JOUBERT, LANAE M.; MANORE, MELINDA M.
Homocysteine (Hcy) is an amino acid intermediate metabolite in the methionine pathway. Circulating levels of plasma Hcy increase when methionine catabolism is elevated. Methionine is considered the most important methyl group source in humans (37) because it donates its methyl groups for many functional molecules such as neurotransmitters, DNA, RNA, and creatine. Physical activity (PA) increases methionine catabolism (13) and induces a higher turnover for many of these methyl-containing molecules, particularly creatine, during high-intensity activities. Thus, it is possible that high-intensity-induced methionine turnover could increase circulating levels of plasma Hcy.
Elevated plasma concentrations of Hcy are associated with an increased risk of cardiovascular disease (CVD) (6), whereas lowering Hcy is associated with reduced incidence of heart attack and stroke (40). Identifying modifiable factors that influence Hcy is important to reduce CVD risk. Nutritional factors such as low dietary intake of folate, vitamin B6, and vitamin B12 are known to be major contributors of elevated blood Hcy (39). Less is known about the relationship of PA and plasma Hcy levels.
The Institute of Medicine's (IOM) Food and Nutrition Board set PA recommendations for weight maintenance at 1h·d−1 based on studies of how much energy is expended on average each day by individuals who maintain a healthy weight (20). These guidelines suggest that activities can be cumulative throughout the day with daily PA to reach the recommended 60 min. Examples include activities of daily living, such as housecleaning, and more moderate-intensity aerobic activities, such as walking at 4 mph, or higher intensities, such as jogging 30 min at a pace >4 mph. These moderate- and high-intensity aerobic activities can help to develop or maintain a high level of cardiorespiratory fitness, which is associated with reduced CVD risk.
B-vitamin (B6, B12, folate)-dependent metabolic reactions in the body, such as energy production and the rebuilding and repairing of muscle tissue, are in high demand during moderate- and high-intensity activities (25). Individuals who participate in PA regularly at these intensity levels may have fewer B-vitamins available for methionine metabolism, induced methionine turnover, and increased creatine production and therefore may have higher plasma Hcy concentrations in comparison to their less active counterparts (23), particularly if dietary intakes of folate, vitamin B12, and vitamin B6 are inadequate.
In general, males have higher levels of plasma Hcy than females (22), perhaps due to higher amounts of fat-free mass (FFM) in males and protective estradiol influences in females. Plasma Hcy concentrations commonly increase with age, with levels in postmenopausal females converging with those of males at older ages (29), supporting the notion that estrogen may protect younger females from higher plasma Hcy levels.
Researchers have examined the impact of PA on plasma Hcy without much consensus. Some cross-sectional studies have found low plasma Hcy concentrations associated with higher levels of PA or fitness (7,27,32,34), yet others reportthe opposite relationship (8,31) or no relationship at all (19,33,36). Although a few studies have shown that one bout of vigorous activity (acute) may increase plasma Hcy levels (15,18,23,24,30), others have found no significant changes (9,10,26,41). Furthermore, evidence of long-term (chronic) exercise training, which includes moderate- and high-intensity levels, is equivocal (5,15,23,28). Research on plasma Hcy and exercise does not permit general conclusions primarily due to poorly described fitness and/or PA levels, limited statistical power with low sample sizes, no control for B-vitamin status, and use of diverse study designs.
The purpose of this study was to determine whether plasma Hcy levels are higher, independent of blood B-vitamin levels, in nonsupplementing (no B-vitamin supplements for ≥30 d) very active young individuals (19-35 yr) participating in consistent moderate- and high-intensity activities (>5 yr; >420 min·wk−1) when compared with less active counterparts.
