Vitamin D is an important determinant of bone health at all ages(Reference Schoenmakers, Goldberg and Prentice1). Vitamin D increases the absorption of Ca and phosphate from the gastrointestinal tract for mineralization of the skeleton. In utero and during childhood, vitamin D deficiency can cause growth retardation and skeletal deformities and is associated with an increased risk of hip fracture later in life(Reference Schoenmakers, Goldberg and Prentice1–Reference Holick3). In addition to its role in bone health, vitamin D has also been reported as a relevant factor in decreasing the risk of many chronic illnesses including common cancers and autoimmune, infectious and cardiovascular diseases(Reference Holick3). Recently, Gilbert-Diamond et al.(Reference Gilbert-Diamond, Baylin and Mora-Plazas4) demonstrated an inverse association between vitamin D serostatus and the development of adiposity in Colombian school-aged children.
Vitamin D deficiency and insufficiency are still very common, especially in high-risk groups such as young children(Reference Holick3, Reference Lips5). Humans get vitamin D from exposure to sunlight, from their diet and from dietary supplements. Previous studies showed that the dietary vitamin D intake of Belgian children and adolescents is very low compared with the Belgian recommendations(Reference Huybrechts and de Henauw6, Reference Sioen, Matthys and De Backer7). However, no recent data on vitamin D status in young children living in Belgium are available. Moreover, most studies describing the vitamin D status of European children start at the age of 8 years and older(Reference Lehtonen-Veromaa, Mottonen and Irjala8–Reference Cashman, Fitzgerald and Viljakainen13). Additionally, different anthropometric variables have been shown to significantly influence vitamin D status. Recently, Rodríguez-Rodríguez et al.(Reference Rodríguez-Rodríguez, Navia-Lomban and Lopez-Sobaler11) suggested that the amount of visceral and not subcutaneous fat determines the serum level of vitamin D in children (9–13 years old), but concluded that more studies are needed to test this hypothesis and to confirm their findings.
The study presented here describes cross-sectional data on the vitamin D status of Belgian children aged between 4 and 11 years, based on the serum concentration of 25-hydroxyvitamin D (25(OH)D) in blood samples taken in winter and spring, and aims to assess the influence of non-nutritional determinants on vitamin D status.
Experimental methods
Participants
Participating children were drawn from the Belgian control region cohort of the EU 6th Framework Programme IDEFICS Study, residents from the city of Aalter (51°05′N) in the northern Dutch-speaking part of Belgium. The IDEFICS (Identification and prevention of Dietary- and lifestyle-induced health EFfects In Children and infantS; http://www.idefics.eu) multicentre study is a European project in eight European countries. IDEFICS is a longitudinal study; however, in the present paper only the cross-sectional data collected in 2010 are used. The children were contacted by random cluster sampling (all children from a selection of classes from all schools in the control city)(Reference Ahrens, Bammann and de Henauw14–Reference Ahrens, Bammann and Siani16). During the fieldwork period, which ran from February 2010 to June 2010, a venous blood collection was received from 509 children in the Belgian IDEFICS control region. Vitamin D analysis was performed on the samples of 358 children. Blood drawn from the rest of the children was not sufficient to allow analysis, since the first six aliquots were analysed for other purposes of the IDEFICS Study. For one child, information on sex was missing. Therefore, the data of 357 children were used for the purposes of the present analysis. Concerning the ethnicity of the children, of the 357, only seven were not born in Belgium (four of the seven were born within the EU). For the children who were born in Belgium, only two had one parent born outside Belgium (one from Cuba and one from the Philippines). The study was conducted according to the guidelines laid down in the Helsinki Declaration of the World Medical Association and the project protocol was approved by the Ethical Committee of Ghent University Hospital. All parents of the participating children gave written informed consent.
Questionnaire
A self-administered parental questionnaire was used to obtain information on the following variables: number of hours playing outside during weekdays and weekend days, use of vitamin supplements, birth weight and fractures. Questions assessing the number of hours playing outside were based on those used in Burdette et al.(Reference Burdette, Whitaker and Daniels17).
