lower extremity arterial disease ( lead ) is a common peripheral arterial disease , which seriously affects the patient 's functional capacity and quality of life . it is one of the factors that contribute to the progressive and the critical courses of foot ulceration and amputation in type 2 diabetes mellitus ( t2 dm ) patients , and it is frequently associated with coronary , cerebral , and renal artery diseases . vitamin d is a secosteroid , which is obtained from exposure to sunlight and through dietary sources including food and supplements . it is hydroxylated in the liver to 25-hydroxyvitamin d [ 25(oh)d ] and further hydroxylated in the kidney to form 1,25-dihydroxyvatamin d [ 1,25(oh)2d , calcitriol ] . although 1,25(oh)2d is considered to be the active form of vitamin d , its level in the serum does not correlate with overall vitamin d status , whereas the 25(oh)d level is a more clinically relevant marker . in addition to the traditional involvement of vitamin d in bone metabolism , several lines of evidence suggest a role for vitamin d in glucose levels , insulin resistance ( ir ) , and prevalence of t2 dm [ 58 ] , and there are more and more studies that have found that it participated in systemic inflammation , immune , and lipid metabolism to reduce the risk of cardiovascular diseases , but few investigations suggest the association between vitamin d and t2 dm complicated with lead . hence , demonstrating a relationship between 25(oh)d and t2 dm complicated with lead is necessary . retinol binding protein 4 ( rbp4 ) is a new adipokine identified by yang et al .   using gene chip technology , which involves the occurrence of ir , t2 dm , and macrovascular complications . several researches   have shown that vitamin d is correlated with adiponectin , leptin , and other adipokines , but there are few studies analyzing a relationship between 25(oh)d and rbp4 in t2 dm patients complicated with lead . therefore , we realize the importance of further investigation in this area . in this effort , we conducted a clinic - based case - control study to explore the relationship among 25(oh)d , rbp4 , and t2 dm complicated with lead and analyze the intervention effect of vitamin d for lead . 107 patients ( 63 males and 44 females ) who were recruited from outpatient department were in line with the t2 dm diagnosis standard delivered by world health organization ( who ) in october 1999 . they were hospitalized from october 2012 to january 2013 in the department of endocrinology in anhui provincial hospital . this study was conducted in accordance with the tenets of the world medical association 's declaration of helsinki and had been approved by the china ethics committee . written informed consent was obtained from all participants . the subjects who met the following criteria were excluded : ( 1 ) individuals who had acute complications of t2 dm , retinopathy , nephropathy , and other chronic complications , cancer , vitamin a deficiency , liver and kidney dysfunction , osteoporosis , and other history of bone metabolic disorders ; ( 2 ) those taking drugs including vitamins , calcium , lipid lowering drugs , estrogen , and other drugs affecting bone metabolism . all the patients were assigned into 2 groups : the one that is without complications ( group dm1 : 25 males , 20 females ) and the other that is complicated with lead ( group dm2 : 38 males , 24 females ) ; then subjects in group dm2 were further divided into group dm3 ( 16 males , 15 females ) and group dm4 ( 22 males , 9 females ) . patients in these 2 groups were both treated with hypoglycemic drugs , and additionally patients of group dm4 were orally administered vitamin d3 1000  iu daily . in this period 2 subjects in group dm3 were out of touch and 4 subjects in group dm4 could not insist on the intervention therapy and quitted . the relevant characteristics were tested and abi was reexamined in groups dm3 and dm4 after 12 weeks of therapy . 