Volunteers were recruited using flyers posted on campus and e-mail sent to local athletic clubs. Participants were first screened via telephone interview and then invited to participate in the study if they met the following inclusion criteria: 1) participated in >7 h·wk−1 of moderate- and high-intensity PA plus were recreational or competitive athletes (HighPA) or 2) participated in <7 h·wk−1 (LowPA) for at least 5 yr. This 7-h·wk−1 criterion was based on the IOM's PA recommendation to maintain healthy weight and reduce risks for chronic disease (20). Eighty young volunteers (19-35 yr; 50% male) were invited to participate in this cross-sectional investigation during 2005-2006. Four participants did not follow appropriate 7-d recording procedures and were excluded from all analyses; thus 76 participants were included in demographic andblood analyses. For the analysis of dietary intake, anadditional 12 participants were eliminated due to underreporting or abnormal dietary patterns. Participants were considered underreporters if their total energy intake was 200 kcal·d−1 less than that calculated by estimated resting metabolic rate multiplied by Goldberg et al.'s (16) factor of 1.3. Therefore, 64 participants were included in the dietary analysis portion of the study. All participants were nonsupplement users and agreed not to use any form of B-vitamin supplements, such as multivitamin/mineral pills, or any form of creatine supplements at least 30 d before the blood test. In addition, they were asked to refrain from consuming sport-type products with a daily value of 50% or higher for folate, vitamin B12, or vitamin B6, such as Powerbars® and/or Lunabars® for at least 30 d before the scheduled blood test. Exclusion criteria were as follows: not within young age range (19-35 yr), history or current status of menstrual dysfunction or disordered eating (assessed by questionnaire), cigarette smoking within past year, body mass index (BMI) >28 kg·m−2, currently taking medications known to alter plasma Hcy, currently injured, or had a health-related problem such as kidney disease, diabetes, or heart disease. This study was approved by the Institutional Review Board of Oregon State University, and written informed consent was obtained from all participants.
Participants visited the research laboratory twice. The first visit included questionnaires and learning how to keep detailed weighed food and PA records, assessment of anthropometrics, and a treadmill V˙O2max fitness test. Participants were assigned specific dates to record concurrent food and PA records. These dates were to represent a typical week (i.e., no special events like weddings, holidays). Females were instructed to complete their 7-d records and schedule their blood draw during the follicular phase of their menstrual cycle. The second visit transpired on the morning of the eighth day; after completion of the 7-d recording period, a fasting blood draw was done and activity and diet records were reviewed.
Individuals completed a health history questionnaire to screen for injuries and/or contraindicated health status and an eating attitudes questionnaire (EAT-26) (14) to screen for disordered eating behaviors. Additionally, all females completed a menstrual history questionnaire to screen for abnormal menstrual cycles (missed more than six menstrual periods within the past 12 months or not menstruated within the past 3 months) because menstrual cycle may influence Hcy levels (9). No participants had an eating disorder or abnormal menstrual cycles.
Energy and dietary composition was assessed using 7-d weighed dietary intake records. Participants attended a 30-min instruction course on how to keep accurate food and PA records. Calibrated food scales were issued to each participant to weigh the amounts of food items. Consistent detailed instruction was provided by the same trained research assistant for all participants. Food models were used to show examples of how to estimate amounts of food if the scale was unavailable (i.e.,restaurant dining). Participants were asked to describe in detail all foods and beverages, including the type of food, how it was prepared, manufacturer if possible, and the amount consumed on the provided record forms, and personal recording dates were assigned. They were instructed to maintain normal food and PA patterns during the 7 d of recording. Energy intake and dietary composition was assessed using Food Processor (version 8.3; ESHA, Salem, OR). Recipes were analyzed when provided, and manufacturer food labels were turned in with records to help select closest foods to Food Processor's database.
7-d PA records.
Each participant recorded the time spent in resting, sitting, standing, moderate- and high-intensity activities over seven consecutive 24-h periods (420 min·wk−1) based on the IOM's PA recommendations (20). Intensity of activity was described to each participant based on descriptive physical activities or METs (1) as follows: resting (laying down; sleeping), very light (all sitting activities; studying, driving, computer work), light (all standing activities; standing around, teaching, housework, walking below 4 mph pace; <3.0 METs), moderate (i.e., fast walk >4 mph pace, jogging, swimming, biking; 3.0-6.0 METs), and heavy (competitive sport event, jogging or running >5 mph; >6.0 METs). Each minute of the day (1440 min) needed to be accounted for, and individuals were instructed to keep their records with them at all times during the recording period. Examples of completed food and PA records were issued to each participant to reduce recording errors. Participants were encouraged to contact researchers if they had questions during their 7-d recording period. Time spent in each category of intensity for each day was tallied, and totals were then recorded as minutes spent in each intensity category for each of the 7 d. Total minutes for all 7 d, recorded in both moderate- and heavy-intensity categories, were used to assign PA groups (HighPA >420 min·wk−1; LowPA <420 min·wk−1).
