The B-vitamins are particularly important for physical exercise because they are involved in regulation of energy metabolism by modulating the synthesis and degradation of carbohydrates, fats, and proteins. Folate, one of the B-vitamins, is reduced to tetrahydrofolate, serving as an essential cofactor in methylation reactions, including the vitamin B₁₂-dependent formation of methionine from homocysteine, and as a carrier of one-carbon units involved in the synthesis of purines and pyrimidines. The roles of folate and vitamin B₁₂ in assisting with cell division make them critical nutrients for growth, synthesis of new cells, such as red blood cells, and for the repair of damaged cells and tissues [1]. Many methylated molecules (e.g., acetylcholine, creatine, and DNA) are essential for physical activity [234]. The exercise-induced metabolic demand possibly exerts an increased turnover of many of these substrates. It can be supposed that regeneration of these substrates is accompanied by considerable stimulation of the methionine metabolism [5]. In the degradation of amino acids such as valine, isoleucine, methionine, and threonine and fatty acids, these amino acids are converted to propionyl-CoA and fatty acids are oxidized to acetyl-CoA and propionyl-CoA. Acetyl-CoA enters directly into the tricarboxylic acid (TCA) cycle. Propionyl-CoA is carboxylated to methylmalonyl-CoA and finally converted to succinyl-CoA by methylmalonyl-CoA mutase. This enzyme requires vitamin B₁₂ as a cofactor [6]. Succinyl-CoA is used for energy production in the TCA cycle. Thus, folate and vitamin B₁₂ status and requirements may be altered by energy production and rebuilding and repair of muscle tissue induced by physical activity.

Acute exercise may cause muscular damage and accelerate protein catabolism, which will lead to an increase of muscular amino acid pools and homocysteine production in the methionine metabolism [7]. β-oxidation of fatty acids is a minor component of energy expenditure early in exercise, however sustained exercise at low or moderate intensity requires a substantial contribution of energy from fat oxidation. Physical exercise demands a significant amount of energy turnover and the metabolic pathways for energy production are stressed during exercise. Exercise training induces adaptations to stress during exercise and plays a key role in homeostasis related to an increase in aerobic metabolism [89]. Endurance exercise training resulted in increased plasma free fatty acids and free fatty acid oxidation and reduced glucose oxidation [10]. The effects of exercise training in increases of body turnover or requirement of several micronutrients including thiamin, riboflavin, and vitamin B₆ have been reported [11121314]. However, little information is available on the effects of exercise training and acute physical activity on folate and vitamin B₁₂ status.

The aim of this study was to examine the influence of 5-week regular moderate exercise training and duration of acute aerobic exercise on plasma folate and vitamin B₁₂ concentrations in rats. We hypothesized that acute aerobic exercise can decrease plasma folate and vitamin B₁₂ concentrations since folate and vitamin B₁₂ are used as co-factors of methionine and lipid metabolism and the magnitude of this decrease might differ according to regular moderate exercise training and duration of acute exercise.

This study showed that regular aerobic exercise training for 5 weeks altered plasma folate and vitamin B₁₂ concentrations. However, plasma folate and vitamin B₁₂ concentrations were not altered under different durations of acute aerobic exercise in both NT and ET groups.

Physical activity influences protein metabolism and turnover and thus the concentration of certain amino acids, including methionine [16]. Methionine concentration in plasma increased significantly during exercise and methionine concentration was shown to decrease below basal levels hours after exercise in both humans and rats [1819]. This finding suggests that decreased plasma methionine after exercise may be due to increased muscle anabolism requiring methionine thus leading to reduced substrate for transmethylation reactions and homocysteine concentration [1820]. Thus, individuals who participate in regular exercise training may have less folate and vitamin B₁₂ available for methionine metabolism. In this study, we hypothesized that regular moderate aerobic exercise training significantly reduced plasma folate concentration in rats, which may be due to an increased use of folate for methylation including methionine metabolism by physical activity. However, vitamin B₁₂ level was significantly higher in the ET group than in the NT group. In this study, folate and vitamin B₁₂ intakes were similar in the NT and ET groups. Thus, other adaptation mechanisms for exercise training may affect plasma vitamin B₁₂ concentration.

