There is a lack of studies that analyze the association between micronutrient-related biomarker status and physical fitness in adolescents. In the present study, biochemical parameters for iron and vitamin status were studied, along with objective measures of physical fitness in healthy male and female European adolescents. One thousand eighty-nine adolescents (580 girls, 12.5–17.5 yr) from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) cross-sectional study were included. Hierarchical linear models were performed to determine the associations between micronutrient biomarkers and physical fitness. Age, seasonality, latitude, body mass index, menarche (in girls), and physical activity were used as covariates. For cardiorespiratory fitness, concentrations of hemoglobin, retinol, and vitamin C in male adolescents and β-carotene and 25(OH)D in female adolescents were associated with maximal oxygen consumption. For muscular fitness, concentrations of hemoglobin, β-carotene, retinol, and α-tocopherol in male adolescents and β-carotene and 25(OH)D in female adolescents were associated with better performance of the standing long jump test. In summary, concentrations of hemoglobin and most antioxidant vitamins in male adolescents and β-carotene and 25(OH)D in female adolescents were positively associated with cardiorespiratory and muscular fitness, after controlling for relevant confounders. The associations between physical fitness and iron or vitamin status observed in this cross-sectional study in adolescents should be followed up by a study specifically designed to evaluate causal relationships.
- iron status
- 25-hydroxyvitamin D
- physical performance
adolescence is a critical period of growth and development and for the acquisition of healthy behaviors (24). Appropriate nutrition during this period is a basic requirement to express genetic potential that, together with physical activity (PA), will influence later adult and elderly health outcomes. Physical fitness is a set of attributes related to a person's ability to perform physical activities that require aerobic capacity, endurance, strength, or flexibility and is mainly determined by a combination of regular PA and genetically inherited ability (6). Physical fitness is frequently evaluated in adolescents, and it is known to be a powerful marker for present (youth) and future (adult) health (32). Scientific evidence shows that the adolescents' performance in these tests has declined in the last three decades (41, 42). For some micronutrients, an at least marginally deficient nutritional status has also been identified (1, 45). It has been stated in the literature that physical fitness interacts with the nutritional status of the individuals (23), which, at the same time, can differ according to sex, age, latitude, ethnicity, climate, seasonality, genetic background, adiposity, and lifestyle factors (2, 5, 11, 17, 33, 39, 43).
It is known that hemoglobin is the oxygen carrier to the muscles. Some studies have shown that iron status is associated with physical fitness, mainly in its cardiorespiratory dimension (47). Some vitamins, such as vitamin C, have biological effects, which are associated with sports performance and recovery from intense training, such as antioxidant function, immunocompetence, collagen metabolism, and carnitine biosynthesis (23). 25-Hydroxyvitamin D [25(OH)D] has been shown to affect skeletal muscle strength and function, acting on calcium mechanisms and cell proliferation and differentiation, preventing insulin resistance and arachidonic acid mobilization and other mechanisms (4, 10), and being also related to fitness performance (11, 15). Nevertheless, the association between status of some micronutrients and physical performance remains still controversial. In addition, limited information is available concerning this association in the adolescent population, taking into account the important effect of the above-mentioned confounders.
In this study, we hypothesized that iron and vitamin status are directly associated with cardiorespiratory fitness (CRF) and muscular fitness in adolescents.
MATERIAL AND METHODS
The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross-sectional Study (HELENA-CSS) is a European Union-funded project conducted on adolescents from 10 European cities: Stockholm (Sweden), Athens and Heraklion (Greece), Rome (Italy), Zaragoza (Spain), Pecs (Hungary), Ghent (Belgium), Lille (France), Dortmund (Germany), and Vienna (Austria) (26). Detailed descriptions of the HELENA sampling and recruitment approaches, standardization and harmonization processes, data collection, analysis strategies, quality control activities, and inclusion criteria have been described in detail elsewhere (25). An extended and detailed manual of operations was designed for, and thoroughly read by, every researcher involved in the field work before the data collection started. Parents and adolescents signed an informed consent, the protocol was approved by Research Ethics Committees of each city involved, and the study has been performed following the ethical guidelines of the Declaration of Helsinki 1964 (revision of Edinburgh 2000), Convention of Oviedo (1997), the Good Clinical Practice, and the legislation about clinical research in humans in each of the participating countries.