Height in meters was measured with no shoes using a stadiometer; a digital scale weighed the subjects wearing only swimsuits. BMI (kg·m2) was calculated. Body composition was measured using the Bod Pod (Life Measurements Instruments, Concord, CA). Fitness was assessed by a graded V˙O2max (mL·kg−1·min−1) running test on a treadmill (Trackmaster, model TMX22; JAS Manufacturing Co., Inc., Newton, KS). Respiratory rate was measured using indirect calorimetry (TrueOne 2400; Parvo Medics, Salt Lake City, UT). Heart rate was monitored (Polar USA, NY) during the entire fitness testing procedure. Verbal encouragement was provided throughout the test. Respiratory exchange ratio (>1.1), RPE, and maximum heart rate (95% of age predicted max) were used to provide objective evidence of leveling of V˙O2 and maximal effort end point for V˙O2max testing.
Blood sampling and preparations.
Participants were instructed to refrain from vigorous PA for 12 h and to fast at least 8 h before arrival at the laboratory for the second visit. Blood was collected during the first 10 d of the menstrual cycle for all female participants. Fasting venous blood was collected in EDTA tubes. Serum lipids and blood chemistry analyses were performed at a regional medical laboratory (Good Samaritan Health Services) to rule out major health problems. All other blood analyses were performed at the University Nutrition Science Laboratory. Blood was immediately centrifuged at 2000g for 15 min at10°C. Plasma was removed and aliquoted (0.6 mL) into 2-mL cryovials and stored in the freezer (−80°C) until the day of analysis.
Reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection was used to measure fasting plasma Hcy concentrations according to the method described by Durand et al. (12). Each sample was reduced by tri-n-butyl phosphine, followed by precipitation of plasma proteins with perchloric acid. Precipitates were then combined with 7-fluoro-2,1,3-benzodiazole-4 sulfonamide and incubated in a heat block at 60°C for 1 h then filtered and injected into an integrated HPLC system (Waters Separations Module; Waltham, MA) and separated on a reversed-phase C18 column (Alltech ODS-25 μ 150 × 4.6 mm; Alltech, Deerfield, IL). Plasma Hcy was detected by fluorescence (Waters Fluorescence Detector; Waltham, MA) at 385-nm excitation and 515-nm emission. The intra- and interassay CV were 1.3% and 1.8%, respectively.
Reversed-phase HPLC with fluorescence detection was used to measure fasting plasma pyridoxal-5′-phosphate (PLP) to determine blood vitamin B6 levels according to the method described by Rybak and Pfeiffer (35). Metaphosphoric acid was used to precipitate plasma proteins from each sample. The supernatant was then filtered and injected into an integrated HPLC system (Waters Separations Module; Milford, MA) and separated on a reversed-phase C18 column (ThermoElectron BDS Hypersil 150 × 3 mm; Waltham, MA). Sodium chlorite was introduced into the HPLC postcolumn effluent flow using an automated postcolumn pump (Waters Reagent Manager; Waltham, MA). The combined flow was passed through a heated (75°C) coiled tubing (300 μL stainless steel) for improved detection. PLP was then detected by fluorescence (Waters Fluorescence Detector) at 300-nm excitation and 400-nm emission. The intra- and interassay CV were 1.3% and 6.7%, respectively.
Fasting plasma folate and vitamin B12 were measured using RIA kit (SimulTRAC-SNB folate/B12; MP Biomedicals, Solon, OH). Radioactivity of 125I and 57Co was counted simultaneously in the pellets using an autogamma counter (Cobra II 5002; Packard Instrument Co). Interassay CV for this method was <12%. Red blood cell folate levels were analyzed using fasting red blood cell folate in participants reporting the lowest plasma folate levels (n = 9; range = 17.63-23.57 nmol·L−1) to eliminate the possibility of folate tissue deficiencies (<3.4 nmol·L−1). Red blood cell folate (nmol·L−1) was measured using the same RIA kit. Radioactivity of 125I was counted in the pellets using an autogamma counter (Cobra II 5002; Packard Instrument Co). No participants had deficient levels (<272 nmol·L−1). Mean red blood cell folate values were measured in those with the lowest plasma folate levels and were found to be normal 1123 ± 180 nmol·L−1 (n = 9).
