatographic detector was set at 0.7 V vs an Ag/AgCl reference electrode.
Protein carbonyl derivatives determination
Protein carbonyl derivatives were measured in erythrocytes by an adaptation of the method of Levine et al. (1994) using the precipitates of deproteinised samples. Precipitates were resuspended with 2,4-dinitrophenylhydrazine (DNPH) 10 mM, and incubated for 60 min at 37°C. Then, samples were precipitated with 20% trichloroacetic acid, and centrifuged for 10 min at 1000 g and 4°C. The precipitate was washed twice with ethanol:ethyl acetate (1:1). Guanidine 6 M in phosphate buffer 2 mM, pH 2.3 was added to the precipitate, and samples were incubated for 40 min at 37°C. Finally, samples were centrifuged for 5 min at 3000 g at 4°C to clarify the supernatant and the absorbance was measured at 360 nm. The molar absorption of 22 000 M−1 cm−1 was used to quantify the levels of protein carbonyl. Samples were analyzed against a blank of guanidine solution.
Nitrite levels were measured in erythrocytes, lymphocytes and plasma by the acidic Griess reaction (Ridnour et al., 2000) using a spectrophotometric method. Lysed cells were deproteinised with 1.5 volumes of acetone and kept at −20°C overnight. Samples were centrifuged for 10 min at 15 000 g at 4°C, and supernatants were recovered. A 96-well plate was loaded with neutrophil samples or nitrite standard solutions (100 μl). Sulfanilamide (50 μl) (2% w/v) in 5% HCl was added to each well, and 50 μl N-(1-napthyl)-ethylenediamine (0.1% w/v) in water was then added. The absorbance at 540 nm was measured following an incubation of 30 min. All determinations were carried out in triplicate.
Inducible nitric oxide synthase (iNOS) protein levels
iNOS protein levels were determined in lymphocytes by ELISA using polyclonal antibody Anti-human iNOS (Stressgen). We followed an adaptation of the method previously described (Tauler et al., 2002). Suitable dilutions of lymphocyte suspensions, or standard dilutions of iNOS (50 μl) were placed in each well of the plate per duplicate (Polystyrene Assay Plate, Costar). The plate was then incubated at 37°C for 3 h. A solution of 1% bovine albumin was added into each well and the plate was incubated (37°C for 3 h) in order to saturate all binding protein sites. The plate was then washed four times with NaCl 0.9%–Tween 20. The commercial antibody (diluted 1000-fold) was placed into each well and the plate was newly incubated for 3 h at 37°C. The plate was then washed as above. The secondary antibody against the IgG chain, conjugated to alkaline phosphatase (diluted 500-fold) was added and the plate was incubated in the same conditions as above. The wells were newly washed and the phosphatase substrate solution was added. Finally, absorbance was measured at 405 nm.
Statistical analysis was carried out by using a statistical package for social sciences (SPSS 9 for windows). Results are expressed as means±s.e.m. and a value of P<0.05 was considered statistically significant. The statistical significance of the data was assessed by the two-way analysis of variance (ANOVA). The ANOVA factors were antioxidant diet supplementation (S) and the diving apnea session (D). The sets of data in which there were significant effects of supplementation, diving or a significant interaction between the two factors were tested by the ANOVA one-way test.
The two diving apnea sessions were very similar with no significant differences between groups in the number and in the characteristics of the immersions. Apnea sessions were about 55 immersions to a depth of 17 m, 68.9 s per immersion and 3.0 min on the surface between immersions. In a similar diving apnea experience, divers participating in the study performed around 55 immersions at a depth of 17 m in 4 h, the divers presented a mean 91.5 pulses/min, with a maximal heart rate of 134.
The analysis of the nutritional habits showed no differences between placebo and supplemented group because the study design (double-blind crossover study) ensured that the two groups were made up of the same divers. The total energy intake (2541±251 kcal/day, carbohydrates 42%, proteins 16%, fat 42%) and nutrient intake were in the range of recommended dietary allowances (RDA) (data not shown). The daily vitamin C intake previous to the diet supplementation (110±4 mg/day) was about 184% higher than the recommended daily allowance (RDA) (Moreiras et al., 2003).
Table 1 shows the effects of vitamin C supplementation on the changes in erythrocyte antioxidant and arginase enzyme activities induced by diving apnea. Activities are expressed per cell. No influence of vitamin C supplementation on the basal enzyme activities was found. A significant influence of the diving apnea was found in the CAT activity, with a higher erythrocyte CAT activity in the placebo group after the recovery than in basal conditions. Cellular oxidative damage measured as the carbonyl derivatives in erythrocytes was unaffected by apnea diving and by the diet supplementation with vitamin C.
Lymphocyte antioxidant enzyme activities expressed per cell are shown in Table 2. Superoxide dismutase activity was influenced by the diving apnea. Superoxide dismutase activity significantly increased about 35% during recovery in the placebo group, whereas maintained the initial values in the supplemented one. No effects of the two ANOVA factors were observed in the CAT, GPx and GRd activities. Neither was lymphocyte arginase activity affected by either diving apnea or by the vitamin C supplementation.
