progressive disorders of the large and small vessels are common and serious complications of diabetes mellitus ( dm ) . studies over the last two decades have shown that hyperhomocysteinemia is associated with several macrovascular diseases , such as coronary artery disease , cerebrovascular disease , peripheral arterial disease , and deep - vein thrombosis [ 13 ] . relatively few studies have investigated the association between hyperhomocysteinemia and diabetic retinopathy ( dr ) with yet inconsistent results . in addition , the vitamin status and its contribution to the development of dr have not been examined . in our study , we aimed to investigate whether there is an association between serum levels of homocysteine and those of folic acid and b - vitamins with dr in patients with type 2 diabetes mellitus ( t2 dm ) . the study group included 140 patients with t2 dm , 69 men and 71 women , aged from 41 to 83 years ( mean age 64.4  9.9 years ) , all of greek descent . all patients were recruited from the ophthalmology outpatient clinics as well as the diabetes center of the  attikon  university hospital ( medical school of athens university ) in athens , greece . we excluded patients with one or more of the following conditions : patients with a history of vascular disease ( myocardial infarct or angina , stroke , peripheral arterial disease , and deep - venous thrombosis ) , renal , hepatic , chronic gastroenterologic , thyroid or blood disease , dementia , and neoplasm , since all these conditions are known to affect homocysteine blood concentrations;patients receiving vitamin supplementation or medications known to affect serum homocysteine concentrations , such as theophylline , statins , fibrates , levodopa , protons pump inhibitors , anticonvulsives , and contraceptives , and patients consuming good amounts of alcohol and heavy smokers;patients with uncontrolled arterial hypertension , defined as a systolic blood pressure  140  mmhg , and/or a diastolic blood pressure  90  mmhg;patients with uncertain diabetes duration . patients with a history of vascular disease ( myocardial infarct or angina , stroke , peripheral arterial disease , and deep - venous thrombosis ) , renal , hepatic , chronic gastroenterologic , thyroid or blood disease , dementia , and neoplasm , since all these conditions are known to affect homocysteine blood concentrations ; patients receiving vitamin supplementation or medications known to affect serum homocysteine concentrations , such as theophylline , statins , fibrates , levodopa , protons pump inhibitors , anticonvulsives , and contraceptives , and patients consuming good amounts of alcohol and heavy smokers ; patients with uncontrolled arterial hypertension , defined as a systolic blood pressure  140  mmhg , and/or a diastolic blood pressure  90  mmhg ; patients with uncertain diabetes duration . the study was in accordance with the declaration of helsinki and was approved by the local ethics committee . retinopathy was assessed by dilated ophthalmoscopy , fundus photography , and fluorescein angiography , when was indicated . retinopathy was defined as the presence of any of such characteristic lesions : microaneurysms , hemorrhages , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , new vessels , laser scars , or a history of vitrectomy . grading of the retinal findings was assessed according to the system used by the early treatment diabetic retinopathy study research group . patients with diabetes were classified into two groups according to the findings of the  worst eye  : 65 with dr ( 46.4% ) and 75 without dr ( 53.6% ) . of the 65 patients with dr , 36 ( 55.4% ) had nonproliferative diabetic retinopathy ( npdr ) and 29 ( 44.6% ) had proliferative diabetic retinopathy ( pdr ) . overnight fasting blood samples were taken from all subjects and were centrifuged within 1 hour following collection . the samples for homocysteine estimation were immediately chilled on ice and then were centrifuged and the serum was stored at 20c . serum glucose concentration was measured with a colorimetric assay on an automated analyzer modular p800 ( roche diagnostics gmbh , mannheim , germany ) and glycosylated hemoglobin ( hba1c ) with an immunoturbidimetric assay on an analyzer integra 400plus ( roche diagnostics gmbh , mannheim , germany ) . serum creatinine concentration was measured with the colorimetric assay ( jaff ) on the automated analyzer modular p800 . folic acid and vitamin b12 levels were estimated by electrochemiluminescence immunoassay on the analyzer modular analytics e170 ( roche diagnostics gmbh , mannheim , germany ) and those for vitamins b2 and b6 by high performance liquid chromatography ( hplc ) with fluorescence detection ( chromsystem diagnostics gmbh , mnchen , germany ) . estimation of serum homocysteine concentration was carried out by fluorescence polarization immunoassay ( axsym system , abbott diagnostics , mannheim , germany ) . estimation of body weight is made by using the body mass index ( bmi ) = kg / height(m ) . differences between patients with and without dr in continuous variables were tested using t - test for two independent samples and , when normality assumption was violated , using the mann - whitney test . for categorical variables , association of homocysteine with folic acid and vitamin b12 was investigated using spearman correlation coefficient , while when hba1c and diabetes duration groups were considered , one - way anova or kruskal - wallis test ( with bonferroni correction when needed ) was used . differences by gender and type of retinopathy were tested by t - test for two independent samples or the mann - whitney test . the association of homocysteine with possible prognostic variables was also tested in multivariable regression models . in the first stage , using generalized additive models ( gams ) , the shape of the association with continuous variables was explored . when a threshold was identified , this was estimated using appropriate methodology . in the final regression model , those variables identified in the first step were included in the model using two linear parts , one below and one above the threshold ( piecewise linear ) . finally , multivariable logistic regression models were used to identify prognostic variables for dr ( binary variable , yes / no ) in the same way that was described above for homocysteine . the age of patients with dr was statistically higher than that of patients without dr ( 68.0 ( 60.075.0 ) versus 61.0 ( 55.067.0 ) , resp . , p = 0.001 ) , as was also their positive family history of diabetes ( 86.2% versus 54.7% resp . , p < 0.001 ) , and their bmi ( 29.2 ( 27.931.2 versus 27.8 ( 26.130.2 ) , resp . , patients with dr had a significantly longer diabetes duration ( 15.0 ( 10.022.0 ) versus 6.0 ( 3.09.0 ) , p < 0.001 ) , higher serum glucose ( 154.0 ( 126.0188.0 ) versus 119.0 ( 103.0136.0 ) , p < 0.001 ) , hba1c ( 7.4 ( 6.68.9 ) versus 6.7 ( 6.07.6 ) , p < 0.001 ) , and serum creatinine level ( 0.9 ( 0.81.1 ) versus 0.9 ( 0.71.0 ) , p = 0.024 ) , but in both groups creatinine levels were well within the normal range . a significantly higher serum homocysteine level was observed in patients with dr than in those without dr ( 16.3 ( 14.719.8 ) versus 11.1 ( 9.513.6 ) , resp . mol / l ) was noted in 70.8% of individuals with dr compared to 14.7% without dr ( p < 0.001 ) . as for the serum vitamins , patients with dr had , though in the normal range , significantly lower folic acid ( 8.7 ( 7.210.3 ) versus 11.0 ( 8.317.0 ) , p < 0.001 ) and vitamin b12 ( 288.8 ( 209.5416.5 ) versus 361.5 ( 255.6471.2 ) , p = 0.014 ) levels , whereas there was no significant difference for vitamin b2 ( 9.1 ( 7.012.7 ) versus 10.8 ( 7.813.2 ) , p = 0.179 ) and vitamin b6 ( 11.0 ( 8.014.6 ) versus 11.6 ( 9.913.2 ) , p = 0.481 ) levels between dr and no dr groups , respectively . systolic blood pressure was higher in patients with dr ( 130.0 ( 120.0135.0 ) versus 125.0 ( 115.0130.0 ) , p = 0.016 ) , but no significant difference in diastolic blood pressure was noted between the two groups ( 75.0 ( 70.080.0 ) versus 75.0 ( 70.080.0 ) , p = 0.409 ) . however , in all our diabetic patients blood pressure was within the normal range . in table 2 , the data on the association between serum homocysteine levels and two significant parameters are shown . serum homocysteine levels of diabetic patients were significantly increased with increasing diabetes duration ( 5 versus 16 years , homocysteine : 11.2 ( 8.713.9 ) versus 16.9 ( 14.818.9 ) , p < 0.001 , 615 versus 16 years , homocysteine : 12.2 ( 10.016.2 ) versus 16.9 ( 14.818.9 ) , p = 0.001 , overall p < 0.001 ) . however , serum homocysteine levels did not change significantly with increments in hba1c levels ( hba1c < 6.5 homocysteine : 11.2 ( 9.515.8 ) , 6.57.4 : 12.3 ( 10.618.4 ) , 7.58.4 : 15.0 ( 11.116.2 ) , 8.5 : 15.1 ( 11.317.4 ) , p = 0.126 ) . there was a statistically significant difference in serum homocysteine levels between the npdr group compared to the pdr group ( npdr : 15.5 ( 11.817.4 ) , pdr : 18.7 ( 16.522.0 ) , p = 0.001 ) . a statistically significant negative linear relationship was found between serum homocysteine and folic acid levels ( spearman r = 0.261 , p = 0.002 ) , as well as between serum homocysteine and vitamin b12 levels ( spearman r = 0.219 , p = 0.009 ) . multiple linear regression analysis revealed a significant association between serum homocysteine level and age ( coef . : 0.099 , 95% ci : 0.02 , 0.18 , p = 0.018 ) , diabetes duration ( coef . : 0.106 , 95% ci : 0.02 , 0.20 , p = 0.020 ) , and an inverse relationship with folic acid ( coef . : 0.220 , 95% ci : 0.37 , 0.07 , p = 0.005 ) and vitamin b12 ( coef . : 0.027 , 95% ci : 0.04 , 0.01 , p = 0.003 ) . no associations with sex ( coef . : 0.859 , 95% ci : 0.56 , 2.28 , p = 0.233 ) , bmi ( coef . : 0.149 , 95% ci : 0.06 , 0.36 , p = 0.166 ) , and glucose ( coef . : 0.005 , 95% ci : 0.01 , 0.02 , p = 0.523 ) were observed ( table 3 ) . there is a threshold in the exposure - response association of serum homocysteine