38 age - sex - matched healthy people with no hypertension or impaired glucose tolerance were chosen at the same period as a control group ( group nc ) ( 20 males and 18 females ) . the doppler instrument all the abi measurements were performed with the subjects in the supine position , a blood pressure cuff was placed on patients ' upper arms , and it was inflated until no brachial pulse was detected by the doppler device . the cuff was then slowly deflated until the doppler - detected pulse returned to measure brachial systolic blood pressure ( bsbp ) . this maneuver was repeated on the leg , with the cuff being wrapped around the distal calf and the doppler device being placed over the dorsalis pedis or the posterior tibial artery to measure ankle systolic blood pressure ( asbp ) . ankle brachial index ( abi ) = asbp / bsbp and abi < 0.9 indicated the presence of lead . all the subjects took balanced diets for 3 days and then fasted overnight for 12 hours . clinical characteristics including sex , age , diabetes duration , height , weight , hip circumference , blood pressure , and history of smoking were collected . body mass index ( bmi ) and waist - hip ratio ( whr ) were calculated as weight divided by height squared ( kg / m ) and waist circumference divided by hip circumference , respectively . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured using a digital automatic blood pressure monitor . fasting plasma glucose ( fpg ) , total cholesterol ( tc ) , triglycerides ( tg ) , low density lipoprotein ( ldl ) , and high density lipoprotein ( hdl ) were detected by hitachi 7600  2020 automatic biochemical analyzer . hba1c was conducted by high - pressure liquid chromatography ( bio - rad variant turbo ii analyzer ) . charles , usa ) . the homeostasis model assessment for insulin resistance ( homa - ir ) was calculated according to the formula homa - ir = fins ( mu / l )  fpg ( mmol / l)/22.5 . serum 25(oh)d concentrations were detected via radioimmunoassay ( diasorin stillwater , mn , usa ) . sufficiency was indicated when 25(oh)d  30  ng / ml , insufficiency when 20  ng / ml  25(oh)d < 30  ng / ml , and deficiency when 25(oh)d < 20  ng / ml . the level of rbp4 was measured by enzyme - linked immunoassay ( assaypro , mo , usa ) . continuous variables are presented as mean  standard deviation ( x-s ) , or median and interquartile range ( 25th and 75th percentile ) in cases of skewed distributions , while categorical variables are presented as percentages . these groups were compared using two - sample t - test , mann whitney u test , or chi - square test . paired sample t - test was used to compare the differences before and after treatment . pearson 's correlation coefficient or spearman 's rank correlation coefficient was used to assess the relationship between 25(oh)d and other markers . multivariable logistic regression analysis was used to calculate the ors and 95% confidence intervals for lead ; statistical significance was accepted at p < 0.05 . the indexes of the subjects were listed in table 1 . there were no differences of sex between the 3 groups ( p > 0.05 ) . participants in group dm2 had a longer duration than in group dm1 ; the differences were statistically significant ( p < 0.05 ) . serum 25(oh)d concentrations were 24.77  5.9  ng / ml , 17.32  7.42  ng / ml , and 12.63  7.83  ng / ml in groups nc , dm1 , and dm2 , respectively ; compared with group nc , 25(oh)d decreased in groups dm1 and dm2 and the decrease in group dm2 was more significant ( p < 0.05 ) . rbp4 level was lower in t2 dm patients with lead compared to the patients without lead and healthy people ( p < 0.05 ) . raised fpg , fins , hba1c , tc , tg , ldl , homa - ir , sbp , and whr and decreased hdl in groups dm1 and dm2 were exhibited as compared with group nc ( p < 0.05 ) ; age , fpg , ldl , sbp , hba1c , and the rate of smoking increased and hdl decreased in group dm2 compared with the other 2 groups ( p < 0.05 ) . as depicted in table 2 , approximately 18.4% of subjects were classified as 25(oh)d sufficient and 65.8% were classified as 25(oh)d insufficient ; only 15.8% of subjects were classified as 25(oh)d deficient in group nc . in group dm1 , only 8.9% of subjects had normal 25(oh)d level and 26.7% had insufficiency 25(oh)d level while 64.4% were 25(oh)d deficient . the percentages of 25(oh)d deficiency , insufficiency , and sufficiency were 77.4% , 