Abstract

Introduction

Cardiovascular diseases (CVD) are at the top of mortality and morbidity indices in developed countries ¹1. Gersh BJ, Sliwa K, Mayosi BM, Yusuf S. Novel therapeutic concepts: the epidemic of cardiovascular disease in the developing world: global implications. Eur Heart J 2010; 31:642-8.. Individuals with CVD typically become symptomatic only in their adult life; however, the CVD underlying process, atherosclerosis, is often initiated in childhood, with inflammatory processes ²2. McGill Jr. HC, McMahan CA, Herderick EE, Malcom GT, Tracy RE, Strong JP. Origin of atherosclerosis in childhood and adolescence. Am J Clin Nutr 2000; 72 (5 Suppl):1307S-15S.. Low-grade inflammation has a pivotal role for the development of CVDs starting in childhood and adolescence ³3. Hansson GK. Mechanisms of disease: Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352:1685-95..

C-reactive protein (CRP) is a marker of acute inflammatory process ⁴4. Yu H, Rifai N. High-sensitivity C-reactive protein and atherosclerosis: From theory to therapy. Clin Biochem 2000; 33:601-10.^,55. Zwaka TP, Hombach V, Torzewski J. C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis. Circulation 2001; 103:1194-7.; it is produced in the liver in response to interleukin 6 (IL-6), which, in turn, is stimulated by the tumor necrosis factor alpha (TNF-α) ⁵5. Zwaka TP, Hombach V, Torzewski J. C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis. Circulation 2001; 103:1194-7.^,66. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340:115-26.. CRP is an independent cardiovascular risk fator ⁷7. Lagrand WK, Visser CA, Hermens WT, Niessen HW, Verheugt FW, Wolbink GJ, et al. C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon? Circulation 1999; 100:96-102., and has shown to be a powerful predictor of cardiovascular risk, even more powerful than the classic markers⁸8. Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detection and prevention. Circulation 2003; 107:363-9., not only in adults, but also in children and adolescents ⁹9. Cook DG, Mendall MA, Whincup PH, Carey IM, Ballam L, Morris JE, et al. C-reactive protein concentration in children: relationship to adiposity and other cardiovascular risk factors. Atherosclerosis 2000; 149:139-50.^,1010. Kapiotis S, Holzer G, Schaller G, Haumer M, Widhalm H, Weghuber D, et al. A proinflammatory state is detectable in obese children and is accompanied by functional and morphological vascular changes. Arterioscler Thromb Vasc Biol 2006; 26:2541-6.. An investigation that compared CRP and LDL-C (Low-density lipoprotein-cholesterol) provided evidence that CRP has higher independent predictive power for future cardiovascular diseases than LDL-C ¹¹11. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med 2002; 347:1557-65.. In addition, associations between CRP levels and the development of type-2 diabetes mellitus ¹²12. Herder C, Schneitler S, Rathmann W, Haastert B, Schneitler H, Winkler H, et al. Low-grade inflammation, obesity, and insulin resistance in adolescents. J Clin Endocrinol Metab 2007; 92:4569-74., early arterial abnormalities in healthy young individuals of both genders ¹³13. Järvisalo MJ, Harmoinen A, Hakanen M, Paakkunainen U, Viikari J, Hartiala J, et al. Elevated serum C-reactive protein levels and early arterial changes in healthy children. Arterioscler Thromb Vasc Biol 2002; 22:1323-8., and adiposity ¹⁴14. Steene-Johannessen J, Kolle E, Andersen LB, Anderssen SA. Adiposity, aerobic fitness, muscle fitness, and markers of inflammation in children. Med Sci Sports Exerc 2013; 45:714-21. were also found.

