the study included 225 boys aged 716 years , recruited from two centers of a football academy in the area of tunis ( latitude , 35n ) .
children with liver , renal , or bone disease ; intestinal malabsorption ; or cancer ; and those taking vitamin d supplements , anticonvulsant drugs , or systemic corticosteroids were not included .
the study was carried out from january to march 2014 , a period during which temperatures varied between 10c and 22c and the humidity ranged from 70 to 75% . besides 2 h per week of outdoor physical activity as part of the school program , each child attends three weekly outdoor sessions in the football academy .
the training sessions are scheduled from 5 pm to 6 pm on friday and saturday , and from 9 am to 10 am on sunday .
skin color of each participant was determined by two investigators ( ib and mf ) , together with one parent of the child , and classified as fair , corresponding to fitzpatrick skin types 1 and 2 , or dark , corresponding to fitzpatrick skin types 3 and 4 ( 19 ) . written parental permission was obtained for each participant .
weight , height , and sitting height were measured with the subjects barefooted and lightly clothed .
body mass index ( bmi ) was calculated as weight per height squared ( kg m ) .
participants were divided according to the world health organization ( who ) child growth standards for bmi in three groups ; normal - weight group ( bmi85th percentile ) , overweight group ( 85th percentile < bmi < 97th percentile ) , and obese group ( bmi>97th percentile ) ( 20 ) .
triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers ( baty international , west sussex , england ) .
biologic maturity was assessed by incorporating anthropometric variables ( weight , standing height , and sitting height ) and was calculated using the equation of mirwald ( 22 ) ; maturity offset=9.236+(0.0002708*leg length*sitting height)+(0.001663*age*leg length)+(0.007216*age*sitting height)+(0.02292*weight by height ratio ) .
this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity ( phv ) as a measure of maturity offset . for the purpose of data analysis
, children were divided into three groups : pre - phv ( 3 to 1 year from phv ) , around phv ( 1 to + 1 year from phv ) , and post - phv ( + 1 to + 3 years from phv ) .
the daily vitamin d intake in children 's diet was assessed using a 3-day food record ( including 2 weekdays and 1 day over the weekend ) , combined with a food frequency questionnaire ( ffq ) that the parents mostly completed . a 35-items ffq to quantify the consumption of nutrients naturally rich in vitamin d , such as fatty fish , meat , eggs , milk , and other dairy products .
the ffq was modified to estimate vitamin d intake and consumption frequency across nine categories ( never , 1 time / month , 12 times / week , 23 times / week , 34 times / week , 1 time / day , 2 times / day , 3 times / day , and 4 times / day ) .
ease of administration of this ffq was enhanced by the use of a food photograph album of tunisian food products that emphasized portion sizes .
four food groups were selected as follows : fish ( 50 g / serving ) , eggs ( 50 g / serving ) , red meat ( 50 g / serving ) , and milk ( 200 ml / serving ) .
consumption frequencies of milk and egg were standardized into servings per day and into servings per week for fish and red meat .
the data about the mean daily intake of nutrients were processed using the professional nutri pro 7 program ( nutri pro 7 software , cerden , brussels , belgium ) .
blood was centrifuged at 2000g for 20 min and the plasma was frozen at 40c until analysis ( within 3 months ) . plasma glucose , calcium , phosphorus , and c - reactive protein ( crp ) were assessed on architect c8000 analyzer ( abbott laboratories , abbott park , il ) , using the respective reagents kits .
plasma 25-hydroxyvitamin d ( 25-ohd ) and insulin concentrations were measured by chemiluminescence immunoassay methods using the liaison analyzer ( diasorin inc . ,
vitamin d status was evaluated according to the standards of the institute of medicine ( iom ) .
vitamin d deficiency , insufficiency , and sufficiency were defined as plasma 25-ohd concentrations below 12 g / l , 12 to 20 g / l , and over 20 g / l , respectively ( 24 ) .
insulin sensitivity / resistance was assessed using two indexes ; the homeostasis model assessment of insulin resistance ( homa - ir ) and the quantitative insulin sensitivity check index ( quicki ) , according to the following equations ( 25 , 26 ) : homa - ir=[(fasting insulin in u / ml )  ( fasting glucose in mg / dl)/405 ] ; quicki=1/[log(fasting insulin in u / ml)+log(fasting glucose in mg / dl ) ] .
