To examine the association between cardiovascular fitness and homocysteine levels in adolescents.
Madrid, Murcia, Granada, Santander, and Zaragoza, Spain.
One hundred fifty-six Spanish adolescents (76 boys and 80 girls) aged (mean ± SD) 14.8 ± 1.4 years.
Cardiovascular fitness was measured by the 20-m shuttle run test. Pubertal stage, birth weight, smoking status, and socioeconomic status were determined, and the sum of 6 skinfold thickness measurements, and serum folic acid and vitamin B12 levels were measured. Methylenetetrahydrofolate reductase (MTHFR; 677C>T genotype) polymorphism was done by DNA sequencing.
Main Outcome Measure
Fasting homocysteine levels.
Mean values of homocysteine were significantly higher in the MTHFR 677CT and TT genotype subgroups compared with the CC genotype subgroup in adolescent boys, whereas in adolescent girls, mean values of homocysteine were significantly higher in the MTHFR 677CT and TT genotype subgroup compared with the CC and CT genotype subgroups. Multiple regression analyses showed that cardiovascular fitness was significantly associated with homocysteine levels in female adolescents after controlling for potential confounders including the MTHFR 677C>T genotype (β = −0.40; semipartial correlation = −0.35; P = .007). No associations were found between cardiovascular fitness and homocysteine levels in male adolescents (β = 0.12; semipartial correlation = 0.08; P = .51).
The results suggest that cardiovascular fitness is negatively associated with homocysteine levels in female adolescents after controlling for potential cofounders including MTHFR 677C>T genotype.
Homocysteine has been suggested to be an independent risk factor for several multisystem diseases,1 including coronary heart disease,2,3 stroke,4 dementia, and Alzheimer disease,5 as well as for risk of hip fracture6 and pregnancy complications.7 Moreover, elevated homocysteine levels have been associated with increased oxidative stress and endothelial damage,8,9 although the mechanisms are not yet clarified. In children, elevated homocysteine levels are positively associated with cardiovascular disease in their parents,10,11 grandparents,12,13 and other relatives.14
Homocysteine levels are influenced by modifiable and nonmodifiable factors. Among the nonmodifiable factors, age and sex seem to have a specific role. Levels of homocysteine are higher in adolescent boys than in adolescent girls, and this sex effect seems to be enhanced during and after puberty.15 Genetic factors also seem to affect homocysteine levels.16- 19 Elevated levels of homocysteine can be caused by mutations in enzymes involved in homocysteine metabolism, which give dysfunctional enzymes, for example, the single-nucleotide polymorphism at position 677 in the methylenetetrahydrofolate reductase (MTHFR) gene for MTHFR.17 Methylenetetrahydrofolate reductase is a key enzyme in homocysteine metabolism. The common polymorphism 677C>T gives a thermolabile form of the enzyme. Subjects homozygous for this mutation (or TT genotype) have higher levels of homocysteine compared with subjects with CC or CT genotypes.18,19
Deficient serum levels of both folic acid and vitamin B12 have been associated with elevated homocysteine levels in children,18 adults,20 and elderly persons.21 Lifestyle factors such as smoking, lack of physical activity, excessive alcohol intake, and obesity have been associated with elevated levels of homocysteine in adults.20,22- 24
Poor cardiovascular fitness (CVF) is another important risk factor for cardiovascular disease and is a predictor of morbidity and all-cause mortality.25,26 Kuo et al27 have recently described a significant negative association between CVF and homocysteine levels in women. Cardiovascular fitness has been negatively associated with features of metabolic syndrome in children and adolescents28,29 and with plasma lipid profile in both overweight and nonoverweight adolescents.30 However, studies examining the association between CVF and homocysteine levels in adolescents are lacking. We hypothesized that there would be a negative correlation between CVF and homocysteine levels in adolescents. For public health strategies and preventive purposes, it is of interest to understand the relative influence of modifiable factors on homocysteine levels from an early age.
