Homocysteine (Hcy) is a sulfur-containing amino acid derivative of methionine, which has been argued as a risk factor for the development of cardiovascular disease (CVD) (6,35). Moderate elevations in plasma total homocysteine (tHcy) concentrations have been involved in atherosclerosis through different mechanisms (16,37): a prothrombotic effect due to the interaction with procoagulant factors, a direct cytotoxic effect on endothelial cells, or an in vivo endothelial dysfunction due to the promotion of inflammation or oxidative stress.
Regular physical activity is now well established as a key component in the maintenance of good health and disease prevention, and there has been specifically recognized to reduce the risk for the development of CVD (2). This effect is generally associated with more favorable novel and traditional cardiovascular biomarkers levels (19), but for plasma tHcy, the results are contradictory. There is a spectrum of studies directly investigating the effect of physical exercise on plasma tHcy. Lower plasma concentrations of tHcy have been observed in subjects practicing regular physical activity (23) and were inversely related to cardiorespiratory fitness (V˙O2max) (31), although these observations have not been a constant finding. On the contrary, acute intense exercise has been demonstrated to increase plasma tHcy concentrations (15), in an extent somehow related with the type, duration, and intensity of exercise.
The exact mechanism by which plasma tHcy concentrations increase after an acute intense exercise is unknown. It has been shown that acute exercise increases oxygen uptake and free radical production (14), which in turn could influence thiol-disulfide exchange and thiol redox reactions leading to increased oxidized forms in plasma and, therefore, increased tHcy concentrations. Two additional mechanisms for the hyperhomocysteinemic effect of strenuous exercise may involve the nonenergetic exercise-induced metabolic stress. First, exercise may involve an increased demand of several methylated substrates, such as creatine or acetylcholine (10), giving rise to an accelerated consumption of the methyl donor S-adenosylmethionine, converted into S-adenosylhomocysteine, which is reversible hydrolyzed to Hcy and adenosine (28). Second, acute physical activity may cause muscular damage and accelerate protein catabolism, which will produce an increase of muscular amino acid pools and, in turn, of the Hcy production in the methionine metabolism (10,32). In both situations, the primary synthesis of Hcy occurs in the reduced form, and the excess of reduced Hcy (rHcy) for the capacity of remethylation and transsulfuration pathways undergoes export to the extracellular space and oxidation (3). As a result, it could be hypothesized that an increase in plasma rHcy could be detected along with the increase in plasma tHcy. On the contrary, a possible finding of increased tHcy concentrations after exercise without concomitant higher concentrations of rHcy would be related to substantial differences in the clearance rates of the oxidized and reduced forms from blood, as it has been previously referred (12).
Exercise has long been associated with an increased amino acid turnover and with several plasma changes in amino acid concentrations. Quantitatively, most important changes are due to increased muscular capture of plasma branched-chain amino acids for energetic metabolism and export to circulation of alanine (Ala) and glutamine for gluconeogenesis in liver cells, but minor effects on plasma concentrations of other amino acids have also been identified (32). Changes in plasma concentrations of Hcy and other aminothiols in exercise may also respond to this enhanced metabolic stress. On the other hand, the metabolism of sulfur amino acids requires the coenzyme function of vitamin B12 and folate in the remethylation of Hcy to methionine (Met), as well as pyridoxal-5′-phosphate (PLP), the active form of vitamin B6, in the transsulfuration pathway through cystathionine and cysteine (Cys). Likewise, PLP is a coenzyme for transaminases, decarboxylases, and other enzymes used in metabolic transformations of amino acids, and folate participates in some of these processes as part of the one-carbon pool. Thus, in acute exercise, altered vitamin availability may contribute to the hyperhomocysteinemia and to other plasma amino acid changes.
On the other hand, a potential relationship between the exercise-induced changes in plasma tHcy concentrations and renal function has not received as much attention as was to be expected of the interaction showed in the general population (23). Therefore, the aim of this study was to investigate the effect of incremental bouts of two aerobic intense exercises, cycling and kayaking, which represent very different muscular demands, on plasma reduced and total concentrations of Hcy and Cys (rCys, tCys) and on the metabolically related vitamins B6 and B12, folate, amino acids, and creatinine. Plasma changes were studied in young healthy athletes before and after a single bout of specific exercise during a less intense training period at the beginning of the season.
