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1 From the Immunonutrition Group, Department of Metabolism and Nutrition, Consejo Superior de Investigaciones Científicas, Madrid, Spain (JW, EN, JR and AM); the Unit for Preventive Nutrition, Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Stockholm, Sweden (JW, JRR, FBO, and MS); the Escuela Universitaria Ciencias de la Salud, University of Zaragoza, Spain (LAM, MIM and MB); and the Department of Physiology, School of Medicine, University of Granada, Spain (JRR and FBO)
2 Supported by the Spanish Ministry of Health (00/0015) and by grants from the Consejo Superior de Deportes (05/UPB32/01, 09/UPB31/03, and 13/UPB20/04), the Spanish Ministry of Education (AP2003-2128 and AP2004-2745), Panrico SA, Madaus SA, and Procter & Gamble SA. 3 Address reprint requests and correspondence to A Marcos, Immunonutrition Group, Department of Metabolism and Nutrition, Instituto del Frío, Consejo Superior de Investigaciones Científicas, Calle José Antonio Novais, 10, Madrid 28040, Spain. E-mail: amarcos{at}if.csic.es.
ABSTRACT
Background: In adults, obesity is characterized by a state of chronic low-grade inflammation accompanied by moderately high concentrations of acute phase inflammatory proteins. Recent results regarding C-reactive protein (CRP) point to a similar status in adolescents; however, studies of associations of the serum inflammatory proteins CRP, ceruloplasmin, and complement factors C3 and C4 with body fat distribution remain scarce.
Objective: We aimed to establish the possible relations of serum inflammatory proteins with body fat estimates and body fat distribution in an apparently healthy adolescent population.
Design: This report included 472 adolescents (248 males and 224 females) aged 13–18.5 y who were recruited from the Spanish cross-sectional multicenter AVENA Study for whom anthropometric and immunologic data were complete. The concentrations of the serum proteins and the in vitro production of cytokines (interleukin 6 and tumor necrosis factor ) by isolated and stimulated white blood cells were measured. Relations with anthropometric measurements were explored by using simple and partial correlations.
Results: CRP, C3, and C4 were correlated with central obesity (as measured by waist circumference) and total body fat in both sexes (P < 0.01) and with ceruloplasmin in females only. After further adjustment for BMI, C3 remained independently associated with central obesity (P < 0.05). Production of the cytokines by white blood cells did not seem to be affected by an excess of body fat.
Conclusions: Total body fat seems to be associated with a chronic low-grade systemic inflammation in apparently healthy adolescents. Central obesity is independently associated with C3 concentrations, which makes this marker especially interesting for further studies of obesity-related diseases.
Key Words: Adolescents • inflammation • complement factors • C-reactive protein • CRP • central obesity
INTRODUCTION
Adipose tissue is a rich source of many immune-related mediators that are involved in the inflammatory response. Adipose tissue produces proinflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor- (TNF-), and complement factors. The hepatic synthesis of acute phase inflammatory proteins—eg, C-reactive protein (CRP), complement factors C3 and C4, and ceruloplasmin—are in turn under the control of these proinflammatory cytokines, and, therefore, adipose tissue is a noteworthy agent of the high circulating concentrations of both cytokines and proteins (1).
Moderately high concentrations of inflammatory markers observed in obesity have led to the view that obesity is characterized by a state of chronic low-grade inflammation (1, 2). Obesity-induced inflammation has been thought to accelerate vascular damages (3), and a potentially important issue is how adiposity regulates inflammation during childhood and adolescent obesity. The answer to this question could help reverse the damage due to adiposity in time to prevent future disease.
Population-based cohort studies of American children and adolescents showed higher concentrations of CRP, the major acute phase inflammatory protein, in overweight subjects than in normal-weight subjects (4, 5). However, these studies did not evaluate different proinflammatory actions of body fat (BF) distribution. BF patterning shows distinctly different metabolic activities, which are believed to predispose a person toward obesity complications such as diabetes and cardiovascular disease [(CVD) 6, 7]. A recent study in adults showed that central obesity is one of the most important determinants of the low-grade chronic inflammation present in the metabolic syndrome (8). However, it remains to be clarified whether central obesity is a stronger predictor of inflammation in healthy adults than is peripheral adiposity and whether a similar relation occurs as early as adolescence.