Power analysis indicated that sample sizes of 17 and 9 per group were needed to provide 90% and 80% power, respectively, to detect a plasma Hcy concentration difference of 3 μmol·L−1. The assumptions of normality for sampling distributions, linearity, homogeneity of variance, homogeneity of regression, and reliability of covariates were all satisfactory. PA group (LowPA, HighPA) comparisons for demographic data were conducted for each biological sex (M, F) by independent samples t-tests. ANCOVA was performed on plasma Hcy concentrations with biological sex and PA groups as independent variables. Plasma folate, PLP, and B12 were included in the full model as covariates to account for their impact on Hcy levels. Only significant covariates (P < 0.05) were included in the final model. For subsample ANCOVA analysis with the same B-vitamins as covariates, participants were divided into two groups based on extreme upper and lower PA percentile criteria. The description of these groups is given below:
1. Extremely High PA (ExHighPA; n = 9; 4 M, 5 F). This group's criteria were statistical Tukey's Hinge >75th percentile for moderate- and high-intensity PA (min·wk−1) plus age- and sex-appropriate V˙O2max (mL·kg−1·min−1) >90th percentile values for maximal aerobic power (2)
2. Extremely Low PA (ExLowPA; n = 11; 6 M, 5 F). This group's criteria were statistical Tukey's Hinge <25th percentile for moderate- and high-intensity PA (min·wk−1) plus age- and sex-appropriate V˙O2max (mL·kg−1·min−1) <70th percentile values for maximal aerobic power (2)
Results are expressed as mean ± SD; statistical significance was determined at P < 0.05. All analyses were performed by SPSS statistical software package (version 15.0; SPSS, Inc., Chicago, IL).
Characteristics of participants (N = 76) are shown in Table 1. Mean age for all participants was 26 ± 4 yr. Both males and females in the HighPA group were significantly taller and older compared with the LowPA group (P < 0.05). Males in the HighPA group had more FFM (kg) than the LowPA group (P < 0.05). By design, combined weekly minutes of moderate- and high-intensity PA were significantly different between HighPA and LowPA groups (P<0.01). On average, the LowPA group (n = 36) completed 219 ± 130 min·wk−1 (V˙O2max = 42.8 ± 8.8 mL·kg−1·min−1), whereas the HighPA group (n = 40) completed 652 ± 191 min·wk−1 (V˙O2max = 54.2 ± 9.7 mL·kg−1·min−1).
PA and Hcy.
The primary objective of this research was to determine whether plasma Hcy concentrations were different in healthy young nonsupplementing adults compared with their less active counterparts. Overall, accounting for blood B-vitamin levels, plasma Hcy levels (μmol·L−1) were not significantly different between HighPA (7.7 ± 1.6; n = 40) and LowPA groups (7.5 ± 1.6; n = 36; P = 0.36) regardless of biological sex. Although males (n = 38) had higher Hcy values than females (n = 38), they were not significantly different (7.9 ± 1.7 vs 7.4 ± 1.4 μmol·L−1, respectively; P = 0.13).
Blood B-vitamins and Hcy.
Because Hcy metabolism is supported by B-vitamin cofactors, we used plasma folate, vitamin B12, and vitamin B6 (PLP) concentrations as covariates in our analysis. Plasma folate was the only statistically significant B-vitamin covariate (P < 0.01) and explained 19% of the variance in plasma Hcy in our final model. Table 2 shows plasma values for Hcy and each B-vitamin by group. No participant was deficient in plasma folate (<3.4 nmol·L−1). Mean plasma vitamin B12 and PLP levels were in the normal range; however, 13% of the participants had low PLP values (<30 nmol·L−1). The following individuals had low plasma PLP values (nmol·L−1) expressed as mean ± SD by group: two males inLowPA had 26.1 ± 2.4; two males in HighPA had 28.4± 0.9; four females in LowPA had 24.3 ± 5.1; two females in HighPA 28.3 ± 0.9 nmol·L−1; and one female in LowPA was considered deficient (<20 nmol·L−1) with a PLP value of 17.9 nmol·L−1. No participants had low plasma vitamin B12 (<120 pmol·L−1).