The geographical distribution of the 10 cities (>100,000 inhabitants) was not random and not represented by the strata, but it was decided according to the following criteria: representation of territorial units (countries) of Europe, according to geographical location (North/South/East/West), cultural reference and socioeconomic situation; and selection of a territorial unit (city) in the country that had an experienced research group to perform the study. The age range considered valid for the HELENA study was 12.5–17.5 yr. All of the analyses conducted on the HELENA data are adjusted by a weighing factor to balance the sample according to the theoretical age distribution foreseen. A total of 3,528 adolescents, 1,683 boys and 1,845 girls, were considered eligible for the HELENA analyses.
In the HELENA-CSS protocol, it was established that blood samples were obtained randomly in one-third of the population sample. Therefore, a total of 1,089 adolescents (509 boys and 580 girls) were included in this report. To make maximum use of the data, all valid data on physical fitness components were included in this report. Consequently, sample sizes vary for the different physical fitness tests and biomarkers.
International guidelines for anthropometry in adolescents were used in the HELENA study (28). With the subjects barefoot and in light indoor clothing, body weight (kg) and height (cm) were measured with an electronic scale (type SECA 861), precision 100 g, range 0–150 kg, and a stadiometer (type Seca 225), precision 0.1 cm, range 70–200 cm, respectively. Body mass index was calculated as body weight (kg) divided by height (m) squared.
Physical fitness tests.
The physical fitness components, i.e., muscular fitness and aerobic capacity, were assessed by the physical fitness tests previously described in detail (30). The scientific rationale for the selection of all of these tests, as well as their reliability in young people, was previously published (31). Lower body muscular strength was assessed with the standing long jump (SLJ) test, which has shown to be a good indicator of overall muscular fitness in youth (7). This test was performed twice, and the best score was retained. CRF was assessed with the 20-m shuttle run test (stage). A stage is the period of time in which the speed is maintained constant. In this test, the initial speed is 8.5 km/h, which is increased by 0.5 km·h−1·min−1 (1 min equals one stage) (18). Léger equation was used to estimate the maximal oxygen consumption (V̇o2max) from the 20-m shuttle run test (21). This test was performed only once.
Specimen collection and biochemical analyses.
The blood sampling procedure and sample logistics have been described in detail elsewhere (16). Briefly, fasting blood samples (24.3 ml) were collected by venipuncture at school between 8 and 10 o'clock in the morning after a 10-h overnight fast. Samples for the different analyses were manipulated in situ, as described below, and transported according to the protocol to the central laboratory in Bonn, Department of Nutrition and Food Sciences, for further manipulation. Whole blood samples for the red blood parameters, including hemoglobin, were sent directly to the local laboratory of each country to be analyzed.
A specific handling, transport, and traceability system for biological samples was developed for the HELENA study and was already described by González-Gross et al. (16). Blood samples were obtained between October 2006 and June 2007 and in October 2007. Blood sampling date was dependent on local field work planning, agreement of the school, and availability and capacity of the central laboratory.
Iron status assessment.
Blood sampling procedure and laboratory measures for iron status indicators [soluble transferrin receptor (STfR) and serum ferritin] have been described elsewhere (14, 16). Briefly, sTfR and serum ferritin were measured using ELISA (enzyme-linked immunosorbent assay) (13) in the Human Nutrition Laboratory of the National Research Institute on Food and Nutrition (Rome, Italy). A commercially available control sample from Bio-Rad Liquichek Immunology Control Level 3 (Bio-Rad, Milan, Italy) was used to obtain a calibration curve on each plate.
Provitamin A (β-carotene), vitamin A (retinol), and vitamin E (α-tocopherol) measurements.