Complement factors and ceruloplasmin in healthy populations have been studied much less than have lipid-related CVD risk factors, although several studies have shown that these proteins strongly predict cardiovascular events in adults (9, 10).
It is particularly interesting to study relations between obesity measurements and low-grade inflammation in an adolescent population, in whom interaction biases due to any ongoing inflammatory pathology that could be present in an apparently healthy adult population are minimized. The aim of this study was thus to evaluate the relations of selected inflammatory markers with BF estimates and patterning in a national population-based cohort of healthy adolescents from the Alimentación y Valoración del Estado Nutricional en Adolescentes (ie, Food and Nutritional Status in Adolescents) multicenter study conducted in Spain (the AVENA Study).
SUBJECTS AND METHODS
Study design
This research was conducted in the context of the AVENA Study, which was designed to evaluate the nutritional and health status of Spanish adolescents to identify risk factors for chronic diseases in adulthood (11). Briefly, this multicenter study involving a representative sample of Spanish adolescents aged 13–18.5 y was performed during 2000–2002. The population was selected by multiple-step, simple random sampling, first according to geographic location (ie, Madrid, Murcia, Granada, Santander, and Zaragoza) and then by random selection of classrooms from public and private schools in each city. This population was then stratified by age and sex. The variable with the greatest variance for this age group—ie, body mass index (BMI; in kg/m2)—was selected for calculation of the sample size. Subjects were included if they had no history of CVD, were free of disease and not taking any medication at the time of the study, and were not pregnant.
A detailed oral description of the nature and purpose of the study was given to the adolescents and their teachers. This information was also sent to their parents by letter, and written informed consent was obtained from both the parents and the subjects. The study was conducted in accordance with the ethical rules of the Helsinki Declaration (Hong Kong revision, September 1989), following the European Community's guidelines for Good Clinical Practice (document EEC 111/3976/88 of July 1990) and current Spanish law regulating clinical research in humans (Royal Decree 561/1993 regarding clinical trials). The study protocol was approved by the Review Committee for Research Involving Human Subjects of the Hospital Universitario Marqués de Valdecilla (Santander, Spain).
Study population
For this study, 472 adolescents (248 males and 224 females) from the AVENA Study, for whom anthropometric data and blood samples for immunologic evaluation were complete, were included. Mean (±SD) ages were 15.28 ± 1.19 y in males and 15.24 ± 1.33 y in females. According to pubertal maturity, most of the males were at Tanner stage 4 (41.1%) or 5 (38.1%), and 0%, 5.6%, and 15.2% were at stages 1, 2, and 3, respectively. Most of the females also were at stage 4 (55.5%) or 5 (35.0%), and 0%, 1.4%, and 8.1% were at stages 1, 2, and 3, respectively. BMI did not differ significantly between the subgroup from which blood samples were obtained (n = 472) and all subjects recruited for the AVENA Study (n = 2278). In this report, the prevalence of overweight (including obesity) in males was almost 30%, and that in females was 21%. Detailed data on overweight and obesity prevalence in the complete AVENA Study were reported elsewhere (12). Socioeconomic (eg, ethnicity and age) data were obtained by using questionnaires. Before blood sampling, each subject was evaluated, and all anamnestic data were recorded and reviewed to judge the adolescent's medical status. Only healthy adolescents were included in this report. Thus, subjects presenting with chronic diseases thought to have a possible effect on immune function (eg, asthma) or taking any medications with known immunologic effects were excluded from the study. Subjects with any acute medical conditions, such as minor infections (et, upper respiratory illness), were also excluded. After completion of the fieldwork, the subjects who did not fulfill the inclusion criteria were excluded.