Table 3 shows energy and dietary intake data for participants with completed 7-d weighed dietary intake records (n = 64). As one would expect, energy intake was significantly higher in the HighPA versus LowPA group (P < 0.05), with the HighPA male group consuming ∼400 kcal·d−1 more than the LowPA male group and the HighPA female group consuming ∼ 240 kcal·d−1 more than the LowPA female group. Although mean dietary intakes of both vitamin B6 and folate were above the recommended dietary allowance (RDA), not all participants consumed recommended levels. One male in the LowPA group did not meet the RDA for dietary vitamin B6 (RDA = 1.3 mg·d−1), and 13 participants (22%) did not meet the RDA for dietary folate (RDA = 400 μg·d−1; nine females in LowPA, one female in HighPA, two males in HighPA, one male in LowPA). All individuals met the RDA for vitamin B12 (2.4 μg·d−1). Furthermore, one female in the LowPA group was below the estimated average requirement (EAR) for folate (320 μg·d−1), one LowPA male was below the EAR for vitamin B6 (1.1mg·d−1), but all were above the EAR for vitamin B12 (2.0 μg·d−1) (21). Analysis of plasma Hcy in this subgroup reporting complete diet data (n = 64) was similar to that seen in the whole group (N = 76).
Extreme PA and Hcy.
If PA increases plasma Hcy concentrations, it is most likely to occur in those who are consistently highly active. Thus, we compared the most active and fit (ExHighPA; n = 11) participants to the least active and unfit participants (ExLowPA; n = 9) based on percentile criteria for both fitness level (V˙O2max; mL·kg−1·min−1) and minutes of PA (min·wk−1). Table 4 shows the data and criteria for these extreme groups. Participants in ExHighPA (PA range = 750-1085 min·wk−1) participated in competitive triathlons, cycling, running, or rowing events consistently for at least 5 yr, whereas ExLowPA were sedentary (PA range = 9-130 min·wk−1). As shown in Figure 1, there were significant differences in Hcy (μmol·L−1) between ExLowPA (n = 9) and ExHighPA groups (n= 11; P = 0.007) and between males (n = 10) and females (n = 10; P = 0.014). Three male participants in ExHighPA had Hcy values >10 μmol·L−1. Plasma B12 was the only significant covariate (P = 0.042). All subsample participants had normal plasma B12 (>120 pmol·L−1) and plasma folate (>3.5 nmol·L−1) values, whereas plasma PLP was low in one male and three females (<30 nmol·L−1). Of the subsample, 14 of the 20 participants had complete the 7-ddietary records. Although plasma folate levels wereadequate in this subsample, three males and three females consumed less than the RDA for dietary folate (400μg·d−1), and one male ate less than the RDA for vitamin B6 (1.3 mg·d−1). All participants consumed the RDA for dietary vitamin B12 (2.4 μg·d−1) (21).
The primary objective of this study was to determine whether plasma Hcy concentrations were higher in active versus less active individuals while controlling for plasma B-vitamin levels. We did not find a significant difference in plasma Hcy concentrations between PA groups as defined by >420 or <420 min·wk−1 of moderate- and high-intensity activity in young participants. However, in a secondary analysis, individuals who participate in high levels of PA(>758 min·wk−1) had significantly higher plasma Hcyvalues compared with sedentary individuals (<130 min·wk−1). Although there were statistical significances in plasma Hcy levels between ExLowPA and ExHighPA, mean level was <10 μmol·L−1. Furthermore, the physiological relevance of a mean difference of 1.75 μmol·L−1 between the ExHighPA and the ExLowPA has not been determined. A meta-analysis by Boushey et al. (6) found a5-μmol·L−1 increment of plasma Hcy concentration >10 μmol·L−1 increased risk for CVD by 60-80% in mostly middle-aged populations. Another meta-analysis by Wald et al. (40) suggested that lowering Hcy concentrations by 3μmol·L−1 would reduce the risk of ischemic heart disease by 11-20%, deep vein thrombosis by 8-38%, and stroke by 15-33%. High plasma Hcy concentrations are not desirable due to the associated increased risk for CVD and other chronic diseases. Longitudinal research is needed to explore whether or not keeping plasma Hcy levels low over a lifespan translates into lower risks for CVD and other chronic diseases.