Lack of physical activity (PA) in childhood and adolescence is an important risk factor for CVD ¹⁵15. World Health Organization. Global recommendations on physical activity for health. Geneva: World Health Organization; 2010.^,1616. Andersen LB, Riddoch C, Kriemler S, Hills AP. Physical activity and cardiovascular risk factors in children. Br J Sports Med; 2011; 45:871-6.. In addition, regular PA is associated with potential benefits for health, and is considered crucial for the growth and healthy development of school-age youths ¹⁷17. Strong WB, Malina RM, Blimkie CJ, Daniels SR, Dishman RK, Gutin B, et al. Evidence based physical activity for school-age youth. J Pediatr 2005; 146:732-7.. A number of studies found associations between PA or cardiorespiratory fitness with CRP in adults ¹⁸18. Plaisance EP, Grandjean PW. Physical activity and high-sensitivity C-reactive protein. Sports Med 2006; 36:443-58.^,1919. Lavie CJ, Church TS, Milani RV, Earnest CP. Impact of physical activity, cardiorespiratory fitness, and exercise training on markers of inflammation. J Cardiopulm Rehabil Prev 2011; 31:137-45.^,2020. Hamer M, Sabia S, Batty GD, Shipley MJ, Tabák AG, Singh-Manoux A, et al. Physical activity and inflammatory markers over 10 years: follow-up in men and women from the Whitehall II cohort study. Circulation 2012; 126:928-33.. In adolescents, however, such associations are not well established. Recently, Martinez-Gomez et al. ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7., in the HELENA Study, investigated the combined association between PA, cardiorespiratory fitness and inflammatory markers in adolescents, and found significant associations between CRP and cardiorespiratory fitness, but not with PA measured objectively ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.. However, Owen et al. ²²22. Owen CG, Nightingale CM, Rudnicka AR, Sattar N, Cook DG, Ekelund U, et al. Physical activity, obesity and cardiometabolic risk factors in 9- to 10-year-old UK children of white European, South Asian and black African-Caribbean origin: the Child Heart and health Study in England (CHASE). Diabetologia 2010; 53:1620-30., in the CHASE study, reported a strong association between PA measured objectively and CRP in a sample of Caucasian-European, Southern-Asiatic, and Afro-Caribbean children. While some studies with young subjects find negative associations between PA or cardiorespiratory fitness and CRP ²2. McGill Jr. HC, McMahan CA, Herderick EE, Malcom GT, Tracy RE, Strong JP. Origin of atherosclerosis in childhood and adolescence. Am J Clin Nutr 2000; 72 (5 Suppl):1307S-15S.^,2222. Owen CG, Nightingale CM, Rudnicka AR, Sattar N, Cook DG, Ekelund U, et al. Physical activity, obesity and cardiometabolic risk factors in 9- to 10-year-old UK children of white European, South Asian and black African-Caribbean origin: the Child Heart and health Study in England (CHASE). Diabetologia 2010; 53:1620-30.^,2323. Harmse B, Kruger HS. Significant differences between serum CRP levels in children in different categories of physical activity: the PLAY study. Cardiovasc J Afr 2010; 21:316-22.^,2424. Parrett AL, Valentine RJ, Arngrimsson SA, Castelli DM, Evans EM. Adiposity, activity, fitness, and C-reactive protein in children. Med Sci Sports Exerc 2010; 42:1981-6.^,2525. Kwon S, Burns TL, Janz K. Associations of cardiorespiratory fitness and fatness with cardiovascular risk factors among adolescents: the NHANES 1999-2002. J Phys Act Health 2010; 7:746-53.^,2626. Llorente-Cantarero FJ, Pérez-Navero JL, De Dios Benitez-Sillero J, Muñoz-Villanueva MC, Guillén-Del Castillo M, Gil-Campos M. Non-traditional markers of metabolic risk in prepubertal children with different levels of cardiorespiratory fitness. Public Health Nutr 2012; 15:1827-34.^,2727. Puder JJ, Schindler C, Zahner L, Kriemler S. Adiposity, fitness and metabolic risk in children: a cross-sectional and longitudinal study. Int J Pediatr Obes 2011; 6:e297-306., others do not ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.^,2828. Ischander M, Zaldivar Jr. F, Eliakim A, Nussbaum E, Dunton G, Leu SY, et al. Physical activity, growth, and inflammatory mediators in BMI-matched female adolescents. Med Sci Sports Exerc 2007; 39:1131-8.^,2929. Ruiz JR, Ortega FB, Warnberg J, Sjöström M. Associations of low-grade inflammation with physical activity, fitness and fatness in prepubertal children; The European Youth Heart Study. Int J Obes 2007; 31:1545-51.^,3030. Thomas N, Baker J, Graham M, Cooper S, Davies B. C-reactive protein in schoolchildren and its relation to adiposity, physical activity, aerobic fitness and habitual diet. Br J Sports Med 2008; 42:357-60.^,3131. Metcalf BS, Jeffery AN, Hosking J, Voss LD, Sattar N, Wilkin TJ. Objectively measured physical activity and its association with adiponectin and other novel metabolic markers: a longitudinal study in children (EarlyBird 38). Diabetes Care 2009; 32:468-73.^,3232. Martinez-Gomez D, Eisenmann JC, Wärnberg J, Gomez-Martinez S, Veses A, Veiga DL, et al. Associations of physical activity, cardiorespiratory fitness and fatness with low-grade inflammation in adolescents: the AFINOS Study. Int J Obes (Lond) 2010; 34:1501-7.^,3333. Sadeghipour HR, Rahnama A, Salesi M, Rahnama N, Mojtahedi H. Relationship between C-reactive protein and physical fitness, physical activity, obesity and selected cardiovascular risk factors in schoolchildren. Int J Prev Med 2010; 1:242-6.^,3434. Loprinzi P, Cardinal B, Crespo C, Brodowicz G, Andersen R, Sullivan E, et al. Objectively measured physical activity and C-reactive protein: National Health and Nutrition Examination Survey 2003-2004. Scand J Med Sci Sports 2013; 23:164-70.^,3535. Hayes HM, Eisenmann JC, Pfeiffer K, Carlson JJ. Weight status, physical activity, and vascular health in 9- to 12-year-old children. J Phys Act Health 2013; 10:205-10.^,3636. Ruiz JR, Ortega FB, Wärnberg J, Moreno LA, Carrero JJ, Gonzalez-Gross M, et al. Inflammatory proteins and muscle strength in adolescents: the Avena study. Arch Pediatr Adolesc Med 2008; 162:462-8.. Moreover, in many of these studies, the results are not presented independently from the body mass index (BMI), a potentially confounding variable of the relationship between CRP and PA or cardiorespiratory fitness ³⁷37. Tam C, Clement K, Baur L, Tordjman J. Obesity and low-grade inflammation: a paediatric perspective. Obes Rev 2010; 11:118-26..