with vitamin b12 , below which changes in serum vitamin b12 levels significantly affect serum homocysteine concentration ( turning point : 248.4 , standard error : 43.8 ) ( figure 1 ) . multiple logistic regression analysis also showed that variables that independently affect dr risk were diabetes duration ( or : 1.18 , 95% ci : 1.08 , 1.28 , p < 0.001 ) , hba1c ( or : 2.30 , 95% ci : 1.49 , 3.54 , p < 0.001 ) , and homocysteine concentrations ( or : 1.66 , 95% ci : 1.24 , 2.23 , p = 0.001 ) . folic acid conferred a protective effect on the dr risk ( or : 0.73 , 95% ci : 0.62 , 0.86 , p < 0.001 ) ( table 4 ) . there is a threshold in the association of homocysteine with dr ( turning point : 13.7 , standard error : 1.4 ) . for every increase of serum homocysteine by 1 mol / l above the threshold , there is an increased risk of about 66% for the development of dr ( figure 2 ) . the results of our study revealed significantly higher concentration of serum homocysteine as well as a higher prevalence of hyperhomocysteinemia ( serum homocysteine > 15 mol / l ) in patients with t2dr compared to those without dr and hyperhomocysteinemia as an independent risk factor for dr . our results also revealed a threshold in the association of serum homocysteine level with dr risk of around 14.0 mol / l , above which a 1  mol / l increase in serum homocysteine concentration was an independent predictor of increased dr risk of 66% . a higher serum homocysteine level in patients with pdr than in those with npdr was also found ; a result that may suggest that homocysteine is related not only to the development but also to the progression of dr . there are a number of studies that have evaluated the association between hyperhomocysteinemia and type 2 dr but have so far yielded inconsistent results [ 69 ] . the differences in the findings among studies are most likely related to differences in type of diabetes or to inadequately controlling for confounding factors , such as nephropathy and hypertension , but also lifestyle ( e.g. , vitamin intake ) and genetic factors . in addition , the cutoff level for hyperhomocysteinemia differed substantially among studies ( from 11.0 to 16.0 the exact pathogenesis of dr is multifactorial and remains largely unclear but may involve ( 1 ) endothelial dysfunction and ( 2 ) low - grade chronic inflammation of the retinal capillaries . it has long been recognized that oxidative stress is associated with the progression of diabetes and its complications . the adverse effects of hyperhomocysteinemia on the endothelium may be triggered by increased oxidative stress in the diabetic vasculature . hyperhomocysteinemia increases nadph oxidase activity , promotes uncoupling of endothelial nitric oxide synthase , and inhibits the function of intracellular antioxidant enzymes , such as glutathione peroxidase and superoxide dismutase . accumulating ros reacts with nitric oxide ( no ) to form peroxynitrite radicals , leading to decreased no bioavailability and activity and subsequent endothelial dysfunction . in addition , upregulation of ros leads to activation of proinflammatory mediators , such as the nuclear factor- , in retinal capillary cells , which further increasing the expression of cytokines . these inflammatory events result in increased endothelial cell expression of adhesion molecules , which contribute to leucocyte accumulation and attachment to the retinal capillaries ( leucostasis ) . leucostasis , which is believed to be an early event in dr , may lead to the blood - retinal barrier breakdown and eventually to chronic leucocyte mediated cell damage and death . with regard to the risk factors for dr , regression analysis of our data confirmed that poor glycemic control and longer diabetes duration are independent risk factors for the development and progression of dr [ 18 , 19 ] . in our study , the group of patients without dr succeeded in achieving the target mean hba1c level of  7.0% or less   ( hba1c6.9  1.1% ) but this was not the case with the group of patients with dr ( hba1c  7.8  1.5% ) . however , it is true that perfect glycemic control is very difficult to attain for most diabetic patients and glucose control has the tendency to deteriorate with time . a significant finding in our study was the association of increasing serum homocysteine concentration with longer diabetes duration . thus , we may infer that the known harmful effect of dm duration on the development of retinopathy and its progression is not only related to the long - standing hyperglycemia but also to the increased homocysteine concentrations with time and its subsequent adverse effects on retinal capillaries . on the contrary this may be due either to an actual lack of such an association between the two factors or to the inconsistency of hyperglycemia during the progression of dm . besides , evidence from both healthy and diabetic twins indicates that hba1c levels are determined for a significant part by genetic factors . this provides evidence that hba1c is in part determined