Figure 1 shows that the lymphocyte ascorbate concentration was significantly influenced by the diving apnea. The placebo and supplemented groups had similar lymphocyte basal values of ascorbate. This concentration increased after diving apnea but the increase was significant only in the supplemented group. After recovery the lymphocyte ascorbate concentration in the supplemented group remained high and placebo maintained the same postdiving value.
Plasma nitrite levels, as marker of endothelial NO synthesis, are shown in Figure 2a. Nitrite levels maintained basal values after diving, but these significantly increased after 1 h of recovery in both placebo and supplemented groups. When we analyzed erythrocyte nitrite levels (Figure 2b) a significant effect of the diving apnea session and diet supplementation and an interaction between the two ANOVA factors were found. Basal nitrite values were similar in erythrocytes from both groups. Nitrite basal levels were maintained in the placebo group after diving apnea and at recovery. However, erythrocyte nitrite levels increased significantly after diving apnea in the supplemented group and it followed increasing during recovery. Lymphocyte nitrite levels were influenced by the diving session, rising in both groups (Figure 2c). This increase in lymphocyte nitrite levels was only significant in the placebo group during recovery, without differences between placebo and supplemented groups.
The nitrite ratios between erythrocytes, lymphocytes and plasma are presented in Table 3. Both erythrocyte/plasma and lymphocyte/plasma ratios were influenced by diving apnea session. Erythrocyte/plasma ratio increased after diving only in the supplemented group, whereas the ratio decreased during recovery in both groups. The response of lymphocyte/plasma ratio was similar than the erythrocyte/plasma ratio in the supplemented group, increasing after exercise and decreasing during recovery, but it was unchanged in the placebo group.
Lymphocyte iNOS protein levels are shown in Figure 3. iNOS levels were influenced by vitamin C supplementation, following the same pattern as lymphocyte nitrite levels. The supplemented group presented lower values than the placebo one, but these were only significantly lower during recovery.
A diving apnea session subjects the organism to successive episodes of hypoxia and reoxygenation and this, combined with physical activity could induce oxidative stress (Sureda et al., 2004a, 2004b). Diving apnea increases circulating neutrophils and plasma CK levels suggesting the presence of an APIR similar to that which takes place after exercise, after ischemia/reperfusion or after infection (Shek and Shephard, 1998; Tauler et al., 2003; Sureda et al., 2004a). The low values of maximal heart rate and the mean heart rate measured in divers during a diving apnea session indicate exercise of a moderate intensity, even taking into account the bradycardia induced by apnea. The exercise intensity during diving apnea does not seem to be enough to completely explain the APIR as we have shown in a previous study (Sureda et al., 2004a). However, the little changes in the antioxidant enzyme activities in erythrocytes and lymphocytes, together with the lack of oxidative damage in erythrocytes after diving reflect a production of ROS, which is balanced with their deactivation. Our modest changes in antioxidant system agrees with the results obtained in previous studies after static and dynamic apnoeas (Joulia et al., 2002; Joulia et al., 2003). Increased ROS production can activate the CAT in erythrocytes (Tauler et al., 1999) and induce the synthesis of antioxidant enzymes such as SOD in lymphocytes (Khassaf et al., 2003; Tauler et al., 2003). We previously reported that exhaustive exercise induced a general increase in the lymphocyte antioxidant enzyme activities, which could be influenced by vitamin C supplementation (Tauler et al., 2003). Diet supplementation with vitamin C protect both erythrocytes and lymphocytes from the ROS overproduction induced by hypoxia/reoxygenation and avoid the antioxidant enzyme activation.
Ascorbate levels determined in lymphocytes are believed to reflect the tissue concentrations of this vitamin (Levine et al., 1996). Diet supplementation with vitamin C for 1 week did not alter the basal lymphocyte ascorbate levels, probably as result of the high vitamin C intake of subjects in their normal diet before supplementation. They consumed 110 mg/day of vitamin C in their normal diet. It has been suggested that lymphocytes become saturated at vitamin C intakes of 100–200 mg per day (Levine et al., 1996). Therefore, we can suppose that the lymphocytes were already near to saturation with ascorbate before the supplementation. However, a general increase in lymphocyte ascorbate concentration was observed after diving apnea and recovery; mainly in the supplemented group. These results are in agreement with previous studies, which showed increases in lymphocyte ascorbate after different exercises (Thompson et al., 2003). Lymphocytes respond to the oxidative stress generated by exercise increasing ascorbate uptake. However, as the ascorbate availability in the supplemented group is higher than in the placebo one, the increase observed in ascorbate concentration was greater in this group. Thus, the lymphocyte requirements for ascorbate increase in the oxidative stress situation induced by hypoxia/reoxygenation and these could be satisfied in the supplemented group, as a consequence of the supplementation, but not in the placebo group.