Evidence is scarce and the results are confusing in relation to the association between PA and CRF with CRP in adolescentes ³⁸38. Thomas NE, Williams DRR. Inflammatory factors, physical activity, and physical fitness in young people: review. Scand J Med Sci Sports 2008; 18:543-56.^,3939. Stolzman S, Bement MH. Inflammatory markers in pediatric obesity: health and physical activity implications. Infant Child Adolesc Nutr 2012; 4:297-302., and previous studies in this population faced limitations due to methodology, small and heterogeneous samples, and the use of subjective PA assessment measures (such as questionnaires, for instance) ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.^,3838. Thomas NE, Williams DRR. Inflammatory factors, physical activity, and physical fitness in young people: review. Scand J Med Sci Sports 2008; 18:543-56.. Epidemiological studies that use questionnaires to measure PA are poorer in terms of methodology when compared with accelerometers, particularly in young populations ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.^,4040. Shephard RJ. Limits to the measurement of habitual physical activity by questionnaires. Br J Sports Med 2003; 37:197-206.^,4141. Hendelman D, Miller K, Baggett C, Debold E, Freedson P. Validity of accelerometry for the assessment of moderate intensity physical activity in the field. Med Sci Sports Exerc 2000; 32 (9 Suppl):S442-9.. Thus, by estimating the level of PA objectively assessed with the use of accelerometers, which provide valid and reliable results ⁴¹41. Hendelman D, Miller K, Baggett C, Debold E, Freedson P. Validity of accelerometry for the assessment of moderate intensity physical activity in the field. Med Sci Sports Exerc 2000; 32 (9 Suppl):S442-9., this study may, perhaps, report the actual relationship between PA and CRP.

