serum 25-hydroxy - vitamin d3 ( 25(oh)d ) is a better indicator of vitamin d sufficiency than the active hormone , that is , 1,25-dihydroxy - vitamin d3 . therefore , the serum concentration of 25(oh)d is widely accepted as a good indicator of the status of vitamin d in a given subject . the main biological actions of vitamin d include the maintenance of mineral homeostasis and the regulation of bone remodeling . however , there is a vast array of pleiotropic actions of this vitamin that were already recognized more than two decades ago . this area of investigation led to improved knowledge on the potential role of vitamin d on glucose homeostasis and in the pathogenesis of type 2 diabetes . multiple studies have previously shown that vitamin d deficiency is highly prevalent in type 1 and type 2 diabetes . additionally , there is a growing interest on the potential role of vitamin in the development of diabetic micro- and macroangiopathic complications [ 4 , 5 ] . further , the potential role of vitamin d in the development of diabetic retinopathy has been a matter of specific interest in recent years . there is some experimental evidence on the preventive effect of vitamin d in the development of diabetic retinopathy in a rodent model . however , the evidence behind the involvement of vitamin d is really scarce . a recent pubmed search ( accessed on april 24 , 2015 ) , with the use of terms  vitamin d  and  diabetic retinopathy ,  yielded only 31 publications . thus , other studies have already addressed the question of the potential implication of vitamin d in the pathogenesis of diabetic retinopathy [ 715 ] . among these studies there were only 7 publications that included the study of the potential association between vitamin d and the presence of this microangiopathic complication in patients with type 2 diabetes [ 713 ] . because of the cross - sectional design of these studies , only an association between lower serum 25(oh)d concentrations and/or vitamin d deficiency and the presence of diabetic retinopathy in type 2 diabetes has been described . additionally , there are also conflicting data on the role of vitamin d in retinopathy in type 1 diabetes [ 4 , 14 , 15 ] . in a cross - sectional study , shimo et al . showed an association between vitamin d deficiency and the presence of diabetic retinopathy in a small sample of young adults with type 1 diabetes . however , the only prospective study in this field was performed also in type 1 diabetes and did not confirm vitamin d deficiency as a risk factor for diabetic retinopathy or any other microvascular complications . to our knowledge , there are no data on the possible association of vitamin d and diabetic retinopathy in type 2 diabetic patients in any european population , including spain . thus , the primary objective of the current study was to assess whether there is an association of vitamin d status and diabetic retinopathy in type 2 diabetes . additionally , as a secondary outcome , we aimed to assess the association of vitamin d concentrations and the frequency of its deficiency with the severity of retinopathy . study subjects were selected from a group of participants in a study on differential characteristics of type 2 diabetic patients with ( cases ) and without retinopathy ( controls ) in our center . the study was done at the university hospital arnau de vilanova in lleida , spain . a detailed description of the study population has been recently published . from a total number of 299 subjects , we could assess a total of 283 subjects with complete data for the current main study outcomes , as explained below . we took advantage of using the previous study design to assess the association of diabetic retinopathy and vitamin d status in subjects without other advanced diabetic late complications . all study subjects had normal kidney function ( calculated glomerular filtration rate > 60  ml / min ) . by study design , all subjects without retinopathy had normal albumin - to - creatinine ratio ( < 30  mg / g ) and those with retinopathy had no macroalbuminuria ( albumin - to - creatinine ratio < 300  mg / g ) . all study subjects had no known cardiovascular complications ( coronary disease , cerebrovascular disease , or peripheral artery disease ) . also , none of the patients had a history of diabetic foot disease . we took advantage from the design and performance of a study on quality of life in patients with diabetic retinopathy . in that study from the original study population , 16 patients could not be included because of the following reasons : 2 patients did not accept biobanking of blood samples at recruitment , serum samples were not available from 8 additional subjects , and 6 were not included because they were taking oral supplements containing vitamin d and/or calcium . thus , a final number of 139 patients with retinopathy and 144 without this complication were included . demographic and clinical data relevant to the proper conduct of the present study were recorded and are shown in table 1 . blood pressure was measured in the sitting position after resting for 10 minutes using a blood pressure monitor ( hem-7001e , omron , spain ) . hypertension or dyslipidemia were considered present when the patient was being treated with antihypertensive or lipid - lowering drugs , respectively . weight and height were measured by standardized methods , and the body mass index was then calculated ( expressed as kg / m ) . standard laboratory procedures were used to determine the basic blood and urine biochemistry variables on a hitachi modular ddpp analyzer ( roche diagnostics , indianapolis , usa ) . glycated hemoglobin was measured with a hplc analyzer variant ii turbo ( bio - rad , hercules , usa ) . intact parathormone was determined by electrochemiluminescence immunoassay in an elecsys e170 analyzer ( roche diagnostics , indianapolis , usa ) , with an intra- and interassay variability of 2.2% and 6% , respectively . serum concentrations of 25(oh)d were measured by a chemiluminescent microparticle immunoassay in an architect i2000sr analyzer ( abbott diagnostics , lake forest , usa ) , with an intra- and interassay variability of 2.3% and 6.2% , respectively . the date of blood testing was used to classify the season of assessment , as follows : winter ( january  march ) , spring ( april  june ) , summer ( july  september ) , and autumn ( october  december ) . an experienced ophthalmologist explored the retina and classified the retinopathy status according to an internationally accepted classification . to assess physical activity , we used the concept of active leisure time , which defines a sedentary person as one who spends less than 10% of his / her daily energy expenditure performing any physical activity that requires at least 4 mets ( equal or greater physical activity expenditure than brisk walking for 30 minutes ) ; this allowed us to classify each subject as sedentary or active . dietary intake of vitamin d and calcium was measured by a validated 101-food - item semiquantitative food frequency questionnaire ( available at http://bibliodieta.umh.es/files/2011/07/cfa101.pdf ) . nutrient values for vitamin d and calcium were obtained from food composition tables of the us department of agriculture and supplemented with spanish sources [ 19 , 20 ] . the study protocol was approved by the local ethics committee , in accordance with the declaration of helsinki . as this is an ancillary study of our previous one , in which only patients with relevant data were included , before the analysis we assumed a difference of at least 3  ng / ml in 25(oh)d concentrations between patients with and without retinopathy with a standard deviation of 8 units . the inclusion of 139 with and 144 patients without retinopathy allowed the achievement of a statistical power of 93% ( beta = 0.07 ) to detect such difference , using a one - sided t - test to assess the differences with 5% of significance ( alpha = 0.05 ) . therefore , we were able to address the analysis of the primary outcome with sufficient post hoc statistical power . mean ( and standard deviations ) or absolute and relative frequencies ( in percentages ) were computed for quantitative or qualitative variables , respectively . differences between groups were assessed by means of mann - whitney tests or chi - squared tests as adequate . vitamin d deficiency was defined for different thresholds ( at each unit from 15 to 40  pg / ml ) of the serum concentrations of 25(oh)d , and the relative risk was computed to assess the increased risk of vitamin d deficiency in patients with retinopathy versus those without this complication . to further analyze the association of retinopathy with vitamin d , linear regression models were used to assess serum concentrations of 25(oh)d and logistic regression models to assess vitamin d deficiency , establishing an appropriate threshold in the serum levels for this purpose . multivariate regression analyses were also performed including age , sex , race , hemoglobin a1c , creatinine , disease duration , body mass index , hypolipidemic treatment , and physical activity as potential predictors in addition to patient group . the final models were obtained by applying a stepwise algorithm to minimize akaike 's information criterion . the interaction of the patient group ( dr and non - dr ) with the creatinine serum was included in the models as it was shown to be a potential and significant predictor . statistical analyses were conducted with r 3.0.1 ( r foundation for statistical computing , vienna , austria : http://www.r-project.org/ ) . table 1 shows the results of the different study variables and the differences between patients with and without retinopathy . as previous studies that compared subjects with and without this diabetic late complication , those with retinopathy had longer diabetes duration , had higher glycated hemoglobin concentrations , and were more frequently on antihypertensive and insulin treatments . interestingly , the dietary intake of vitamin d and calcium was very similar in both groups of patients . no differences were observed in other clinical and laboratory parameters in the unadjusted analysis of the data . although 25(oh)d concentrations were lower in patients with retinopathy , this difference reached only borderline statistical difference ( p = 0.05 ) . additionally , the frequency of vitamin d deficiency , defined as serum 25(oh)d below 20  ng / ml , was higher in subjects with retinopathy , but this difference did not reach statistical significance ( p = 0.07 ) . nevertheless , the prevalence of retinopathy for 25(oh)d thresholds below 20  mg / ml was significantly higher ( thresholds 15 to 19 ; p  0.05 ) , and the associated relative risk of retinopathy was higher for all these thresholds ( figure 1 ) . for the purpose of the multivariate analysis , a concentration of 15  ng / ml was chosen as the threshold to define vitamin deficiency ; in the previous analysis , this threshold yielded the highest relative risk of retinopathy ( rr : 1.43 ; p = 0.03 ) . the multivariate analyses showed that there was a significant association of retinopathy and 25(oh)d , even when considering other variables