Seminars in Ophthalmology, Early Online, 1–5, 2014 ! Informa Healthcare USA, Inc. ISSN: 0882-0538 print / 1744-5205 online DOI: 10.3109/08820538.2014.912338
ORIGINAL ARTICLE
Plasma Homocysteine is Associated with Retinopathy in Type 1 Diabetic Patients in the Absence of Nephropathy
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Tomislav Bulum, Kristina Blaslov, and Lea Duvnjak Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic Diseases, University Hospital Merkur, Medical School, University of Zagreb, Zagreb, Croatia
ABSTRACT Introduction: Previous cross-sectional studies suggested that plasma total homocysteine (tHcy) is associated with retinopathy in patients with type 1 diabetes (T1DM) only in cases of impaired renal function. The objective of this study was to examine whether there is an independent relationship between tHcy and retinopathy in normoalbuminuric T1DM patients with normal estimated glomerular filtration rate (eGFR). Methods: The study included 163 normoalbuminuric patients with T1DM and normal renal function (eGFR 460 125 ml min 1 1.73 m 2). Urinary albumin excretion rate (UAE) was measured from at least two 24 h urine samples. Photodocumented retinopathy status was made according to the EURODIAB protocol. tHcy level was measured with the chemiluminescent immunoassay. Results: Retinopathy was present in 48% of normoalbuminuric patients. Patients with retinopathy were older (49 vs 42 years, p = 0.001), had higher systolic blood pressure (130 vs 120 mmHg, p = 0.001), triglycerides (0.89 vs 0.77 mmol/L, p = 0.01), tHcy (9.8 vs 9.1 mmol/L, p = 0.04), and lower eGFR (100 vs 106 ml min 1 1.73 m 2, p = 0.03). In multivariate logistic regression analysis, after adjustment for variables that reached statistical significance in the univariate analysis, only tHcy was significantly associated with a risk of retinopathy in our subjects (p = 0.02), with odds ratios of 1.02 to 1.43. Conclusion: These data suggest that tHcy is independently associated with retinopathy in normoalbuminuric T1DM with normal eGFR. The mechanisms relating tHcy and retinopathy in T1DM are not clear. Prospectives studies are needed to confirm whether higher tHcy in normoalbuminuric T1DM patients has predictive value for development of retinopathy. Keywords: Homocysteine, microvascular complications, retinopathy, type 1 diabetes
INTRODUCTION
increased levels of tHcy and cardiovascular disease was reported in T1DM.7,8 tHcy is independently associated with glomerular filtration rate (GFR) and albuminuria.1,2 The independent relationship between GFR and tHcy is based on a relationship between declining GFR and increasing tHcy levels in T1DM.9 In addition, experimental findings suggested that Hcy may directly impair glomerular filtration.10 Diabetic patients with microalbuminuria and normal GFR also have mild hyperhomocysteinaemia.6,11 On the other hand, it is assumed that relative hyperfiltration in younger normoalbuminuric T1DM is responsible for similar
Moderate hyperhomocysteinaemia has been observed in studies of patients with type 1 diabetes (T1DM) in which the plasma total homocysteine (tHcy) levels are independent of vitamin status and reflect the heterogeneous nature of the patients, including a variety of microvascular and macrovascular complications.1,2 Most previous cross-sectional studies exploring the relationship between tHcy and microvascular complications reported positive associations between tHcy and nephropathy,1,3,4 but not for retinopathy.5,6 In addition, no independent relationship between
Received 9 January 2014; accepted 1 April 2014; published online 2 May 2014 Correspondence: Tomislav Bulum, MD, PhD, Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic Diseases, University Hospital Merkur, School of Medicine, Dugi dol 4a, University of Zagreb, Zagreb, Croatia. E-mail: [email protected]
1
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tHcy levels in those with and without microvascular complications.12 Identification of the determinants of the onset of diabetic retinopathy is essential for reducing the morbidity and mortality associated with diabetes because diabetic retinopathy is one of the leading causes of visual impairment and blindness in T1DM.13 It is assumed that retinopathy and nephropathy, as the most important microvascular complications in diabetes, occur at the same time and that retinovascular pathology reflects renal disease.14 However, it is suggested that higher tHcy is associated with retinopathy in T1DM only in cases of impaired renal function measured with GFR and/or albuminuria,1,2,5,6,12 although recent studies including type 2 diabetic patients found higher tHcy in individuals with retinopathy, independent of renal dysfunction and other major risk factors.15,16 The objective of this study was to examine whether there is an independent relationship between tHcy and retinopathy in normoalbuminuric T1DM patients with normal GFR.
