as shown by epidemiological studies , the prevalence of type 2 diabetes has a significant rise worldwide , which resulted in an increased burden on individuals and health care systems . for diabetic patients , the development of vascular complications related to microangiopathy and macroangiopathy is one of the most severe problems . in particular , diabetic retinopathy ( dr ) , a kind of serious microvascular complication , is the leading cause of visual impairment in adults aged 30 to 65 years , which draws wide attention for public healthy institution . it is reported that almost all patients develop background retinopathy with time , and 4050% progress to proliferative retinopathy within 25 years of diabetes onset . thus , we need effective diagnostic methods and therapeutic tool to prevent the occurrence of dr for diabetic patients . at present , the diagnosis of retinopathy still depended on ophthalmoscopy and fluorescein angiography . however , it is generally acknowledged that only the pathological changes which occurred at the severe stages of the retinopathy can be discovered using this diagnostic method . therefore , it is of tremendous importance to find out the markers that can be used for the screening and prediction of retinopathy . several related factors including oxidative stress , microalbuminuria , gene polymorphisms [ 57 ] , vascular endothelial growth factor receptor-1 , thrombospondin-2 , and angiopoietin-2   and obesity   have been reported to contribute to dr . but diagnosis markers with high precision for dr in individuals with diabetes are still lacking , especially markers that are easy to be measured . it is well known that purine metabolic pathway may be strongly associated with the development of diabetic microvascular complication . adenosine is reported to play an important role in water - electrolyte metabolism , such as retinal blood flow [ 11 , 12 ] , because of its vasoactive property . in purine metabolic cycle , adenosine is phosphorylated into adenine and deaminated rapidly into inosine . then inosine is converted to hypoxanthine , which is converted to xanthine with the effect of xanthine oxidase . for diabetic patients , superoxide plays a major role in microvascular dysfunction and exerts direct tissue damage , leading to lipid and protein peroxidation . at last , xanthine is metabolized to uric acid , the final product of purine degradation in humans . it was reported that the high level of uric acid was associated with diabetic microvascular complications , such as nephropathy , retinopathy , and neuropathy [ 1416 ] , but it is usually considered a marker of tissue dysfunction rather than a risk factor for progression . recent studies have reported that uric acid might be a true mediator of microvascular and macrovascular disease . some researchers considered that uric acid might affect the function of vascular smooth muscle cells , which is related to diabetic retinopathy . although the purine metabolism may be associated with diabetic complication , to the best of our knowledge , there has been no detailed report about the relationship between several purine metabolites and dr . in this paper , we measured six purine metabolites including adenosine , adenine , inosine , xanthine , hypoxanthine , uric acid , and xanthine oxidase ( reflected by the uric acid  :  xanthine ) in the plasma of patients with dm and dr . we also observed the relationship between the related metabolites and clinical parameters . the current study included 74 type 2 diabetes patients ( 41 males and 33 females , age : 56.20  6.22 years ) in beijing , china . type 2 diabetes was defined in accordance with the criteria of the american diabetes association . 40 plasma samples of healthy people ( 22 males and 18 females , age : 54.41  5.36 years ) in the same area were collected as controls . among these patients , 39 patients had a diagnosis of nonproliferative dr according to international clinical diabetic macular edema disease severity scale . the dr was determined by ophthalmoscopy and fluorescein angiography through dilated pupils by a retinal specialist prior to the examination of the blood samples , and the patients were classified according to the presence or absence of dr , regardless of its degrees of severity . the other 35 patients were diagnosed to be having type ii diabetes without retinopathy by clinician . and the diabetic patients with and without dr were not diagnosed to have nephropathy , neuropathy , or other microvascular or macrovascular complications . all study participants had given their informed consent , and the institutional ethics committee approved the study . the related information such as age and duration of diabetes and other details of diabetic therapy were recorded . the clinical and biochemical parameters of 74 patients were reviewed and approved by the clinical medicinal research institute , sino - japanese friendship hospital , beijing , china . the diabetic duration was defined as the duration from the first diagnosis of type 2 dm to the time of blood sampling . the dates of first diagnosis of dm2 were obtained from the patients ' medical records . exclusion criteria included acute myocardial