by factors other than glycemic control and may account for the variation in hba1c levels among people [ 20 , 21 ] . methionine and homocysteine metabolism depends upon adequate stores of folic acid and vitamins b2 , b6 , b12 , which act as cofactors or substrates in the metabolism and are important nutritional determinants of serum homocysteine . a number of studies have shown an inverse association between blood folic acid and homocysteine concentration [ 22 , 23 ] . such an inverse relationship between folic acid and homocysteine levels was also observed in our study . in regard with vitamin b12 , the reason for the limited number of studies with this vitamin is that its deficiency is rather unusual , especially in western countries , due to the fact that the vitamin is present in food of animal origin . a recent study from india , where the prevalence of micronutrient deficiency is reported to be high , mentions a high prevalence of vitamin b12 deficiency in the diabetic population ( 5467% ) . mean vitamin b12 level in the dr group was below the normal range and higher homocysteine levels were significantly associated with lower vitamin b12 levels . our study illustrates lower levels of folic acid and vitamin b12 in patients with dr . it is noteworthy that low , but not necessarily deficient , levels of these two vitamins contributed to the development of hyperhomocysteinemia in our study . regression analysis also showed that folic acid confers a protective effect on the development of dr . another significant observation in our study was the finding of a threshold of about 250  pg / ml of serum vitamin b12 , below which there is a sharp rise in serum homocysteine with decreasing vitamin b12 levels . vitamin b6 deficiency is extremely unusual , because this vitamin is present in a wide range of food of animal and plant origin . in our study one study has reported an association between low vitamin b6 and vascular disease that was unrelated to hyperhomocysteinemia , but it was explained by a relationship between low vitamin b6 status and chronic inflammation , which is known to promote dr . the results of our study show that vitamin b6 levels were well within the normal range in patients with diabetes irrespective of the presence of retinopathy . vitamin b2 acts in the form of flavin adenine dinucleotide ( fad ) , which is a substrate for the enzyme methylene tetrahydrofolate reductase ( mthfr ) . this enzyme is the most important genetic determinant of blood homocysteine in the general population . in our study material , no cases of biochemical vitamin b2 deficiency were observed , neither do we know any such studies , and the levels of this vitamin were also well within the normal range in all of our diabetic patients . increased dietary folic acid and vitamin b2 requirements have been observed mainly in persons with the genetic variant of the enzyme mthfr , c677 t . studies confirm that vitamin b2 is an independent determinant of homocysteine in tt homozygotes of the c677 t polymorphism . in conclusion , among the many factors , some of them are yet unidentified , that affect the development and progression of dr , hyperhomocysteinemia seems to be associated with dr , at least as a biomarker . longer diabetes duration and lower folic acid and vitamin b12 status appear to be important determinants for hyperhomocysteinemia in dr patients . monitoring serum homocysteine concentration , as well as folate and vitamin b12 status in t2 dm patients , could be used as an indicator for assessing microvascular risk in dm . treatment of existing hyperhomocysteinemia with folic acid and vitamin b12 may be useful in reducing the risk of microvascular complications in t2 dm .
we investigated the association of serum homocysteine levels and vitamin status with type 2 diabetic retinopathy . 
 this study included 65 patients with and 75 patients without diabetic retinopathy . 
 patients with diabetic retinopathy had significantly higher serum homocysteine levels ( p < 0.001 ) , higher prevalence of hyperhomocysteinemia ( p < 0.001 ) , lower serum folic acid ( p < 0.001 ) , and vitamin b12 ( p = 0.014 ) levels than those without diabetic retinopathy . 
 regression analysis revealed that homocysteine was an independent risk factor for diabetic retinopathy and there was a threshold in its serum level ( 13.7  mol / l ) , above which the risk of diabetic retinopathy greatly increases ( or = 1.66 , p = 0.001 ) . 
 folic acid was associated with decreased odds for diabetic retinopathy ( or = 0.73 , p < 0.001 ) . 
 there was a threshold in serum vitamin b12 level ( 248.4  pg / ml ) , below which serum homocysteine concentration significantly increases with decreasing serum vitamin b12 ( p = 0.003 ) . 
 our findings suggest that hyperhomocysteinemia is an independent risk factor for the development and progression of diabetic retinopathy . 
 decreased serum levels of folic acid and vitamin b12 , through raising serum homocysteine concentrations , may also affect the diabetic retinopathy risk .