To our knowledge, this is the first study in young men and women examining the relationship between PA levels and plasma Hcy concentrations while also accounting for blood and dietary intakes of the three primary B-vitamins that influence plasma Hcy concentrations. Several factors known to influence plasma Hcy were controlled for in thisstudy. We used young men and women within a smallage range. We measured all females during the same phase of the menstrual cycle and screened for menstrual dysfunction, eating disorder behaviors, and underreporters of energy intake. We used detailed 7-d weighed food records to examine dietary intakes of B-vitamin and account for other dietary factors, such as alcohol, that have been shown to influence Hcy levels. Finally, fitness levels and specific PA intensity levels of the participants were well described.
PA and Hcy.
Considering the hypothesis that high creatine synthesis and protein turnover increase plasma Hcy levels, it is possible that only highly intense or exhaustive activities, relying on anaerobic or protein-derived energy sources, produce significant increases in plasma Hcy concentrations. Plasma Hcy levels were only significantly different when individuals who participated in high levels of PA and were physically fit (ExHighPA >758 min·wk−1; 57.2 mL·kg−1·min−1) were compared with thosethat were sedentary and unfit (ExLowPA <130 min·wk−1; 35.1 mL·kg−1·min−1). If only PA (min·wk−1) or V˙O2max (mL·kg−1·min−1) was used independently to define these groups, there were no significant differences in plasma Hcy concentrations. This suggests that fit individuals who chronically participate in PA have higher plasma Hcy levels than their unfit sedentary counterparts. This was supported in the present study because both males and females in the ExHighPA groups had significantly higher plasma Hcy levels than the ExLow PA groups. Similarly, other cross-sectional studies report higher plasma Hcy values in the groups most physically active (8,31), yet others have found the opposite (3,7,23,27,32,34) or no relationship (19,33), possibly due to poor distinction between PA amounts or intensities.
Discrepancies in previous research may be due to poorly defined fitness levels of the participants, unaccounted consistency of PA, or the different modes and intensities of PA. As reported by Herrmann et al. (18), marathon runners had higher postrace plasma Hcy levels than 100-km runners or mountain bike competitors, which they explain by different load profiles of the three disciplines.
Participants in the present study performed a variety of moderate- and high-intensity exercises. Collectively, only 30% of the HighPA participants (mean PA = 11 h·wk−1; n= 40) completed 15 min or more of high-intensity PA daily (>75% V˙O2max), which was attributed to interval/sprint training and resistance weight training. Yet approximately 75% of the subsample ExHighPA participants (mean PA = 15 h·wk−1; n = 11) completed more than 15min of high-intensity PA daily, including interval training for swimming, cycling, running, rowing, and weightlifting. Duncan et al. (11) reported that plasma Hcy levels in sedentary participants increased significantly in response to 25 wk of high-intensity walking (65-75% heart rate reserve) but not with moderate-intensity walk training (45-55% heart rate reserve). Moreover, Rousseau et al. (32) reported significant differences in Hcy values between intermittent sport athletes (10.6 ± 2.6 μmol·L−1) competing in soccer, water polo, and rugby compared with athletes in aerobic-type sports (9.2 ± 2.0 μmol·L−1), such as running and swimming, suggesting that the mode and/or intensity ofthe activity may influence Hcy.
Acute exercise has also been shown to significantly increase plasma Hcy levels, and these levels remain high several hours after the activity (15,18,24,30). In the present study, we controlled for these acute effects by asking participants to refrain from any moderate- to high-intensity PA at least 12 h before the blood draw.
Blood B-vitamins and Hcy.