Endurance exercise training results in sparing of plasma glucose and liver glycogen by increasing the oxidation of fat during submaximal exercise [21]. After training, the decreased carbohydrate utilization during exercise is compensated for by an increase in fat oxidation [22]. Mild- or moderate-intensity exercises are associated with a 5-10-fold increase in fat oxidation above resting amounts [23]. The increased capacity to use fat following exercise training results from an enhanced ability to mobilize free-fatty acids from fat depots and an improved capacity to oxidize fat consequent to the increase in the muscle enzymes responsible for fat oxidation [24]. Choi and Cho [25] reported that aerobic exercise trained rats had significantly higher plasma free-fatty acid compared to sedentary rats. The function of vitamin B₁₂ is different from that of folate, which is involved in fat oxidation by acting as a cofactor of methylmalonyl-CoA mutase. Therefore, in this study, an increase in plasma vitamin B₁₂ concentration in the ET group may be due to increased fat oxidation as an exercise adaptation.

In this study, plasma folate and vitamin B₁₂ concentration were also determined by different durations of acute aerobic exercise in the NT and ET groups. Plasma folate concentrations in both groups were not changed by acute exercise duration although there were significant alterations in folate concentration after 5-week training. The acute effect of exercise on protein turnover is catabolic, thus total plasma concentration of amino acids and sulfur-containing amino acids as well as homocysteine is increased after acute physical activity [26]. Thus, during acute exercise, the metabolism requiring folate may not be promoted by increasing exercise duration. However, long-term effects are an overall increase in protein synthesis and lean body mass [27]; therefore, after exercise training, the folate requirement may be increased for formation of methionine from increased homocysteine by acute exercise for protein synthesis and lean body mass. Although vitamin B₁₂ level of each group was not altered significantly by acute exercise duration, vitamin B₁₂ level of the NT group tended to increase by duration. Before exercise and after 0.5-hour acute aerobic exercise, vitamin B₁₂ concentrations were significantly lower in the NT group compared with those of the ET group; however there were no differences in vitamin B₁₂ concentrations after 1-hour exercise. The study conducted by Choi and Cho [25] reported that after 1-hour acute aerobic exercise, plasma free-fatty acids in both exercise trained and sedentary rats were similar although the concentration in exercise trained rats was higher than that in sedentary rats before exercise. Thus, in the current study, an increase in plasma free-fatty acid by aerobic exercise may demand a vitamin B₁₂ influx into plasma from the stored vitamin B₁₂ in the liver for fat oxidation in the NT group.

However, the results of human studies regarding the effects of exercise on plasma folate and vitamin B₁₂ levels are equivocal. Plasma folate was increased during a 3-week training (swimming) period, but dropped after 5 days of recovery training [7]. In physically active subjects, erythrocyte folate and serum vitamin B₁₂ were stable in both a normoxia- and hypoxia-training for 4 weeks [28]. In soccer players, plasma folate concentrations before and after acute high-intensity sprint exercise were similar, but vitamin B₁₂ concentration after exercise was significantly higher than that before exercise [29]. Individuals involved in high intensity physical activity for at least 5 years had slightly higher plasma vitamin B₁₂ concentrations than sedentary individuals [30]. The plasma B₁₂ levels at 6 months in exercising (brisk walking for 20-60 min) women did not differ from those in a control group of similarly afflicted women who did not exercise [31]. Also, vitamin B₁₂ concentrations were not significantly influenced either by 4 weeks of strenuous endurance training or by differences in duration of acute exercise in trained male triathletes [32]. Most human studies were conducted with an uncontrolled diet during experiments and the subjects were already trained before exercise training or acute exercise for the studies, which might have induced the different results. No rat study to find the effects of exercise training and acute exercise on plasma folate and vitamin B₁₂ has been reported.

In conclusion, regular moderate exercise training decreased plasma folate level, but increased vitamin B₁₂ concentration, which might result from the adaptation for exercise training. By increasing duration of acute aerobic exercise, no significant alterations of plasma folate and vitamin B₁₂ concentrations were observed in the NT and ET groups. Further research is needed in order to examine change of folate and vitamin B₁₂ requirements and necessity for supplementation of the vitamins during exercise training and to determine the association of vitamin B₁₂ and fat oxidation for body adaptations of aerobic exercise training.