Anthropometric measurements and body composition
All anthropometric measurements were done by two trained researchers. Height and weight were measured respectively with a standard clinical Seca 225 stadiometer (Seca GmbH & Co. KG, Hamburg, Germany) to the nearest 0·1 cm and a balance (Tanita BC 420 SMA; Tanita, Amsterdam, The Netherlands) to the nearest 0·1 kg, without shoes and in light clothing. The Tanita balance was calibrated and did not need further calibration; also the Seca stadiometer needed neither maintenance nor further calibration. BMI was calculated according to the formula: BMI = weight (kg)/[height (m)]2. For each child, weight, height and BMI Z-score and the International Obesity Taskforce grade was determined using the LMS method (with British reference population) which summarizes the distribution of weight, height and BMI at each age by its median and CV, plus a measure of skewness based on the Box–Cox power required to transform the data to normality(Reference Cole, Freeman and Preece18). Waist and hip circumferences were measured using a Seca 200 inelastic tape (Seca GmbH & Co. KG; precision 0·1 cm, range 0–150 cm). This tape did not need any calibration. Skinfold thickness was measured at the previously marked points using Holtain Tanner/Whitehouse skinfold callipers (Holtain Ltd, Crosswell, UK; range 0–40 mm). The callipers were calibrated every morning and additionally when dropped by means of a calibration block of 20 mm. Skinfold thickness was measured at two sites (triceps and subscapular) according to the international standards for anthropometric assessment (International Society for the Advancement of Kinanthropometry)(Reference Marfell-Jones, Olds and Stewart19). Skinfold thickness was measured twice at each site and the mean of both measurements was calculated. For the different body composition parameters, mean Z-scores were calculated using the formula: Z = (X − μ)/sd, with X the measured value, μ the mean and sd the standard deviation of the parameter.
Biochemical analysis
Fasting blood samples (10 ml) were drawn by venepuncture by a medical doctor. The serum was separated, aliquoted and stored at −80°C until analysis. 25(OH)D concentrations were measured by RIA (DiaSorin 25(OH)D – 125I RIA kit, Stillwater, MN, USA). The inter-assay CV for low and higher 25(OH)D controls was 6·2 % and 6·5 %, respectively, whereas the intra-assay CV was 8·4 % and 7·3 % for low and higher 25(OH) controls, respectively. For validation of the vitamin D assay, the laboratory participates in the External Quality Assessment Scheme of SKML (Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek) from The Netherlands, which six times a year gives two samples of vitamin D controls.
Statistical analysis
Pearson correlation coefficients were calculated to investigate correlations between vitamin D status and other continuous variables. Differences in vitamin D status between two groups (sex, (non-)use of vitamin D supplements and (not) having had fractures in the past) were examined by the independent-samples t test. When more than two groups were considered, ANOVA was used. In addition, as exploratory analysis, the post hoc Tukey Honestly Significant Difference (HSD) test was performed to make pairwise comparisons.
The independent contribution of non-nutritional determinants (age, gender, month of sampling, number of hours playing outside per week) to the variance in vitamin D status was examined by analysis of covariance (ANCOVA). Two ANCOVA models were constructed using two different indicators of body composition: (i) the sum of two skinfold thicknesses, reflecting the amount of subcutaneous fat; and (ii) waist circumference, reflecting the amount of abdominal fat. Both body composition parameters were log-transformed to obtain normality in these variables. Only significant variables associated with vitamin D status were included in the final model. The effect of clustering was investigated using mixed model analyses, with ‘school’ being the nested variable and taking on board the same covariates and interaction factors as was done in the ANCOVA model.
Relationships between vitamin D status and related anthropometric variables (weight, height, BMI Z-score, waist circumference, waist:hip ratio, waist:height ratio, triceps skinfold thickness, subscapular skinfold thickness, sum of two skinfold thicknesses) were quantified using linear regression, controlling for age, month of sampling and number of hours playing outside per week. For these analyses, the month of sampling was dummy coded (March and April = 0; May and June = 1; the cases of February (n 29) were excluded).
All analyses were performed using the SPSS for Windows statistical software package version 15·0 (SPSS Inc., Chicago, IL, USA), and values of P < 0·05 were considered statistically significant.
