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Serum 25-Hydroxyvitamin D, Plasma Lipids, and Associated Gene Variants in Prepubertal Children

Serum 25-Hydroxyvitamin D, Plasma Lipids, and Associated Gene Variants in Prepubertal Children Abstract Context The associations of serum 25-hydroxyvitamin D [25(OH)D] with plasma lipids remain controversial in children. Objective To examine the associations and interactions of 25(OH)D and related gene variants with lipids in children. Design Cross-sectional. Setting Kuopio, Finland. Participants Population sample of 419 prepubertal white children aged 6 to 8 years. Main Outcome Measures 25(OH)D, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. Results Serum 25(OH)D was negatively associated with total cholesterol (β = –0.141, P = 0.004), LDL cholesterol (β = –0.112, P = 0.023), HDL cholesterol (β = –0.150, P = 0.002), and triglycerides (β = –0.104, P = 0.035) adjusted for age and sex. Associations of 25(OH)D with total cholesterol, LDL cholesterol, and HDL cholesterol remained after adjustment for adiposity, physical activity, sedentary behavior, diet, daylight time, and parental education. Children in the highest quartile of 25(OH)D had the lowest total cholesterol (P = 0.022) and LDL cholesterol (P = 0.026) adjusted for age and sex. Cytochrome P450 family 2 subfamily R member 1 (CYP2R1) rs12794714, CYP2R1 rs10741657, and vitamin D binding protein (DBP) rs2282679 were associated with 25(OH)D adjusted for age and sex. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and C10orf88 rs6599638 with HDL cholesterol adjusted for age, sex, and 25(OH)D. The gene variants did not explain or modify the associations of 25(OH)D with lipids. Conclusions 25(OH)D was independently and inversely associated with total cholesterol, LDL cholesterol, and HDL cholesterol. CYP2R1 rs12794714, CYP2R1 rs10741657, and DBP rs2282679 were associated with 25(OH)D. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and chromosome 10 open reading frame 88 (C10orf88) rs6599638 with HDL cholesterol independent of 25(OH)D. None of the gene variants modified the associations of 25(OH)D with lipids. Further studies are needed to detect the mechanisms for the associations of 25(OH)D with lipids. Vitamin D regulates calcium, phosphorus, and bone metabolism, and its deficiency is associated with rickets in children and osteomalacia in adults (1). The knowledge of the other health effects of vitamin D is increasing, and low serum 25-hydroxyvitamin D [25(OH)D] has been associated with the components of metabolic syndrome and the increased risk of cardiovascular diseases in adults (2, 3). In children, decreased serum 25(OH)D has been associated with cardiometabolic risk factors in some studies, but the results have been inconsistent (4–8). Abnormalities in lipid and lipoprotein metabolism, especially increased plasma low-density lipoprotein (LDL) cholesterol, are risk factors for atherosclerosis already in childhood (9). In adults, serum 25(OH)D has been positively associated with plasma high-density lipoprotein (HDL) cholesterol and inversely associated with plasma triglycerides, but the associations with plasma total cholesterol and LDL cholesterol have been inconsistent (10). In children and adolescents, the associations of 25(OH)D with plasma levels of these lipids have been conflicting, and both positive and inverse associations have been found (4–6, 8, 11–13). The knowledge on the effects of vitamin D supplementation on lipid metabolism and cardiovascular health obtained from intervention studies in children and in adults is also insufficient and inconclusive (3, 4, 10). Increased serum 25(OH)D may be due to a healthy lifestyle, including regular exercise, spending plenty of time outdoors resulting in increased vitamin D production in the skin, and a healthy diet, all of which may also be associated with a more favorable plasma lipid profile. Therefore, causality between serum 25(OH)D and cardiovascular risk factors and diseases is not clear. Many studies in children lack information on several potential confounding factors for the associations of serum 25(OH)D with plasma lipids, such as pubertal status, adiposity, physical activity, socioeconomic status, and dietary factors. Also genetic factors may affect the associations of serum 25(OH)D with plasma lipids. Genome-wide association studies (GWASs) have identified several single nucleotide polymorphisms (SNPs) in genes linked with vitamin D metabolism to be associated with serum 25(OH)D (14, 15). However, there are few studies on the associations of SNPs related to serum 25(OH)D with plasma lipids (16–18). Vitamin D and cholesterol have a common precursor, 7-dehydrocholesterol (1), and vitamin D receptor complexes have been suggested to regulate cholesterol metabolism (19). We therefore hypothesized that genetic factors related to vitamin D metabolism may partly explain or modify the association between serum 25(OH)D and plasma lipids. As the process of atherosclerosis begins already in childhood (9), and cardiometabolic risk factors track from childhood to adulthood (20), it is important to understand the associations, mechanisms, and potential confounding factors between serum 25(OH)D and plasma lipids. We therefore studied the associations of serum 25(OH)D with plasma lipids, adjusting for a number of possible confounding factors in a population sample of prepubertal children 6 to 8 years of age. Moreover, we investigated whether SNPs previously related to serum 25(OH)D modify the associations of serum 25(OH)D with plasma lipids. Subjects and Methods Study design and participants The current study is based on the baseline data of the Physical Activity and Nutrition in Children (PANIC) study, which is a physical activity and dietary intervention study in a population sample of children 6 to 8 years of age from the city of Kuopio, Finland (ClinicalTrials.gov no. NCT01803776). Altogether 736 children from the primary schools of Kuopio were invited to participate in the baseline examinations from 2007 to 2009. Of the invited children, 512 (70%) participated in the baseline examinations. The participants did not differ in age, sex distribution, or body mass index standard deviation score (BMI-SDS) from all children who started the first grade in the city of Kuopio in 2007 to 2009 based on data from the standard school health examinations. We excluded children who had chronic diseases or medications that could affect serum 25(OH)D or plasma lipids, had entered puberty, or had race other than white to avoid confounding in statistical analyses. Complete data on the main variables were available for 419 children (195 girls, 224 boys) and valid data on dietary factors for 377 children (179 girls, 198 boys). The study was conducted according to the ethical guidelines laid down in the Declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of the Hospital District of Northern Savo. Both children and their parents gave their written informed consent. The data that has been used is confidential, and therefore the data sources are not shared. Measurement of serum 25(OH)D and plasma lipids Venous blood samples for the measurement of 25(OH)D and lipids were taken after 12-hour overnight fasting. For 25(OH)D analyses, blood was immediately centrifuged and stored at a temperature of –75°C until biochemical analyses. Serum 25(OH)D concentration was analyzed by a chemiluminescence immunoassay called the LIAISON® 25 OH Vitamin D TOTAL Assay (DiaSorin, Stillwater, MN) using an automatic immunoanalyser (DiaSorin). Total variation, including intra-assay and interassay variation, for the assay was 8.2% to 11.0% in the concentration range of 21 to 123 nmol/L. The 25(OH)D analyses were performed in Eastern Finland Laboratory Centre Joint Authority Enterprise (ISLAB), which has been participating in the Vitamin D External Quality Assessment Scheme (DEQAS) since 2008 with DiaSorin LIAISON 25(OH)D assay meeting the performance targets, as described earlier (21). Lipids were measured from nonfrozen plasma samples. A colorimetric enzymatic assay was used to analyze plasma total cholesterol and triglyceride concentrations (Roche Diagnostics, Mannheim, Germany). The intra-assay and interassay coefficients of variation were 1.0% to 1.4% and 1.2% to 3.1% for total cholesterol and 0.9% to 4.2% and 1.5% to 1.8% for triglycerides, respectively. Homogeneous enzymatic colorimetric assays were used to analyze plasma HDL and LDL cholesterol concentrations (Roche Diagnostics). The intra-assay and interassay coefficients of variation were 1.1% to 1.3% and 1.3% to 4.3% for HDL cholesterol and 0.9% to 1.2% and 1.7% to 2.7% for LDL cholesterol, respectively. Genotyping and selection of SNPs DNA was isolated from the blood mononuclear cells using the QIAamp® DNA Blood Kit (Qiagen, Hilden, Germany). Genotyping was performed in the Institute for Molecular Medicine Finland (FIMM) using the Infinium® HumanCoreExome BeadChip (Illumina, San Diego, CA). The genotypes were determined using the GenomeStudio® software (Illumina). The final quality control was done using the PLINK® software, version 1.07. We selected SNPs that are located in genes involved in vitamin D metabolism and have been associated with 25(OH)D in two GWASs (14, 15). SNPs characterized by a minor allele frequency (MAF) <0.15 based on the 1000 Genomes data from the Single Nucleotide Polymorphism database (22) or a high linkage disequilibrium (R2 ≥ 0.8) based on the SNP Annotation and Proxy Search (23) were not considered. Overall seven SNPs from five regions were found in our data set: rs12785878 and rs3829251 in NAD synthetase 1 (NADSYN1), which is near a locus coding 7-dehydrocholesterol reductase (DHCR7) that converts 7-dehydrocholesterol to cholesterol but also is a substrate for vitamin D; rs6599638 in chromosome 10 open reading frame 88 (C10orf88) near gene coding acyl-Coenzyme A dehydrogenase involved in producing substrate for cholesterol synthesis; rs10741657 and rs12794714 