data were analyzed using spss for windows ( version 18.0 ; spss inc . , chicago , il ) .
values are expressed as mean ( sd ) or median ( inter quartile range , iqr ) for continuous variables and as a percent for categorical variables . comparisons between groups were performed using analysis of variance or the mann 
whitney test for continuous variables and pearson chi - square test or fisher 's exact test for categorical variables as appropriate .
unadjusted and multi - adjusted odd - ratios with 95% confidence intervals were calculated as an estimate of the risk of vitamin d deficiency / insufficiency for several potential risk factors .
a binary logistic regression model was used to identify predictors for vitamin d deficiency , while adjusting for possible confounding factors .
adjustment was performed on maturation status ( pre - phv / around and post - phv ) ; body mass ( normal - weight / overweight and obese ) ; skin color ( fair / dark ) ; and dichotomous variables for fat mass ( < 20%/20% ) , homa - ir ( < 1.5/1.5 ) ; and the daily intake of vitamin d ( < 8.5/8.5 g ) , milk ( < 500/500 ml ) , red meat ( < 100/100 g ) , fish ( < 100/100 g ) , and eggs ( < 50/50 g ) , defined as the respective continuous variables split at the median .
the study included 225 boys aged 716 years , recruited from two centers of a football academy in the area of tunis ( latitude , 35n ) .
children with liver , renal , or bone disease ; intestinal malabsorption ; or cancer ; and those taking vitamin d supplements , anticonvulsant drugs , or systemic corticosteroids were not included .
the study was carried out from january to march 2014 , a period during which temperatures varied between 10c and 22c and the humidity ranged from 70 to 75% . besides 2 h per week of outdoor physical activity as part of the school program , each child attends three weekly outdoor sessions in the football academy .
the training sessions are scheduled from 5 pm to 6 pm on friday and saturday , and from 9 am to 10 am on sunday .
skin color of each participant was determined by two investigators ( ib and mf ) , together with one parent of the child , and classified as fair , corresponding to fitzpatrick skin types 1 and 2 , or dark , corresponding to fitzpatrick skin types 3 and 4 ( 19 ) . written parental permission was obtained for each participant .
weight , height , and sitting height were measured with the subjects barefooted and lightly clothed .
body mass index ( bmi ) was calculated as weight per height squared ( kg m ) .
participants were divided according to the world health organization ( who ) child growth standards for bmi in three groups ; normal - weight group ( bmi85th percentile ) , overweight group ( 85th percentile < bmi < 97th percentile ) , and obese group ( bmi>97th percentile ) ( 20 ) .
triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers ( baty international , west sussex , england ) .
biologic maturity was assessed by incorporating anthropometric variables ( weight , standing height , and sitting height ) and was calculated using the equation of mirwald ( 22 ) ; maturity offset=9.236+(0.0002708*leg length*sitting height)+(0.001663*age*leg length)+(0.007216*age*sitting height)+(0.02292*weight by height ratio ) .
this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity ( phv ) as a measure of maturity offset . for the purpose of data analysis , children were divided into three groups : pre - phv ( 3 to 1 year from phv ) , around phv ( 1 to + 1 year from phv ) , and post - phv ( + 1 to + 3 years from phv ) .
weight , height , and sitting height were measured with the subjects barefooted and lightly clothed .
body mass index ( bmi ) was calculated as weight per height squared ( kg m ) .
participants were divided according to the world health organization ( who ) child growth standards for bmi in three groups ; normal - weight group ( bmi85th percentile ) , overweight group ( 85th percentile < bmi < 97th percentile ) , and obese group ( bmi>97th percentile ) ( 20 ) .
triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers ( baty international , west sussex , england ) .
biologic maturity was assessed by incorporating anthropometric variables ( weight , standing height , and sitting height ) and was calculated using the equation of mirwald ( 22 ) ; maturity offset=9.236+(0.0002708*leg length*sitting height)+(0.001663*age*leg length)+(0.007216*age*sitting height)+(0.02292*weight by height ratio ) .
this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity ( phv ) as a measure of maturity offset . for the purpose of data analysis , children were divided into three groups : pre - phv ( 3 to 1 year from phv ) , around phv ( 1 to + 1 year from phv ) , and post - phv ( + 1 to + 3 years from phv ) .