In this scenario, the goal of this study was to investigate the association between the concentration levels of CRP with PA measured objectively and CRF in Portuguese adolescents. The established premise is that the association between CRP and PA or CRF is negative for both genders.

Methods

Study design and sampling

The data of the following study were obtained from an investigation carried out by the Center for Research on Physical Activities, Health and Leisure (Centro de Investigação em Atividade Física, Saúde e Lazer – CIAFEL), University of Porto, Portugal. This study investigated a sample of adolescents enrolled in the 7th grade (primary education) and in the 10th grade (secondary education), in five schools in the North of Portugal, with age ranging from 12 to 18 years.

The participating schools had already established collaboration agreements with the University’s research center, and were therefore selected for convenience, mainly for logistic and budgetary reasons.

All participants were informed about the goals of the study; they and their parents/guardians signed the informed consent form. The study was approved by School of Sports. Authorization was also given by the National Data Protection Committee (number: 12434/2011), by the Ministry of Education (number: 0246200001/2011), and by the schools’ principals’ office. The study was conducted in accordance with the World Medical Association Declaration of Helsinki for investigations involving human subjects.

In the course of the study, no exclusion criteria were applied to avoid discrimination. However, for this analysis, only apparently healthy adolescents were assessed, i.e., subjects with no medical diagnosis of physical or mental impairment.

Considering potential refusals to participate in the study, due to blood sample collection and the use of accelerometers, a “consent in parts” was allowed. This means that participants could consent to only some parts of the study protocol, and not to other parts. An adolescent could, for instance, be submitted to the physical fitness assessment without the use of accelerometers, or refuse to provide blood samples.

All students enrolled in the 7th and 10th grade of the participating schools were invited to take part in the study (n = 1,678). Data collection was standardized, and took place over the 2011/2012 school year; a total of 1,229 subjects took part, and 534 agreed to provide blood samples. The study was conducted by the research center (CIAFEL), and included filling out questionnaires; performing the physical fitness assessment battery tests (Fitnessgram and Alpha); objective assessment of physical activity (accelerometry); assessment of anthropometric measures; blood pressure measurement, and blood sample analysis. The participants were evaluated during physical education classes by physical education teachers specifically trained for data collection (anthropometric measures, batteries of physical fitness tests, physical activity, questionnaire completion, and blood pressure measurement). Blood samples were collected by nurses who went to the schools for that purpose.

For the present study, a subsample of 386 adolescents (207 females) with all (100%) the information about PA, cardiorespiratory fitness and CRP was considered.

Instruments and variables

• Anthropometry

a) Height

Height was measured with the use of a stadiometer (Seca 213, Seca Medical Scales and Measuring Systems, UK), with accuracy in millimeters. Measurements were taken in the anthropometric position, with subjects in bare feet or wearing socks. After placing the subjects in the proper position, the horizontal plastic rod was moved until it reached the vertex, and the figure in centimeters of the height was recorded.

b) Weight

Weight was measured with the adolescents wearing only t-shirts and shorts, on a portable electronic scale (Tanita BF 350, Tanita Corp., Arlington Heights, U.S.A.); later, the body mass index (BMI) was calculated as weight/height², and expressed as kg/m².

• Sexual maturity status

To establish the sexual maturity status (ranging from 1 to 5), each subject was asked to self-assess their secondary sexual characteristics. Breast development stage, for girls, and genital development, for boys, were assessed according to the criteria established by Tanner & Whitehouse ⁴²42. Tanner JM, Whitehouse RH. Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty. Arch Dis Child 1976; 51:170-9..

• Objective measures of physical activity

The PA standards were objectively assessed with the use of accelerometers Actigraph GT1-M (Actigraph, Pensacola, U.S.A.). The subjects of the study used the accelerometer for 7 consecutive days (5 week day and 2 weekend days). The subjects were instructed to use the accelerometer at their waist throughout the day, and to remove them only during water activities and to sleep. The epoch length was 2 seconds. For this study, only participants with valid accelerometry data (at least 3 days, with 10 hours or more of monitored data) were included in the analysis. For this study, the proportion of time was split into moderate, vigorous and very vigorous PA in accordance with the cutoff points established by Freedson (1998, apud Trost et al.) ⁴³43. Trost SG, Pate RR, Sallis JF, Freedson PS, Taylor WC, Dowda M, et al. Age and gender differences in objectively measured physical activity in youth. Med Sci Sports Exerc 2002; 34:350-5..