Results
Characteristics and anthropometric variables of the population sample are summarized in Table 1. Serum 25(OH)D ranged from 13·6 nmol/l to 123·5 nmol/l, with a mean concentration of 47·2 (sd 14·6) nmol/l.
Characteristics, serum 25(OH)D concentrations and anthropometric data of the study population: Belgian children aged 4–11 years participating in the IDEFICS Study, 2010
25(OH)D, 25-hydroxyvitamin D; IDEFICS, Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants.
No significant difference in vitamin D status was found between boys and girls (P = 0·263). Children using vitamin D-containing supplements (5·9 %) had a mean 25(OH)D concentration of 49·5 (sd 11·2) nmol/l compared with 47·0 (sd 15·2) nmol/l for those not using vitamin D-containing supplements; however, the difference was not significant (P = 0·464). On the same line, no significant difference (P = 0·302) was found between those children who reported having had a fracture (12·6 %) so far (25(OH)D = 44·9 (sd 13·7) nmol/l) and those without any reported fractures (25(OH)D = 47·3 (sd 13·7) nmol/l).
ANOVA did not show any significant difference in 25(OH)D concentration for different birth months (P = 0·160). However, a significant difference in 25(OH)D concentration between the months of sampling (February to June) was found (P < 0·001). The post hoc Tukey HSD test indicated a difference between March and May (P < 0·001), March and June (P < 0·001), April and May (P = 0·007) and April and June (P = 0·002), as illustrated in Fig. 1.
Box plot of serum 25-hydroxyvitamin D (25(OH)D) concentrations in Belgian children aged 4–11 years participating in the IDEFICS (Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants) Study, 2010, according to month of sampling. a,bMean values with unlike superscript letters were significantly different (P < 0·05); ○, outlier; *, extreme value
Table 2 shows the correlation coefficients between 25(OH)D concentration and several covariates of interest. A positive significant correlation (r = 0·140) was found between 25(OH)D concentration and the number of hours playing outside per week. Moreover, negative correlations were found between 25(OH)D concentration and weight (r = −0·121), triceps (r = −0·112) and subscapular (r = −0·119) skinfold thickness, the sum of two skinfold thicknesses (r = −0·125) and waist circumference (r = −0·108).
Pearson correlation coefficients between 25(OH)D concentration and several covariates: Belgian children aged 4–11 years participating in the IDEFICS Study, 2010
25(OH)D, 25-hydroxyvitamin D; IDEFICS, Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants.
*Significant at level P = 0·05.
The results of the ANCOVA models are shown in Table 3. The adjusted R 2 (coefficient of determination) of the first (with skinfold thickness) and second (with waist circumference) model was 0·126 and 0·118, respectively. Both models indicated a significant contribution of the month of sampling, the number of hours playing outside per week and the included body composition parameter (reflecting respectively subcutaneous and abdominal fat) to the variance in 25(OH)D concentration.
Results of the two ANCOVA models assessing the independent contributions of determinants to the variance in 25(OH)D concentration: Belgian children aged 4–11 years participating in the IDEFICS Study, 2010
ANCOVA, analysis of covariance; 25(OH)D, 25-hydroxyvitamin D; IDEFICS, Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants.
*Significant at level P = 0·05.
The mixed model analyses indicated that the cluster random sampling design of the study (via schools) did not have an influence on the study results (P = 0·198).
Using linear regression analyses with serum 25(OH)D concentration as the dependent variable and controlling for age, month of sampling and number of hours playing outside per week, it was found that weight, BMI Z-score, waist circumference and triceps and subscapular skinfold thickness (as well as the sum of both) independently influenced these concentrations (Table 4).
Regression results using 25(OH)D concentration as the dependent variableFootnote †: Belgian children aged 4–11 years participating in the IDEFICS Study, 2010
25(OH)D, 25-hydroxyvitamin D; IDEFICS, Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants.
* Significant at level P = 0·05.
† Confounding variables (age, month of sampling and number of hours playing outside per week) have been taken into account in the statistical analysis.
