The dietary requirements for this study were based on the Dietary Reference Intake for the Japanese (2020) and the guidelines for athletes (Thomas et al. 2016). Hcy is produced from methionine, which is an essential amino acid. Therefore, dietary protein is a source of methionine. Although standards for protein intake have been established, there are no clear intake standards for methionine. Therefore, we set the limit to 1,500 mg/day or less, based on the results of previous studies (Chambers et al. 1999; Verhoef et al. 2004). Two previous studies have investigated the changes in total Hcy (tHcy) concentrations with oral intake of free methionine and dietary intake of a high-protein diet (methionine intake 3,100–3,500 mg/day, 30 mg/kg body weight/day) (Chambers et al. 199; Verhoef et al. 2004). However, the tHcy concentration increase is much lower with dietary intake. Furthermore, a study comparing a methionine intake of 4,100–4,500 mg/day as a high-protein diet and 1,600–1,700 mg/day as a low-protein diet reported that the high-protein diet increased tHcy concentrations throughout the day, but did not affect fasting tHcy concentrations, which returned to baseline the next morning (Vehoef et al. 2005). In summary, the methionine intake in this study (< 1,500 mg/day) is a reasonable level that does not cause much change in tHcy concentration throughout the day and does not affect the fasting plasma Hcy concentration.
The exercise conditions implemented in this study did not have a set intensity. However, previous studies have shown that, by calculating the average heart rate and comparing it to both the maximum heart rate and the resting heart rate of the individual, it is possible to calculate the relative heart rate to the unworkable intensity (%HRmax) (Karvonen and Vuorimaa 1988). The exercise performed was a run of 10,000 m over 40 minutes which maintained a heart rate of 166 ± 11 beats/min (exercise intensity, 75 ± 8% HRmax). Therefore, the acute exercise performed in this study can be considered a high-intensity endurance exercise.
In this study, the plasma Hcy concentration in the Res was significantly lower than that in the Exe per participant and for the overall average of all participants. Furthermore, in comparison to the reported cut-off value of 5.4 µmol/L for stress fractures, this was significantly lower in the Res and significantly higher in the Exe. These results indicate that, in female long-distance runners, acute endurance exercise increases fasting plasma Hcy concentrations the following day when the dietary conditions are held constant.
Previous studies have reported that acute exercise (both endurance and resistance exercises) increases plasma Hcy concentrations. Hermann et al. (2003) reported that three different types of acute endurance exercise (marathon, 100 km run, and long-distance bicycle race) can induce an increase in plasma Hcy concentration that lasts for 24 hours after the end of exercise. A study by Venta et al. (2009) also reported a significant increase in plasma Hcy concentration after acute endurance exercise (21–28 minutes of incremental exercise load to exhaustion). These reports are based on amateur athletes, i.e., participants who perform exercise daily. In contrast, studies by Bizheh et al. (2011) and Iglesias-Gutierrez et al. (2012) included participants with sedentary lifestyles who did not exercise daily. A previous study reported a significant increase in plasma Hcy levels after acute circuit resistance exercise (Chrysohoou et al. 2004). Acute endurance exercise was performed at a high intensity (40% VO2 peak) for approximately 36 minutes and at low intensity (80% VO2 peak) for approximately 70 minutes, and they reported a significant increase in plasma Hcy concentration under both exercises (Chrysohoou et al. 2004).
Although we observed an increase in plasma Hcy concentrations under acute exercise, different results have been reported for plasma Hcy concentrations with habitual exercise. Chrysohoou et al. (2004) observed that plasma Hcy concentrations were significantly lower in those who habitually performed endurance exercise than in those with sedentary lifestyles. Similarly, Unt et al. (2008) found a significant negative correlation between MET hours per week and plasma Hcy concentrations. However, caution should be exercised when interpreting these results, since they were based on cross-sectional studies, and not those of exercise studies or trials. When considering the effect of habitual physical activity on Hcy concentrations, it is difficult to establish a causal relationship between daily physical activity and plasma Hcy concentrations, since participants with an active lifestyle tend to be more aware of other factors with health benefits.