SUBJECTS, MATERIALS, AND METHODS This cross-sectional study included 163 patients with T1DM, defined as an onset of diabetes before the age of 35 years, positive autoantibodies, and permanent insulin treatment initiated within one year of diagnosis. The study included patients with the following characteristics: age of 18–65 years, minimum duration of type 1 diabetes of one year, no medical history of cardiovascular diseases or electrocardiogram (ECG) evidence of ischemic heart disease, absence of any systemic disease, and absence of any infections in the previous month. Those with microalbuminuria (urinary albumin excretion rate (UAE) 305300 mg/24h), macroalbuminuria (UAE 300 mg/24h), impaired estimated GFR (less than 60 ml min 1 1.73 m 2), and hyperfiltration (estimated GFR over 125 ml min 1 1.73 m 2) were also excluded from the study. All subjects were studied in the morning after an overnight fast. Basic anthropometric measurements were performed on all study subjects, including body mass index (BMI) and waist to hip ratio (WHR). Blood pressure was measured on both arms twice in the sitting position with a mercury sphygmomanometer, after a resting period of 10 minutes, and was expressed in mmHg. UAE was measured from at least two 24 h urine samples and determined as the mean of 24 h urine collections. Patients performed collections on two consecutive days to minimize variability. Normoalbuminuria was defined as a UAE530 mg/24h. Serum creatinine was measured in a fasting blood sample. Data on serum creatinine levels, age, sex, and race were used to calculate the
estimated GFR using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula, which has been shown to be accurate in determining renal function in diabetic patients with normal renal function.17,18 All subjects were confirmed to be free of urinary tract infections. Retinopathy was diagnosed by binocular indirect slit lamp fundoscopy and fundus photography after mydriasis with eye drops containing 0.5% tropicamide and 5% phenylephrine. Color fundus photographs of two fields of both eyes were taken with a suitable 45 fundus camera (VISUCAM, Zeiss) according to the EURODIAB retinal photography methodology.19 In each patient, the ‘‘worse’’ eye was graded for retinopathy using fundus photographs. Fasting venous blood samples were collected in the morning between 08:00 and 09:30 hours after an overnight fast for the determination of HbA1c, total LDL, HDL cholesterol, triglycerides, and tHcy. Microalbumin and HbA1c were measured spectrophotometrically by turbidimetric immuno-inhibition (Olympus AU600, Beckman-Coulter, USA). Results of HbA1c (%) are expressed in the DCCT-equivalent. Serum tHcy level was measured with the Chemiluminescent Immunoassay (CLIA). Glucose, cholesterol, and triglycerides in serum were measured by an enzymatic colorimetric method (Olympus AU600, Beckman-Coulter, USA). Complete blood count was determined on an automatic blood counter (Advia 120, Siemens Diagnostic Solutions, USA). The study protocol complies with the Declaration of Helsinki as well as with local institutional guidelines, and was approved by the local ethics committees. Data are expressed as means ± SD for normally distributed values, as median with range for nonnormally distributed values, and percentage. Differences between groups were examined, depending on the nature of the data, parametric (t-test), or nonparametric tests (Mann-Whitney). All of the variables that reached statistical significance in the univariate analysis were included in multivariate logistic regression in order to assess associations of tHcy and risk of retinopathy. Level of statistical significance was chosen to be = 0.05. Statistical analysis was performed by statistical package SPSS, version 17.0 for Windows.
RESULTS The characteristics of the study subjects are listed in Table 1. The average age was approximately 47 years, most were not overweight, and 51% of subjects were male. Mean/median values of BMI, WHR, systolic blood pressure, diastolic blood pressure, LDL, HDL cholesterol, and triglycerides were within the normal range for patients with T1DM, with slightly elevated Seminars in Ophthalmology
Homocysteine and Retinopathy in Type 1 Diabetes TABLE 1. Clinical and metabolic characteristics of all patients.