β-Carotene, retinol, and α-tocopherol were analyzed by reversed-phase high-performance liquid chromatography using UV detection (RP-HPLC) (Sykam Gilching Germany) in serum. The vacutainer was centrifuged for 15 min at 3,500 rpm at 4°C. Standards (β-carotene, retinol, α-tocopherol), hexane, and isopropanol were obtained from Sigma Aldrich (Germany) and had all HPLC grade. The variation of the method is <3% for all of the vitamins. The samples were stable over 24 h at room temperature [coefficient of variation (CV) vitamin E = 4.6%; vitamin A = 3.2%].
Vitamin C measurement.
For vitamin C measurements, the heparin tubes were put immediately on ice and centrifuged within 30 min (3,500 rpm for 15 min). For stabilization, heparin plasma was precipitated with a 6% (wt/wt) perchloric acid solution spiked with metaphosphoric acid (1:1). The precipitated samples were transported at a stable temperature of 4–7°C within 24 h to the central laboratory and stored at −80°C until analysis. Plasma vitamin C was analyzed by RP-HPLC using UV detection (Sykam Gilching Germany). The CV of the method was 1.7%.
Vitamin B6, B12 (cobalamin and holo-transcobalamin), and folate [plasma and red blood cell (RBC)] measurements.
For the measurement of vitamin B6 (pyridoxal 5′-phosphate), aliquots of EDTA whole blood were sent by cooled transport to the central laboratory and stored at −80°C until bunched analysis. Pyridoxal 5′-phosphate was measured by HPLC (Varian Deutschland, Darmstadt, Germany; CV = 1%) with a modified method of Kimura et al. (19). For vitamin B12 status, cobalamin and holo-transcobalamin were determined. For folate status, plasma folate and RBC folate were determined. For the measurement of cobalamin and plasma folate, blood was collected in heparinized tubes, immediately placed on ice, and centrifuged within 30 min (3,500 rpm for 15 min). The supernatant fluid was transported at a stable temperature of 4–7°C to the central laboratory and stored there at −80°C until assayed. After the hematocrit was measured in situ, EDTA whole blood was sent to the central laboratory for the RBC folate analysis. EDTA whole blood was diluted 1:5 with freshly prepared 0.1% ascorbic acid for cell lysis incubated for 60 min in the dark before storage at −80°C. Cobalamin, plasma, and RBC folate were measured by competitive immunoassay (Immulite 2000, DPC Biermann, Bad Nauheim, Germany) (CV for plasma folate = 5.4%, RBC folate = 10.7%, cobalamin = 5.0%). Sera for measuring holo-transcobalamin were obtained by centrifuging blood collected in evacuated tubes without anticoagulant at 3,500 rpm for 15 min within 1 h. Once sent to the central laboratory, the sera were aliquoted and stored at −80°C until transport in dry ice to the biochemical laboratory at the Universidad Politécnica de Madrid for analysis (Laboratorio número 242 de la Red de Laboratorios de la Comunidad de Madrid). Holo-transcobalamin was measured by microparticle enzyme immunoassay (Active B12 Axis-Shield, Dundee, Scotland, UK; CV = 5.1%) with the use of AxSym (Abbot Diagnostics, Abbott Park, IL).
For vitamin D status, plasma 25(OH)D was measured. Blood was collected in EDTA tubes transported at room temperature to the central laboratory at IEL within 24 h. There it was centrifuged at 3,500 rpm for 15 min at 4°C, and the supernatant stored at −80°C until analysis.
Plasma 25(OH)D was analyzed by ELISA using a kit (OCTEIA 25-Hydroxy Vitamin D) from Immunodiagnostic System (Germany) and measured with a Sunrise Photometer by TECAN (Germany). The IDS OCTEIA 25(OH)D kit is an enzyme immunoassay intended for the quantitative determination of 25(OH)D and other hydroxylated metabolites in human serum or plasma. The sensitivity of this method is 5 nmol/l 25(OH)D, and the variation is <6%. The mean recovery of 25(OH)D is 101%. The CV for the method was <1%.