Anthropometric method
All anthropometric measurements were made while the subjects were barefoot and wearing only their underwear. Body weight was measured to the nearest 0.05 kg by using a standard beam balance. Skinfold thicknesses (STs) were measured at the left side of the body to the nearest 0.2 mm with the use of Holtain skinfold calipers (Holtain, Crymych, United Kingdom), at the following 6 sites: 1) triceps, halfway between the acromion process and the olecranon process; 2) biceps, at the same level as the triceps skinfold, directly above the center of the cubital fossa; 3) subscapular, 20 mm below the tip of the scapula, at an angle of 45° to the lateral side of the body; 4) suprailiac, 20 mm above the iliac crest and 20 mm toward the medial line; 5) thigh, in the midline of the anterior aspect of the thigh, midway between the inguinal crease and the proximal border of the patella; and 6) calf, at the level of maximum calf circumference, on the medial aspect of the calf. Circumferences were measured with an inelastic tape (range: 0–150 cm; precision of 1 mm). In general, for these measurements, the subject was in a standing position. For measurement of the waist circumference (WC), the tape was applied horizontally midway between the lowest rib margin and the iliac crest, after gentle expiration. The hip circumference measurement was taken at the point yielding the maximum circumference over the buttocks, and the tape was held in a horizontal plane. The complete set of anthropometric measurements was performed 3 times, but not consecutively; we measured all the anthropometric variables in order, and then we repeated the same measurements twice.
Before carrying out the field work, we conducted a pilot study in 101 subjects from the 5 cities to study the reliability of the measurements that would be assessed in the multicenter AVENA Study. Intraobserver reliability for STs was >95% in almost all cases; interobserver reliability for STs ranged from 83.05 for biceps to 96.38 for calf (13). As an index of total adiposity, we calculated the sum of the 6 measured STs. The percentage of BF (%BF) was calculated by the formula described by Slaughter et al (14) because this equation showed the best agreement with total %BF measured by dual-energy X-ray absorptiometry (DXA) in a subsample of 280 subjects from the Zaragoza cohort of the AVENA Study (15). Central BF accumulation was described as WC alone and waist-hip ratio (WHR), together with the ratio of truncal (subscapular + suprailiac) to total STs (sum of biceps, triceps, subscapular, and suprailiac), expressed as a percentage.
During anthropometric measurements, pubertal maturity was classified according to one of the 5 stages defined by Tanner (16). The standard staging of pubertal maturity has to do with breast and pubic hair development in girls and genital and pubic hair development in boys.
Laboratory method
After overnight fasting, blood samples were collected between 0800 and 0930 by venipuncture. Blood for serum samples was collected in evacuated tubes and allowed to clot. Serum was separated by centrifugation, divided into aliquots, and frozen and stored at –80 °C until withdrawn for analysis. C3, C4, highly sensitive CRP (hs-CRP), and ceruloplasmin were measured in serum by using immunoturbidimetry (AU2700 biochemistry analyzer; Olympus, Rungis, France). The capacity of cytokine production was assessed in cultured mitogen–stimulated peripheral blood mononuclear cells (PBMCs). Mononuclear cells were isolated from heparinized peripheral blood on Ficoll-Hypaque (Lymphoprep; Hyegaard, Oslo, Norway) and washed twice in RPMI-1640 medium (BioWhittaker, Verviers, Belgium). The PBMCs were resuspended in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin and streptomycin. The concentration was adjusted to 106 viable cells/mL, and 1 mL cell suspension was incubated per well with 2 mitogens—phytohemagglutinin (3.5 µL/mL) and lipopolysaccharide (1.5 µL/mL)—in 24-well plates for 48 h at 37 °C in 5% CO2. After incubation, the cells were removed by centrifugation and the supernatant fluid was stored at –80 °C before analysis. Cytokine concentrations in supernatant fluids were assessed by using the human Th1/Th2 cytokine CBA II kit (BD Biosciences Pharmingen, San Diego, CA) and analyzed by flow cytometry.
All laboratory measurements were analyzed at the same time to minimize systematic variation. The CVs (interassay precision) were <2% for CRP, ceruloplasmin, and C3 and C4 and <7% for cytokines. Data on glucose, cholesterol, HDL and LDL cholesterol, and triacylglycerols are provided here for descriptive purposes; complete methods were reported previously (17).