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Variable
Value
Age (years) Sex (m/f, %) Body mass index (kg/m2) Waist to hip ratio HbA1c (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L) Triglycerides (mmol/L) Serum creatinine (mmol/L) eGFR (ml min 11.73 m 2) Urinary albumin excretion (mg/24 h) Retinopathy (no/yes, %) Homocysteine (mmol/L)
47 (23–65) 54/46 25 (18–38) 0.82 ± 0.07 6.7 (4.2–10.6) 120 (90–180) 80 (55–105) 5.1 ± 0.9 2.8 ± 0.8 1.7 (0.6–3.4) 0.84 (0.3–4.3) 73 (42–120) 104 (60–124) 12.2 (1.2–29.8) 52/48 9.5 (1.7–19.7)
eGFR, estimated glomerular filtration rate.
No (n = 85)
Yes (n = 78)
Age (years) 42 (23–65) 49 (26–65) Sex (m/f) 45/40 43/35 24 (18–38) 25 (18–34) Body mass index (kg/m2) Waist to hip ratio 0.81 ± 0.07 0.83 ± 0.07 HbA1c (%) 6.5 (4.6–10.6) 6.8 (4.2–10.2) SBP (mmHg) 120 (90–180) 130 (105–160) DBP (mmHg) 80 (60–105) 80 (55–100) Total cholesterol 5.0 ± 0.8 5.2 ± 1.0 (mmol/L) LDL cholesterol 2.7 ± 0.7 3.0 ± 0.9 (mmol/L) HDL cholesterol 1.8 (0.6–3.4) 1.7 (0.8–2.7) (mmol/L) Triglycerides (mmol/L) 0.77 (0.2–4.3) 0.89 (0.4–2.8) Serum creatinine 73 (56–105) 73 (42–120) (mmol/L) 106 (71–125) 100 (60–123) eGFR (ml min 1 1.73 m 2) UAE (mg/24 h) 11.5 (2.1–29.8) 13.2 (1.2–29.6) Homocysteine (mmol/L) 9.1 (1.7–19.7) 9.8 (6.0–19.0)
p 0.001 0.7 0.6 0.1 0.05 0.001 0.6 0.2 0.5 0.2 0.01 0.7 0.03 0.2 0.04
SBP, systolic blood pressure; DBP, diastolic blood pressure; UAE, urinary albumin excretion rate.
levels of total cholesterol. The majority of patients had no retinopathy (48% with retinopathy). The mean GFR estimated by the CKD-EPI was 104 ml min 1 1.73 m 2. Clinical and metabolic characteristics of patients with and without retinopathy are presented in Table 2. Patients with retinopathy were older, had higher systolic blood pressure, triglycerides, tHcy, and lower estimated GFR (p50.05 for all). BMI, WHR, diastolic blood pressure, total LDL, HDL cholesterol, serum creatinine, and UAE levels did not differ between the two groups. !
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TABLE 3. Multivariate logistic regression analysis of risk factors for development of retinopathy in normoalbuminuric type 1 diabetic patients. Independent variable
Odds ratio (95% CI)
Age Homocysteine eGFR Systolic blood pressure Triglycerides
0.96 1.21 1.00 1.01 1.05
(0.91–1.03) (1.02–1.43) (0.7–1.03) (0.98–1.05) (0.99–3.3)
p 0.3 0.02 0.8 0.2 0.5
eGFR, estimated glomerular filtration rate.
In multivariate logistic regression analysis, after adjustment for variables that reached statistical significance in the univariate analysis, only tHcy was significantly associated with risk of retinopathy in our subjects (p = 0.02), with odds ratios of 1.02 to 1.43 (Table 3).