• Assessment of the cardiorespiratory fitness

To assess the cardiorespiratory fitness, the 20-meter Pacer test was used⁴⁴44. Leger LA, Mercier D, Gadoury C, Lambert J. The multistage 20 metre shuttle run test for aerobic fitness. J Sports Sci 1988; 6:93-101.. This test predicts the maximum cardiorespiratory capacity, and provides significant associative evidences so that it can be used to assess cardiorespiratory fitness in children and adolescentes ⁴⁵45. Boreham CA, Paliczka VJ, Nichols AK. A comparison of the PWC170 and 20-MST tests of aerobic fitness in adolescent schoolchildren. J Sports Med Phys Fitness 1990; 30:19-23.. For this test, the participants have to run between two lines set 20 meters apart, and must run in synchronization with signal beeps. The initial speed at the initiation of the test is 8.5km/h which is gradually increased by 0.5km/h increments at each minute, reaching 18km/h at minute 20. A signal beep would mark each increment. The participants were asked to keep the pace until exhaustion. The test finished when the participant failed to reach the line at the end concurrently with the beep for two consecutive times. Otherwise, the test finished when the participant stopped due to fatigue. The participants were encouraged by the investigators to perform as best as they could, and to keep on in the test for as long as possible. The total number of laps of each participant was recorded. Later, the Vo_2max of each participant was calculated by the equation of Léger et al. ⁴⁴44. Leger LA, Mercier D, Gadoury C, Lambert J. The multistage 20 metre shuttle run test for aerobic fitness. J Sports Sci 1988; 6:93-101..

• Measures of high-sensitivity CRP

All blood samples were collected from participants who were fasting for at least 10 hours, early in the morning. Blood sample was collected from a vein at the antecubital fossa, with the subjects in a sitting position. For that purpose, vacuum tubes manufactured by Sarstedt (Sarsdedt Ag & Co, Nümbrecht, Germany), with gel barrier to separate serum form the blood clot. After rest at room temperature for about 30 minutes, the samples were centrifuged for 10 minutes at 3000 rot/min for serum to be obtained. The high-sensitivity CRP levels were measured by latex-enhanced immunoturbidimetric assay using Siemens Advia 1600/1800 (Siemens AG, Erlangen, Germany).

• Statistical analyses

For the data statistical analyses, the software IBM-SPSS (IBM Corp., Armonk, U.S.A.) version 20.0 for Windows, was used, with 95% confidence level (95%CI) and 5% significance level (p < 0.05). Gender differences were established by the t test for independent samples. CRP levels were normalized with the use of natural logarithm, as they did not present normal distribution. For each gender, partial correlations were made (Pearson’s correlation coefficient) to check the association between CRP with the cardiorespiratory fitness and PA variables adjusted for age, sexual maturity and BMI. To calculate the power of the sample, the software G*Power 3.1.9.2 (http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpow er3) (was used, and post-hoc power calculation was made separately for genders (p < 0.05) and established for each significant correlation in the models used. For females, the results were 0.99 and 0.88 for correlation models 1 and 2, respectively; for males, 0.61 for model 1.

Results

The descriptive characteristics of this study and gender differences are presented in Table 1. The mean CRP values in Table 1 are presented in mg/L and not normalized. No significant differences were seen between CRP concentration for male (0.90 ± 1.55) and female (0.68 ± 1.40) adolescents. There were significant differences between genders for weight, height and Vo_2max (p < 0.005 for all).

Significant partial correlations were found between cardiorespiratory fitness and CRP for both females (r = -0.313; p = 0.00) and males (r = -0.163; p = 0.02) when adjusted for age and sexual maturity, as presented in Table 2. However, when BMI is included as a control variable, this association remained significant for females only (r = -0.215; p = 0.00). No significant association was found for the PA measured objectively.