the daily vitamin d intake in children 's diet was assessed using a 3-day food record ( including 2 weekdays and 1 day over the weekend ) , combined with a food frequency questionnaire ( ffq ) that the parents mostly completed . a 35-items ffq to quantify the consumption of nutrients naturally rich in vitamin d , such as fatty fish , meat , eggs , milk , and other dairy products .
the ffq was modified to estimate vitamin d intake and consumption frequency across nine categories ( never , 1 time / month , 12 times / week , 23 times / week , 34 times / week , 1 time / day , 2 times / day , 3 times / day , and 4 times / day ) .
ease of administration of this ffq was enhanced by the use of a food photograph album of tunisian food products that emphasized portion sizes .
four food groups were selected as follows : fish ( 50 g / serving ) , eggs ( 50 g / serving ) , red meat ( 50 g / serving ) , and milk ( 200 ml / serving ) .
consumption frequencies of milk and egg were standardized into servings per day and into servings per week for fish and red meat .
the data about the mean daily intake of nutrients were processed using the professional nutri pro 7 program ( nutri pro 7 software , cerden , brussels , belgium ) .
blood was centrifuged at 2000g for 20 min and the plasma was frozen at 40c until analysis ( within 3 months ) .
plasma glucose , calcium , phosphorus , and c - reactive protein ( crp ) were assessed on architect c8000 analyzer ( abbott laboratories , abbott park , il ) , using the respective reagents kits .
plasma 25-hydroxyvitamin d ( 25-ohd ) and insulin concentrations were measured by chemiluminescence immunoassay methods using the liaison analyzer ( diasorin inc . ,
vitamin d status was evaluated according to the standards of the institute of medicine ( iom ) .
vitamin d deficiency , insufficiency , and sufficiency were defined as plasma 25-ohd concentrations below 12 g / l , 12 to 20 g / l , and over 20 g / l , respectively ( 24 ) .
insulin sensitivity / resistance was assessed using two indexes ; the homeostasis model assessment of insulin resistance ( homa - ir ) and the quantitative insulin sensitivity check index ( quicki ) , according to the following equations ( 25 , 26 ) : homa - ir=[(fasting insulin in u / ml )  ( fasting glucose in mg / dl)/405 ] ; quicki=1/[log(fasting insulin in u / ml)+log(fasting glucose in mg / dl ) ] .
data were analyzed using spss for windows ( version 18.0 ; spss inc . , chicago , il ) .
values are expressed as mean ( sd ) or median ( inter quartile range , iqr ) for continuous variables and as a percent for categorical variables .
whitney test for continuous variables and pearson chi - square test or fisher 's exact test for categorical variables as appropriate .
unadjusted and multi - adjusted odd - ratios with 95% confidence intervals were calculated as an estimate of the risk of vitamin d deficiency / insufficiency for several potential risk factors .
a binary logistic regression model was used to identify predictors for vitamin d deficiency , while adjusting for possible confounding factors .
adjustment was performed on maturation status ( pre - phv / around and post - phv ) ; body mass ( normal - weight / overweight and obese ) ; skin color ( fair / dark ) ; and dichotomous variables for fat mass ( < 20%/20% ) , homa - ir ( < 1.5/1.5 ) ; and the daily intake of vitamin d ( < 8.5/8.5 g ) , milk ( < 500/500 ml ) , red meat ( < 100/100 g ) , fish ( < 100/100 g ) , and eggs ( < 50/50 g ) , defined as the respective continuous variables split at the median .
serum crp and insulin concentrations and homa - ir and total energy intake were significantly higher and quicki lower in obese and overweight children compared to normal - weight children .
plasma 25-ohd concentrations ranged between 3.80 and 31 g / l , and did not differ according to body mass ( fig .
vitamin d inadequacy was noted in about 85% of the children , with 40.9% having deficiency and 44% having insufficiency .
plasma 25-ohd was positively correlated with dietary intake of proteins ( r=0.407 , p<0.001 ) , milk ( r=0.542 , p<0.001 ) , red meat ( r=0.282 , p<0.001 ) , fish ( r=0.502 , p<0.001 ) , and eggs ( r=0.512 , p<0.001 ) ( fig .