The study participants were a subsample of the AVENA (Alimentación y Valoración del Estado Nutricional de los Adolescentes Españoles [Food and Assessment of the Nutritional Status of Spanish Adolescents]) study, which was designed to assess the health and nutritional status of adolescents. The AVENA study design has been reported in detail elsewhere.31- 33 Data were collected from November 6, 2000, to June 28, 2002, in 5 Spanish cities: Madrid, Murcia, Granada, Santander, and Zaragoza. Data in the present article are from adolescents in whom both homocysteine levels and MTHFR genotypes were measured (n = 156; 76 boys and 80 girls).
A comprehensive verbal description of the nature and purpose of the study was given to both the adolescents and their teachers. Written consent to participate was requested from parents and adolescents. Adolescents with a personal history of cardiovascular disease, who were taking medication at the time of the study, or who were pregnant were excluded. The study protocol was performed in accord with the ethical standards established in the 1961 Declaration of Helsinki (as revised in Hong Kong in 1989 and in Edinburgh, Scotland, in 2000) and was approved by the Review Committee for Research Involving Human Subjects of the Hospital Universitario Marqués de Valdecilla, Santander.
Before any testing was performed, the parents completed a questionnaire, part of which addressed the adolescent's previous and current health status. Socioeconomic status was also assessed in the questionnaire and was defined by the educational achievement and occupation of the father. According to this information and following the recommendation of the Spanish Society for Epidemiology,34 the adolescents were classified into 5 socioeconomic categories: low, medium low, medium, medium high, and high. Smoking status at the time of the study was reported via questionnaire completed by the adolescents, and they were categorized as smoker, nonsmoker, and occasional smoker (ie, once a week).
Anthropometric measurements were obtained as described elsewhere.35- 37 In brief, skinfold thickness was measured to the nearest 0.2 mm at the biceps, triceps, subscapular, suprailiac, thigh, and calf on the left side of the body. The sum of the 6 skinfold thicknesses was used as an indicator of body fat. These measurements correlate highly with measured body fat percentage in adolescents of similar ages as measured with dual-energy x-ray absorptiometry.38
Identification of pubertal stage was assessed according to the method of Tanner and Whitehouse.39 Self-reported breast development in adolescent girls and genital development in adolescent boys was used for pubertal stage classification.
Cardiovascular fitness was assessed by the 20-m shuttle run test as previously described.40 In brief, participants were required to run between 2 lines 20 m apart while keeping pace. Running pace was determined by audio signals emitted from a prerecorded cassette tape. The initial speed was 8.5 km/h, which was increased by 0.5 km/h per minute (1 minute equal to 1 stage). The tape used was calibrated over 1 minute. Subjects were instructed to run in a straight line, to pivot on completing a shuttle, and to pace themselves in accord with the audio signals. The test was finished when the subject failed to reach the end lines concurrent with the audio signals on 2 consecutive occasions or when the subject stopped because of fatigue. All measurements were carried out under standardized conditions on an indoor rubber-floored gymnasium. Constant vocal encouragement was given to participants throughout the test. All participants were familiar with the test because the 20-m shuttle run test is one of the fitness tests included in the physical education curriculum in Spain. Adolescents were instructed to abstain from strenuous exercise in the 48 hours preceding the test.
Cardiovascular fitness was considered as the number of stages completed (precision of 0.5 steps) for being the most direct measurement obtained. Moreover, for the purpose of comparing the results with those of previous publications, maximal oxygen consumption (V̇O2max, milliliters per kilogram per minute) was estimated by the Leger equation40: V̇O2max = 31.025 + [(3.238S − 3.248A) + 0.1536SA], where A is age and S is final speed (S = [8 + 0.5] × number of stages completed). The reliability and validity of this test has been shown in young persons.41,42
With the subject in the supine position, blood samples were obtained by venipuncture after an overnight fast, using vacuum tubes (Vacutainer; Becton, Dickinson and Co, Franklin Lakes, NJ), and placed on ice immediately. The fasting state was verbally confirmed by the subject before blood sampling. All samples were processed within 1 hour by centrifugation, divided into aliquots, and the portions stored at −80°C until withdrawn for analysis.