Discussion

Low-grade inflammation seems to play an important role in the development of CVD starting in childhood and adolescence ¹³13. Järvisalo MJ, Harmoinen A, Hakanen M, Paakkunainen U, Viikari J, Hartiala J, et al. Elevated serum C-reactive protein levels and early arterial changes in healthy children. Arterioscler Thromb Vasc Biol 2002; 22:1323-8.. Over the past decade, a number of scientific investigations were conducted that provided enough evidence showing the power of CRP as predictive of CVD ¹⁸18. Plaisance EP, Grandjean PW. Physical activity and high-sensitivity C-reactive protein. Sports Med 2006; 36:443-58..

There are CRP concentration cutoff points that represent the CVD risk for adults: < 1.0mg/L (low risk), 1.0 until 3.0mg/L (intermediate risk), and > 3.0mg/L (high risk) ⁴⁶46. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon 3rd RO, Criqui M, et al. Markers of inflammation and cardiovascular disease application to clinical and public health practice: a statement for healthcare professionals from the centers for disease control and prevention and the American Heart Association. Circulation 2003; 107:499-511.. However, for children and adolescents no such scale exists. In a study with 1,617 Finnish children and adolescents, age 3 to 18 years, assessed in 1980 and reassessed in 2001 for high-sensitivity CRP, no significant association was found between CRP concentration in childhood and in adult life ⁴⁷47. Juonala M, Viikari JSA, Rönnemaa T, Taittonen L, Marniemi J, Raitakari OT. Childhood C-reactive protein in predicting CRP and carotid intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study. Arterioscler Thromb Vasc Biol 2006; 26:1883-8.. In our sample, we found 42 children and adolescents that had CRP concentration higher than 3.0mg/L. Cross-sectional studies are necessary to determine the meaning of these concentration levels in children and adolescents.

In this study, despite CRP concentration differences between genders, these differences are not statistically significant. The results we found are in accordance with those found in some studies ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.^,3030. Thomas N, Baker J, Graham M, Cooper S, Davies B. C-reactive protein in schoolchildren and its relation to adiposity, physical activity, aerobic fitness and habitual diet. Br J Sports Med 2008; 42:357-60.^,4848. Isasi CR, Deckelbaum RJ, Tracy RP, Starc TJ, Berglund L, Shea S. Physical fitness and C-reactive protein level in children and young adults: the Columbia University BioMarkers Study. Pediatrics 2003; 111:332-8., but are contrary to those of other studies ⁹9. Cook DG, Mendall MA, Whincup PH, Carey IM, Ballam L, Morris JE, et al. C-reactive protein concentration in children: relationship to adiposity and other cardiovascular risk factors. Atherosclerosis 2000; 149:139-50.^,1414. Steene-Johannessen J, Kolle E, Andersen LB, Anderssen SA. Adiposity, aerobic fitness, muscle fitness, and markers of inflammation in children. Med Sci Sports Exerc 2013; 45:714-21.. For Thomas et al. ³⁰30. Thomas N, Baker J, Graham M, Cooper S, Davies B. C-reactive protein in schoolchildren and its relation to adiposity, physical activity, aerobic fitness and habitual diet. Br J Sports Med 2008; 42:357-60., due to the great CRP concentration variation in children and adolescents, and the small sample of the studies, it is unlikely that gender differences may be explained. Large-scale epidemiological studies are clearly necessary to fully clarify the role of gender in CRP concentration, in these populations.