Statistical analysis
Statistical analyses were performed by using SPSS for WINDOWS software (version 13.0; SPSS Inc, Cary, NC). Sex and weight status effects and sex x weight-status interactions were analyzed by 2-factor ANOVA. As previously reported by Moreno et al (18), the differences in BF composition between the sexes in this population were, as expected, significant. Moreover, because male and female adolescents are metabolically different, sex effects were always studied, and all analysis were made separately in the sexes. To reflect the correlations studied separately in subjects with normal weight and overweight, groups were formed by applying age- and sex-specific cutoffs (extrapolated from the adult cutoffs for overweight) set at a BMI > 25, according to recommendations of the International Obesity Task Force (19). Overweight and obese subjects were studied together. P < 0.05 was considered significant. Data are presented as means ± SD. CRP, IL-6, and TNF- were transformed logarithmically, and C3, C4, and ceruloplasmin were square-rooted to stabilize variability and to achieve normality in the residuals. Raw values are presented in the tables and figures. Partial correlations between protein concentrations and anthropometric variables and indexes of BF and distribution were calculated after adjustment for age and Tanner stage. Tanner stages were analyzed as dummy variables. Further relations between central obesity and each of the acute phase proteins, after adjustment for BMI, were studied separately by partial correlations.
RESULTS
Physical characteristics of the population are described in Table 1. Concentrations of the studied inflammatory proteins and cytokines are summarized in Table 2. There was a sex effect on CRP values. Weight status affected CRP, C3, and C4 values in both males and females. A sex x weight status effect was found for ceruloplasmin only, and overweight girls had the highest values. Neither sex nor weight status had an observed effect on the capacity of isolated and mitogen-stimulated white blood cells for in vitro production of IL-6 and TNF-.
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TABLE 1. Physical characteristics of the subjects1
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TABLE 2. Concentrations of inflammation markers in the subjects1
Partial correlations, adjusted for age and Tanner stages, between each of the inflammatory proteins and adiposity measures are shown in Table 3. CRP, C3, and C4 correlated significantly with all estimates of total adiposity (ie, BMI, sum of 6 STs, and %BF) in both sexes. Ceruloplasmin showed these clear associations in females only. Accumulation of central adiposity assessed by WHR was significantly associated only with C3. WC was correlated with CRP, C3, and C4 in both sexes and also with ceruloplasmin in females. Truncal:total STs (in %) correlated significantly with C3 and C4 only in females. No significant correlations were found between the capacity of IL-6 and TNF- production and directly measured or calculated body-composition variables (data not shown). We found no sex x weight-status interaction for each anthropometric variable and inflammatory protein (2-factor ANOVA) except %BF for all proteins and the sum of 6 STs for the square root of ceruloplasmin. Correlations of these interactions according to weight status are presented in Table 4.
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TABLE 3. Partial correlations between inflammatory proteins and obesity measures after adjustment for age and Tanner stage1
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TABLE 4. Partial correlations between inflammatory proteins and obesity measures that present a significant sex x weight-status interaction in normal-weight and overweight subjects after adjustment for age and Tanner stage1
To analyze further whether the associations found between inflammatory proteins and WC are merely a reflection of higher total adiposity, we tested whether the observed correlations remained after additional adjustment for BMI in the partial correlations (Table 5). In both sexes, only C3 remained independently associated with WC. This independent association was further analyzed to check for possible differences between relations in normal-weight or overweight subjects (separately), but no differences were observed (data not shown).