infarction , pregnancy , liver disease , stroke , current use of cholesterol lowering agents , and uncertained diabetic duration . body mass index ( bmi ) was defined as weight ( kg ) divided by squared height ( m ) ; that is , bmi = w/(h ) . the clinical and biochemical analyses of all the cases were carried out in 2007 and 2008 . ( hba1c ) , and lipid levels were measured using an automatic biochemical analyzer ( hitachi7180 , tokyo , japan ) . blood samples were collected into edta from 6 to 9 o'clock after an overnight fast . all blood samples were centrifuged to obtain plasma in the hospital and sent to our laboratory , where they were stored at 80c until sample preparation . before analysis , 800  l of methanol was added to 200  l aliquots of plasma , kept in vortex for 2  min , and then centrifuged at 10000  rpm for 15  min at 4c . the clear supernatant was transferred to a 1.5  ml polypropylene tube and dried under a gentle stream of nitrogen at room temperature . the residue was reconstituted with 100  l of a mixture of methanol and water ( 1  :  1 , by volume ) and stored at 4c before analysis . using high - performance liquid chromatography coupled to ultraviolet and tandem mass spectrometry method ( hplc - ms / ms ) , plasma concentrations of adenosine , adenine , inosine , xanthine , hypoxanthine , and uric acid were simultaneously measured . the hplc - ms / ms includes an applied biosystems ( toronto , canada ) api 3000 triple quadrupole tandem mass spectrometer equipped with a turbo ionspray interface for determination and an agilent 1100 binary hplc system for separation . samples were separated on an agilent tc - c18 column with an alltech guard column . the column temperature was maintained at 25c , the uv detector was set at 254  nm , and the injection volume was 20  l . the method has been validated according to the requirement of analytical chemistry [ 19 , 20 ] . calibration curves suitable for the analysis of plasma were linear ( r > 0.999 ) with limits of detection ( lod ) from 10 to 50  ng / ml . the recovery for every metabolite is from 79.1% to 102.0% with the relative standard deviation ( rsd ) less than 10% . figure 2 shows the typical multiple extracted ion chromatograms of these six purine metabolites in a real plasma sample . all statistical analyses were performed with spss statistical package ( version 14.0 , spss inc . the  test and fisher 's exact test were used for the comparison of the clinical characteristics in dm and dr subjects . the data of related clinical parameters and metabolites ' concentration were log - transformed to achieve normality . then the kolmogorov - smirnov test was used to verify the normality of the transformed data . the differences between the groups were calculated with student 's t - test , whereas linear regression was used to adjust these comparisons for age , duration ( years ) of dm and bmi . the spearman or the pearson correlations were also examined for the research on correlations between metabolites and clinical parameters . in this study , the metabolites , the levels of which have statistically significant differences between two groups of the three groups ( normal , dm , and dr ) , were defined as potential biomarkers . receiver operating characteristics ( roc ) graph was performed . a total of 74 patients were enrolled in this study .   table 1 shows the clinical characteristics of the study groups . from the table , we can see that dm patients with retinopathy ( dr patients ) have significantly ( p = 0.034 ) longer duration of the disease compared to dm patients without retinopathy ( dm patients ) , ( 13.63  2.08 years versus 10.47  1.48 years ) . for age , bmi , fasting blood glucose , and hba1c , the differences between the dm patients and dr patients were not statistically significant ( p = 0.971 , 0.481 , 0.311 , and 0.296 ) . and lipid profiles such as total cholesterol , triglyceride , high density lipoprotein ( hdl ) , and low density lipoprotein ( ldl ) of dm patients are not significantly changed compared to dr patients . for blood pressure , systolic blood pressure is significantly ( p = 0.011 ) increased in dr patients compared with dm patients ( 143.07  6.44  mm  hg versus 131.87  4.21  mm  hg ) whereas diastolic blood pressure is not changed . in addition , urea nitrogen is significantly ( p < 0.001 ) increased in dr patients compared with dm patients ( 14.71  3.33  mmol / l versus 8.23  1.77 the levels of uric acid and xanthine in the group of dr were significantly higher as compared with dm ( 70.55  3.97 versus 53.81  2.36 , p < 0.001 ; 1.01  0.21 versus 0.54  0.05 , p = 0.009 ) and control group ( 70.55  3.97 versus 46.63  2.41 , p < 0.001 ; 1.01  0.21 versus 0.47  0.06 , p < 0.001 ) . additionally , also , group of dm had significantly higher level of uric acid as compared to the healthy subjects ( 53.81  2.36 versus 46.63  2.41 , p = 0.012 ) . for level of uric acid  :  xanthine , the levels of inosine and adenosine in the group of dr were significantly higher as compared with dm ( 0.297  0.078 versus 0.077  0.010 , p < 0.001 ; 0.94    0.17 versus 0.17  0.01 , p < 0.001 ) and control group ( 0.297  0.078 versus 0.077  0.010 , p < 0.001 ; 0.94  0.17 