14.5% , and 8.1% , respectively , in group dm2 . the percentages of sufficient 25(oh)d level in groups dm1 and dm2 were both lower than the percentage in group nc , and the difference was statistically significant ( p < 0.05 ) . after 12 weeks of vitamin d supplementation in group dm4 , 25(oh)d level increased and ldl , rbp4 , and hba1c decreased significantly when compared with baseline ( p < 0.05 ) , while , following 12-week basic therapy of hypoglycemic drugs in group dm3 , only rbp4 and hba1c decreased ( p < 0.05 ) . supplementation with vitamin d for 12 weeks in diabetics with lead significantly raised 25(oh)d level and lowered ldl , rbp4 , and hba1c relative to baseline and unsupplemented diabetics ( p < 0.05 ) . reexamination of abi by doppler ultrasonography for subjects in group dm3 and group dm4 after 12 weeks showed that there were , respectively , 5 cases in group dm3 and 10 cases in group dm4 whose values increased to 0.9 , and the difference was not statistically significant ( p > 0.05 ) ( table 3 ) . as depicted in table 4 the correlation analysis results suggested that the level of 25(oh)d was inversely associated with duration ( r = 0.663 , p < 0.05 ) , hba1c ( r = 0.482 , p < 0.05 ) , rbp4 ( r = 0.538 , p < 0.05 ) , fpg ( r = 0.229 ) , and homa - ir ( r = 0.267 , p < 0.05 ) , while there was no significant correlation between 25(oh)d and the remaining indicators ( p > 0.05 ) . 25(oh)d had a positive correlation trend with hdl , but the difference was not statistically significant ( p > 0.05 ) . as shown in table 5 , the presence of lead in t2 dm patients was used as the dependent variable , while 25(oh)d , tc , tg , hdl , ldl , bmi , rbp4 , homa - ir , sbp , fins , fpg , whr , age , sex , duration , and smoking were used as the independent variables . multivariable logistic regression analysis showed that smoking ( or = 5.565 , 95% ci = 1.37922.458 ) , fpg ( or = 1.818 , 95% ci = 1.0273.217 ) , sbp ( or = 1.167 , 95% ci = 1.0811.260 ) , and ldl ( or = 2.746 , 95% ci = 1.1226.721 ) were significant negative predictors . hdl ( or = 2.746 , 95% ci = 00.432 ) and 25(oh)d ( or = 0.892 , rbp4 is a molecule found in the circulation , thought to be secreted mainly by adipose tissue and the liver . increased serum rbp4 levels have been reported in subjects with obesity , ir , and t2 dm and in other insulin - resistant states , such as metabolic syndrome and vascular complications of dm . pointed out that rbp4 would increase in patients of t2 dm complicated with coronary heart disease , and when the level of rbp4 increased by 1/4 , the risk of cardiovascular disease would increase by 2.5 times . in this study , the level of rbp4 was higher in patients of t2 dm with lead than in patients of t2 dm and healthy people . metheniti et al .   reported that 25(oh)d was low in ultra obese young females and it was significantly associated with rbp4 and neutrophil gelatinase - associated lipocalin ( ngal ) . we found that there was a negative correlation between 25(oh)d and rbp4 ; the concentrations of rbp4 decreased after vitamin d supplementation in the study . vitamin d may reduce peripheral ir by inhibiting the expression of peroxisome proliferator - activated receptor- ( ppar- ) and the differentiation from preadipocytes to mature adipocytes . this result suggests that one of the reasons for ir led by vitamin d deficiency in t2 dm patients is the regulation of rbp4 . the third national health and nutrition examination survey ( nhanes - iii ) found that , compared with residents with normal concentrations of 25(oh)d , residents with low concentrations of 25(oh)d had a higher incidence of peripheral arterial disease . in this study , the level of 25(oh)d in t2 dm group was lower than the one in control group , and it was the lowest in t2 dm with lead group ; 25(oh)d was a factor to protect dm patients from the pathogenesis of lead . ( 1 ) vitamin d can promote pancreatic  cells to secrete insulin by adjusting vitamin d receptor ( vdr ) and vitamin - d - dependent calcium - binding protein ( dbp ) in pancreatic tissue . it can also reduce ir in peripheral tissues by regulating inflammatory cytokines and inhibiting the expression