Homocysteine in acidified citrated plasma43 was assayed using a fluorescence polarization immunoassay on an IMx unit (Abbott Laboratories, Abbott Park, Ill). Serum folic acid and vitamin B12 levels were measured using the fluorometric method with an IMx automatic analyzer (Abbott Laboratories).
Total blood DNA was extracted and purified from 500 μL of whole blood anticoagulated with EDTA using the Quiagen procedure described by Higuchi.44 Genotyping of the 677C>T variant in the human MTHFR gene was performed by means of polymerase chain reaction and allele-specific restriction digestion of the amplified products with the restriction enzyme HinfI (GE Healthcare, Buckinghamshire, England), as previously described by Frosst et al.17
Data are given as mean ± SD unless otherwise indicated. After serum folic acid and vitamin B12 concentrations were normalized by natural logarithm transformation, all of the residuals showed a satisfactory pattern.
The effect on homocysteine levels of sex and MTHFR 677C>T were analyzed by 1-way analysis of variance because there was a significant interaction between sex and MTHFR 677C>T. The subgroup means were compared using the Tukey test.
After bivariate correlation analysis, multiple regression analyses were used to study the relation between homocysteine levels and CVF after controlling for potential confounders. We used an extended-model approach: Model 1 examined the influence of CVF on homocysteine levels after controlling for age, pubertal stage, birth weight, smoking status, socioeconomic status, and the sum of 6 skinfold measurements. Model 2 examined the influence of CVF on homocysteine levels after controlling for the confounders included in model 1 plus serum folic acid and vitamin B12 levels. Model 3 examined the influence of CVF on homocysteine levels after controlling for the cofounders included in model 1 and model 2 plus the MTHFR 677C>T genotype. Semipartial correlation was used as a measure of the relationship between CVF and homocysteine levels after controlling for the effect that 1 or more additional variables (eg, age or birth weight) had on one of those variables. The analyses were performed using Statistical Package for Social Sciences software (version 14.0 for Windows; SPSS Inc, Chicago, Ill), and the level of significance was set at P = .05.
Both homocysteine levels and the MTHFR genotype were measured in 156 adolescents (76 boys and 80 girls). Of these, 23% of the adolescents refused to continue the 20-m shuttle run test because of discomfort or distress, and their results are not included in the final data sample. The observed power for the sample size was 0.40. Pubertal stage was obtained from 96% of the subjects, and skinfold thickness data from 94%. Birth weight, socioeconomic status, and smoking status were available for 93%, 87%, and 71% of the subjects, respectively.
The descriptive characteristics of the study sample are given in Table 1. Adolescent boys were significantly heavier and taller than adolescent girls, and girls had significantly higher skinfold thicknesses. Adolescent boys had significantly higher levels of homocysteine, lower levels of serum vitamin B12, and significantly higher CVF levels (Table 1).
Mean values for homocysteine levels were significantly higher in the CT and TT genotype subgroups compared with the CC genotype subgroup in adolescent boys (CC, 61.6 ± 10.0 mg/L [8.3 ± 1.4 μmol/L]; CT, 81.9 ±40.0 mg/L [11.1 ± 5.4 μmol/L]; TT, 94.5 ± 40.5 mg/L [12.8 ± 5.5 μmol/L]; CT vs CC, P = .01; TT vs CC, P = .003), whereas in adolescent girls, mean values for homocysteine were significantly higher in the TT subgroup compared with the CC and CT subgroups (CC, 55.5 ± 16.8 mg/L [7.5 ± 2.3 μmol/L]; CT, 61.6 ± 12.7 mg/L [8.3 ± 1.7 μmol/L]; TT, 75.3 ± 16.6 mg/L [10.2 ± 2.2 μmol/L]; P = .001). Bivariate correlations between homocysteine levels and the studied independent variables are given in Table 2.