in the locus of vitamin D-25-hydroxylase, cytochrome P450 family 2 subfamily R member 1 (CYP2R1), which converts vitamin D into 25(OH)D in liver; rs6013897 in the locus of vitamin D-24-hydroxylase, cytochrome P450 family 24 subfamily A member 1 (CYP24A1), which catabolizes both 25(OH)D and 1,25(OH)2D to prevent accumulation of toxic levels; and rs2282679 in the locus coding vitamin D binding protein (DBP). The genotype distributions of all these SNPs were within the Hardy-Weinberg equilibrium. Rs6013897 had a call rate <95% and was therefore excluded from the analyses. Other assessments Body weight was measured twice, with the children having fasted for 12 hours and emptied the bladder and standing in light underwear by the InBody® 720 bioelectrical impedance device (Biospace, Seoul, Korea) to accuracy of 0.1 kg. Body height was measured three times, with the children standing in the Frankfurt plane using a wall-mounted stadiometer to an accuracy of 0.1 cm. BMI-SDS was calculated using national reference values (24). Waist circumference was measured three times after expiration at middistance between the bottom of the rib cage and the top of the iliac crest with an unstretchable measuring tape to an accuracy of 0.1 cm. The means of the nearest two values of weight, height, and waist circumference were used. Body fat percentage was measured with the children being in the supine position, having emptied the bladder, and being in light clothing by dual-energy X-ray absorptiometry using the Lunar Prodigy Advance® dual-energy X-ray absorptiometry device (GE Medical Systems, Madison, WI) and the Encore® software, version 10.51.006 (GE Company, Madison, WI), using standardized protocols. Energy and nutrient intakes were assessed using food records as described in detail earlier (25). Valid food records of consecutive 3 (7.8%) or 4 (92.2%) days, including at least 1 weekend day, were accepted. Supplemental intakes of vitamin D or other nutrients are not included in the total intakes. Intakes of saturated, monounsaturated, and polyunsaturated fatty acids and carbohydrates were calculated as percentages of energy intake. Energy and nutrient intakes were assessed using Micro Nutrica® dietary analysis software, version 2.5 (Social Insurance Institution of Finland). Physical activity and sedentary behavior were assessed by the PANIC Physical Activity Questionnaire filled out by the parents with their children (26). The average daylight time from sunrise to sunset in Kuopio, Finland, at latitude 62·89°N, during 3 months before the blood sampling was obtained from the Almanac Office, University of Helsinki, Finland. Chronic diseases and allergies diagnosed by a physician, the use of medications, parental education, and annual household income were assessed using questionnaires administered by the parents. Parental education was defined as the highest completed or ongoing degree of the parents (vocational school or less; polytechnic or university). Annual household income was categorized as ≤30,000€ or >30,000€. A research physician carried out a medical examination and defined central puberty as breast development at Tanner stage ≥2 for girls and testicular volume ≥4 mL assessed using an orchidometer for boys. Statistical methods We performed statistical analyses using the IBM SPSS Statistics® software, version 23 (IBM Corp., Armonk, NY). The normality of distributions of the variables was verified visually and by the Kolmogorov-Smirnov test, and logarithmic transformation was performed when appropriate. The t test for independent samples and the Pearson χ2 test were used to examine differences in the basic characteristics between sexes. We selected factors that have earlier been associated with 25(OH)D or lipids (25, 27–33) as potential confounders for the associations of 25(OH)D with lipids. Of these factors, body fat percentage, parental education, physical activity, sedentary behavior, average daylight time before blood sampling, and the dietary intakes of fiber and carbohydrates correlated statistically significantly with at least one of the lipid variables and were included in the multivariate models. Of other dietary factors, the quality of dietary fat has been associated with lipids (29) and vitamin D intake with 25(OH)D (25), but the intakes of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and vitamin D were not correlated with lipids and were not included in the multivariate models. We did not include the use of vitamin D supplements in the analyses, because this information was missing for many children. Linear regression analysis was used to investigate the associations of 25(OH)D and possible confounding factors with plasma lipids. In Model 1, 25(OH)D and each confounding factor was entered separately with age and sex. In Model 2, age, sex, 25(OH)D, and all selected potential confounding factors were entered simultaneously using backward procedure. The means of total, LDL, and HDL cholesterol and triglycerides in the quartiles of serum 25(OH)D were compared using covariance analysis adjusted for age and sex. The pairwise comparisons of means in the quartiles were performed using the Sidak post hoc correction. These covariance analyses were repeated after additional adjustment for confounding factors that were statistically significantly associated with plasma lipids in backward linear regression analyses. The associations of the SNPs with 25(OH)D and lipids were studied using covariance analysis adjusted for age and sex. Moreover, the associations of SNPs with lipids were adjusted for 25(OH)D, and the associations of 25(OH)D with lipids were adjusted for the SNPs. The interaction between 25(OH)D and each SNP on lipids was studied using linear regression analysis. When dietary factors were used in the analyses, data on only 377 children with valid food records were included. Associations, differences, and interactions with P values <0.05 were considered statistically significant. Results Characteristics of children The boys were heavier and taller, had a higher waist circumference, a lower body fat percentage, and lower LDL cholesterol, were physically more active, and had a higher dietary intake of vitamin D than the girls (Table 1). Mean serum 25(OH)D was 68.1 nmol/L (Table 1). Of the children, 86 (20.5%) had serum 25(OH)D levels below 50 nmol/L, and only 4 (1.0%) had serum 25(OH)D below 30 nmol/L. Table 1. Characteristics of Children All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 The values are means (standard deviations) or numbers (percentages) of children and P values for differences between girls and boys. Differences between girls and boys were tested with independent samples t test for continuous variables and Pearson χ2 test for categorical variables. Logarithmic transformation was performed for triglycerides before analysis. Abbreviations: E%, percentage of energy intake; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. a Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, waist, weight, height, BMI-SDS, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 407, 192 girls and 215 boys: household income; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of vitamin D, fiber, saturated fatty acid percentage of energy intake, monounsaturated fatty acid percentage of energy intake, polyunsaturated fatty acid percentage of energy intake, and carbohydrate percentage of energy intake. View Large Table 1. Characteristics of Children All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 The values are means (standard deviations) or numbers (percentages) of children and P values for differences between girls and boys. Differences between girls and boys were tested with independent samples t test for continuous variables and Pearson χ2 test for categorical variables. Logarithmic transformation was performed for triglycerides before analysis. Abbreviations: E%, percentage of energy intake; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. a Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, waist, weight, height, BMI-SDS, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 407, 192 girls and 215 boys: household income; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of vitamin D, fiber, saturated fatty acid percentage of energy intake, monounsaturated fatty acid percentage of energy intake, polyunsaturated fatty acid percentage of energy intake, and carbohydrate percentage of energy intake. View Large Associations of serum 25(OH)D and other factors with plasma lipids Higher 25(OH)D was associated with lower total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides adjusted for age and sex (Table 2, Model 1). These negative associations of 25(OH)D with total, LDL, and HDL cholesterol, but not that with triglycerides, remained statistically significant after additional adjustment for other confounding factors (Table 2, Model 2). Table 2. Associations of Serum 25(OH)D and Other Factors With Plasma Lipids Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 The values are standardized regression coefficients (β) and P values from linear regression models. Model 1: Each variable was entered separately in linear regression analysis with age and sex. Model 2: Age, sex, and all variables listed in the table were entered simultaneously in linear regression analysis using backward procedure. Abbreviation: E%, percentage of energy intake. Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, sex, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of fiber and carbohydrate percentage of energy intake. View Large Table 2. Associations of Serum 25(OH)D and Other Factors With Plasma Lipids Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 The values are standardized regression coefficients (β) and P values from linear regression models. Model 1: Each variable was entered separately in linear regression analysis with age and sex. Model 2: Age, sex, and all variables listed in the table were entered simultaneously in linear regression analysis using backward procedure. Abbreviation: E%, percentage of energy intake. Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, sex, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of fiber and carbohydrate percentage of energy intake. View Large Children in the highest quartile of 25(OH)D (>79 nmol/L) had the lowest total cholesterol adjusted for age and sex and after additional adjustment for body fat percentage and average daylight time [Fig. 1(a)]. Children in the highest quartile of 25(OH)D also had the lowest LDL cholesterol adjusted for age and sex and after further adjustment for body fat percentage [Fig. 1(b)]. The differences in HDL cholesterol [Fig. 1(c)] or triglycerides [Fig. 1(d)] across the quartiles of 25(OH)D were not statistically significant. Figure 1. View largeDownload slide Mean (95% CI) plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides in quartiles of serum 25(OH)D. (a) Mean (95% CI) plasma total cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.244, P = 0.022; difference between first and fourth quartile: P = 0.022. Gray: adjusted for age, sex, body fat percentage, and average daylight time 3 months before blood sampling. Difference across quartiles: F = 3.477, P = 0.016; difference between first and fourth quartile: P = 0.015. (b) Mean (95% CI) plasma LDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.122, P = 0.026; difference between first and fourth quartile: P = 0.020. Gray: adjusted for age, sex, and body fat percentage. Difference across quartiles: F = 2.881, P = 0.036; difference between first and fourth quartile: P = 0.029. (c) Mean (95% CI) plasma HDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.079, P = 0.102. Gray: adjusted for age, sex, body fat percentage, physical activity, and fiber intake. Difference across quartiles: F = 1.895, P = 0.130. (d) Mean (95% CI) plasma triglycerides in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.235, P = 0.084. Gray: adjusted for age, sex, body fat percentage, parental education, physical activity, average daylight time, and carbohydrate intake as percentage of energy intake. Difference across quartiles: F = 1.678, P = 0.171. Figure 1. View largeDownload slide Mean (95% CI) plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides in quartiles of serum 25(OH)D. (a) Mean (95% CI) plasma total cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.244, P = 0.022; difference between first and fourth quartile: P = 0.022. Gray: adjusted for age, sex, body fat percentage, and average daylight time 3 months before blood sampling. Difference across quartiles: F = 3.477, P = 0.016; difference between first and fourth quartile: P = 0.015. (b) Mean (95% CI) plasma LDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.122, P = 0.026; difference between first and fourth quartile: P = 0.020. Gray: adjusted for age, sex, and body fat percentage. Difference across quartiles: F = 2.881, P = 0.036; difference between first and fourth quartile: P = 0.029. (c) Mean (95% CI) plasma HDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.079, P = 0.102. Gray: adjusted for age, sex, body fat percentage, physical activity, and fiber intake. Difference across quartiles: F = 1.895, P = 0.130. (d) Mean (95% CI) plasma triglycerides in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.235, P = 0.084. Gray: adjusted for age, sex, body fat percentage, parental education, physical activity, average daylight time, and carbohydrate intake as percentage of energy intake. Difference across quartiles: F = 1.678, P = 0.171. Of other factors, higher body fat percentage was associated with higher total and LDL cholesterol, higher triglycerides and lower HDL cholesterol, higher levels of physical activity with higher HDL cholesterol and lower triglycerides, longer average daylight time with higher total cholesterol and triglycerides, a lower intake of dietary fiber with higher HDL cholesterol, and a higher intake of carbohydrates and lower parental education with higher triglycerides adjusted for confounding factors (Table 2, Model 2). Associations of gene variants with 25(OH)D and lipids The G allele of rs2282679 in DBP and the A allele of rs12794714 in CYP2R1 were negatively associated and the A allele of rs10741657 in CYP2R1 was positively associated with 25(OH)D adjusted for age and sex (Table 3). The G allele of rs6599638 in C10orf88 was positively associated with HDL cholesterol adjusted for age and sex (Table 3) and after additional adjustment for 25(OH)D (P for linear trend = 0.021). The A allele of rs12794714 in CYP2R1 was negatively associated with total and LDL cholesterol adjusted for age and sex (Table 3). The associations of rs12794714 in CYP2R1 with total cholesterol (P for linear trend <0.001) and LDL cholesterol (P for linear trend = 0.007) remained after further adjustment for 25(OH)D. The associations of 25(OH)D with total, LDL, and HDL cholesterol and triglycerides remained after further adjustments for the SNPs. There was no interaction between any SNP and 25(OH)D on lipids. Table 3. Associations of Gene Variants With Serum 25(OH)D and Plasma Lipids SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNPs, nearest genes, genotypes, and MAFs. The values are numbers of subjects (percentages) and means (95% CIs) from analysis of variances adjusted for age and sex. p1 signifies P value for the difference across groups, p2 signifies P value for linear trend. All SNPs were in Hardy-Weinberg equilibrium. Abbreviation: chr, chromosome. View Large Table 3. Associations of Gene Variants With Serum 25(OH)D and Plasma Lipids SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNPs, nearest genes, genotypes, and MAFs. The values are numbers of subjects (percentages) and means (95% CIs) from analysis of variances adjusted for age and sex. p1 signifies P value for the difference across groups, p2 signifies P value for linear trend. All SNPs were in Hardy-Weinberg equilibrium. Abbreviation: chr, chromosome. View Large Discussion In our population study among prepubertal children, higher serum 25(OH)D was associated with lower plasma total, LDL, and HDL cholesterol and triglycerides. The associations of 25(OH)D with total, LDL, and HDL cholesterol but not with triglycerides remained after controlling for all confounding factors. The A allele of rs12794714 in CYP2R1 was negatively associated and the A allele of rs10741657 in CYP2R1 was positively associated with 25(OH)D, and the G allele of rs2282679 in DBP was negatively associated with 25(OH)D. However, these SNPs did not explain or modify the associations of 25(OH)D with lipids. Moreover, the allele A of rs12794714 in CYP2R1 was negatively associated with total and LDL cholesterol, and the G allele of rs6599638 in C10orf88 was positively associated with HDL cholesterol even when adjusted for 25(OH)D. Serum 25(OH)D levels below 30 to 50 nmol/L have been determined as vitamin D deficiency (34, 35), and some authors have suggested that the lower limit for the sufficient level could be as high as 75 nmol/L (35). A review and meta-analysis in adults found an inverse association between 25(OH)D and the risk of cardiovascular diseases at 25(OH)D of 20 to 60 nmol/L but not above this level (3). We found the lowest total and LDL cholesterol levels above 79 nmol/L, representing the highest quartile of 25(OH)D, that is consistent with the higher suggested limit for the sufficient level of 25(OH)D (35). Many studies in children have not found an association between 25(OH)D and total cholesterol (7, 11, 36). An inverse association between 25(OH)D and total cholesterol has been reported in some studies among children (8, 37), whereas one study observed a positive relationship in girls (13). In most of the studies among children and adolescents, there has been no association between 25(OH)D and LDL cholesterol (4–7, 13, 36, 37). However, 25(OH)D has been inversely associated with LDL cholesterol in some pediatric studies (8, 11) and was positively related to LDL cholesterol in one study among obese female adolescents (38). A review and meta-analysis including mainly children and adolescents found weak inverse associations of 25(OH)D with total and LDL cholesterol (39). Our study confirms the inverse associations of 25(OH)D with total and LDL cholesterol among children. Importantly, the associations remained even though several confounding factors, including body fat percentage, dietary factors, physical activity, sedentary behavior, daylight time, and socioeconomic status, were taken into account. One reason for the discrepancy between the results of some previous studies may be that confounding factors have not been taken into account in all studies. In addition, some studies have not measured serum lipids using fasting samples. The association between 25(OH)D and HDL cholesterol has been positive in many studies among children and adolescents (5, 6, 12, 40), but several studies have not observed such an association (8, 11, 13, 36). A review and meta-analysis that included mainly children and adolescents found a weak positive association between 25(OH)D and HDL cholesterol (39). The inverse association between 25(OH)D and HDL cholesterol that was found in the current study has previously been reported only in infants (37). It is possible that the association is different in older children with more advanced puberty. In most of the pediatric studies, 25(OH)D has been inversely associated with triglycerides (6–8, 37, 40), but one study found a positive association in girls (13), and several studies have observed no association (5, 11, 12, 36). In line with many previous studies, we found that 25(OH)D was inversely associated with triglycerides, but the relationship was partly explained by confounding factors. Cholesterol and vitamin D are synthesized from a common precursor, 7-dehydrocholesterol. DHCR7 converts 7-dehydrocholesterol to cholesterol. However, in the presence of ultraviolet B radiation in the skin, 7-dehydrocholesterol can be