A variable was computed by recoding the original variable “blood drawing date” into “seasonality”, as follows: winter (from 21st December to 20th March, coded as 1), autumn (from 21st September to 20th December, coded as 2), spring (from 21st March to 20th June, coded as 3), and summer (from 21st June to 20th September, coded as 4), as it was performed in previous studies (11). As the HELENA study was performed during the academic year, few adolescents (N = 25) were assessed in the first days of summer, and they were included along with those assessed during spring. Therefore, the final variable was composed of three groups: winter (coded as 1), autumn (coded as 2), and spring (coded as 3).
Latitude of residence.
The latitude of the study centers was also taken into account as a confounder in the analyses. The latitude of each city was obtained from http://maps.google.es/. Latitudes of the involved cities were as follows: Stockholm (59°33′ N), Dortmund (51°51′ N), Ghent (51°06′ N), Lille (50°63′ N), Vienna (48°21′ N), Pecs (46°07′ N), Rome (41°89′ N), Zaragoza (41°66′ N), Athens (37°98′ N), and Heraklion (35°33′ N). To make use of this data, latitudes were added to the database as numeric variables with two decimals (i.e., Stockholm = 59.55).
Age at menarche.
A quantitative variable was computed as a measure of time (months) as follows: menarche (months) = age (months) at the moment of blood drawing − age (months) at the moment of menarche. Those girls (n = 104) who had no menarche before blood drawing were considered as “0” for the analyses.
A uniaxial accelerometer (Actigraph GT1M, Manufacturing Technology Pensacola, FL) was used to assess PA, as described previously (36). In this study, the interval of time (epoch) was set at 15 s. The time spent (min/day) at moderate PA (MPA) [4–6 metabolic equivalents (METs)] was calculated based on a cutoff of 2,000–3,999 counts/min. The time spent (min/day) at vigorous PA (>6 METs) was calculated based on a cutoff of 4,000 counts/min. Furthermore, MPA to vigorous PA (MVPA) (>4 METs) was calculated as the sum of MPA and vigorous PA. The cutoffs to define the intensity categories are similar to those used in previous studies (12).
Subjects were classified as nonactive adolescents (<60 min/day of MVPA) and active adolescents (≥60 min/day of MVPA), according to the recent guidelines launched by the US Department of Health and Human Services and other medical institutions (44).
After square root transformation of serum ferritin and natural logarithm transformation of sTfR, β-carotene, retinol, vitamin B6, cobalamin, holo-transcobalamin, plasma folate, and RBC folate, all of the residuals showed a satisfactory pattern (normal distribution). Since interactions between sex and the studied variables were observed (P < 0.001), results are given separately by sex. Descriptive data were assessed by one-way ANOVA for normally distributed variables and by U Mann-Whitney for nonnormally distributed variables.
The relationships among micronutrient biomarkers, i.e., iron, hidrosoluble, and liposoluble vitamins, and the performance in the physical fitness tests, i.e., 20-m shuttle run and SLJ, were assessed using hierarchical linear models. The adjusted analysis was conducted according to a previously formulated hierarchical model, including different models: 1) age, seasonality, and latitude; 2) nutritional status and menarche (in girls); 3) MVPA; and 4-7) micronutrient biomarkers. At each model of micronutrient biomarkers, the variables were controlled for those at the same level and the levels above (models 1–3) (46). For a variable to remain in the model, a significance level of P < 0.20 was required.
All of the analyses were performed using the Statistical Package for Social Sciences software (SPSS, version 15.0 for WINDOWS; SPSS, Chicago, IL), and values of P < 0.05 were considered statistically significant. Figures were performed using Sigmaplot (version 10.0 for WINDOWS; Systat Software, San José, CA).
Table 1 shows descriptive characteristics of the sample by sex.
Micronutrient status and CRF in adolescents.
Results from the hierarchical linear models investigating the relationship between the adolescent CRF (assessed by V̇o2max) and their micronutrient-related biomarker status are presented in Tables 2 and 3. In male adolescents, adjusted results showed that hemoglobin (β = 0.192), retinol, and vitamin C (β = 0.148 and β = 0.186, respectively) were associated with V̇o2max (Table 2). In female adolescents, adjusted results showed that β-carotene (β = 0.101) and 25(OH)D (β = 0.091) were associated with V̇o2max (Table 3).