MATERIALS AND METHODS
Twenty-nine healthy male aerobic athletes aged between 14 and 22 yr were included into the study: 15 cyclists younger than 23 yr from a nonprofessional cycling team, performing long-duration aerobic training and 14 kayakers from the Spanish Junior National Team, performing shorter and more intense aerobic training and resistance-strength training. Men were selected as subjects because of the stability of their hormonal status.
The study was performed in accordance with the Helsinki Declaration of the World Medical Association, and informed consent was requested from parents and subjects. Each participant gave written, informed consent, and the study protocol was approved by the external Regional Ethical Committee of Clinical Investigation of the Principado de Asturias. At recruitment, all participants underwent a medical examination.
Participants were studied during a period of low-intensity training and were advised to avoid changes in dietetic or lifestyle habits the week before the experiment. Cyclists and kayakers underwent a bout of incremental specific exercise test to exhaustion, described elsewhere, lasting approximately 28 min in cyclists and 21 min in kayakers (7,20). Tests were carried out in a cycle ergometer (Orion S.T.E., Toulouse, France) and a kayak ergometer (Modest, Odense, Denmark). Expired respiratory gases, V˙O2max, and RER were determined by a metabolic cart (Vmax 29; SensorMedics, Corp, Yorba Linda, CA). HR was continuously monitored by telemetry (Polar, Kempele, Finland), and the capillary blood lactate concentration was analyzed by an electroenzymatic method (Analox GM7, London, England).
Blood Sampling and Processing
After an overnight fast, immediately before and 30 ± 5 min after the exercise test, venous blood samples were collected from antecubital vein in four evacuated tubes for each subject: a) plain tubes for analysis of folate and vitamin B12 and basic metabolites including creatinine (Vacuette; Greiner Bio-one, Kremansmünster, Austria); b) heparinized tubes for amino acids (Vacuette); c) EDTA-containing tubes for hemoglobin, hematocrit, and vitamin B6 (Vacuette); and d) acidified sodium citrate (pH 4.3)-containing tubes for analysis of reduced and total Hcy and Cys (Stabilyte; Biopool, Umeå, Sweden).
For heparinized and acidified citrate plasma samples, tubes were immediately transported to the laboratory in a refrigerated container and then centrifuged at 3000g for 5min at 4°C. Blood without anticoagulant was allowed to clot for 10 min at room temperature and centrifuged at 2000g for 10 min at 4°C. Plasma and serum samples were aliquoted and stored at −80°C until analysis.
Whole-blood EDTA tubes were directly introduced in a Sysmex XE2100 (Roche Diagnostics, Mannheim, Germany) to measure hemoglobin and hematocrit. Both data were used to account for plasma volume change after exercise, according to the formula from Dill and Costill (5). Before statistical analysis, the concentrations of plasma aminothiols, vitamins, amino acids, and creatinine were adjusted for plasma volume change after exercise.
For every metabolite, pre- and postexercise samples were analyzed in the same batch.
Hcy and Cys.
Acidified sodium citrate plasma samples were analyzed within 2 wk after storage at −80°C, a period that allows a satisfactory recovery of the reduced forms of aminothiols (38). Total concentrations were analyzed as described by Pfeiffer et al. (21), and reduced plasma concentrations were assessed using a derived method previously validated in our laboratory (33), with minor modifications.
In short, total thiols were determined in the plasma after reduction with tris(2-carboxyethyl)phosphine and protein precipitation with trichloroacetic acid (TCA). The clear TCA supernatants were incubated for 60 min at 60°C with 4-fluoro-7-sulfobenzofurazan (SBD-F) in sodium borate buffer, pH 10. For reduced thiols analysis, plasma was directly incubated with SBD-F in borate buffer at 60°C for 60 min and further precipitated with TCA. Thiol separations were performed by isocratic reversed-phase high-performance liquid chromatography (RP-HPLC; Breeze Waters, Milford, MA) according to the method of Pfeiffer.
Quantitative analysis was performed with the use of a common Hcy/Cys external standard in both procedures, aswell as an internal standard, cisteamine, in the total concentrations determination. The within-day and between-day coefficients of variation were below 2.8% and 4.1% and below 4.1% and 12.1% for total and reduced concentrations, respectively.