associated with this variable . these results were consistent when analyzing both the concentrations of 25(oh)d and the presence of vitamin d deficiency , as defined by a 25(oh)d < 15  ng / ml ( p values 0.04 and 0.009 , resp . ) in addition to this , caucasian patients and physically active patients had significantly higher 25(oh)d levels ( p values 0.05 and 0.03 , resp . ) ( table 2 ) and significantly lower vitamin d deficiency risk ( p values 0.009 and 0.004 , resp . ) . body mass index was inversely associated with 25(oh)d concentrations and positively associated with vitamin d deficiency ( p values 0.009 and 0.004 , resp . ) associated with lower vitamin d levels ( one as risk value ) , the other seasonal 25(oh)d concentrations during autumn , spring , and summer were statistically higher in the two models ( table 2 ) . interestingly , creatinine concentration interacted with the patient group ( retinopathy versus no retinopathy ) in both models ( p values 0.03 and 0.003 ) . to further illustrate this interaction ( table 3 ) , the stratification of creatinine concentrations by tertiles in each of the 2 study groups revealed that patients with retinopathy had frequencies of vitamin d deficiency that augmented with increasing creatinine concentration tertiles ( 31.9% , 34.8% , and 46.7% , resp . ) further to the main analysis , we explored the association of vitamin d status with the severity of retinopathy . the distribution of subjects with retinopathy according to its severity was as follows : grade 1 : 63 ; grade 2 : 42 ; grade 3 : 28 , and grade 4 : 6 . we analyzed the differences among patients without retinopathy and those with this complication stratified according to its severity ( grade 1 versus grades 24 ) . interestingly , patients with advanced retinopathy ( grades 24 ) were more frequently vitamin d deficient ( 25(oh)d < 20  ng / ml ) and had lower mean concentration of 25(oh)d compared with type 2 diabetic patients without retinopathy ( p values of 0.03 and 0.02 , resp . ) ( table 4 ) . concerning these variables , patients with mild diabetic retinopathy ( grade 1 ) were not significantly different to the group without retinopathy . our study confirms the association of vitamin d deficiency with diabetic retinopathy in type 2 diabetes . as in previous reports , type 2 diabetic patients with retinopathy had lower serum 25(oh)d concentrations . furthermore , a higher proportion of vitamin d deficiency was confirmed in subjects carrying this microvascular complication . in addition to this , there was an increased risk of retinopathy in those with severe vitamin d deficiency ( rr = 1.43 , p = 0.03 ) , with this association being even more significant in the adjusted model that included the interaction of retinopathy with creatinine ( p = 0.009 ) . concerning the severity of diabetic retinopathy , patients with more advanced stages of retinopathy had lower concentrations of 25(oh)d and were more frequently vitamin d deficient . this is the first study to address the potential relationship of vitamin d status with diabetic retinopathy in type 2 diabetes in europe . previous publications analyzed the data according to differences in 25(oh)d concentrations between groups or , alternatively , as the prevalence of retinopathy according to a given 25(oh)d threshold to define deficiency of this vitamin . an initial study on 66 turkish type 2 diabetic subjects ( 46 with retinopathy ) found no differences in serum 25(oh)d between type 2 diabetic patients according to the presence or absence of retinopathy and also to the severity of retinopathy . in contrast , suzuki et al . showed that japanese type 2 diabetic patients with proliferative retinopathy had lower serum 25(oh)d . also , the first study carried out in the usa revealed an association of 25(oh)d concentrations with the severity of diabetic retinopathy . however , the only population - based study on this matter , performed also in the usa , could not demonstrate an association between retinopathy and serum 25(oh)d concentration ; these authors only found a greater prevalence of vitamin d deficiency with increased severity of retinopathy . in lebanese type 2 diabetic patients with retinopathy , lower concentrations of 25(oh)d were also found ; additionally , vitamin d was an independent predictor of retinopathy . a very recent chinese study , with the largest number of patients with diabetic retinopathy included in a single study so far , revealed an independent association between 25(oh)d concentrations and diabetic retinopathy , especially for advanced sight - threatening retinopathy . this study found a twofold increase in sight - threatening retinopathy among subjects with serum 25(oh)d below 15.57  ng / ml . a recent large , population - based , cross - sectional study in korea confirmed that there is an inverse relationship of 25(oh)d concentrations with the presence of any retinopathy and also with proliferative retinopathy . this study did not provide details on the type of diabetes of the included subjects . thus , most of the reports were able to identify an association between 25(oh)d and retinopathy or its deficiency . additionally , those studies that performed a detailed characterization of the severity of retinopathy revealed that proliferative or advanced retinopathy grades were clearly associated with 25(oh)d and/or its deficiency [ 9 , 10 , 12 , 13 ] . it should be noted that the few available studies on this issue in type 1 diabetes show conflicting data [ 14 , 15 ] . in summary , our results are discordant with 2 previous studies that did not show an association