Micronutrient status and muscular fitness in adolescents.
Results from the hierarchical linear models investigating the relationship between the adolescent muscular fitness (assessed by SLJ) and their micronutrient-related biomarker status are presented in Tables 4 and 5. In male adolescents, adjusted results showed that hemoglobin (β = 0.203), β-carotene (β = 0.160), retinol (β = 0.128), and α-tocopherol (β = −0.135) were associated with SLJ (Table 4). In female adolescents, adjusted results showed that β-carotene (β = 0.177) and 25(OH)D (β = 0.125) were associated with SLJ (Table 5).
In the present study, we examined cross-sectional associations of micronutrient-related biomarker status with the performance in physical fitness tests in a large sample of European adolescents from 10 cities. The studied cities were equivalent and comparable among countries, but the samples were representative for the cities and not for the countries (25). To the best of our knowledge, there is limited literature concerning the association among micronutrient status and physical fitness in adolescents.
In our study, hemoglobin concentration was positively associated with CRF (assessed through V̇o2max) in male adolescents after controlling for relevant confounders. The influence of iron status (i.e., hemoglobin) on physical fitness has been studied and demonstrated during decades (9, 29). A recent study showed that severe iron deficiency (boys, <0.75 mg/l and girls, <0.73 mg/l) impaired aerobic capacity, assessed through V̇o2max (47). Our finding of a positive association between blood hemoglobin and muscular fitness (assessed through SLJ) in male adolescents is novel, but previous studies found positive associations between serum ferritin and the performance in SLJ in boys (3), enhancing the importance of iron status, in relation to not only CRF, but also muscular fitness.
The concentrations of antioxidant vitamins in adolescents participating in the HELENA study were recently described and published (5). In our study, we found that concentrations of retinol and vitamin C in male adolescents and β-carotene in female adolescents were positively associated with CRF, after controlling for relevant confounders. Our results agree with those shown in the study of Suboticanec-Buzina et al. (40), who reported that vitamin C supplementation resulted in a significant increase in CRF (V̇o2max) in adolescents with initially lower values. In contrast, studies performed in young adults showed that vitamin C was not associated with CRF (8, 49). Therefore, there is no clear evidence of a benefit of vitamin C supplementation on physical performance, suggesting further research. However, vitamin C may exert permissive effects on physiological functions, such as antioxidant, immunocompetence, and collagen repair, which are associated with recovery from intense training and, as a consequence, promoting performance (23). Our results support the positive correlations found in female adolescents between β-carotene concentrations and CRF (V̇o2max) in the study of Lloyd et al. (22); however, a later review showed that any positive effects of β-carotene on performance remains to be determined (23). Regarding muscular fitness (SLJ), we found positive associations with β-carotene (in both sexes) and retinol in male adolescents, after controlling for relevant confounders. In addition, α-tocopherol was negatively associated with muscular fitness (SLJ) in male adolescents. Some studies showed that the performance in standard exercise tests (37), CRF (V̇o2max) (37), muscular fitness (handgrip test) (38), swimming endurance (20, 37), or blood lactate concentrations (20) was not affected by long-term α-tocopherol supplementation. However, there are no studies analyzing the relationship between α-tocopherol and the lower limbs strength and, therefore, the physiological factors behind cannot be elucidated, suggesting the need for more studies.
In recent years, vitamin D has been the most widely studied micronutrient in relation to physical fitness. The concentrations of 25(OH)D in adolescents participating in the HELENA study were recently described and published (17). In our study, we found positive associations between 25(OH)D and cardiorespiratory and muscular fitness only in female adolescents, after controlling for relevant confounders. Our results support previous evidence that shows a positive association between circulating 25(OH)D and CRF (V̇o2max) (11, 27, 34). In addition, our results support previous studies that showed positive associations between 25(OH)D and muscular fitness (assessed through handgrip test and/or pliometric tests) in girls (15, 34, 48). It has been shown that vitamin D affects skeletal muscle strength and function, acting on calcium mechanisms and cell proliferation and differentiation, preventing against insulin resistance and arachidonic acid mobilization or other mechanisms (4, 10). However, with this study, it is not possible to disentangle the mechanisms by which 25(OH)D affects cardiovascular and muscle function in our population.