Significant associations between CRP and BMI were also found in both genders (preliminary data not shown), which is in accordance with other studies ²⁴24. Parrett AL, Valentine RJ, Arngrimsson SA, Castelli DM, Evans EM. Adiposity, activity, fitness, and C-reactive protein in children. Med Sci Sports Exerc 2010; 42:1981-6.^,2525. Kwon S, Burns TL, Janz K. Associations of cardiorespiratory fitness and fatness with cardiovascular risk factors among adolescents: the NHANES 1999-2002. J Phys Act Health 2010; 7:746-53.^,2929. Ruiz JR, Ortega FB, Warnberg J, Sjöström M. Associations of low-grade inflammation with physical activity, fitness and fatness in prepubertal children; The European Youth Heart Study. Int J Obes 2007; 31:1545-51.^,3030. Thomas N, Baker J, Graham M, Cooper S, Davies B. C-reactive protein in schoolchildren and its relation to adiposity, physical activity, aerobic fitness and habitual diet. Br J Sports Med 2008; 42:357-60.^,3232. Martinez-Gomez D, Eisenmann JC, Wärnberg J, Gomez-Martinez S, Veses A, Veiga DL, et al. Associations of physical activity, cardiorespiratory fitness and fatness with low-grade inflammation in adolescents: the AFINOS Study. Int J Obes (Lond) 2010; 34:1501-7.. The current literature shows body fat as the main influencing factor of CRP concentration ³⁷37. Tam C, Clement K, Baur L, Tordjman J. Obesity and low-grade inflammation: a paediatric perspective. Obes Rev 2010; 11:118-26.. Cook et al. ⁹9. Cook DG, Mendall MA, Whincup PH, Carey IM, Ballam L, Morris JE, et al. C-reactive protein concentration in children: relationship to adiposity and other cardiovascular risk factors. Atherosclerosis 2000; 149:139-50., in 2000, were the first to demonstrate the close relationship between CRP and body fat in a sample of 699 children age 10 and 11 years ⁹9. Cook DG, Mendall MA, Whincup PH, Carey IM, Ballam L, Morris JE, et al. C-reactive protein concentration in children: relationship to adiposity and other cardiovascular risk factors. Atherosclerosis 2000; 149:139-50., which was further confirmed in the NHANES III study with 3,512 children and adolescents age between 8 and 16 years ⁴⁹49. Visser M, Bouter LM, McQuillan GM, Wener MH, Harris TB. Low-grade systemic inflammation in overweight children. Pediatrics 2001; 107:E13.. Thus, due to the close relationship found between BMI and CRP in this and in other studies, BMI was also used as an adjusted variable in the association of PA and cardiorespiratory fitness with CRP.

This study found significant partial correlations between CRP and cardiorespiratory fitness for both genders, adjusted for age and sexual maturity; however, when BMI was included as a variable for adjustment, these associations were no longer significant for males, but only for females. This may perhaps be explained by the difference in Vo_2max found between both genders. In the HELENA Study, conducted in ten European countries with adolescents age 12.5 to 17.5 years ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7., a negative association was found between CRP and cardiorespiratory fitness (β = 0.188; p < 0.001) adjusted for age, gender and city; when BMI was included as a variable of adjustment, the values decreased, but still remained significant (β = -0.124; p < 0.011). Kwon and collaborators, in the 2010 NHANES study with 3,202 youths, age 12 to 19 years, found CRP concentration levels significantly higher in subjects who presented low cardiorespiratory fitness ²⁵25. Kwon S, Burns TL, Janz K. Associations of cardiorespiratory fitness and fatness with cardiovascular risk factors among adolescents: the NHANES 1999-2002. J Phys Act Health 2010; 7:746-53.. However, in a study with 416 Spanish adolescents, age 13 to 18.5 years, with samples adjusted for gender, sexual maturity and body fat, no significant association was found between CRP and cardiorespiratory fitness ³⁶36. Ruiz JR, Ortega FB, Wärnberg J, Moreno LA, Carrero JJ, Gonzalez-Gross M, et al. Inflammatory proteins and muscle strength in adolescents: the Avena study. Arch Pediatr Adolesc Med 2008; 162:462-8..

In most studies that investigated the same variables examined in this one, the results are not presented according to gender. Tam et al. ³⁷37. Tam C, Clement K, Baur L, Tordjman J. Obesity and low-grade inflammation: a paediatric perspective. Obes Rev 2010; 11:118-26. reinforce that potential CRP-related influences could be observed were the results disaggregated according to gender. In the model presented in this study, in addition to possible confounding variables, such as age, sexual maturity status and BMI, the results were analyzed by gender, based on the theoretical assumption of the possible association between body fat and CRP concentration, and hormonal differences between genders, as suggested by Kwon ⁵⁰50. Kwon S. C-reactive protein in children: confounding by sex and linear regression modeling. Med Sci Sports Exerc 2011; 43:740..