In the total group (N = 76), plasma folate was the only significant covariate involved in the modulation of plasma Hcy concentrations. However, in the subsample analysis (n = 20), only plasma B12 was a significant modulator of plasma Hcy. Unfortunately, several diet records in this subsample were not complete enough to allow the examination of this observation in more detail. It is possible that the subsample group (n = 20) ate more meat and thus had higher dietary vitamin B12 intakes than the group as a whole (N = 76).
Selhub et al. (38) suggest that nutrient deficiencies of vitamin B6, folate, and vitamin B12 contribute to two thirds of all cases of hyperhomocysteinemia. In our study, 22% of the participants consumed less than the RDA for the B-vitamins. Furthermore, 2% of the participants consumed less than the EAR for folate and vitamin B6. In general, these participants consumed less energy or selected less nutrient-dense foods (such as potato chips and soda) and ate higher amounts of processed foods. Although energy intake was higher in our very active participants, they did not necessarily consume higher amounts of B-vitamins (see Table 3 for B-vitamin/1000 kcal). Other dietary factors can also affect plasma Hcy levels. High dietary intake of methionine could lead to high Hcy concentrations if adequate dietary folate intake is not consumed, yet mean intakes between initial groups (N = 76) for methionine and folate were similar. Moreover, plasma Hcy concentrations are reported to be higher in alcoholics (4) and heavy coffee drinkers (17). We had no known alcoholics in our study. We monitored coffee consumption and found that ∼ 55% of our participants consumed coffee during the 7-d diet record session but did not control for this in the analyses.
Due to the cross-sectional design of this study, causality cannot be established. However, the increased levels of plasma Hcy in our ExHighPA subsample warrant further research to determine whether people who participate in PA >758 min·wk−1 have higher B-vitamin needs to keep blood Hcy levels as low as possible. In addition, the majority of our participants were healthy young white college students. Results may be different in older adults who have subsequent CVD risk factors. Another limitation was using 7-d PA records in active and sedentary individuals because the individual interpretation of PA intensity can vary depending on an individual's fitness level. What may be reported as moderate-intensity activity for an active individual might be reported as vigorous activity for a sedentary person. Without the use of heart rate monitors, pedometers, or some other objective measure of intensity during the 7-d recording period, we relied on personal interpretation of PA intensity levels. We minimized error and exaggeration by supplying participants with a detailed description of appropriate PA categories (i.e., moderate = walking faster than 4 mph; high = sprinting that cannot carry on for more than 1-2 min), such as those suggested by Ainsworth et al. (1). Participants completed V˙O2max fitness testing as an objective measure of fitness levels between groups that also supported their reported PA.Finally, it is well recognized that the methylene tetrahydrofolate reductase genotype is a major determinant of plasma Hcy, which we did not measure. However, because we did not have any participants with extremely high Hcy levels, the probability of this being a confounder is low.
This study accounted for several discrepancies found in previous research. Because blood Hcy concentrations increase with age (22), we used a young and discrete age range (19-35 yr). Menstrual influences were accounted for by drawing blood during the follicular phase in all female participants, which has been shown to influence blood Hcy levels in previous research (9). We used both males and females, whereas many of the previous studies have used only male participants. All three key B-vitamins involved in Hcy metabolism were measured and accounted for, whereas previous studies have mainly only focused on blood folate levels. This research contributes to the small amount of existing dietary intake data for highly active individuals, especially B-vitamin intake.
To our knowledge, this is the first study to examine the relationship between PA and plasma Hcy while controlling for B-vitamin intake and B-vitamin blood levels. Overall, there were no significant differences in plasma Hcy concentrations between PA groups as defined by >420 min·wk−1 or <420 min·wk−1 of moderate- and high-intensity activity in young nonsupplementing men and women, unless PA of moderate and high intensities was extremely high (>758 min·wk−1). These data suggest that much strenuous PA may increase Hcy metabolism and thus plasma levels. It is possible that a higher dietary intake of B-vitamins may be necessary to keep plasma Hcy levels low in extremely active young individuals, particularly vitamins B12 and folate, to reduce CVD risk later in life.
This work was supported in part by the Northwest Health Foundation student grant and the American College of Sports Medicine (ACSM) EAS grant. The results of the present study are not endorsed by the ACSM.
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