metabolic syndrome ( mets ) , a collection of cardiometabolic risk factors that includes obesity , insulin resistance , hypertension , and dyslipidemia , confers increased risks of coronary heart disease and cardiovascular disease ( cvd ) [ 15 ] . in terms of mechanism , both adipocytokine and vitamin d a low adiponectin level has been reported to be correlated with obesity , mets , and cvd [ 713 ] , while a high leptin level has been indicated to be associated with cvd that has close relation to obesity [ 6 , 1416 ] . to be integrated , adiponectin / leptin ratio ( a / l ratio ) has been identified to be highly associated with insulin resistance , mets , and cvd and even is able to predict cv outcome [ 1721 ] . in addition , vitamin d deficiency has also been reported to be highly associated with mets [ 6 , 2225 ] and to increase the risk of developing cvd , including hypertension , heart failure , and ischemic heart disease . vitamin d3 has been found to be highly associated with adiponectin [ 2729 ] , while leptin has been reported to suppress the synthesis of renal 1,25(oh)2d3 . in the formation of an active form of vitamin d3 , 1,25(oh)2d3 , two key enzymes of the cytochrome p450 superfamily , sterol 27-hydoxylase ( cyp27a1 ) in the liver and 25-hydroxyvitamin d3 1-alpha - hydroxylase ( cyp27b1 ) in the kidney , are needed . however , in the view of genetics , little about these two vitamin d3 metabolic genes with adipocytokines and mets is studied . therefore , our purpose was to explore the potential associations of vitamin d3 metabolism genes , cyp27a1 and cyp27b1 , with adiponectin and leptin and , subsequently , with mets . in this cross - sectional study , 694 taiwanese males ( age : 4477 years ) living in kaohsiung city were recruited from a free health screening program held by our institution [ 1 , 32 ] . ethical approval following the declaration of helsinki was authorized by the institutional research ethics committee of kaohsiung medical university hospital . the men who had a previous diagnosis of hypertension , diabetes mellitus ( dm ) , or hyperlipidemia controlled by regular medication were included in this study , but those who were diagnosed with labile hypertension , labile dm , and advanced liver and/or renal disease were excluded . entire records of medical , surgical , and psychosexual history , as well as the results of physical examinations including measurements of body weight , height , and blood pressure for each participant , were collected ( figure 1 ) . the subjects were classified as alcohol drinkers , cigarette smokers , or betel nut chewers if they had regularly consumed any alcoholic beverage 1 time per week , smoked 10 cigarettes per week , or chewed 7 betel quids per week for at least the last 6 months [ 33 , 34 ] . hypertension was defined as a systolic blood pressure of 140  mmhg or diastolic blood pressure of 90  mmhg . hyperlipidemia was defined as a total cholesterol level of 200  mg / dl or triglyceride level of 200  mg / dl [ 1 , 35 ] . dm was diagnosed when the fasting blood glucose ( fbg ) was 126  mg / dl . in accordance with the modified criteria proposed by the bureau of health promotion in taiwan , mets was diagnosed once meeting at least three of the following criteria : ( 1 ) waist circumference ( wc ) 90  cm ; ( 2 ) triglycerides ( tg ) 150  mg / dl ; ( 3 ) high density lipoprotein ( hdl ) cholesterol < 40  mg / dl ; ( 4 ) blood pressure ( bp ) 130/85  mmhg or current use of antihypertensive medications ; ( 5 ) fbg > 100  mg / dl , previously diagnosed as dm , or current use of oral hypoglycemic agents or insulin [ 1 , 5 , 36 ] . for further biochemical analysis and hormone profiling , each peripheral venous blood sample was drawn after more than 8 hours of fasting overnight into pyrogen - free tube . diasorin radioimmunoassay kits ( northwestern avenue stillwater , usa ; intra - assay cv : 6.8%~11.3% ; interassay cv : 12.3%~15.3% ) were used to determine the expression of 1,25(oh)2d3 . millipore radioimmunoassay kits ( missouri , usa ) were used to measure the leptin and adiponectin ( intra - assay cv : 3.4%~8.3% and 1.78%~6.21% , resp . ; interassay cv : 3.0%~6.2% and 6.90%~9.25% , resp . ) . tag single nucleotide polymorphisms ( snps ) were selected by searching data on han chinese in the hapmap project ( http://www.hapmap.org/ ) using haploview software . afterward the tag snps were identified by the following criteria : ( 1 ) snps located in the cyp27a1 and cyp27b1 genes or within the flanking 5  kb regions ; ( 2 ) a minor allele frequency of 0.10 ; and ( 3 ) other unselected snps that could be captured by one of the tag snps with a linkage disequilibrium of r  0.80 . selection of snps had been completed in march 2006 , and only 1 tag snp had been identified for each of the cyp27a1 and cyp27b1 genes , namely , rs4675344 on the intron 1 region and rs10877012 on the promoter region ( figure 1 ) . dna was extracted from the peripheral whole blood using a puregene dna isolation kit ( gentra systems inc . , the two snps were determined using a taqman 5 allelic discrimination assay performed on an assays - on - demand snp genotyping kit ( applied biosystems , foster city , ca ) . snp amplification assays including 10  ng of sample dna in 25  l of reaction solution contained in 12.5  l of 2x taqman universal pcr mix ( applied biosystems ) and 1.25  l of predeveloped assay reagent from the snp genotyping product ( applied biosystems ) contained in two primers and two mgb - taqman probes were performed according to the manufacturer 's instructions . reaction conditions consisted of preincubation at 50c for 2  min and 95c for 10  min , followed by 40 cycles at 92c for 15  s and 60c for 1  min . amplifications were performed in an abi prism 7500 sequence detection system ( applied biosystems ) . our genotyping results were also validated in accordance with the identical results from the 25 patients and 25 controls ( about 10% of our participants ) who were randomly selected for regenotyping by direct sequencing . the demographic and laboratory data are presented as mean  standard deviation ( sd ) . for the controls , the categorical variables and continuous variables were examined by the contingency tables ( pearson chi - square test ) and the student 's t - test , respectively . the ancova test was used to evaluate the associations between the cyp27a1 rs4675344 and adiponectin / leptin ratio ( a / l ratio ) controlling for other covariates . multiple linear regression analysis was used to examine the associations between the a / l ratio and mets score for the cyp27a1 rs4675344 . since the established cardiovascular risk factors including hypertension and dm are the components of mets , we were only adjusted for sex , age , coronary artery disease ( cad ) , stroke , alcohol consumption , and smoking status . all statistical analyses were performed by spss 18.0 software ( chicago , il , usa ) with significance levels of  = 0.05 . the baseline characteristics and biochemical data of our subjects with respect to cyp27a1 rs4675344 were summarized in table 1 . a total of 649 patients had adequate qualities for analysis , of whom 447 ( 68.9% ) , 179 ( 27.6% ) , and 23 ( 3.5% ) patients had the aa , at , and tt genotypes , respectively . due to the low minor allele frequency , the patients with tt and patients with at were united into the t - carriers for analysis . compared with the patients with the aa genotype , the t - carriers had significantly higher wc ( 87.1  6.9 versus 85.7  7.2  cm , p = 0.003 ) , positive history of dyslipidemia , total and hdl cholesterol ( 4.3  1.0 versus 4.1  1.0 , p = 0.006 ) , leptin ( 6.5  28.2 versus 4.0  3.3 , p = 0.005 ) , and a / l ratio ( 3.7  4.0 versus 5.1  6.0 , p = 0.001 ) . the t - carriers showed nominal significant number of mets components ( 2.1  1.4 versus 1.9  1.4 , p = 0.04 ) , lower hdl ( 46.5  11.3 versus 48.5  11.0  mg / dl , p = 0.04 ) , adiponectin ( 10.9  6.2 versus 12.1  7.2  ng / ml , p = 0.03 ) , and increased number of mets components and greater percentages of each mets component were also noted in the t - carriers compared to those with the aa genotype ( table 2 ) . four models were established for our regression analysis , namely , model 1 that was adjusted for baseline characteristics including age , bmi , wc , hip circumference , systolic blood pressure , and diastolic blood pressure ; model 2 that was then adjusted for exsmoker , current smoker , ex - betel nut chewer , current betel nut chewer , exdrinker , and current drinker ; model 3 that was further adjusted for comorbidities including dm , hypertension , dyslipidemia , cad , and stroke ; and model 4 that was farther adjusted for biochemistry data including fasting glucose , total cholesterol , low density lipoprotein ( ldl ) , hdl , and triglycerides ( tg ) . negative associations between the cyp27a1 rs4675344 snp and the a / l ratio were found consistently from model 1 to model 4 . in addition , obesity and the parameters related to mets , including bmi and wc , were also negatively related to the a / l ratio . in model 4 , ldl and fasting glucose were also significantly , negatively associated with the a / l ratio ( table 3 ) . the a / l ratio showed greatest association with the cyp27a1 rs4674344 snp , and the mets also had significant association , too . therefore , we further examined the influence of mets and its components on the associations between the cyp27a1 rs4674344 snp and a / l ratio . greater association between a / l ratio and the cyp27a1 rs4674344 snp was noted in the low mets score group when compared to the high mets score group ( figure 2 ) . significant