versus 0.13  0.02 , p < 0.001 ) . but no statistically significant differences of levels of inosine and adenosine between the group of control and dm were observed . the concentrations of the metabolites with significant changes were shown in figure 3 . for levels of hypoxanthine and adenine as shown in table 3 , the relationship between levels of metabolites and clinical parameters including age , bmi , systolic blood pressure ( sbp ) , hba1c , and various other variables in all patients of both the dm and dr groups . uric acid , xanthine , inosine , and adenosine correlated positively with sbp and urea nitrogen . in particular , the pearson correlation coefficient between any of these four metabolites and urea nitrogen was above 0.568 . roc curve analysis ( uric acid - dr , inosine - dr , xanthine - de , and adenosine - dr ) was used to estimate the optimal numerical value of related metabolites to predict the risk of dr in patients with type 2 diabetes ( figure 4 ) . as a result , the area under curve ( auc ) of adenosine was 0.913  0.031 and significantly higher than that of null hypothesis ( true area was 0.5 , p < 0.001 ) . this means that the plasma adenosine level could serve as a potential diagnostic marker for dm to dr . plasma uric acid yielded an auc of 0.832 with 71.3% sensitivity and 97.2% specificity in discriminating dr . plasma xanthine yielded auc of 0.751 with 68.2% sensitivity and 81.3% specificity in discriminating dr . plasma inosine yielded an auc of 0.824 with 63.7% sensitivity and 100% specificity in discriminating dr . to find an optimal cutoff point to discriminate dichotomize  diseased  or , youden 's index was used in this study . according to youden 's index , the optimal operating point of adenosine was 0.32  mg / l . at this cutoff point , the sensitivity was 94.7% and specificity was 100% . mg / l was the optimal numerical value for predicting the risk of dr in patients with type 2 diabetes . the researchers have reported that both genetic and environmental factors determine the susceptibility for the development of dr [ 21 , 22 ] . it is shown that the development of dr in type 2 diabetes patients was associated with baseline glycemia , glycemic exposure over several years , poor lipid control , higher blood pressure , and smoking . despite the rapid research progress , robust predictors for the diagnosis of dr in individuals with diabetes are still lacking . thus , it is necessary to set out the study of biomarker discovery , especially for the low molecular weight metabolites , which are important and easy to be measured . in the present study , four potential biomarkers ( adenosine , inosine , xanthine , and uric acid ) of retinopathy were shown . and with the roc curve of these four markers , the plasma adenosine level could serve as a potential diagnostic marker for dm to dr . adenosine = 0.32  mg / l was the optimal numerical value for predicting the risk of dr in patients with type 2 diabetes ( sensitivity was 94.7% and specificity was 100% ) . as reported , serum uric acid concentration was found to be independently correlated with insulin resistance [ 24 , 25 ] . and uric acid has some physiologic functions including activation of the rennin angiotensin system and direct actions on endothelial cells and vascular smooth muscle cells . but in the past researches , uric acid was usually considered as a marker rather than a risk factor for the progression of disease . there is always a controversy that plasma uric acid concentration is a cause or a result of microvascular complications . in this study , we found significantly increased concentration of uric acid in the plasma of dm patients ( p = 0.011 ) . it demonstrated that the high level of uric acid might be a cause of microvascular complications . in addition , we found that the level of plasma uric acid correlated positively with urea nitrogen ( r = 0.622 , p < 0.001 ) and sbp ( r = 0.382 , p there was also a statistically significant difference of the level of plasma uric acid between the group of control and dr ( p < 0.001 ) , as well as the group of dm and dr ( p < 0.001 ) . in addition , several lines of evidence suggest that increased plasma uric acid may be a significant risk factor of vascular disease . reported that serum uric acid was associated with microvascular diseases and macrovascular diseases in men with type 2 diabetes mellitus . the evidence is that hyperuricemia stimulated human vascular muscle cells proliferation   and increased c - reactive protein expression in these cells . animal model experiments demonstrate that high level of uric acid in normal rats induced by the uricase inhibitor , oxonic acid , results in hypertension and vascular disease . this led to the hypothesis that uric acid may contribute to progressive vascular disease . and tseng have reported that increased uric acid concentration is a significant and independent risk factor for peripheral arterial diseases in taiwanese patients with type 2 dm . hence , strategies to control and decrease plasma uric acid level may have a beneficial effect on slowing the progression of retinal diseases in clinical practice . in this study , compared to dm and normal group , the