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TABLE 5. Partial correlations between waist circumference and C-reactive protein (CRP), C3, C4, and ceruloplasmin after adjustment for age and Tanner stages and further adjustment for BMI1
DISCUSSION
The current study of apparently healthy Spanish adolescents shows that BF seems to be associated with a higher production of inflammatory proteins. Associations between inflammatory proteins, proinflammatory cytokines, and adiposity measurements were examined, and we found that acute phase inflammatory proteins CRP, C3, C4, and ceruloplasmin were positively correlated with most measurements of BF. Increasing concentrations of inflammatory markers accompanied by increasing BF thus designate this apparently healthy, young, overweight population as being in a state of chronic low-grade inflammation. In the past few years, CRP has had a large effect as a predictor of CVD. Several important studies in children have shown increases in serum CRP as BMI increases (4, 20-22). The results are convincing and now have been reproduced in Spanish adolescents. The presence of early precursors of atherosclerosis and early-stage atherosclerotic lesions was documented in children and young adults (23-25), and, indeed, the atherosclerosis process is thought to begin in childhood and to progress slowly into adulthood (23, 26). Elevated serum CRP has been associated with early arterial changes in American (27) and Finnish (28) children and adolescents, which supports the hypothesis that inflammation plays a role in the pathogenesis of early atherosclerosis (3, 29). There is also increasing evidence that chronic subclinical inflammation is associated with metabolic dysfunction, which is linked causally to insulin resistance and the metabolic syndrome (30). Thus, adolescence is a period of critical importance with respect to the onset of obesity and obesity-associated morbidity in later life. The strong relations of BF with C3 and C4 found in this study suggest that this increase could be due to an increased production in adipocytes, apart from other complement-producing tissues (ie, hepatic cells, fibroblast, mononuclear cells, and endothelial cells; 1, 31). Indeed, both C3 and C4 have been related to obesity in adults (9. 10, 32). Further data supporting the actions of BF on serum complement factors came from anorexic patients, in whom C3 concentrations are low, but they normalize with weight gain; on the other hand, obese adults have higher C3 concentrations than do control subjects, but the concentrations decrease after weight loss (33, 34). However, to our knowledge, studies of complement factors in younger populations have been conducted only in subjects with pathologic conditions (35, 36), so that this is probably the first population-based study to show this association in apparently healthy adolescents.
Central obesity is widely recognized to be a strong predictor of the presence of the metabolic syndrome (7), and this relation first appears during childhood (37, 38). Several studies have also associated WC with CRP in childhood and adolescence (39, 40) However, investigations of the BMI-independent proinflammatory action of central obesity in healthy young subjects are still scarce. The importance of these research topics is highlighted by the fact that waist circumference in the Spanish adolescent population is growing (41). In the current study, WC has special sensitivity for predicting the observed low-grade status of inflammation; the values are similar to the associations with total measures of adiposity. Considerations to bear in mind are that BMI and WC are strongly correlated and that both measures are good predictors of fat content in children and adolescents, and thus they reflect total BF. However, C3 remained independently associated with WC in both sexes after further adjustment for pubertal maturity and BMI. This is an interesting finding, which agrees with the results of a recent study by Onat et al (9), in which, unlike CRP, C3 was preferentially associated with WC. Some other studies also found positive correlations between C3 and WHR (42-44), and one study reported high gene expression of complement components in omental adipose tissue in obese men (30). Similarities must be interpreted carefully, because these studies were conducted in adults with a high prevalence of metabolic syndrome. Whether a slight chronic elevation in baseline C3 concentrations predicts future diseases or is merely a practical marker of BF is beyond the scope of the current study.
It has been suggested that different complement factors have different preferential associations with traditional risk factors (eg, insulin, blood lipids, early atherosclerotic lesions). These differences may be due to the factors' involvement in various cellular activations and metabolic processes (9, 10, 32, 45). In the current study, C3 was associated significantly more than were the other inflammatory proteins with abdominal obesity. However, the deposition of C3 and the activation of complement in arteries could potentially promote atherogenesis (45), and the implication of higher values with central obesity in adolescents should be studied further. Three of the 4 acute phase proteins assessed in the current study showed clear linear associations with adiposity in both males and females. However, the relations were different for ceruloplasmin in males, which had a less clear association with BF. Although inflammation is mediated by all acute phase proteins, the relations of these proteins to adiposity in healthy adolescents differ, as shown by the results of this study, and these different roles require further study.