Amino acids, vitamins, and creatinine.
Measurements of amino acid concentrations in heparinized plasma samples were performed by gradient RP-HPLC using the Pico Tag method (Waters) according to the manufacturer's specifications. Methionine sulphone was used as internal standard as well as a calibration mixture of two commercial solutions (Sigma-Aldrich, St. Louis, MO) of acid-neutral and basic amino acids in HCl 0.1 mol·L−1. The within-day and between-day coefficients of variation ranged between 5.4% and 9.9%.
The assay for plasma vitamin B6, as PLP, on the basis of precolumn derivatization and fluorometric detection, was performed by isocratric RP-HPLC as described by Talwar et al. (30). A commercially certified calibrator (Chromsystems, Martinsried, Germany) was used. The within-day and between-day coefficients of variation were 2.3% and 5.3%, respectively, at the concentration of 55.9 nmol·L−1.
Serum concentrations of creatinine and vitamin B12 and folate were determined with a Jaffe kinetic method and electrochemiluminescence immunoassays, respectively, in an SWA Modular Analytics from Roche Diagnostics.
Data are given as mean ± SEM or median and percentiles (P90), according to their distribution. Gaussian distribution was assessed by the Shapiro-Wilk test and by the verification of the straightness of a normal plot. Log transformations were needed for PLP and folate in the comparison tests. Main effects were analyzed by two-way ANOVA (exercise × group) for repeated measures with exercise as the repeated measure. The relation between variables was evaluated by Pearson correlation coefficient or Spearman rank-order correlation coefficient, as appropriate. Two-tailed P < 0.05 values were considered statistically significant. The statistical analysis was carried out with the SPSS software (11.0 for Windows; SPSS, Inc., Chicago, IL).
Anthropometric, biochemical, and hematological characteristics of subjects before exercise tests are shown in Table 1. Significant differences between kayakers and cyclists were observed in the anthropometric data, in the extension of training period (50% longer in cyclists), and in baseline level of fitness (20% higher V˙O2max in cyclists). However, the most common biochemical and hematological analytes were similar in both groups, with the exception of a significantly higher hemoglobin concentration in cyclists.
After an incremental bout of specific exercise, the concentrations of aminothiols were increased regardless of the group considered, as shown in Table 2. Plasma concentrations were significantly higher than baseline values in tHcy (17.7 ± 1.5%; F = 122, P < 0.001) as well as in tCys (9.9 ± 1.6%; F = 42.7, P < 0.001). Likewise, reduced forms in plasma were increased after exercise 10.6± 1.6% for rHcy (F = 39.2, P < 0.001) and 7.6 ± 2.2% for rCys (F = 9.4, P < 0.01). Simultaneously, kayakers and cyclists showed significant elevations of PLP (F = 15.6, P < 0.01), vitamin B12 concentrations (F = 12.3, P < 0.001), and creatinine (F = 166, P < 0.001) after acute exercises, but no changes were seen in folate. Groups showed significant differences in circulating concentrations of rHcy (F = 5.4, P< 0.05), tCys (F = 8.5, P < 0.01), and vitamin B12 (F = 15.6, P < 0.01), whereas no differences were seen in the change with exercise (exercise × group interaction) in aminothiols and vitamins.
To assess the relative prevalence of aminothiol changes in plasma, we calculated the respective ratios (Table 2). Ratios of rHcy to rCys (F = 4.5, P < 0.05) and tHcy to tCys (F = 59.5, P < 0.001) increased after exercise, although lesser variations were observed in the ratio of tHcy to tCys after cycling, according to the significant exercise × group interaction (F = 8.2, P < 0.01).
The total plasma amino acid concentrations at rest were significantly higher in kayakers than in cyclists as a result of the significant higher concentrations of most amino acids, and these concentrations were modified by the acute bouts of intense exercise (Table 3). In general, the differences were low between before and after exercise, except for Ala, which showed the highest increase (F = 215, P < 0.001). On the contrary, branched-chain amino acids decreased after exercise (F = 6.4, P < 0.05). The total plasma amino acid concentration was increased after exercise (F = 24.6, P < 0.001) mainly due to the increase of nonessential amino acids (F = 86.5, P < 0.001). However, changes in plasma concentrations with exercise were similar between sports, with the exception of a higher increase of taurine (Tau) in cyclists (21.1 ± 4.7%) compared that in kayakers (7.8 ± 3.3%; exercise × group interaction, F = 4.6, P < 0.05).