converted to previtamin D3 and further to vitamin D3 (1). Based on this metabolic pathway, one could have expected lower cholesterol levels in summer when daylight time is longer and that this could be one of the reasons for the inverse association between 25(OH)D and cholesterol. However, we found weak positive associations of daylight time with total cholesterol and triglycerides, and daylight time did not explain the association of 25(OH)D with total, LDL, or HDL cholesterol. One of the reasons for this may be that daylight time is a less important determinant of 25(OH)D than dietary intake of vitamin D in our study population from the northern latitude (25). Moreover, SNPs related to DHCR7 involved in cholesterol and vitamin D synthesis in the skin were not associated with 25(OH)D or lipid levels. Altogether, these findings suggest that vitamin D metabolism in the skin may not explain the association between 25(OH)D and lipids in the current study. The inverse associations of 25(OH)D with total, LDL, and HDL cholesterol could also be related to liver metabolism. Vitamin D receptor has been shown to downregulate the small-heterodimer partner and increase cholesterol 7α-hydroxylase in the liver, leading to a higher metabolism of cholesterol to bile acids and thus lower cholesterol levels (19). Moreover, genetic factors could partly explain the associations of 25(OH)D with lipids. An SNP in APOA5 that is involved in cholesterol metabolism has modified the association between 25(OH)D and HDL cholesterol (41), but this gene variant was not included in the current analyses. Finally, there may also be some indirect mechanisms for the associations of 25(OH)D with lipids, such as the effects of parathyroid hormone and calcium metabolism. Further studies on the mechanisms between 25(OH)D and lipids are needed. We investigated six SNPs in genes in the vitamin D pathway that have been associated with serum 25(OH)D in GWASs (14, 15). Consistent with the GWAS results, rs10741657 and rs12794714 in CYP2R1 and rs2282679 in DBP were associated with 25(OH)D. However, these gene variants did not explain the association between 25(OH)D and lipids. Moreover, we observed that rs12794714 in CYP2R1 was associated with total cholesterol and LDL cholesterol and that rs6599638 near gene C10orf88 was associated with HDL cholesterol even after controlling for 25(OH)D. The associations of 25(OH)D with lipids did not depend on the SNPs, and the associations of rs12794714 and rs6599638 with lipids were independent of 25(OH)D. CYP2R1 is the main enzyme converting vitamin D into 25(OH)D in the liver (1) and is a member of CYP450 family of enzymes, some of which are involved in cholesterol synthesis (42). One of the explanations for the associations of rs12794714 in CYP2R1 with total and LDL cholesterol could be that CYP2R1 is also such an enzyme. C10orf88 is near a gene coding acyl-coenzyme A dehydrogenase involved in producing substrates for cholesterol synthesis. This could be a mechanism for the association between the SNP in C10orf88 and HDL cholesterol in the current study. Gene variants in DBP and NADSYN/DHCR7 were associated with increased risk of dyslipidemia in adults of African descent (16). However, we found no associations of these gene variants with lipids in children. The strength of our study is a population sample of children with a low prevalence of diseases and medications possibly affecting the association between 25(OH)D and lipids. Moreover, we excluded children who had such diseases or medications or had entered puberty to avoid associated confounding. We took several possible confounding factors, including body fat percentage, physical activity, sedentary behavior dietary factors, daylight time, and socioeconomic status, into account in the analyses. However, we cannot exclude residual confounding due to some unmeasured factors. The number of children who were homozygous for the rare allele of rs2282679 in DBP was small, which limited statistical power in the analyses. Finally, our results are based on cross-sectional analyses, and it is therefore not possible to draw a conclusion on the causality of the associations. Conclusion Serum 25(OH)D was associated with lower total, LDL, and HDL cholesterol independent of body fat percentage, dietary factors, physical activity, sedentary behavior, daylight time, and socioeconomic status. Children having serum 25(OH)D over 79 nmol/L had the lowest total and LDL cholesterol. Consistent with earlier findings (15), rs12794714 and rs10741657 in CYP2R1 and rs2282679 in DBP were associated with 25(OH)D. A new observation of the study is that rs12794714 in CYP2R1 was also associated with total and LDL cholesterol and rs6599638 in C10orf88 with HDL cholesterol even after controlling for 25(OH)D. However, none of the gene variants explained or modified the associations of 25(OH)D with lipids. Further studies are needed to confirm our findings and to detect mechanisms for the associations between 25(OH)D and lipids. Abbreviations: Abbreviations: 25(OH)D 25-hydroxyvitamin D BMI-SDS body mass index standard deviation score C10orf88 chromosome 10 open reading frame 88 CYP2R1 cytochrome P450 family 2 subfamily R member 1 DBP vitamin D binding protein DHCR7 7-dehydrocholesterol reductase GWAS genome-wide association study HDL high-density lipoprotein LDL low-density lipoprotein MAF minor allele frequency NADSYN1 NAD synthetase 1 PANIC Physical Activity and Nutrition in Children SNP single nucleotide polymorphism Acknowledgments The authors are grateful to all the children and their parents for participating in the PANIC study. The authors are also indebted to the members of the PANIC research team for their skillful contribution in performing the study. We also thank Sami Heikkinen for help with genetic data and Juuso Väistö for help with editing the figures. Financial Support: This work was supported by grants from Ministry of Social Affairs and Health of Finland, Ministry of Education and Culture of Finland, Finnish Innovation Fund Sitra, Social Insurance Institution of Finland, Finnish Cultural Foundation, Juho Vainio Foundation, Foundation for Pediatric Research, Doctoral Programs in Public Health, Paavo Nurmi Foundation, Paulo Foundation, Diabetes Research Foundation, Yrjö Jahnsson Foundation, Finnish Foundation for Cardiovascular Research, Orion Research Foundation sr, Research Committee of the Kuopio University Hospital Catchment Area (State Research Funding), Kuopio University Hospital [previous state research funding (EVO), funding no. 5031343], and the city of Kuopio. Clinical Trial Information: ClinicalTrials.gov no. NCT01803776 (registered 4 March 2013). Author Contributions: S.S. participated in the collection of data, conducted the statistical analyses, and wrote the draft of the manuscript. A.-M.E., V.L., and A.V. participated in data collection and contributed to the critical revision of the manuscript. G.D., A.E., and V.S. contributed to the critical revision of the manuscript. A.M. contributed to the interpretation of the data and critical revision of the manuscript and provided funding for the study. T.A.L. was responsible for planning the study, funding, statistical analyses, and the interpretation of the data and also contributed to the critical revision of the manuscript. All the authors read and approved the final version of the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Hossein-nezhad A , Holick MF . Vitamin D for health: a global perspective . Mayo Clin Proc . 2013 ; 88 ( 7 ): 720 – 755 . 2. 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Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Serum 25-Hydroxyvitamin D, Plasma Lipids, and Associated Gene Variants in Prepubertal Children

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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
DOI
10.1210/jc.2018-00335
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Abstract

Abstract Context The associations of serum 25-hydroxyvitamin D [25(OH)D] with plasma lipids remain controversial in children. Objective To examine the associations and interactions of 25(OH)D and related gene variants with lipids in children. Design Cross-sectional. Setting Kuopio, Finland. Participants Population sample of 419 prepubertal white children aged 6 to 8 years. Main Outcome Measures 25(OH)D, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. Results Serum 25(OH)D was negatively associated with total cholesterol (β = –0.141, P = 0.004), LDL cholesterol (β = –0.112, P = 0.023), HDL cholesterol (β = –0.150, P = 0.002), and triglycerides (β = –0.104, P = 0.035) adjusted for age and sex. Associations of 25(OH)D with total cholesterol, LDL cholesterol, and HDL cholesterol remained after adjustment for adiposity, physical activity, sedentary behavior, diet, daylight time, and parental education. Children in the highest quartile of 25(OH)D had the lowest total cholesterol (P = 0.022) and LDL cholesterol (P = 0.026) adjusted for age and sex. Cytochrome P450 family 2 subfamily R member 1 (CYP2R1) rs12794714, CYP2R1 rs10741657, and vitamin D binding protein (DBP) rs2282679 were associated with 25(OH)D adjusted for age and sex. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and C10orf88 rs6599638 with HDL cholesterol adjusted for age, sex, and 25(OH)D. The gene variants did not explain or modify the associations of 25(OH)D with lipids. Conclusions 25(OH)D was independently and inversely associated with total cholesterol, LDL cholesterol, and HDL cholesterol. CYP2R1 rs12794714, CYP2R1 rs10741657, and DBP rs2282679 were associated with 25(OH)D. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and chromosome 10 open reading frame 88 (C10orf88) rs6599638 with HDL cholesterol independent of 25(OH)D. None of the gene variants modified the associations of 25(OH)D with lipids. Further studies are needed to detect the mechanisms for the associations of 25(OH)D with lipids. Vitamin D regulates calcium, phosphorus, and bone metabolism, and its deficiency is associated with rickets in children and osteomalacia in adults (1). The knowledge of the other health effects of vitamin D is increasing, and low serum 25-hydroxyvitamin D [25(OH)D] has been associated with the components of metabolic syndrome and the increased risk of cardiovascular diseases in adults (2, 3). In children, decreased serum 25(OH)D has been associated with cardiometabolic risk factors in some studies, but the results have been inconsistent (4–8). Abnormalities in lipid and lipoprotein metabolism, especially increased plasma low-density lipoprotein (LDL) cholesterol, are risk factors for atherosclerosis already in childhood (9). In adults, serum 25(OH)D has been positively associated with plasma high-density lipoprotein (HDL) cholesterol and inversely associated with plasma triglycerides, but the associations with plasma total cholesterol and LDL cholesterol have been inconsistent (10). In children and adolescents, the associations of 25(OH)D with plasma levels of these lipids have been conflicting, and both positive and inverse associations have been found (4–6, 8, 11–13). The knowledge on the effects of vitamin D supplementation on lipid metabolism and cardiovascular health obtained from intervention studies in children and in adults is also insufficient and inconclusive (3, 4, 10). Increased serum 25(OH)D may be due to a healthy lifestyle, including regular exercise, spending plenty of time outdoors resulting in increased vitamin D production in the skin, and a healthy diet, all of which may also be associated with a more favorable plasma lipid profile. Therefore, causality between serum 25(OH)D and cardiovascular risk factors and diseases is not clear. Many studies in children lack information on several potential confounding factors for the associations of serum 25(OH)D with plasma lipids, such as pubertal status, adiposity, physical activity, socioeconomic status, and dietary factors. Also genetic factors may affect the associations of serum 25(OH)D with plasma lipids. Genome-wide association studies (GWASs) have identified several single nucleotide polymorphisms (SNPs) in genes linked with vitamin D metabolism to be associated with serum 25(OH)D (14, 15). However, there are few studies on the associations of SNPs related to serum 25(OH)D with plasma lipids (16–18). Vitamin D and cholesterol have a common precursor, 7-dehydrocholesterol (1), and vitamin D receptor complexes have been suggested to regulate cholesterol metabolism (19). We therefore hypothesized that genetic factors related to vitamin D metabolism may partly explain or modify the association between serum 25(OH)D and plasma lipids. As the process of atherosclerosis begins already in childhood (9), and cardiometabolic risk factors track from childhood to adulthood (20), it is important to understand the associations, mechanisms, and potential confounding factors between serum 25(OH)D and plasma lipids. We therefore studied the associations of serum 25(OH)D with plasma lipids, adjusting for a number of possible confounding factors in a population sample of prepubertal children 6 to 8 years of age. Moreover, we investigated whether SNPs previously related to serum 25(OH)D modify the associations of serum 25(OH)D with plasma lipids. Subjects and Methods Study design and participants The current study is based on the baseline data of the Physical Activity and Nutrition in Children (PANIC) study, which is a physical activity and dietary intervention study in a population sample of children 6 to 8 years of age from the city of Kuopio, Finland (ClinicalTrials.gov no. NCT01803776). Altogether 736 children from the primary schools of Kuopio were invited to participate in the baseline examinations from 2007 to 2009. Of the invited children, 512 (70%) participated in the baseline examinations. The participants did not differ in age, sex distribution, or body mass index standard deviation score (BMI-SDS) from all children who started the first grade in the city of Kuopio in 2007 to 2009 based on data from the standard school health examinations. We excluded children who had chronic diseases or medications that could affect serum 25(OH)D or plasma lipids, had entered puberty, or had race other than white to avoid confounding in statistical analyses. Complete data on the main variables were available for 419 children (195 girls, 224 boys) and valid data on dietary factors for 377 children (179 girls, 198 boys). The study was conducted according to the ethical guidelines laid down in the Declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of the Hospital District of Northern Savo. Both children and their parents gave their written informed consent. The data that has been used is confidential, and therefore the data sources are not shared. Measurement of serum 25(OH)D and plasma lipids Venous blood samples for the measurement of 25(OH)D and lipids were taken after 12-hour overnight fasting. For 25(OH)D analyses, blood was immediately centrifuged and stored at a temperature of –75°C until biochemical analyses. Serum 25(OH)D concentration was analyzed by a chemiluminescence immunoassay called the LIAISON® 25 OH Vitamin D TOTAL Assay (DiaSorin, Stillwater, MN) using an automatic immunoanalyser (DiaSorin). Total variation, including intra-assay and interassay variation, for the assay was 8.2% to 11.0% in the concentration range of 21 to 123 nmol/L. The 25(OH)D analyses were performed in Eastern Finland Laboratory Centre Joint Authority Enterprise (ISLAB), which has been participating in the Vitamin D External Quality Assessment Scheme (DEQAS) since 2008 with DiaSorin LIAISON 25(OH)D assay meeting the performance targets, as described earlier (21). Lipids were measured from nonfrozen plasma samples. A colorimetric enzymatic assay was used to analyze plasma total cholesterol and triglyceride concentrations (Roche Diagnostics, Mannheim, Germany). The intra-assay and interassay coefficients of variation were 1.0% to 1.4% and 1.2% to 3.1% for total cholesterol and 0.9% to 4.2% and 1.5% to 1.8% for triglycerides, respectively. Homogeneous enzymatic colorimetric assays were used to analyze plasma HDL and LDL cholesterol concentrations (Roche Diagnostics). The intra-assay and interassay coefficients of variation were 1.1% to 1.3% and 1.3% to 4.3% for HDL cholesterol and 0.9% to 1.2% and 1.7% to 2.7% for LDL cholesterol, respectively. Genotyping and selection of SNPs DNA was isolated from the blood mononuclear cells using the QIAamp® DNA Blood Kit (Qiagen, Hilden, Germany). Genotyping was performed in the Institute for Molecular Medicine Finland (FIMM) using the Infinium® HumanCoreExome BeadChip (Illumina, San Diego, CA). The genotypes were determined using the GenomeStudio® software (Illumina). The final quality control was done using the PLINK® software, version 1.07. We selected SNPs that are located in genes involved in vitamin D metabolism and have been associated with 25(OH)D in two GWASs (14, 15). SNPs characterized by a minor allele frequency (MAF) <0.15 based on the 1000 Genomes data from the Single Nucleotide Polymorphism database (22) or a high linkage disequilibrium (R2 ≥ 0.8) based on the SNP Annotation and Proxy Search (23) were not considered. Overall seven SNPs from five regions were found in our data set: rs12785878 and rs3829251 in NAD synthetase 1 (NADSYN1), which is near a locus coding 7-dehydrocholesterol reductase (DHCR7) that converts 7-dehydrocholesterol to cholesterol but also is a substrate for vitamin D; rs6599638 in chromosome 10 open reading frame 88 (C10orf88) near gene coding acyl-Coenzyme A dehydrogenase involved in producing substrate for cholesterol synthesis; rs10741657 and rs12794714 in the locus of vitamin D-25-hydroxylase, cytochrome P450 family 2 subfamily R member 1 (CYP2R1), which converts vitamin D into 25(OH)D in liver; rs6013897 in the locus of vitamin D-24-hydroxylase, cytochrome P450 family 24 subfamily A member 1 (CYP24A1), which catabolizes both 25(OH)D and 1,25(OH)2D to prevent accumulation of toxic levels; and rs2282679 in the locus coding vitamin D binding protein (DBP). The genotype distributions of all these SNPs were within the Hardy-Weinberg equilibrium. Rs6013897 had a call rate <95% and was therefore excluded from the analyses. Other assessments Body weight was measured twice, with the children having fasted for 12 hours and emptied the bladder and standing in light underwear by the InBody® 720 bioelectrical impedance device (Biospace, Seoul, Korea) to accuracy of 0.1 kg. Body height was measured three times, with the children standing in the Frankfurt plane using a wall-mounted stadiometer to an accuracy of 0.1 cm. BMI-SDS was calculated using national reference values (24). Waist circumference was measured three times after expiration at middistance between the bottom of the rib cage and the top of the iliac crest with an unstretchable measuring tape to an accuracy of 0.1 cm. The means of the nearest two values of weight, height, and waist circumference were used. Body fat percentage was measured with the children being in the supine position, having emptied the bladder, and being in light clothing by dual-energy X-ray absorptiometry using the Lunar Prodigy Advance® dual-energy X-ray absorptiometry device (GE Medical Systems, Madison, WI) and the Encore® software, version 10.51.006 (GE Company, Madison, WI), using standardized protocols. Energy and nutrient intakes were assessed using food records as described in detail earlier (25). Valid food records of consecutive 3 (7.8%) or 4 (92.2%) days, including at least 1 weekend day, were accepted. Supplemental intakes of vitamin D or other nutrients are not included in the total intakes. Intakes of saturated, monounsaturated, and polyunsaturated fatty acids and carbohydrates were calculated as percentages of energy intake. Energy and nutrient intakes were assessed using Micro Nutrica® dietary analysis software, version 2.5 (Social Insurance Institution of Finland). Physical activity