The mechanisms by which BF mediates inflammation are unfortunately not entirely understood. One direct mechanism is the direct sourcing of proinflammatory cytokines such as IL-6 and TNF- and including complement factors from adipose tissue. In turn, the hepatic synthesis of acute phase proteins such as CRP, C3, C4, and ceruloplasmin is mostly under the control of proinflammatory proteins such as IL-6 and TNF- (1, 46). The current study included the measurement of IL-6 and TNF- production by white blood cells, which are just one of the sources for the overall production in the body. Measurements of circulating serum concentration, derived from all cytokine producing cells, were not available. Despite that lack of difference in cytokines, it seems that the observed chronic low-grade inflammatory state may derive from the action of proinflammatory cytokines, directly produced locally by adipose tissue and not by circulating white blood cells. Adipose tissue, and the visceral depot in particular, is a major contributor of plasma IL-6, and it explains 33% of plasma IL-6 concentrations. TNF- is also expressed in adipose tissue, but it seems not to be released into circulation and thus is unable to signal systemically (47, 48). Direct assessment of serum proinflammatory cytokines is needed in future studies to further test this hypothesis. One limitation, however, is the low serum cytokine concentrations in healthy young populations, which fall below the assay's sensitivity in a large proportion of cases.
Our study has some limitations. Because of its cross-sectional design, we cannot establish the directionality of the associations. Whereas the general assumption is that inflammation is consequent to increased adipose compartment, adipocyte hypoxia, and the stimulation of angiogenesis (1), it has been suggested that obesity is in fact a result of inflammatory disease (49). Furthermore, we used a single blood measurement of inflammation, but that may not accurately reflect long-term inflammatory status. Although we asked the subjects about any clinical infection during the study, and no subject with a known underlying cause of infection was included, we cannot exclude that elevated concentrations were due to the onset of an infection. Nevertheless, to further minimize the confounder of an ongoing infection, subjects with CRP concentrations > 10 mg/L were excluded from this analysis. The effect of any such measurement bias should be attenuated, however, by the large numbers of participants enrolled. In conclusion, the results of this large-scale, national, cross-sectional study showed that BF seems to contribute to circulating concentrations of inflammatory serum proteins, which suggests a proinflammatory action of the adipose tissue in apparently healthy adolescents. Moreover, central obesity seems to especially trigger C3 production.
ACKNOWLEDGMENTS
We gratefully acknowledge all participating adolescents, and their parents and teachers for their collaboration. We thank Laura Barrios and Olle Carlsson for their valuable assistance with the statistical analysis of the data. We also acknowledge the collaborating investigators for their efforts and great enthusiasm during the field work. The AVENA Study group included the following persons: A Marcos (Madrid), Coordinator; MJ Castillo (Granada), A Marcos (Madrid), S Zamora (Murcia), M García Fuentes (Santander), and M Bueno (Zaragoza), Principal Investigators; MJ Castillo, MD Cano, R Sola, A Gutiérrez, JL Mesa, JR Ruiz, FB Ortega, M Delgado, P Tercedor, P Chillón, M Martín, F Carreño, GV Rodríguez, R Castillo, and F Arellano (all: Universidad de Granada, Granada); A Marcos, M González-Gross, J Wärnberg, E Nova, J Romeo, S Gómez, A Álvarez, L Barrios, A Leyva, B Payá, MC Vallejo, F Sánchez Muniz, L Martínez, E Ramos, R Ortiz de Zárate, and A Urzanqui (all: Instituto de Nutrición y Bromatología, Consejo Superior de Investigaciones Científicas, Madrid); S Zamora, M Garaulet, F Pérez-Llamas, JC Baraza, JF Marín, F Pérez de Heredia, MA Fernández, C González, R García, C Torralba, E Donat, E Morales, MD García, JA Martínez, JJ Hernández, A Asensio, FJ Plaza, and MJ López (all: Departmento Fisiología, Universidad de Murcia, Murcia); M García Fuentes, D González-Lamuño, P de Rufino, R Pérez-Prieto, D Fernández, and T Amigo (all: Departmento Pediatría, Universidad de Cantabria, Santander); and M Bueno, LA Moreno, A Sarriá, J Fleta, G Rodríguez, CM Gil, MI Mesana, JA Casajús, V Blay, MG Blay (all: Escuela Universitaria de Ciencias de la Salud, Universidad de Zaragoza, Zaragoza).
AM, EN, LAM, and MB contributed to the study design; JW, EN, LAM, JR, MIM, JRR, and FBO performed the data collection; all authors were involved in data analysis and interpretation of results; and JW, EN, LAM, JR, JRR, FBO, MS, and AM contributed to writing the manuscript. None of the authors had any personal or financial conflict of interest.
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