Changes in plasma concentrations of reduced and total aminothiols after both types of exercise did not reach significant correlation with changes in free amino acids or baseline vitamins. On the other hand, changes in free amino acids showed a high degree of covariation, remaining reciprocally related one to each other. Provided that the extent of metabolic plasma changes with exercise was not significantly different between sports (except for Tau), we also correlated the pooled data of the two groups (Table 4). Accordingly, significant and positive correlations were seen among changes in aminothiols, with the exception of that of rHcy with tCys. Likewise, significant and positive correlations were seen between changes in reduced and total aminothiols with changes in PLP and vitamin B12 and with changes in plasma creatinine concentrations.
The main finding of this study indicates that the acute physical activity increased plasma concentrations of tHcy in association with an increase in rHcy, and this hyperhomocysteinemic effect seems to be independent of the type of exercise and the vitamin status. Moreover, after the specific tests for kayaking and cycling, no significant differences were seen among changes produced in plasma reduced and total Hcy and Cys and in its metabolically related vitamins. In general, these changes in plasma aminothiols were interrelated with each other and correlated positively with changes in PLP, vitamin B12, and creatinine but not with changes in folate or amino acids.
According to the respective reduced and total ratios, plasma changes in Hcy concentrations with exercise exceed those observed in Cys in both groups. On the other hand, total and reduced aminothiol concentrations in plasma were similar to those reported in different studies using SBD-F (26,34) in healthy middle-age and older controls (rHcy 0.09± 0.03 and 0.17 ± 0.06 μmol·L−1; tHcy 11 ± 3 and 14.1 ± 6.6 μmol·L−1, respectively). Likewise, similar plasma concentrations were observed with other derivatization procedures, such as 4,4′-dithiopyridine (1) in healthy elderly subjects (rHcy 0.18 ± 0.08 μmol·L−1; tHcy 14.1 ± 4.9 μmol·L−1), monobromobimane (38) in healthy adults (rHcy 0.18 ± 0.01 μmol·L−1; tHcy 6.5 ± 0.3 μmol·L−1), or 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (4) in healthy controls (rHcy 0.11-0.24 μmol·L−1; tHcy 8.4-14.5 μmol·L−1). However, it has recently been observed that derivatization of reduced thiols by the SBD-F method could be affected by thiol exchange reactions (25); therefore, some artifact in the measured concentrations cannot be excluded in the present study.
Several lifestyle and physiological factors have been related to the variations of the circulating concentrations of tHcy (23), age, sex, diet, tobacco, alcohol, coffee, or regular physical activity. Therefore, the selection of the experimental groups in this study was directed to minimize the influence of those variables and, in fact, the increase in tHcy after kayaking and cycling occurred within the recommended ranges. On the other hand, comparison of both groups concerning anthropometrical data or training level showed significant differences that, however, did not affect the extent of plasma aminothiol changes in both exercises, pointing to hyperhomocysteinemia as an independent effect of the type of acute exercise studied.
The effect of acute exercise on plasma tHcy concentrations in active individuals has been examined in several studies. Increased plasma tHcy concentrations have also been observed in runners on treadmill after 30 min (39), marathon runners (10,22), swimmers after 3 wk of training (11), and athletes of triathlon after competition (15). On the contrary, some studies reporting plasma tHcy after an acute bout of exercise in a cycle ergometer have found no changes (27) or even decreases in trained subjects (9). It has been reported that the extent of change in plasma concentrations of tHcy depends on the type and duration of the exercise (10), as opposite to the present results, but discrepancies in data of tHcy within laboratories may be caused by different methodologies or experimental procedures. Thus, differences in the time of sampling after the cessation of the exercise may show temporal fluctuations in the tHcy concentrations, and there can also be changes in plasma volume when no corrections by appropriate formulas have been applied. In our study, a 30-min delay in sampling results in a prevalence of the hemodilution with plasma volume changes of 4.5 ± 1.2% in kayakers and 8.0 ± 1.1% in cyclists. These values are similar to those reported by Medved et al. (18) after 30 min of recovery of successive bouts of exercise until exhaustion on a cycle ergometer.