and sedentary behavior were assessed by the PANIC Physical Activity Questionnaire filled out by the parents with their children (26). The average daylight time from sunrise to sunset in Kuopio, Finland, at latitude 62·89°N, during 3 months before the blood sampling was obtained from the Almanac Office, University of Helsinki, Finland. Chronic diseases and allergies diagnosed by a physician, the use of medications, parental education, and annual household income were assessed using questionnaires administered by the parents. Parental education was defined as the highest completed or ongoing degree of the parents (vocational school or less; polytechnic or university). Annual household income was categorized as ≤30,000€ or >30,000€. A research physician carried out a medical examination and defined central puberty as breast development at Tanner stage ≥2 for girls and testicular volume ≥4 mL assessed using an orchidometer for boys. Statistical methods We performed statistical analyses using the IBM SPSS Statistics® software, version 23 (IBM Corp., Armonk, NY). The normality of distributions of the variables was verified visually and by the Kolmogorov-Smirnov test, and logarithmic transformation was performed when appropriate. The t test for independent samples and the Pearson χ2 test were used to examine differences in the basic characteristics between sexes. We selected factors that have earlier been associated with 25(OH)D or lipids (25, 27–33) as potential confounders for the associations of 25(OH)D with lipids. Of these factors, body fat percentage, parental education, physical activity, sedentary behavior, average daylight time before blood sampling, and the dietary intakes of fiber and carbohydrates correlated statistically significantly with at least one of the lipid variables and were included in the multivariate models. Of other dietary factors, the quality of dietary fat has been associated with lipids (29) and vitamin D intake with 25(OH)D (25), but the intakes of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and vitamin D were not correlated with lipids and were not included in the multivariate models. We did not include the use of vitamin D supplements in the analyses, because this information was missing for many children. Linear regression analysis was used to investigate the associations of 25(OH)D and possible confounding factors with plasma lipids. In Model 1, 25(OH)D and each confounding factor was entered separately with age and sex. In Model 2, age, sex, 25(OH)D, and all selected potential confounding factors were entered simultaneously using backward procedure. The means of total, LDL, and HDL cholesterol and triglycerides in the quartiles of serum 25(OH)D were compared using covariance analysis adjusted for age and sex. The pairwise comparisons of means in the quartiles were performed using the Sidak post hoc correction. These covariance analyses were repeated after additional adjustment for confounding factors that were statistically significantly associated with plasma lipids in backward linear regression analyses. The associations of the SNPs with 25(OH)D and lipids were studied using covariance analysis adjusted for age and sex. Moreover, the associations of SNPs with lipids were adjusted for 25(OH)D, and the associations of 25(OH)D with lipids were adjusted for the SNPs. The interaction between 25(OH)D and each SNP on lipids was studied using linear regression analysis. When dietary factors were used in the analyses, data on only 377 children with valid food records were included. Associations, differences, and interactions with P values <0.05 were considered statistically significant. Results Characteristics of children The boys were heavier and taller, had a higher waist circumference, a lower body fat percentage, and lower LDL cholesterol, were physically more active, and had a higher dietary intake of vitamin D than the girls (Table 1). Mean serum 25(OH)D was 68.1 nmol/L (Table 1). Of the children, 86 (20.5%) had serum 25(OH)D levels below 50 nmol/L, and only 4 (1.0%) had serum 25(OH)D below 30 nmol/L. Table 1. Characteristics of Children All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 The values are means (standard deviations) or numbers (percentages) of children and P values for differences between girls and boys. Differences between girls and boys were tested with independent samples t test for continuous variables and Pearson χ2 test for categorical variables. Logarithmic transformation was performed for triglycerides before analysis. Abbreviations: E%, percentage of energy intake; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. a Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, waist, weight, height, BMI-SDS, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 407, 192 girls and 215 boys: household income; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of vitamin D, fiber, saturated fatty acid percentage of energy intake, monounsaturated fatty acid percentage of energy intake, polyunsaturated fatty acid percentage of energy intake, and carbohydrate percentage of energy intake. View Large Table 1. Characteristics of Children All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 All (N = 419)a Girls (n = 195)a Boys (n = 224)a P Value Age, y 7.6 (0.4) 7.6 (0.4) 7.6 (0.4) 0.11 Parental education 0.15  Vocational school or less 81 (19.5%) 32 (16.5%) 49 (22.2%)  Polytechnic or university 334 (80.5%) 162 (83.5%) 172 (77.8%) Household income 0.62  ≤30,000 €/y 91 (22.4%) 45 (23.4%) 46 (21.4%)  >30,000 €/y 316 (77.6%) 147 (76.6%) 169 (78.6%) Body weight, kg 26.7 (4.7) 26.3 (4.8) 27.2 (4.6) 0.048 Body height, cm 128.7 (5.5) 127.6 (5.6) 129.6 (5.3) <0.001 BMI-SDS −0.19 (1.04) −0.19 (1.03) −0.19 (1.05) 0.95 Waist circumference, cm 56.5 (5.3) 55.8 (5.4) 57.1 (5.1) 0.008 Body fat percentage, % 19.5 (7.9) 22.1 (7.3) 17.2 (7.6) <0.001 25(OH)D, nmol/L 68.1 (22.5) 66.5 (18.9) 69.5 (25.2) 0.16 Total cholesterol, mmol/L 4.28 (0.61) 4.33 (0.61) 4.23 (0.61) 0.09 LDL cholesterol, mmol/L 2.36 (0.51) 2.42 (0.52) 2.31 (0.49) 0.032 HDL cholesterol, mmol/L 1.61 (0.31) 1.58 (0.31) 1.63 (0.31) 0.08 Triglycerides, mmol/L 0.60 (0.24) 0.62 (0.25) 0.58 (0.24) 0.08 Total physical activity, h/d 1.9 (0.7) 1.7 (0.6) 2.0 (0.72) <0.001 Total sedentary behavior, h/d 3.6 (1.6) 3.7 (1.6) 3.5 (1.6) 0.12 Average daylight time during 3 months before blood sampling, h/d 11.0 (3.8) 11.1 (3.9) 10.8 (3.7) 0.40 Vitamin D intake from food, µg/d 5.87 (2.16) 5.36 (1.66) 6.34 (2.45) <0.001 SFA intake, E% 12.1 (2.7) 12.0 (2.6) 12.2 (2.8) 0.49 MUFA intake, E% 10.0 (1.8) 9.9 (1.8) 10.1 (1.9) 0.29 PUFA intake, E% 4.9 (1.3) 4.9 (1.3) 5.0 (1.3) 0.66 Carbohydrate intake, E% 51.8 (5.0) 52.1 (4.6) 51.6 (5.3) 0.27 Fiber intake, g/1000 kcal 9.0 (2.5) 9.2 (2.4) 8.7 (2.5) 0.09 The values are means (standard deviations) or numbers (percentages) of children and P values for differences between girls and boys. Differences between girls and boys were tested with independent samples t test for continuous variables and Pearson χ2 test for categorical variables. Logarithmic transformation was performed for triglycerides before analysis. Abbreviations: E%, percentage of energy intake; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid. a Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, waist, weight, height, BMI-SDS, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 407, 192 girls and 215 boys: household income; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of vitamin D, fiber, saturated fatty acid percentage of energy intake, monounsaturated fatty acid percentage of energy intake, polyunsaturated fatty acid percentage of energy intake, and carbohydrate percentage of energy intake. View Large Associations of serum 25(OH)D and other factors with plasma lipids Higher 25(OH)D was associated with lower total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides adjusted for age and sex (Table 2, Model 1). These negative associations of 25(OH)D with total, LDL, and HDL cholesterol, but not that with triglycerides, remained statistically significant after additional adjustment for other confounding factors (Table 2, Model 2). Table 2. Associations of Serum 25(OH)D and Other Factors With Plasma Lipids Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 The values are standardized regression coefficients (β) and P values from linear regression models. Model 1: Each variable was entered separately in linear regression analysis with age and sex. Model 2: Age, sex, and all variables listed in the table were entered simultaneously in linear regression analysis using backward procedure. Abbreviation: E%, percentage of energy intake. Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, sex, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of fiber and carbohydrate percentage of energy intake. View Large Table 2. Associations of Serum 25(OH)D and Other Factors With Plasma Lipids Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 25(OH)D −0.141 0.004 −0.130 0.012 −0.112 0.023 −0.109 0.032 −0.150 0.002 −0.143 0.004 −0.104 0.035 Body fat percentage 0.130 0.012 0.115 0.026 0.209 < 0.001 0.216 < 0.001 −0.195 < 0.001 −0.169 0.001 0.175 0.001 0.105 0.041 Parental education −0.024 0.63 −0.063 0.20 0.106 0.031 −0.155 0.002 −0.150 0.003 Total physical activity 0.029 0.57 −0.077 0.13 0.170 0.001 0.150 0.003 −0.158 0.002 −0.106 0.040 Total sedentary behavior 0.021 0.67 0.044 0.37 −0.043 0.39 0.106 0.041 Average daylight time 0.105 0.031 0.120 0.020 0.042 0.39 −0.020 0.69 0.139 0.004 0.113 0.026 Carbohydrate intake, E% −0.016 0.76 −0.024 0.64 −0.078 0.13 0.110 0.034 0.129 0.010 Fiber intake, g/1000 kcal −0.086 0.10 −0.034 0.52 −0.155 0.003 −0.154 0.002 −0.010 0.86 The values are standardized regression coefficients (β) and P values from linear regression models. Model 1: Each variable was entered separately in linear regression analysis with age and sex. Model 2: Age, sex, and all variables listed in the table were entered simultaneously in linear regression analysis using backward procedure. Abbreviation: E%, percentage of energy intake. Number of children (n) varies from 377 to 419 in different variables; n = 419, 195 girls and 224 boys: age, sex, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and average daylight time; n = 408, 191 girls and 217 boys: body fat percentage; n = 415, 194 girls and 221 boys: parental education; n = 415, 194 girls and 221 boys: physical activity and sedentary behavior; and n = 377, 179 girls and 198 boys: intake of fiber and carbohydrate percentage of energy intake. View Large Children in the highest quartile of 25(OH)D (>79 nmol/L) had the lowest total cholesterol adjusted for age and sex and after additional adjustment for body fat percentage and average daylight time [Fig. 1(a)]. Children in the highest quartile of 25(OH)D also had the lowest LDL cholesterol adjusted for age and sex and after further adjustment for body fat percentage [Fig. 1(b)]. The differences in HDL cholesterol [Fig. 1(c)] or triglycerides [Fig. 1(d)] across the quartiles of 25(OH)D were not statistically significant. Figure 1. View largeDownload slide Mean (95% CI) plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides in quartiles of serum 25(OH)D. (a) Mean (95% CI) plasma total cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.244, P = 0.022; difference between first and fourth quartile: P = 0.022. Gray: adjusted for age, sex, body fat percentage, and average daylight time 3 months before blood sampling. Difference across quartiles: F = 3.477, P = 0.016; difference between first and fourth quartile: P = 0.015. (b) Mean (95% CI) plasma LDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.122, P = 0.026; difference between first and fourth quartile: P = 0.020. Gray: adjusted for age, sex, and body fat percentage. Difference across quartiles: F = 2.881, P = 0.036; difference between first and fourth quartile: P = 0.029. (c) Mean (95% CI) plasma HDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.079, P = 0.102. Gray: adjusted for age, sex, body fat percentage, physical activity, and fiber intake. Difference across quartiles: F = 1.895, P = 0.130. (d) Mean (95% CI) plasma triglycerides in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.235, P = 0.084. Gray: adjusted for age, sex, body fat percentage, parental education, physical activity, average daylight time, and carbohydrate intake as percentage of energy intake. Difference across quartiles: F = 1.678, P = 0.171. Figure 1. View largeDownload slide Mean (95% CI) plasma total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides in quartiles of serum 25(OH)D. (a) Mean (95% CI) plasma total cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.244, P = 0.022; difference between first and fourth quartile: P = 0.022. Gray: adjusted for age, sex, body fat percentage, and average daylight time 3 months before blood sampling. Difference across quartiles: F = 3.477, P = 0.016; difference between first and fourth quartile: P = 0.015. (b) Mean (95% CI) plasma LDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 3.122, P = 0.026; difference between first and fourth quartile: P = 0.020. Gray: adjusted for age, sex, and body fat percentage. Difference across quartiles: F = 2.881, P = 0.036; difference between first and fourth quartile: P = 0.029. (c) Mean (95% CI) plasma HDL cholesterol in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.079, P = 0.102. Gray: adjusted for age, sex, body fat percentage, physical activity, and fiber intake. Difference across quartiles: F = 1.895, P = 0.130. (d) Mean (95% CI) plasma triglycerides in quartiles of serum 25(OH)D. Black: adjusted for age and sex. Difference across quartiles: F = 2.235, P = 0.084. Gray: adjusted for age, sex, body fat percentage, parental education, physical activity, average daylight time, and carbohydrate intake as percentage of energy intake. Difference across quartiles: F = 1.678, P = 0.171. Of other factors, higher body fat percentage was associated with higher total and LDL cholesterol, higher triglycerides and lower HDL cholesterol, higher levels of physical activity with higher HDL cholesterol and lower triglycerides, longer average daylight time with higher total cholesterol and triglycerides, a lower intake of dietary fiber with higher HDL cholesterol, and a higher intake of carbohydrates and lower parental education with higher triglycerides adjusted for confounding factors (Table 2, Model 2). Associations of gene variants with 25(OH)D and lipids The G allele of rs2282679 in DBP and the A allele of rs12794714 in CYP2R1 were negatively associated and the A allele of rs10741657 in CYP2R1 was positively associated with 25(OH)D adjusted for age and sex (Table 3). The G allele of rs6599638 in C10orf88 was positively associated with HDL cholesterol adjusted for age and sex (Table 3) and after additional adjustment for 25(OH)D (P for linear trend = 0.021). The A allele of rs12794714 in CYP2R1 was negatively associated with total and LDL cholesterol adjusted for age and sex (Table 3). The associations of rs12794714 in CYP2R1 with total cholesterol (P for linear trend <0.001) and LDL cholesterol (P for linear trend = 0.007) remained after further adjustment for 25(OH)D. The associations of 25(OH)D with total, LDL, and HDL cholesterol and triglycerides remained after further adjustments for the SNPs. There was no interaction between any SNP and 25(OH)D on lipids. Table 3. Associations of Gene Variants With Serum 25(OH)D and Plasma Lipids SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNPs, nearest genes, genotypes, and MAFs. The values are numbers of subjects (percentages) and means (95% CIs) from analysis of variances adjusted for age and sex. p1 signifies P value for the difference across groups, p2 signifies P value for linear trend. All SNPs were in Hardy-Weinberg equilibrium. Abbreviation: chr, chromosome. View Large Table 3. Associations of Gene Variants With Serum 25(OH)D and Plasma Lipids SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNP, Nearest Gene(s), Chromosome Genotypes n 25(OH)D Total Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides Rs12785878 T/T 163 (40.2%) 69.4 (65.9–72.9) 4.25 (4.15–4.34) 2.35 (2.28–2.43) 1.60 (1.55–1.65) 0.59 (0.55–0.63) NADSYN1/DHCR7 T/G 178 (44.0%) 66.5 (63.2–69.8) 4.36 (4.27–4.45) 2.42 (2.35–2.45) 1.62 (1.57–1.66) 0.61 (0.57–0.64) chr 11 G/G 63 (15.8%) MAF: 0.362 67.4 (61.9–73.9) 4.14 (3.99–4.29) 2.23 (2.11–2.36) 1.61 (1.53–1.68) 0.55 (0.49–0.62) p1 = 0.54 p1 = 0.040 p1 = 0.037 p1 = 0.91 p1 = 0.19 p2 = 0.47 p2 = 0.69 p2 = 0.37 p2 = 0.80 p2 = 0.41 Rs3829251 G/G 215 (53.3%) 68.4 (65.3–71.4) 4.25 (4.18–4.34) 2.37 (2.30–2.44) 1.59 (1.55–1.63) 0.59 (0.56–0.63) NADSYN1/DHCR7 G/A 151 (37.5%) 66.9 (63.4–70.5) 4.33 (4.23–4.43) 2.40 (2.32–2.48) 1.61 (1.56–1.66) 0.61 (0.57–0.64) chr 11 A/A 37 (9.2%) MAF: 0.279 68.7 (61.4–76.0) 4.13 (3.93–4.33) 2.13 (1.98–2.31) 1.68 (1.58.1.78) 0.50 (0.42–0.58) p1 = 0.78 p1 = 0.18 p1 = 0.020 p1 = 0.27 p1 = 0.022 p2 = 0.96 p2 = 0.69 p2 = 0.11 p2 = 0.13 p2 = 0.10 rs6599638 A/A 129 (31.9%) 68.7 (64.8–72.6) 4.23 (4.13–4.34) 2.38 (2.29–2.46) 1.56 (1.51–1.61) 0.59 (0.55–0.64) C10orf88 A/G 208 (51.4%) 67.4 (64.4–70.5) 4.30 (4.21–4.38) 2.38 (2.31–2.45) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 10 G/G 68 (16.8%) MAF: 0.377 68.4 (63.1–73.8) 4.29 (4.15–4.44) 2.29 (2.17–2.41) 1.66 (1.59–1.73) 0.60 (0.53–0.65) p1 = 0.85 p1 = 0.61 p1 = 0.46 p1 = 0.07 p1 = 0.93 p2 = 0.73 p2 = 0.39 p2 = 0.36 p2 = 0.020 p2 = 0.75 rs2282679 T/T 277 (66.1%) 70.0 (67.3–72.6) 4.30 (4.23–4.37) 2.38 (2.32–2.44) 1.61 (1.57–1.64) 0.60 (0.57–0.62) DBP T/G 112 (27.7%) 63.7 (59.5–67.9) 4.20 (4.08–4.31) 2.29 (2.20–2.39) 1.62 (1.56–1.68) 0.56 (0.52–0.61) chr 4 G/G 16 (3.8%) MAF: 0.178 65.6 (54.7–76.5) 4.45 (4.14–4.75) 2.61 (2.36–2.86) 1.51 (1.35–1.66) 0.78 (0.66–0.90) p1 = 0.022 p1 = 0.19 p1 = 0.05 p1 = 0.37 p1 = 0.006 p2 = 0.004 p2 = 0.52 p2 = 0.99 p2 = 0.43 p2 = 0.33 rs10741657 G/G 123 (30.4%) 66.0 (62.1–70.0) 4.26 (4.15–4.37) 2.37 (2.28–2.46) 1.60 (1.54–1.65) 0.59 (0.55–0.63) CYP2R1 G/A 214 (52.8%) 66.8 (63.8–69.8) 4.27 (4.19–4.36) 2.35 (2.28–2.42) 1.62 (1.58–1.66) 0.59 (0.56–0.63) chr 11 A/A 68 (16.8%) MAF: 0.432 76.2 (70.7–81.6) 4.33 (4.18–4.48) 2.39 (2.27–2.52) 1.60 (1.52–1.67) 0.60 (0.54–0.66) p1 = 0.004 p1 = 0.75 p1 = 0.82 p1 = 0.81 p1 = 0.97 p2 = 0.006 p2 = 0.60 p2 = 0.94 p2 = 0.90 p2 = 0.80 rs12794714 G/G 152 (36.3%) 72.0 (68.5–75.6) 4.38 (4.28–4.48) 2.44 (2.36–2.52) 1.60 (1.56–1.65) 0.61 (0.58–0.65) CYP2R1 G/A 204 (50.4%) 66.3 (63.2–69.3) 4.25 (4.17–4.33) 2.33 (2.26–2.40) 1.63 (1.59–1.67) 0.58 (0.55–0.62) chr 11 A/A 49 (12.1%) MAF: 0.373 63.0 (56.7–69.3) 4.09 (3.92–4.26) 2.28 (2.13–2.42) 1.52 (1.44–1.61) 0.58 (0.51–0.65) p1 = 0.023 p1 = 0.010 p1 = 0.06 p1 = 0.09 p1 = 0.35 p2 = 0.005 p2 = 0.003 p2 = 0.019 p2 = 0.38 p2 = 0.23 SNPs, nearest genes, genotypes, and MAFs. The values are numbers of subjects (percentages) and means (95% CIs) from analysis of variances adjusted for age and sex. p1 signifies P value for the difference across groups, p2 signifies P value for linear trend. All SNPs were in Hardy-Weinberg equilibrium. Abbreviation: chr, chromosome. View Large Discussion In our population study among prepubertal children, higher serum 25(OH)D was associated with lower plasma total, LDL, and HDL cholesterol and triglycerides. The associations of 25(OH)D with total, LDL, and HDL cholesterol but not with triglycerides remained after controlling for all confounding factors. The A allele of rs12794714 in CYP2R1 was negatively associated and the A allele of rs10741657 in CYP2R1 was positively associated with 25(OH)D, and the G allele of rs2282679 in DBP was negatively associated with 25(OH)D. However, these SNPs did not explain or modify the associations of 25(OH)D with lipids. Moreover, the allele A of rs12794714 in CYP2R1 was negatively associated with total and LDL cholesterol, and the G allele of rs6599638 in C10orf88 was positively associated with HDL cholesterol even when adjusted for 25(OH)D. Serum 25(OH)D levels below 30 to 50 nmol/L