Discussion
The present paper describes the vitamin D status of Belgian children aged 4–11 years. The optimal vitamin D levels for health remain a subject of debate(Reference Holick3). In the present paper four categories were considered: category 1, <25 nmol/l, deficient; category 2, 25–50 nmol/l, insufficient; category 3, 50–75 nmol/l, sufficient; and category 4, ≥75 nmol/l, optimal. These categories are based on recent publications taking into account the full advantage of all the health benefits that vitamin D provides(Reference Holick3, Reference Lips5, Reference Holick20). More specifically for children, the Pediatric Endocrine Society stated that 50 nmol/l is the lower end of the optimal 25(OH)D concentration in children(Reference Wagner and Greer21). Within our study sample of Belgian children, 58 % had an insufficient vitamin D status and only a small proportion (2·2 %) reached the optimal level, based on blood sampled in winter and spring. Until now, recent data on the vitamin D status of Belgian young children had not been available. Evidence on the vitamin D status of other European children up to 10 years is limited as well, most including an older age group.
Comparison with other European data
The data of the present study were compared with other available European data; however, this comparison must be interpreted with caution, as not all the studies used the same method of determining 25(OH)D which can influence the results. At the end of the last millennium, data were published on the vitamin D status of French, Spanish and Finnish children aged 13–17 years, 8–12 years and 9–15 years, respectively(Reference Lehtonen-Veromaa, Mottonen and Irjala8–Reference Guillemant, Taupin and Le10). In the French children (only males), a mean 25(OH)D concentration of 58·5 nmol/l was found after summer and 20·6 nmol/l was found after winter(Reference Guillemant, Taupin and Le10). Similarly, in the Spanish children, a mean of 74·8 and 31·5 nmol/l was determined in October and March, respectively(Reference Docio, Riancho and Perez9). This illustrates a strong effect of the season of sampling, similarly to that observed in our study (e.g. March v. June; Fig. 1). The Finnish study, which included only girls, indicated a mean 25(OH)D concentration of 33·9 nmol/l(Reference Lehtonen-Veromaa, Mottonen and Irjala8), lower than the mean found in the present study. More recent publications describing the vitamin D status in European children, in Spain (9–13 years)(Reference Rodríguez-Rodríguez, Navia-Lomban and Lopez-Sobaler11), in Northern Ireland (12–15 years)(Reference Hill, Cotter and Mitchell12) and in a combined cohort of Finnish and Danish girls (11 years old)(Reference Cashman, Fitzgerald and Viljakainen13), indicated higher concentrations compared with our Belgian sample. For instance, mean 25(OH)D concentrations in Spain were 49·6 nmol/l(Reference Rodríguez-Rodríguez, Navia-Lomban and Lopez-Sobaler11), in Northern Ireland were 56·7 nmol/l (winter) and 78·1 nmol/l (summer), in the Danish and Finnish girls were 57·2 and 56·2 nmol/l, respectively(Reference Cashman, Fitzgerald and Viljakainen13), as opposed to 47·2 nmol/l in our sample. We found no significant differences by gender, unlike the Spanish and Northern Irish populations where significant differences by gender were observed. However our study population was younger and this age difference could have an influence on gender differences.
Determinants of vitamin D status
The ANCOVA yielded three important non-nutritional determinants of the vitamin D status in Belgian young children: month of sampling, number of hours playing outside per week and body composition. The first two parameters are related to the fact that solar UV-B radiation converts and modifies 7-dehydrocholesterol into vitamin D at skin temperature(Reference Cashman2). In northern countries, such as Belgium, there is no UV radiation of the appropriate wavelength from the end of October to the end of March(Reference Lanham-New, Thompson and More22). This is clearly illustrated in Fig. 1, showing a minimum in 25(OH)D in March and an increase from April on. Moreover, the study results showed an interaction effect of season and the weekly number of hours playing outside, since the highest levels of outdoor playtime occur in summer and the lowest in winter(Reference Burdette, Whitaker and Daniels17).