The results of the regression models using the homocysteine level as the outcome variable are given in Table 3. Variation in homocysteine levels was significantly explained by CVF (expressed as number of stages completed) in female adolescents after controlling for age, pubertal stage, birth weight, smoking status, socioeconomic status, and the sum of 6 skinfold thicknesses (model 1). Additional adjustments for serum folic acid and vitamin B12 levels (model 2), and MTFHR 677C>T genotype (model 3) further strengthened the association between the homocysteine level and CVF in adolescent girls. No significant association was found between the homocysteine level and CVF in adolescent boys. The results did not change when the analyses were performed with CVF expressed as V̇O2max, or speed (data not shown).
The results of this study suggest that CVF is negatively associated with homocysteine levels in female adolescents but is not associated with homocysteine levels in male adolescents. The results also suggest that homocysteine levels are higher in adolescent boys than in adolescent girls, that serum folic acid and vitamin B12 levels are negatively associated with homocysteine levels, and that MTHFR 677C>T genotype has an important role in homocysteine levels. To our knowledge, there are no other available data on the association of homocysteine levels with CVF in adolescents.
Cardiovascular fitness is a direct marker of physiologic status and reflects the overall capacity of the cardiovascular and respiratory systems and the ability to carry out prolonged strenuous exercise.45 In theory, disturbances in the peripheral tissues and related vasculature or in the coronary arteries and the heart may decrease CVF. High CVF levels during childhood and adolescence have been associated with a healthier metabolic profile during these years.29,30 Moreover, CVF has recently been associated with arterial compliance in children aged 9 to 11 years, which supports the concept that CVF may exert a protective effect on the cardiovascular system from an early age.46 It is biologically plausible that a high CVF level provides more health protection than a low CVF level, even in healthy adolescents, as has been found in adults.25,26
Homocysteine is metabolized to homocysteine thiolactone by methionyl transfer RNA synthetase. Homocysteine thiolactone acylates lysine residues of proteins, a process called protein homocysteinylation.47 Protein homocysteinylation is a possible mechanism of homocysteine-related protein damage, which in conjunction with the increased oxidative stress and endothelial damage seen in subjects with elevated homocysteine levels may result in impaired CVF.9 However, this cannot explain why the association between the homocysteine level and CVF is found only in female adolescents. Our findings support those of a previous study that examined the relationship between homocysteine levels and CVF in adults.27 Kuo et al27 showed that high homocysteine levels were negatively associated with estimated CVF in women. However, they did not find any association in men, which is in accord with our results. These results suggest that sex hormones may have a role in mediating the CVF-homocysteine association, exerting different effects in female and male subjects; however, further studies to determine whether this is the case are needed. One longitudinal study observed 499 independent community-dwelling elderly persons for 3 years and found that those with elevated homocysteine levels were at increased risk of decline in physical function.48 However, CVF data were not provided and a comparison by sex was not performed.
None of the previous studies included the MTHFR 677C>T genotype, which affects homocysteine levels.16- 19 Balasa et al19 found that the MTHFR 677C>T polymorphism was an independent determinant of homocysteine levels in 197 healthy US children aged 6 months to 16 years. Similarly, Papoutsakis et al18 reported in a sample of healthy Greek children that the TT genotype was associated with homocysteine concentrations. Homocysteine levels in our study sample were significantly higher in the MTHFR 677CT and TT genotype subgroups compared with the CC subgroup in adolescent boys, whereas in adolescent girls, mean values of homocysteine were significantly higher in the TT genotype subgroup compared with the CC and CT genotype subgroups.
In the present study, CVF was objectively measured by the 20-m shuttle run test. We did not have a direct measurement of V̇O2max, the most valid method of measuring CVF. However, from a practical point of view, field tests may be a better option than laboratory testing, especially in epidemiologic studies, because a large number of subjects can be tested at the same time, which enhances the motivation of the participants, and the tests are simple, safe, and often the only feasible choice, especially in school settings. The 20-m shuttle run test meets these criteria. Cardiovascular fitness was considered as the number of stages completed in the 20-m shuttle run test. However, CVF estimated from the Leger equation (V̇O2max, milliliters per kilogram per minute) was also provided for the purpose of making comparisons with other studies possible. When the analyses were performed using V̇O2max or speed (kilometers per hour) rather than the number of stages as the measurement of CVF, similar results were obtained.