2 ) . however , no significant correlation was observed with phv , bmi , fat mass , homa - ir , or quicki .
compared with vitamin - d - sufficient children , those with vitamin d deficiency or insufficiency showed lower intakes of proteins , milk , red meat , fish , and eggs ( fig .
vitamin d deficiency and insufficiency were associated with lower dietary intakes of vitamin d , proteins , milk , red meat , fish , and eggs . however ,
no association was observed with phv , bmi , fat mass , homa - ir , or skin color ( table 2 ) .
. correlations of plasma 25-hydroxyvitamin d with daily intake of red meat , fish , eggs , and milk in tunisian active children ( n=174 )
. daily intake of proteins , red meat , fish , eggs , and milk according to the vitamin d status in tunisian active children ( n=174 ) .
clinical , nutritional , and biochemical characteristics of children according to body mass ( n=225 ) values are expressed as meansd or median ( inter quartile range ) ; homa - ir , homeostasis model assessment of insulin resistance ; quicki , quantitative insulin sensitivity check index .
plasma 25-hydroxyvitamin d and multi - adjusted odd - ratios for vitamin d deficiency / insufficiency in children according to confounding variables phv , pick of high velocity ; or , odd - ratio ; 95% ci , 95% confidence interval ; 25-ohd , 25-hydroxyvitamin d adjusted for maturation status , bmi , skin color , fat mass , homa - ir , and intake of vitamin d , milk , red meat , fish , and eggs
this study showed that vitamin d inadequacy is common among active tunisian children and is associated with a low intake of vitamin d food sources , but not with maturity status , adiposity , or insulin resistance .
the high prevalence of vitamin d inadequacy in tunisian children is consistent with similar findings in other parts of the world ( 410 ) .
this finding is somewhat surprising since these are active children who live in a sunny environment and engage in a reasonable level of outdoor activities .
our study had been conducted during the winter when plasma 25-ohd levels are usually at their nadir ( 3 ) . because of cool environmental temperatures , children were wearing clothes that covered the trunk and limbs , minimizing the skin area exposed to the sun 's rays and preventing vitamin d synthesis .
also , the training sessions were held in the early mornings or late afternoons when the sun 's rays are least efficient for vitamin d synthesis ( 7 , 27 ) .
although dark skin color is a risk factor for hypovitaminosis d , obviously , that distinction is of little importance when most of the skin areas are covered with the clothing .
however , the most important factor behind the observed vitamin d inadequacy in most children is probably the inadequate intake of dietary vitamin d sources .
our data demonstrate that active children living in sunny environments may still have vitamin d inadequacy , which underlines the importance of monitoring plasma 25-ohd concentrations in children .
in all the children participating in the present study , the daily oral vitamin d intake was well below the us institute of medicine recommended daily allowance ( rda ) of 15 mcg ( 24 ) .
there is no dietary recommendation for vitamin d for children and adolescents in tunisia , as it is assumed that sun exposure will ensure an adequate vitamin d status .
our finding of low plasma 25-ohd levels in 85% of the participants highlights the need for evidence - based dietary recommendations for tunisian children .
our study also showed a clear relationship between vitamin d deficiency / insufficiency and low intake of vitamin d food sources ( i.e. fish , meat , milk , and eggs ) .
( 10 ) showing a positive correlation of serum 25-ohd with the consumption of vitamin d food sources in korean adolescents . in sunny areas , although sun exposure is the major source of vitamin d in the body , vitamin d inadequacy may occur .
this suggests that concomitant appropriate dietary intake is required . the assumption that , in sunny environment , sun exposure alone may provide adequate plasma 25-ohd levels is often false .
when sun exposure is limited as a consequence of low - sunshine seasons , pollution , dark skin , or clothing , the dietary intake of vitamin d may be the more significant contributor to vitamin d status . in these conditions ,
the present study showed no association between plasma 25-ohd and adiposity as assessed either by bmi percentile or fat mass .
some previous studies reported an inverse relationship between plasma 25-ohd and adiposity ( 7 , 9 , 11 , 28 ) , while others found no association ( 8 , 13 , 14 , 29 ) . in a sample of quebec youth , bmi
was negatively associated with 25-ohd levels in girls but not in boys ( 30 ) .
hypothetic mechanisms of low plasma 25-ohd levels in obesity include sequestration of vitamin d in the fat depot , impaired mobilization from the fat depot , and reduced skin and dilution throughout the body .
other important factors in the obese include limited sun exposure due to few outdoor activities and reduced exposed skin area because of clothing ( 31 , 32 ) .