concentration of adenosine in the group of dr was significantly higher with p value below 0.001 . in addition , adenosine correlated positively with urea nitrogen ( r = 0.568 ) and sbp ( r = 0.336 ) . therefore , adenosine was supposed to be a potential biomarker for the prediction and diagnosis of diabetic retinopathy , for the guidance and evaluation of treatment . adenosine plays an important role in water - electrolyte metabolism , such as retinal blood flow and renin release , which make the patients with diabetes be susceptible to retinopathy . for the produce of adenosine , there are two kinds of important enzymes , s - adenosylhomocysteine hydrolase and 5-nucleotidase . the extracellular metabolism of adenosine is mediated by two mechanisms . first , via an equilibrative facilitated diffusion system , adenosine is taken up quickly and efficiently by red blood cells . second , adenosine is deaminated rapidly into inosine by adenosine deaminase ( ada ) , which is found in large amounts particularly in mononuclear cells . and ada plays a major role in adenosine concentration regulation in both extracellular   and intracellular spaces [ 33 , 34 ] . it is suggested that , with the decrease of the activity and expression of ada , the production of adenosine increased . then the elevated adenosine , with the vasoactive property , leads to the generation of superfluous no . no , as the important vasoactive substances , affects retinal microcirculation and induces the capillary occlusion , pericytes apoptosis , and matrix membrane thickening , which are closely related to diabetic retinopathy . in this study , the significant difference of xanthine between the group of control and dr ( p < 0.001 ) , as well as the group of dm and dr ( p < 0.001 ) , was presented . when xanthine oxidase converts hypoxanthine to xanthine in the presence of molecular oxygen , superoxide radicals ( o2 ) are released . thereby , reactive oxygen species ( ros ) is generated , which play a major role in microvascular dysfunction and exert direct tissue damage , leading to lipid peroxidation , denaturation of proteins , and oxidation of dna . in addition , ros leads to the increase of vascular endothelial growth factor , which increases vascular permeability and promotes angiogenesis in response to ischemia or hypoxia , resulting in visual impairment . many direct lines of evidence have demonstrated that ros was one of the most important mechanisms of dr . previous studies on the effect of xanthine or xanthine oxidase as inducers of microvascular injury have produced inconsistent results . found that infusion of xanthine increased the generation of oxygen free radicals and induced the vascular injury . another study by linas et al . found that xanthine oxidase depletion improved vascular function after reperfusion . in this study , the level of xanthine increased with the progression of dr , while the level of xanthine oxidase ( reflected by the uric acid  :  xanthine ) did not change . so we suppose that the inducer of vascular injury is not xanthine oxidase but xanthine itself . we also see that there is little change of the level of plasma hypoxanthine while there is an accrescent level of xanthine . thanks to the invariant activity of xanthine oxidase , the increase of xanthine may come from guanine with the catalysis of guanine deaminase , which catalyzes the hydrolytic deamination of guanine to xanthine . in summary , plasma adenosine , inosine , uric acid , and xanthine were higher in subjects with dr . plasma levels of adenosine , inosine , uric acid , and xanthine were also significantly correlated with the urea nitrogen and sbp . the levels of these four metabolites , especially the level of adenosine , may be useful for monitoring the progression of diabetic retinopathy and evaluating the treatment .
aims . 
 the purpose of the study was to investigate the differences of adenosine , adenine , inosine , xanthine , hypoxanthine , and uric acid concentrations in patients with type 2 diabetes mellitus and diabetic retinopathy and assess the relationship between purine metabolites and disease . 
 materials and methods . 
 the study group consisted of 114 subjects which were divided into three groups : control ( n = 40 ) , type 2 diabetes without retinopathy ( n = 35 ) , and type 2 diabetes with retinopathy ( n = 39 ) . 
 levels of metabolites were measured in plasma of all participants . 
 results . 
 there is a significant increase of levels of adenosine ( 0.94  0.17  mg / l versus 0.17  0.01  mg / l , p < 0.001 ) , inosine ( 0.297  0.078 
 mg / l versus 0.086  0.010 
 mg / l , p < 0.001 ) , xanthine ( 1.01  0.21 
 mg / l versus 0.54  0.05  mg / 
 l , p = 0.009 ) , and uric acid ( 70.55  3.97 
 mg / l versus 53.81  2.36 
 mg / l , p < 0.001 ) with diabetic retinopathy compared to diabetes mellitus . 
 the levels of adenine , hypoxanthine , and xanthine oxidase did not change . 
 uric acid , xanthine , inosine , and adenosine correlated positively with systolic blood pressure and urea nitrogen . conclusions . 
 the levels of adenosine , inosine , uric acid , and xanthine may be useful for monitoring the progression of diabetic retinopathy and evaluating the treatment .