Results from cross-sectional studies have shown associations between homocysteine levels and lifestyle-related factors.20,22- 24 However, findings are different when analyzed prospectively.49,50 Duncan et al50 found that 6 months of exercise increased homocysteine levels in sedentary adults, whereas Randeva et al51 showed that 6 months of sustained brisk walking for 20 to 60 minutes 3 days a week significantly decreased homocysteine levels and increased CVF in young overweight and obese women with polycystic ovary syndrome, a group at increased risk of premature atherosclerosis. Similarly, a weight-reduction program that included physical activity had a positive effect on the homocysteine levels in obese children.52 Together, these results suggest that modifications in lifestyle-related factors may influence homocysteine levels in a different manner in children and adolescents than in adults.
The results from the present study should be interpreted with caution because of the limitations of the cross-sectional design; that is, direction of causality cannot be determined. Elevated homocysteine levels may be simply a marker of an unhealthy lifestyle that is associated with poor exercise capacity. The relationship between homocysteine levels and CVF should be studied prospectively. It must be borne in mind that the subjects in this study were healthy adolescents with no previously diagnosed cardiovascular disorders. Also, our study included a moderate number of participants. The observed power for the sample size was low (0.40), which may have masked the association between CVF and homocysteine levels in the adolescent boys. This warrants further investigation. However, we believe that covariates that may confound the measures of association in our study were appropriately considered and controlled for.
The results of this study suggest that CVF is negatively associated with homocysteine levels in adolescent girls after controlling for potential cofounders including the MTHFR 677C>T genotype. These results should stimulate a debate on whether the metabolism of homocysteine could be one way in which the benefits of high CVF levels are exerted.
Correspondence: Jonatan R. Ruiz, BSc, Department of Medical Physiology, School of Medicine, University of Granada, Avda Madrid 12, 18071 Granada, Spain (firstname.lastname@example.org).
Accepted for Publication: September 20, 2006.
Author Contributions:Study concept and design: Ruiz, Sola, Gonzales-Gross, Sjöström, Pietrzik, and Castillo. Acquisition of data: Ruiz, Sola, Gonzales-Gross, Ortega, Garcia-Fuentes, Gutierrez, and Castillo. Analysis and interpretation of data: Ruiz, Sola, Gonzales-Gross, Ortega, and Castillo. Drafting of the manuscript: Ruiz. Critical revision of the manuscript for important intellectual content: Sola, Gonzales-Gross, Ortega, Vicente-Rodriguez, Garcia-Fuentes, Gutierrez, Sjöström, Pietrzik, and Castillo. Statistical analysis: Ruiz. Obtained funding: Gonzales-Gross and Castillo. Administrative, technical, and material support: Gonzales-Gross, Sjöström, and Castillo. Study supervision: Sola, Gonzales-Gross, Sjöström, and Castillo.
Financial Disclosure: None reported.
Funding/Support: This study was supported by fund FIS PI021830 from the Spanish Ministry of Health Instituto de Salud Carlos III. The AVENA study was funded by the Spanish Ministry of Health; FEDER-FSE Funds FIS No. 00/0015; CSD grants 05/UPB32/0, 109/UPB31/03, and 13/UPB20/04; grants (AP2003-2128; AP-2004-2745) from the Spanish Ministry of Education; and scholarships from Panrico SA, Madaus SA, and Procter & Gamble SA.
Acknowledgment: We thank Pilar Carazo, BSc, Remedios Perez, BSc, and Teresa AmigoBSc, for their contributions to the laboratory work; and Olle Carlsson, PhD, from the Unit for Preventive Nutrition, Department of Biosciences and Nutrition at NOVUM, Karolinska Institutet, for statistical assistance.
Ruiz JR, Sola R, Gonzalez-Gross M, Ortega FB, Vicente-Rodriguez G, Garcia-Fuentes M, Gutierrez A, Sjöström M, Pietrzik K, Castillo MJ. Cardiovascular Fitness Is Negatively Associated With Homocysteine Levels in Female Adolescents. Arch Pediatr Adolesc Med. 2007;161(2):166-171. doi:10.1001/archpedi.161.2.166