this may explain the lack of an association between vitamin d and adiposity in our series .
indeed , the overweight or obese and normal - weight children included in this study spend as much time outdoors and wear similar clothes , and thus receive the same dose of sunshine .
the relationship between adiposity and the low vitamin d status described in some studies may be related to short exposure to the sun , rather than an excess of body fat per se . in line with the lack of an association with adiposity , our study showed no association between plasma 25-ohd and insulin resistance , a condition usually associated with obesity .
in fact , the relationship between vitamin d and insulin resistance is still a subject of debate ( 1720 , 33 ) .
our study has focused on a broad sample of children and adolescents , and its findings arose from multivariate analyses adjusting on several potential confounders for vitamin d status .
the study has controlled for the time spent outdoors and thus on the amount of sun exposure , which is an important predictor of vitamin d status .
although sun exposure was not measured with precision , all participants have comparable outdoor activities and equivalent sun exposure .
this group of children is probably typical of urban and suburban children from the mediterranean region .
the trans - sectional design prevents the evaluation of vitamin d status year - round , rendering the findings only suitable for the winter season .
however , based on parents occupation , most participants have average to high socioeconomic status .
nutritional assessment was achieved in only 77% of participants and vitamin d intake was estimated using a non - tunisian food database . because the vitamin d content of foods is not given in the food composition database for tunisia , the presumed concentrations of vitamin d were obtained from european food composition tables , which make estimations less precise . to overcome this issue , we considered the consumption of vitamin d food sources in tunisian diet . the development and validation of a vitamin d database for tunisian food is necessary to allow future estimations of vitamin d intake .
our study did not look for health problems that may be related to vitamin d deficiency , an aspect that is beyond the scope of the study .
further studies should address the health consequences of hypovitaminosis d in exposed populations . in conclusion ,
vitamin d intake is also low in most children , due to little consumption of vitamin d food sources . finally , circulating 25-ohd concentrations
are related to vitamin d food sources intake , suggesting that dietary intake is a key contributor in vitamin d status when sun exposure is limited .
given the key role of vitamin d in growth and health in general , every measure should be undertaken to achieve sufficient vitamin d status in these children .
these measures include education in order to ensure adequate and safe sun exposure and appropriate consumption of vitamin d - rich / fortified foods .
further research is needed to establish an optimal combination of sun exposure and food intake / supplementation ensuring year - round sufficient circulating 25-ohd in children and adolescents living in a sunny climate .
the study was supported by funds of research unit  05/ur08 - 08 and research laboratory lr99es11 , ministry of higher education and scientific research of tunisia .
 backgroundvitamin d inadequacy is widespread in children and adolescents worldwide . 
 the present study was undertaken to assess the vitamin d status in active children living in a sunny climate and to identify the main determinants of the serum concentration of 25-hydroxyvitamin d ( 25-ohd).methodsthis cross - sectional study included 225 children aged 715 years practicing sports in a football academy . 
 anthropometric measures were performed to calculate body mass index ( bmi ) , fat mass , and maturity status . 
 a nutritional enquiry was performed including 3-day food records and food frequency questionnaire . 
 plasma 25-ohd and insulin were assessed by immunoenzymatic methods ensuring categorization of vitamin d status and calculation of insulin sensitivity / resistance indexes . 
 a logistic regression model was applied to identify predictors for vitamin d inadequacy.resultsvitamin d deficiency ( 25-ohd<12 
 g / l ) was observed in 40.9% of children and insufficiency ( 12<25-ohd<20 
 g / l ) was observed in 44% of children . in a multivariate analysis , 
 vitamin d deficiency and insufficiency were associated with a lower dietary intake of vitamin d , proteins , milk , red meat , fish , and eggs . 
 however , no significant relationship was observed with maturation status , adiposity , or insulin resistance.conclusionstunisian children and adolescents are exposed to a high risk of vitamin d inadequacy despite living in a sunny climate . 
 circulating 25-ohd concentrations are related to the intake of vitamin d food sources but not to maturation status or body composition . 
 ensuring sufficient and safe sun exposure and adequate vitamin d intake may prevent vitamin d inadequacy in children from sunny environments . 
Materials and methods
Subjects
Experimental protocol
Anthropometrical and maturity status measures
Dietary intake
Biochemical analyses
Statistical analysis
Results
Discussion
Conflict of interest and funding