have been determined as vitamin D deficiency (34, 35), and some authors have suggested that the lower limit for the sufficient level could be as high as 75 nmol/L (35). A review and meta-analysis in adults found an inverse association between 25(OH)D and the risk of cardiovascular diseases at 25(OH)D of 20 to 60 nmol/L but not above this level (3). We found the lowest total and LDL cholesterol levels above 79 nmol/L, representing the highest quartile of 25(OH)D, that is consistent with the higher suggested limit for the sufficient level of 25(OH)D (35). Many studies in children have not found an association between 25(OH)D and total cholesterol (7, 11, 36). An inverse association between 25(OH)D and total cholesterol has been reported in some studies among children (8, 37), whereas one study observed a positive relationship in girls (13). In most of the studies among children and adolescents, there has been no association between 25(OH)D and LDL cholesterol (4–7, 13, 36, 37). However, 25(OH)D has been inversely associated with LDL cholesterol in some pediatric studies (8, 11) and was positively related to LDL cholesterol in one study among obese female adolescents (38). A review and meta-analysis including mainly children and adolescents found weak inverse associations of 25(OH)D with total and LDL cholesterol (39). Our study confirms the inverse associations of 25(OH)D with total and LDL cholesterol among children. Importantly, the associations remained even though several confounding factors, including body fat percentage, dietary factors, physical activity, sedentary behavior, daylight time, and socioeconomic status, were taken into account. One reason for the discrepancy between the results of some previous studies may be that confounding factors have not been taken into account in all studies. In addition, some studies have not measured serum lipids using fasting samples. The association between 25(OH)D and HDL cholesterol has been positive in many studies among children and adolescents (5, 6, 12, 40), but several studies have not observed such an association (8, 11, 13, 36). A review and meta-analysis that included mainly children and adolescents found a weak positive association between 25(OH)D and HDL cholesterol (39). The inverse association between 25(OH)D and HDL cholesterol that was found in the current study has previously been reported only in infants (37). It is possible that the association is different in older children with more advanced puberty. In most of the pediatric studies, 25(OH)D has been inversely associated with triglycerides (6–8, 37, 40), but one study found a positive association in girls (13), and several studies have observed no association (5, 11, 12, 36). In line with many previous studies, we found that 25(OH)D was inversely associated with triglycerides, but the relationship was partly explained by confounding factors. Cholesterol and vitamin D are synthesized from a common precursor, 7-dehydrocholesterol. DHCR7 converts 7-dehydrocholesterol to cholesterol. However, in the presence of ultraviolet B radiation in the skin, 7-dehydrocholesterol can be converted to previtamin D3 and further to vitamin D3 (1). Based on this metabolic pathway, one could have expected lower cholesterol levels in summer when daylight time is longer and that this could be one of the reasons for the inverse association between 25(OH)D and cholesterol. However, we found weak positive associations of daylight time with total cholesterol and triglycerides, and daylight time did not explain the association of 25(OH)D with total, LDL, or HDL cholesterol. One of the reasons for this may be that daylight time is a less important determinant of 25(OH)D than dietary intake of vitamin D in our study population from the northern latitude (25). Moreover, SNPs related to DHCR7 involved in cholesterol and vitamin D synthesis in the skin were not associated with 25(OH)D or lipid levels. Altogether, these findings suggest that vitamin D metabolism in the skin may not explain the association between 25(OH)D and lipids in the current study. The inverse associations of 25(OH)D with total, LDL, and HDL cholesterol could also be related to liver metabolism. Vitamin D receptor has been shown to downregulate the small-heterodimer partner and increase cholesterol 7α-hydroxylase in the liver, leading to a higher metabolism of cholesterol to bile acids and thus lower cholesterol levels (19). Moreover, genetic factors could partly explain the associations of 25(OH)D with lipids. An SNP in APOA5 that is involved in cholesterol metabolism has modified the association between 25(OH)D and HDL cholesterol (41), but this gene variant was not included in the current analyses. Finally, there may also be some indirect mechanisms for the associations of 25(OH)D with lipids, such as the effects of parathyroid hormone and calcium metabolism. Further studies on the mechanisms between 25(OH)D and lipids are needed. We investigated six SNPs in genes in the vitamin D pathway that have been associated with serum 25(OH)D in GWASs (14, 15). Consistent with the GWAS results, rs10741657 and rs12794714 in CYP2R1 and rs2282679 in DBP were associated with 25(OH)D. However, these gene variants did not explain the association between 25(OH)D and lipids. Moreover, we observed that rs12794714 in CYP2R1 was associated with total cholesterol and LDL cholesterol and that rs6599638 near gene C10orf88 was associated with HDL cholesterol even after controlling for 25(OH)D. The associations of 25(OH)D with lipids did not depend on the SNPs, and the associations of rs12794714 and rs6599638 with lipids were independent of 25(OH)D. CYP2R1 is the main enzyme converting vitamin D into 25(OH)D in the liver (1) and is a member of CYP450 family of enzymes, some of which are involved in cholesterol synthesis (42). One of the explanations for the associations of rs12794714 in CYP2R1 with total and LDL cholesterol could be that CYP2R1 is also such an enzyme. C10orf88 is near a gene coding acyl-coenzyme A dehydrogenase involved in producing substrates for cholesterol synthesis. This could be a mechanism for the association between the SNP in C10orf88 and HDL cholesterol in the current study. Gene variants in DBP and NADSYN/DHCR7 were associated with increased risk of dyslipidemia in adults of African descent (16). However, we found no associations of these gene variants with lipids in children. The strength of our study is a population sample of children with a low prevalence of diseases and medications possibly affecting the association between 25(OH)D and lipids. Moreover, we excluded children who had such diseases or medications or had entered puberty to avoid associated confounding. We took several possible confounding factors, including body fat percentage, physical activity, sedentary behavior dietary factors, daylight time, and socioeconomic status, into account in the analyses. However, we cannot exclude residual confounding due to some unmeasured factors. The number of children who were homozygous for the rare allele of rs2282679 in DBP was small, which limited statistical power in the analyses. Finally, our results are based on cross-sectional analyses, and it is therefore not possible to draw a conclusion on the causality of the associations. Conclusion Serum 25(OH)D was associated with lower total, LDL, and HDL cholesterol independent of body fat percentage, dietary factors, physical activity, sedentary behavior, daylight time, and socioeconomic status. Children having serum 25(OH)D over 79 nmol/L had the lowest total and LDL cholesterol. Consistent with earlier findings (15), rs12794714 and rs10741657 in CYP2R1 and rs2282679 in DBP were associated with 25(OH)D. A new observation of the study is that rs12794714 in CYP2R1 was also associated with total and LDL cholesterol and rs6599638 in C10orf88 with HDL cholesterol even after controlling for 25(OH)D. However, none of the gene variants explained or modified the associations of 25(OH)D with lipids. Further studies are needed to confirm our findings and to detect mechanisms for the associations between 25(OH)D and lipids. Abbreviations: Abbreviations: 25(OH)D 25-hydroxyvitamin D BMI-SDS body mass index standard deviation score C10orf88 chromosome 10 open reading frame 88 CYP2R1 cytochrome P450 family 2 subfamily R member 1 DBP vitamin D binding protein DHCR7 7-dehydrocholesterol reductase GWAS genome-wide association study HDL high-density lipoprotein LDL low-density lipoprotein MAF minor allele frequency NADSYN1 NAD synthetase 1 PANIC Physical Activity and Nutrition in Children SNP single nucleotide polymorphism Acknowledgments The authors are grateful to all the children and their parents for participating in the PANIC study. The authors are also indebted to the members of the PANIC research team for their skillful contribution in performing the study. We also thank Sami Heikkinen for help with genetic data and Juuso Väistö for help with editing the figures. Financial Support: This work was supported by grants from Ministry of Social Affairs and Health of Finland, Ministry of Education and Culture of Finland, Finnish Innovation Fund Sitra, Social Insurance Institution of Finland, Finnish Cultural Foundation, Juho Vainio Foundation, Foundation for Pediatric Research, Doctoral Programs in Public Health, Paavo Nurmi Foundation, Paulo Foundation, Diabetes Research Foundation, Yrjö Jahnsson Foundation, Finnish Foundation for Cardiovascular Research, Orion Research Foundation sr, Research Committee of the Kuopio University Hospital Catchment Area (State Research Funding), Kuopio University Hospital [previous state research funding (EVO), funding no. 5031343], and the city of Kuopio. Clinical Trial Information: ClinicalTrials.gov no. NCT01803776 (registered 4 March 2013). 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Journal of Clinical Endocrinology and Metabolism – Oxford University Press

Published: May 9, 2018

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