of vitamin d with diabetic retinopathy in type 2 diabetes [ 7 , 10 ] . in contrast , our findings are in line with the majority of previous reports that demonstrated that vitamin d status is associated with the presence of diabetic retinopathy [ 9 , 1113 ] . additionally , it is remarkable that , as in our study , most others have found an association of vitamin d status and the severity of retinopathy [ 913 ] . our study shares the most important limitation with previous studies ; that is , all of them were cross - sectional . this design allows only for the identification of an association between study variables . to our knowledge , there is no prospective follow - up study in type 2 diabetes and the only one available in type 1 diabetes did not identify vitamin d as a factor involved in the development of retinopathy . thus , the question on the potential role of vitamin d in the pathogenesis of diabetic retinopathy has not been answered yet . another limitation resides on the fact that only one of the previous studies can be considered as population - based ; thus , the external validity of the findings of all other reports , including ours , is clearly limited . although subjects included in our study may not be fully representative of the background population , the clinical characteristics of patients in our study are very similar to those of recently published population - based cohorts in our region . another limitation of our study is that we did not determine the time of sun exposure . this limitation is also shared with most previous studies , except for the one by ahmadieh et al . who used a questionnaire to assess the time spent outdoors . physical activity may be used as a surrogate of sun exposure as most of this activity is performed outdoors . thus , we used this measure as an additional variable in the multivariate analysis that revealed that this was a variable with an influence on vitamin d status . therefore , future studies should include a measure of sun exposure as an essential contributor to 25(oh)d concentrations . we should point out that none of the subjects in the current study had advanced renal disease and/or impairment . it must be pointed out that renal disease in diabetes could contribute as an important confounder of vitamin d status [ 1 , 22 ] . some of the previous studies either did not provide information on renal function or included patients with advanced renal disease . our findings concerning the strong interaction of serum creatinine and the retinopathy status should be taken into consideration in future studies . the careful revision of the previous publications on this matter confirmed that only the american population - based study by patrick et al . included creatinine as a potential important confounder in the analysis of the association of 25(oh)d and retinopathy . besides , an association between vitamin d and cardiovascular disease has been previously described , also in type 2 diabetes . the presence of macrovascular disease in patients with type 2 diabetes may be a confounding factor when assessing an association of vitamin d with other conditions . in our study , however , the potential effect of the presence of atherosclerotic disease was not controlled for in any of the previous studies . four of them did not provide information on the presence of cardiovascular disease [ 7 , 8 , 10 , 11 ] , and the other 2 included a significant proportion of patients with cardiovascular disease [ 9 , 12 ] . the exclusion of this complication in our study clearly avoided the potential association of vitamin d with cardiovascular disease as a confounding variable . in conclusion , an important strength of the current study is that the patients included did not have important confounding conditions that have an important influence on vitamin d concentrations , that is , renal insufficiency and cardiovascular disease . our study confirms the association of a higher frequency of vitamin d deficiency and lower concentrations of 25(oh)d with diabetic retinopathy in patients with type 2 diabetes . further , these parameters of poor vitamin d status are also associated with the severity of diabetic retinopathy . these findings reveal the potential role of vitamin d in the pathogenesis of diabetic retinopathy . however , we are in great need of well - designed prospective observational studies sufficiently powered to test the role of vitamin d status in the development of diabetic retinopathy and other diabetic microvascular complications .
there is very few evidences on the role of vitamin d in the development of diabetic retinopathy . 
 the aim of the current study was to explore whether there is an association of vitamin d status and diabetic retinopathy in type 2 diabetes . 
 two groups of patients were selected : 139 and 144 patients with and without retinopathy , respectively , as assessed by an experienced ophthalmologist . 
 subjects with advanced late diabetic complications were excluded to avoid confounding biases . 
 25-hydroxy - vitamin d3 ( 25(oh)d ) concentrations and vitamin d deficiency were associated with the presence of diabetic retinopathy . 
 additionally , patients with more advanced stages of retinopathy ( grades 24 ) had lower concentrations of 25(oh)d and were more frequently vitamin d deficient as compared with patients not carrying this eye complication . in conclusion 
 , our study confirms the association of vitamin d deficiency with the presence and severity of diabetic retinopathy in type 2 diabetes . 
 further experimental and prospective studies on this issue are clearly warranted .