Our results are consistent with previous studies which found that acute endurance exercise increases plasma Hcy concentrations (Hermann et al. 2003; Venta et al. 2009; Iglesias-Gutierrez et al. 2012). Furthermore, since the dietary and living conditions in this study were aligned, the results can be considered reliable, i.e., that an increase in Hcy concentrations could be attributed to the effect of exercise. Considering the results of previous studies, this increase may be independent of the type (endurance or resistance) and duration of exercise. However, the results are inconsistent regarding whether the increase in plasma Hcy concentration after acute exercise is temporary or lasts for 24 hours after the end of exercise. By excluding the type of exercise as a confounding factor, the exercise habits and fitness levels of the participants could be considered. In our sample, the participants exercised daily and participated in competitive events. In comparison, the participants included in two previous studies were sedentary (Bizheh et al. 2011; Iglesias-Gutierrez et al. 2012).
The present study could not determine whether habitual exercise led to lower plasma Hcy concentrations. However, the impact of habitual exercise among athletes is still unclear. This is because of the problem of high plasma Hcy concentrations in some athletes, which leads to a deterioration in bone quality. Athletes (especially endurance athletes such as long-distance runners) may have a gradually increasing baseline plasma Hcy concentration during continuous high-intensity endurance exercise. To understand the effects of exercise on plasma Hcy concentrations, it is necessary to examine not only the changes caused by acute endurance and resistance exercise, but also the chronic changes caused by habitual exercise training.
Various studies have also been conducted on the mechanism of acute exercise-induced increase in plasma Hcy concentration. During exercise, the demand for creatine synthesis increases because of energy production in the muscles. For creatine synthesis, the liver increases its consumption of the methyl group donor S-adenosylmethionine by demethylation, which in turn increases the production of S-adenosyl Hcy (SAH). Thus, the cleavage of SAH yields Hcy, leading to an increase in its concentration. Sotgia et al. (2007) demonstrated that exercise-induced changes in the concentration of plasma Hcy were associated with changes in the concentration of plasma creatine, an important methylated compound. Additionally, the tricarboxylic acid cycle and amino acid metabolism are activated during exercise. This increases the requirement for folic acid, vitamin B6, and B12 (Bolander-Gousille 2002)., which are also associated with Hcy metabolism. Vitamin B6 is necessary as a cofactor for transaminase, decarboxylase, and glycogen phosphorylase (Joubert and Manore 2006). Therefore, there is insufficient vitamin B6 available for Hcy metabolism, especially for conversion to cysteine in the sulfation pathway, resulting in elevated Hcy concentrations. These two factors are thought to be responsible for the increase in plasma Hcy concentrations caused by acute exercise. Furthermore, these increases in plasma Hcy concentrations may be temporary, and levels can return to baseline within 24 hours. However, the results of the present study show that the fasting plasma Hcy concentration was elevated the day after acute high-intensity endurance exercise; therefore, the exact mechanism remains unclear. To elucidate the above mechanisms, it is necessary to track changes over time not only in Hcy, but also in vitamin B, folic acid, and creatine concentrations in the blood.
This study had several important limitations. First, only “acute endurance exercise” was examined, and the effects of resistance exercise, is unknown. Second, the exact intensity of the acute endurance exercise is unknown. Third, the effects of continuous training are unknown. Fourth, although the conditions of dietary and lifestyle factors (drinking, smoking) were matched, other confounding factors, such as genetic mutations and other inborn factors, were not controlled for. Lastly, there was no information on plasma concentrations of folic acid and other B vitamins.
Stress fractures can be difficult to diagnose during the early stages of the condition and can lead to prolonged withdrawal from competition. Therefore, it is important to examine the relationship between plasma Hcy concentrations and risk for the development of stress fractures. This study investigated whether increased plasma Hcy is a risk factor for stress fractures in female long-distance runners under a range of dietary and exercise conditions. Ours is one of the first studies to clarify the effect of “exercise” on plasma Hcy concentrations.
The present study showed that, in female long-distance runners who are at a high risk of stress fracture, and who are expected to have large changes in plasma Hcy concentrations due to diet and exercise, early morning fasting plasma Hcy concentrations on the following day were increased by the performance of acute endurance exercise when dietary conditions were aligned. Mild to moderate hyperhomocysteinemia in female endurance athletes is a risk factor for stress fractures. More observational and intervention studies are necessary to demonstrate the effects of habitual exercise training on plasma Hcy concentrations in contrast to acute endurance exercise as the outcome of interest in the present research.