a / l ratio was only found when most of the mets components were in the preclinical stage ( not meeting individual criteria ) . however , there was no significant association with the bp component after adjusting for age , bmi , smoking , alcohol drinking , betel nut chewing , cad , total cholesterol , ldl , hdl , and the other 4 components for the cyp27a1 rs4674344 snp ( table 4 , figure 3 ) . this study selected the snps by searching han chinese data from the hapmap project ( http://www.hapmap.org/ ) using haploview software . these snps were located in the cyp27a1 genes and cyp27b1 genes or within the flanking 5  kb regions with a minor allele frequency 0.10 . accordingly , the other unselected snps could be captured by one of the tag snps with a linkage disequilibrium of r  0.80 . as a result , only 1 tag snp was obtained for each of the cyp27a1 and cyp27b1 genes , namely , the rs4675344 on intron 1 region and the rs10877012 on the promoter region ( figure 1 ) . the mets and its hdl component are significantly associated with the cyp27a1 rs4674344 snp . the hdl cholesterol has been reported to have a strong correlation with the adiponectin and to affect the adipocyte metabolism and adiponectin expression [ 39 , 40 ] . and it is known that mutations in cyp27a1 are associated with cerebrotendinous xanthomatosis , a rare lipid storage disease with the deposition of a form of cholesterol ( cholestanol ) in the brain and other tissues [ 4144 ] . our study provides further novel information of the impact of cyp27a1 rs4674344 snp on both of them . however , like previous document , no cyp27 snps in our study were associated with 1,25(oh)(2)d levels . a / l ratio is the best surrogate for inflammatory burden and insulin resistance [ 46 , 47 ] . adiponectin acts as an insulin - sensitizing adipocytokine and an anti - inflammatory factor especially with regard to atherosclerosis . leptin is known to be a satiety factor regulating body weight by suppressing appetite and stimulating energy expenditure . leptin is also a proinflammatory adipocytokine , which induces the development of t helper 1 cells that contribute to the progress of inflammation [ 4951 ] . for in - depth analysis in our study , a / l ratio was not only significantly higher in the cyp27a1 rs4674344 snp t - carriers than in those with the aa genotype ( 3.7  4.0 versus 5.1  6.0 , p = 0.001 ) , but was also most correlated with this snp . against other study parameters for the a / l ratio , the cyp27a1 rs4675344 snp consistently had significantly negative association with the a / l ratio from model 1 to model 4 after adjusting for baseline characteristics , life habits , comorbidities , and biochemistry profiles . in addition , obesity and mets - related parameters including bmi , wc , and hip circumference were also negatively associated with the a / l ratio . in model 4 , ldl and fasting glucose were significantly negatively associated with the a / l ratio , too ( table 3 ) . these results all indicated that the cyp27a1 rs4675344 snp , a / l ratio , and mets are closely associated with each other . another set of novel in - depth information provided by our study related to the cyp27a1 rs4674344 snp was that the a / l ratio was only significantly different in the preclinical stage of most individual mets criteria , except for the bp component , but not in the clinical stage of mets . the snps of genes are imprinted in the germ - cell stage and one previous investigation had also reported that the changes of a / l ratio and development of the mets partly accorded with one 's own genetics . therefore , our novel findings indicated that those t - carriers with the cyp27a1 rs4674344 snp tend to have a higher risk of developing mets , owing to an adipocytokine imbalance . however , the cyp27a1 rs4674344 snp did not seem to play a role in the development of bp component . based on our findings and the previous evidence , the cyp27a1 rs4674344 might participate in lipid and adipocytokine homeostasis and in the crosstalk of hdl with adiponectin . however , unequal number of subjects with the polymorphism of cyp27a1 rs4674344 was the main limitation in this study , which might lead to bias in the results . a large - scale cohort study with equal numbers of subjects may be dedicated to investigate the refined causality , including the snp of cyp27a1 rs4674344 for the major cardiovascular events . in vitro studies this study reported the novel finding that vitamin d3 metabolism gene cyp27a1 rs4674344 was closely associated with a / l ratio and mets . in addition , for the cyp27a1 rs4674344 snp , the a / l ratio was only significantly different in the preclinical stage of the most individual mets criteria except the bp component . this finding indicated that the t - carriers were at a higher risk of developing mets .