Cellular nitrite concentration has been used as an indicator of NO production (Dejam et al., 2004). Nitric oxide synthesized by endothelial NOS plays a crucial role in the regulation of vascular tone and blood flow. In the lumen, NO is either oxidized by oxygen, resulting in the formation of nitrite, or is taken up by erythrocytes. Plasma nitrite levels mainly reflect endothelial NO production (Dejam et al., 2004). The diving apnea session increased the nitrite levels in plasma both in the placebo and supplemented groups. The high plasma nitrite levels were attained during recovery, after 3 h of the cessation of the repetitive episodes of hypoxia/reoxygenation, when oxygen was fully available. As the half-life of NO is very short, we can suspect that the high rate of NO production is attained after diving. Hypoxic conditions are known to induce NO synthesis by endothelial cells (Barer et al., 1993; Swenson et al., 2005) in accordance with the increased plasma nitrite levels after diving apnea session. The lack of differences between placebo and supplemented groups could indicate that endothelial cells are saturated with ascorbate in both groups. It has been suggested that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis (Levine et al., 1996; Heller and Werner, 2002). The high ascorbate intake, in the divers, with the diet could maintain tetrahydrobiopterin in the reduced state in endothelial cells (Shi et al., 2004). The high NO synthesis after diving reflects vasodilatation and participates in a mechanism to protect the divers to suffer from decompression sickness. We have evidenced that apnea divers are also at risk of suffering from decompression sickness like scuba divers (Batle, 2002). The vasodilatation induced by NO could avoid the possible concentration of gas bubbles in a main vessel and, hence, minimize the risk of damage. The high increase in plasma nitrite in both groups after apnea diving is in accordance with this hypothesis.
Nitrite increased in erythrocytes after diving apnea and recovery but only in the supplemented group as is evidenced with a higher values in the relation between erythrocytes and plasma nitrite levels. Recent studies suggest blood flow control requires the transport of NO from the arteries to the arterioles (Nagababu et al., 2003). In the arteries, with high oxygen partial pressure, NO rapidly reacts with oxygen limiting its transport and its availability for vasodilatation. Nitric oxide is taken up by erythrocytes and reacts quickly with hemoglobin providing a pool of NO bound to hemoglobin. Hypoxic conditions in arterioles stimulate nitrite reduction by hemoglobin producing vasoactive NO and vasodilatation. As a result, the hemoglobin is oxidized to methemoglobin. Vitamin C could enhance this transport of NO by erythrocytes participating in the regeneration of hemoglobin from methemoglobin and facilitating the vasodilatation in peripheral vessels. It has been evidenced in ‘in vitro’ studies that reduced glutathione enhances the erythrocyte capability to produce NO (Crawford et al., 2003; Gladwin and Schechter, 2004). Ascorbate could play a role in the provision of NO to hypoxic tissues during the episodes of hypoxia/reoxygenation, when the availability of oxygen is limited. On the other hand, more dilated vessels could be important in order to reduce the possibility of suffering from decompression sickness.
Lymphocyte nitrite levels tend to increase in both groups but are significantly different only in the placebo one. This differential response induced by vitamin C is manifested when the lymphocyte/plasma ratio is calculated. iNOS protein levels in lymphocytes also tend to be higher in the placebo group than in the supplemented one. The lymphocyte response to oxidative stress induced by hypoxia/reoxygenation could be mediated by the induction of the iNOS expression by ROS and the consequent NO production. Nitric oxide plays a role in the activation of the lymphocyte response (Cifone et al., 2001; Roozendaal et al., 2002). Moreover, iNOS also produces superoxide anion when there is a limited arginine availability (Xia et al., 1996). It has been evidenced that, under stress situations such as intense exercise or hypoxic conditions, the immune functions of lymphocytes are inhibited and the apoptotic rate increased. Vitamin C supplementation could avoid the increase in the iNOS expression in the supplemented group by removing the excess of ROS produced and also it can reduce the superoxide anion synthesis by iNOS.
In summary, we have shown that vitamin C supplementation influences the response of lymphocytes and erythrocytes to oxidative stress induced by hypoxia/reoxygenation. Erythrocyte NO levels, which are involved in the control of the blood flow, could be dependent of ascorbate availability. Vitamin C availability in erythrocytes could facilitate the peripherical vasodilatation and consequently reduce the effects of the decompression sickness. Lymphocyte response to oxidative stress is mediated by the iNOS expression, which could be regulated by the ROS and antioxidants. Vitamin C supplementation in lymphocytes could interfere with the ROS function as cellular messengers.
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This work has been Granted by the Spanish Ministry of Health (Program of Promotion of Biomedical Research and Health Sciences, Project PI021593).
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Sureda, A., Batle, J., Tauler, P. et al. Vitamin C supplementation influences the antioxidant response and nitric oxide handling of erythrocytes and lymphocytes to diving apnea. Eur J Clin Nutr 60, 838–846 (2006). https://doi.org/10.1038/sj.ejcn.1602388
- vitamin C
- oxidative stress
- nitric oxide
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