Body composition is another important determinant of vitamin D status. Previous studies have suggested that vitamin D deficiency is associated with excess body weight mainly due to the fact that body fat could function as a sink for vitamin D, which is fat soluble(Reference Bischof, Heinze and Vierhapper23–Reference Konradsen, Ag and Lindberg25). Moreover, inadequate vitamin D status could also be a risk factor for childhood obesity, since it affects lipolysis and adipogenesis in human adipocytes(Reference Zemel, Shi and Greer26–Reference Kong and Li29). In the present study, it was found that parameters of whole-body obesity (defined by the BMI), parameters of subcutaneous fat (skinfold thickness) as well as parameters of abdominal fat (waist circumference) independently influenced vitamin D status in young Belgian children (4–11 years old). The study of Spanish children (9–13 years)(Reference Rodríguez-Rodríguez, Navia-Lomban and Lopez-Sobaler11) also concluded that BMI and abdominal obesity influenced the appearance of vitamin D insufficiency in children. However, the Spanish study did not find a difference in terms of the amount of subcutaneous fat between the children with insufficient vitamin D levels and the children with adequate levels. The difference concerning this finding between both studies can possibly be influenced by the difference in age (on average 8·1 years in Belgium v. 10·9 years in Spain), resulting in lower weight, height, BMI, triceps skinfold thickness and waist circumference in Belgium compared with Spain.
Strengths and limitations
The availability of various anthropometric parameters measured in a standardized way in a relatively large sample is one of the strengths of the present study. In this way the authors had the opportunity to address the influence of various anthropometric indicators on vitamin D status and explore possible relationships and interactions. The present study is one of the first addressing this issue in young children, based on the hypothesis suggested by Rodriguez-Rodriguez et al.(Reference Rodríguez-Rodríguez, Navia-Lomban and Lopez-Sobaler11). To assess vitamin D status, the serum concentration of 25(OH)D was measured, currently the best parameter for vitamin D status(Reference Lips5, Reference Holick20). Next, the questions assessing the number of hours playing outside were based on Burdette et al.(Reference Burdette, Whitaker and Daniels17), concluding that parent-reported measures of outdoor playtime were significantly correlated to a direct measure of physical activity in children of pre-school age and are worthy of future evaluation as a survey measure. Although the analyses controlled for several potential confounders we cannot be certain that other unmeasured confounders such as genetic variation or dietary intake have not influenced our observations. Nevertheless, concerning dietary intake, the number of food items on the Belgian market that are fortified with vitamin D is rather limited, i.e. margarine (mandatory fortified), growth milk and some normal milks as well as breakfast cereals (depending on the brand). More detailed figures about the dietary intake of vitamin D in Belgian pre-school children have recently been published(Reference Huybrechts, Lyn and De Keyzer30).
Conclusions
The majority of Belgian children (4–11 years old) have a suboptimal vitamin D status, with more than half having an insufficient status during winter and spring months. Month of the year, number of hours playing outside per week as well as body composition were identified as important determinants of vitamin D status in this group of children. The vitamin D status of these children was independently influenced by parameters of whole-body obesity, parameters of subcutaneous fat as well as parameters of abdominal fat. The impact of recommendations to optimize the vitamin D status of young children should be investigated in order to prevent associated diseases.
Acknowledgements
This work was done as part of the IDEFICS Study (www.idefics.eu) and was supported by the European Community within the Sixth RTD Framework Programme Contract No. 016181 (FOOD). I.S., B.V. and K.V. are financially supported by the Research Foundation Flanders (grant no. 1.2.683.11.N.00, 1.1.894.11.N.00 and 1.1.905.10.N.00, respectively). The information in this document reflects the authors’ views and is provided as is. None of the authors has a conflict of interest. I.S. performed the statistical analyses and wrote the manuscript. All co-authors revised the manuscript. T.M. and I.P. provided support for the statistical analyses. K.B., I.P. and S.D.H. were involved in the design of the IDEFICS Study and the development of the questionnaires. N.M., B.V. and K.V. conducted the fieldwork. J.-M.K. was responsible for the laboratory analyses. The authors gratefully acknowledge the financial support of the European Commission and thank all participants of the IDEFICS Study in Aalter, Belgium for their involvement.