of ppar- . besides , it can affect insulin sensitivity by stimulating ( ppar ) and the gene expression of insulin receptor . ( 2 ) calcitriol can regulate rennin angiotensin - aldosterone system ( raas ) , inhibit the expression of renin , and adjust the synthesis and secretion of atrial natriuretic peptide . ( 3 ) vitamin d can upregulate the expression of related protein of delaying arterial calcification including vascular endothelial growth factor and matrix metalloproteinase-9 . ( 4 ) vitamin d can reduce the occurrence of arterial calcification and atherosclerosis by inhibiting angiogenesis , smoothing muscle cells proliferation , and playing a role in immune regulation with vdr mediation of immune cells . ( 5 ) a lack of calcitriol results in an increase in the serum parathyroid hormone ( pth ) levels . excess pth levels may at least in part promote cardiovascular disease by increasing the cardiac contractility and myocardial calcification . ( 6 ) vitamin d can improve the inflammation state [ 23 , 24 ] and reduce chronic inflammatory reaction of the arterial wall   by inhibiting the secretion of inflammatory cytokines , such as il-6 , tnf- , and c - reactive protein ( crp ) . ( 7 ) calcitriol can inhibit foam cells formation in vascular wall by reducing acetylation and reduce the level of oxidized ldl in t2 dm patients . a 20-year retrospective study has found that   the incidence of dm complicated with cardiovascular diseases reduced by 33% in population with 800  iu vitamin d and 1200  mg calcium of daily intake compared to those with 400  iu vitamin d and 600  mg calcium of daily intake , which suggests that vitamin d supplementation might become an effective measurement to prevent the occurrences of t2 dm complicated with macrovascular diseases . major et al .   suggested that oral supplementation of vitamin d could regulate blood lipid . in this study , the levels of ldl and rbp4 reduced in t2 dm group after the intervention of vitamin d , and , simultaneously , the incidence rate of lead in t2 dm group also decreased after the intervention of vitamin d. therefore , vitamin d supplementation can protect t2 dm patients from being complicated with lead ultimately by improving ir and lipid metabolism and inhibiting inflammation . the sample size was relatively small and we did not consider the influence of outdoor activities and seasonal variation on the level of vitamin d. however , the study was conducted in winter so that the impact of sun exposure was minimized . in conclusion , the phenomenon of vitamin d deficiency amongst t2 dm patients was more and more prevalent , and , with the advantages of low price and validated long - term safety , the supplementation of vitamin d would be one of the intervening measurements for the presentation of t2 dm and its vascular complications .
the aim of this study was to explore the relationship between serum 25-hydroxyvitamin d [ 25(oh)d ] concentrations and lower extremity arterial disease ( lead ) in type 2 diabetes mellitus ( t2 dm ) patients and to investigate the intervention effect of vitamin d. 145 subjects were assigned to a control group ( group nc ) , t2 dm group ( group dm1 ) , and t2 dm complicated with lead group ( group dm2 ) ; then group dm2 were randomly divided into group dm3 who received oral hypoglycemic agents and group dm4 who received oral hypoglycemic drugs and vitamin d3 therapy . 
 compared to group nc , 25(oh)d was significantly lower in group dm2 and marginally lower in group dm1 . 
 in contrast to baseline and group dm3 , 25(oh)d rose while low density lipoprotein ( ldl ) , retinol binding protein 4 ( rbp4 ) , and hba1c significantly lowered in group dm4 . 
 statistical analysis revealed that 25(oh)d had a negative correlation with rbp4 , duration , hba1c , homeostasis model assessment for insulin resistance ( homa - ir ) , and fasting plasma glucose ( fpg ) . 
 ldl , systolic blood pressure ( sbp ) , fpg , and smoking were risk factors of lead while high density lipoprotein ( hdl ) and 25(oh)d were protective ones . 
 therefore , we deduced that low level of 25(oh)d is significantly associated with the occurrence of t2 dm complicated with lead .