The association between PA and CRP in youths has been examined particularly with the use of questionnaires; indeed, studies that use objective assessment of PA are limited ³⁸38. Thomas NE, Williams DRR. Inflammatory factors, physical activity, and physical fitness in young people: review. Scand J Med Sci Sports 2008; 18:543-56.. Data collected from adolescents by means of questionnaires are poorer methodologically when compared with objective measures obtained with the use of accelerometers ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.. In this study, we have observed the association between CRP and the proportion of moderate, strenuous and very strenuous PA. Our results did not find significant association between CRP and the proportion of moderate, strenuous and very strenuous PA adjusted for age, sexual maturity and BMI. These findings are consistent with those reported in the NHANES study, with 1,643 children and adolescents, age 6 to 17 years, in which no association was found between CRP and PA measured objectively ³⁴34. Loprinzi P, Cardinal B, Crespo C, Brodowicz G, Andersen R, Sullivan E, et al. Objectively measured physical activity and C-reactive protein: National Health and Nutrition Examination Survey 2003-2004. Scand J Med Sci Sports 2013; 23:164-70.. Similarly, in two other recent studies, no association was found between PA measured objectively and CRP ²¹21. Martinez-Gomez D, Gomez-Martinez S, Ruiz JR, Diaz LE, Ortega FB, Widhalm K, et al. Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study. Atherosclerosis 2012; 221:260-7.^,3232. Martinez-Gomez D, Eisenmann JC, Wärnberg J, Gomez-Martinez S, Veses A, Veiga DL, et al. Associations of physical activity, cardiorespiratory fitness and fatness with low-grade inflammation in adolescents: the AFINOS Study. Int J Obes (Lond) 2010; 34:1501-7.. However, Parrett et al. ²⁴24. Parrett AL, Valentine RJ, Arngrimsson SA, Castelli DM, Evans EM. Adiposity, activity, fitness, and C-reactive protein in children. Med Sci Sports Exerc 2010; 42:1981-6. explored a joint association between body fat, CRP and PA (measured by pedometers) and found a negative association (r = -0.49, p < 0.05) in a group of 44 children.

It is possible that childhood and adolescence is too early for one to observe a direct and independent relationship between PA and CRP concentration ³⁰30. Thomas N, Baker J, Graham M, Cooper S, Davies B. C-reactive protein in schoolchildren and its relation to adiposity, physical activity, aerobic fitness and habitual diet. Br J Sports Med 2008; 42:357-60.. It is possible that when CRP concentration is normal, PA may have little influence ³⁴34. Loprinzi P, Cardinal B, Crespo C, Brodowicz G, Andersen R, Sullivan E, et al. Objectively measured physical activity and C-reactive protein: National Health and Nutrition Examination Survey 2003-2004. Scand J Med Sci Sports 2013; 23:164-70.. However, there is no CRP reference value established for children and adolescents, which may lead these results to be re-assessed.

One of the strengths of the study was that it investigated the association between CRP concentration and objective measures of PA, and observed the assessments independently from the BMI, and disaggregated by gender. Our results should be interpreted taking into consideration some limitations. First, one must acknowledge the cross-sectional nature of the study, and because of that no causal directionality could be inferred. Another important factor to be observed is the 0.61 power of the sample for males. Longitudinal and intervention studies may provide a different perspective, and perhaps present more robust results regarding the association between CRP with PA and cardiorespiratory fitness in adolescents.

The results of this study, therefore, suggest that CRP is negatively associated with cardiorespiratory fitness, but with gender-related differences: it seems less dependent on BMI in females. Thus, more studies with representative samples are necessary to clarify the relationship between PA and cardiorespiratory fitness with CRP concentration in children and adolescents, as well as longitudinal epidemiological studies.

Acknowledgments

Centro de Investigação em Atividade Física, Saúde e Lazer, (“Center for Research on Physical Activities, Health and Leisure”); students and teachers of the participating schools.

References

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