The effect of acute exercise on plasma rHcy has not received a deal of attention. Zinellu et al. (40) found lower concentrations of rHcy immediately after incremental cycle ergometer tests in two groups, athletes and sedentary subjects, whereas they did not observe equivalent changes in tHcy and they concluded that the change in Hcy after physical activity could be due to metabolic effects rather than redox thiol imbalances. Furthermore, in the same subjects, Sotgia et al. (27) have related these changes in plasma rHcy levels with the increase in plasma creatine concentrations observed.
These findings are apparently opposed to those observed in our study, showing increases of rHcy and tHcy 30 min after acute exercises, but different recovery periods after exercise in both experiments may explain the discrepancies. Dynamic changes in tHcy and tCys concentrations during incremental exercise and recovery has been described (9) with a significant fall in plasma during exercise and return to baseline values from the 2nd min of recovery until the 15th min. Conceivably, the longer period of recovery in our study could be responsible for the higher-than-baseline tHcy and rHcy concentrations observed. Thus, significant equivalent increases of 10% in plasma tHcy have been observed 30 min after running on a treadmill (39) or 19% in marathon runners 24 h after finishing the race (22).
Resting plasma amino acid concentrations were within the range of previously reported values (17). These resting concentrations varied significantly between sports and differences may be due to minor variations in protein content of the diet before the overnight fast and to different body mass index or physical training. Acute tests of kayaking and cycling caused an increase in concentrations of nonessential and total amino acids, an effect that has also been observed in other studies (32) in the recovery period of a test in cycle ergometer. Interestingly, in both groups, changes in amino acid concentrations were equivalent, and no consistent correlations were shown among postexercise concentrations of reduced or total aminothiols and baseline or postexercise amino acids. These results seem not to support our assumption of the relationship between exercise-induced metabolic stress on amino acid metabolism and enhanced Hcy production.
Resting concentrations of folate, PLP, and vitamin B12 seemed to be adequate in both groups of athletes, although consistently higher vitamin B12 concentrations were observed in cyclists. Likewise, an increase in concentrations of PLP and vitamin B12 occurred after respective bouts of physical exercise, the extent of which directly correlated with change in reduced and total aminothiols when the pooled data were considered to overcome the small number of athletes enrolled. The association of the increased postexercise tHcy with baseline concentrations of metabolically related B vitamins has been examined in previous studies, and contradictory inverse relationships have been reported, either with baseline folate after a sprint triathlon (15) or with resting vitamin B12 after a high-intensity training in swimming (11). Differences in physical activities and experimental models may explain those discrepancies. On the other hand, increased concentrations of PLP have been described after exercise in marathon runners and attributed to the mobilization of the hepatic and erythrocytic reservoirs (24); therefore, an association of independent effects of acute exercise on changes in plasma B vitamins and aminothiols cannot be excluded in our study.
Of note, changes in plasma creatinine concentration after physical activity have been related with a temporal reduction in renal blood flow and glomerular filtration rate (36). Likewise, the removal of Hcy by the kidney seems to be dependent on renal plasma flow (8) and, in fact, higher concentrations of reduced and total aminothiols (13,26) and increased ratios of tHcy to tCys (29) have been described in chronic renal disease. Accordingly, our observation of an association of plasma changes in aminothiols and creatinine with exercise could indicate an important role for the renal function in the increased aminothiols concentrations in the postexercise period.
In conclusion, our results show that higher plasma concentrations of tHcy after an acute intense exercise seems to be related with higher concentrations of rHcy presumably driven by cellular export, and this effect is independent of the type of exercise, vitamin status, or amino acid metabolic stress but could be related to potential changes in the renal function.
The authors thank the subjects who participated in the study and J. Muñiz of the Hospital San Agustín, for the technical assistance. We wish to express appreciation to J. Fernández Comins and J. Cousins for the manuscript language revision. This research was supported by a grant from the Fondo de Investigación Sanitaria to RV (FIS 02/0665). The results of the present study do not constitute endorsement by ACSM.
Conflict of interest statement: All authors declare that they have no conflict of interest.
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