Limitations and strengths.
Although we controlled for several potential confounders, we cannot be certain that other unmeasured confounders have not influenced our observations. Cross-sectional studies provide evidence of associations. However, in this specific case, it seems reasonable to think that micronutrient status can influence physical fitness, whereas it is not so clear the mechanisms by which physical fitness could determine higher or lower micronutrient status. It should also be considered that sexual maturation was not included as a covariate in our analyses, due to the fact that it was not available on the whole sample. However, the age of the adolescents was used instead, which showed a slight and stronger association with the dependent variables.
Despite the aforementioned, this is the first study reporting the association between different physical fitness components (i.e., CRF and muscular fitness) and a large number of micronutrient biomarkers in a large sample of European adolescents. The fitness tests used in the present report showed a good criterion-related validity in adolescents (35). In addition, this study includes important sets of confounders, i.e., age, seasonality, latitude, body mass index, menarche (in girls), and MVPA, which is crucial to analyze the association among micronutrient-related biomarker status and physical fitness.
In summary, concentrations of hemoglobin and most antioxidant vitamins in boys and β-carotene and 25(OH)D in girls were positively associated with cardiorespiratory and muscular fitness in a large sample of European adolescents, after controlling for relevant confounders. The associations between physical fitness and iron or vitamin status observed in this cross-sectional study in adolescents should be followed up by a study specifically designed to evaluate causal relationships.
The HELENA Study takes place with the financial support of the European Community Sixth RTD Framework Programme (Contract FOOD-CT-2005-007034). This study was also supported by a grant from the Spanish Ministry of Health: Maternal, Child Health and Development Network (number RD08/0072), grants from the Spanish Ministry of Science and Innovation (EX-2008-0641), and the Swedish Heart-Lung Foundation (20090635). Additional support was from the Spanish Ministry of Education (AGL2007-29784-E/ALI; AP-2005-3827), Axis-Shield Diagnostics Ltd. (Oslo, Norway), and Abbot Científica S.A. (Spain). J. Valtueña is financially supported by the Universidad Politécnica de Madrid (CH/018/2008). Finally, this study was also supported by a grant from Fundación MAPFRE, Spain (L. Gracia-Marco, G. Vicente-Rodríguez, L. A. Moreno).
The writing group takes sole responsibility for the content of this article.
No conflicts of interest, financial or otherwise, are declared by the author(s).
L.G.-M., J.V., F.B.O., G.V.-R., C.B., L.A.M., and M.G.-G. conception and design of research; L.G.-M. and F.B.O. analyzed data; L.G.-M., F.B.O., F.R.P.-L., G.V.-R., and L.A.M. interpreted results of experiments; L.G.-M. prepared figures; L.G.-M., J.V., F.B.O., F.R.P.-L., G.V.-R., and L.A.M. drafted manuscript; L.G.-M., J.V., F.B.O., F.R.P.-L., G.V.-R., C.B., M.F., D.M., K.W., S.D.H., A.K., L.E.D., F.G., G.M., P.S., M.J.C., L.A.M., and M.G.-G. edited and revised manuscript; L.G.-M., J.V., F.B.O., F.R.P.-L., G.V.-R., C.B., M.F., D.M., K.W., S.D.H., A.K., L.E.D., F.G., G.M., P.S., M.J.C., L.A.M., and M.G.-G. approved final version of manuscript.
Many thanks to Christel Bierschbach, Adelheid Schuch, Anke Berchtold, Petra Pickert, Miriam Segoviano, Jasmin Benser, Miriam Pedrero, and Ulrike Albers for contribution to laboratory work and to Laura Barrios for statistical support. We gratefully acknowledge all participating adolescents and their parents for collaboration.
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