DISCUSSION
TABLE 2. Clinical and metabolic characteristics of patients without and with retinopathy. Retinopathy
3
Previous cross-sectional studies suggested that higher tHcy is associated with retinopathy in T1DM only in cases of impaired renal function measured with GFR and/or albuminuria.1,2,5,6,12 Our patients with retinopathy had significantly higher metabolic risk factors including age, systolic blood pressure, triglycerides, tHcy, and lower estimated GFR. The difference in the mean tHcy concentration between subjects with and without retinopathy was relatively small but significant, and the median tHcy concentration for both subjects was below the upper limit for adults, as previously documented in diabetic and nondiabetic subjects with retinopathy.15,20 In multiple logistic regression analysis, only tHcy was independently related to retinopathy in our normoalbuminuric T1DM with normal renal function. In support of these data, recent studies, including a similar number of type 2 diabetic patients, found higher tHcy in individuals with retinopathy, independent of renal dysfunction and other major risk factors.15,16 Furthermore, hyperhomocysteinemia is an independent risk factor for retinal venous occlusion, retinopathy of prematurity, age-related macular degeneration, and macular edema.21 24 Higher homocysteine levels were observed not only in blood plasma, but also in aqueous of eyes with proliferative diabetic retinopathy.25 The mechanisms relating tHcy and retinopathy in T1DM are not clear, although proposals include oxidative stress, endothelial damage, and decreased nitric oxide bioavailability.2 Homocysteine impairs retinal endothelial function regarding nitric oxidemediated vasodilatation in the retinal arterioles and reduces vasodilation of the retinal arterioles in response to the calcium, indicating that hyperhomocysteinemia is implicated in the pathogenesis of
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ocular vascular disorders.26 Moreover, it has been shown that the nitric oxide concentration can decrease after acute and chronic exposure of homocysteine to human vein endothelial cells.27 Homocysteine is a sulfur-containing amino acid that forms during metabolism of methionine, an essential amino acid, and hyperhomocysteinemia induced by oral administration of methionine impaired vascular endothelial function in human brachial arteries.28 Homocysteine can also induce endothelial toxicity by reactive oxygen species (ROS) generated via both enzymatic and nonenzymatic mechanisms.29 Furthermore, accumulating evidence suggests that homocysteine activates vascular inflammation through inflammatory cytokines.30,31 Renal failure is an important cause of hyperhomocysteinaemia because the kidneys play a major role in the metabolism of Hcy.1,2,9,10,32 In our study, although it included normoalbuminuric patients with normal estimated GFR, those with retinopathy had slightly higher UAE and significantly lower estimated GFR. Higher tHcy in patients with retinopathy may be due to (although non-significant) higher UAE because previous studies demonstrated a strong correlation between UAE and retinopathy, even in the absence of chronic kidney disease.6,11,33 On the other hand, lower estimated GFR probably results from the synthesis of the precursor of creatinine and donation of methyl groups formed in the transformation of methionine to Hcy.34 It can be argued that we should measure kidney function with gold standard methods like cystatin C in this specific population (with special regard to hyperfiltration). However, cystatin C is impractical in studies with large numbers of subjects; it should not be used for assessment of GFR in patients with thyroid disorders, which are common in patients with T1DM, and it has yet to be shown whether estimating GFR from cystatin C compared with creatinine-based methods improves risk stratification for adverse vascular and renal outcomes in subjects with diabetes.35,36 In the early stages of T1DM and in those with normoalbuminuria and without retinopathy, hyperfiltration may cause lower plasma tHcy, but we excluded patients with hyperfiltration from the study.37 Vitamins B12 and B6 and especially folate are important determinants of plasma tHcy.38 Determinations of these factors are needed in evaluating hyperhomocysteinemia and its possible etiology. However, the majority of our patients had tHcy in the reference range, and previous studies documented that, in most studies of plasma tHcy levels and T1DM serum, B12 and folate levels were in the normal range and those with poor folate status did not account for the tHcy-retinopathy relationship.2,15 In addition, the most common genetic cause of elevated tHcy is the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene, and a previous study on a
large cohort of T1DM found that C677T mutation did not significantly affect plasma tHcy levels.39 Furthermore, the MTHFR polymorphism has been observed more frequently in diabetic patients with nephropathy and it is not associated with retinal vein occlusion, an important cause of vision loss.4,40–42 The present study and the results have a number of potential limitations, in addition to those previously mentioned. First, our study was cross-sectional, which limited our ability to infer a causal relation between tHcy and risk for the development of retinopathy. Second, our analyses were based on a single measurement of tHcy and serum creatinine that may not reflect the relationship over time. Third, the methods used for diagnosis of retinopathy, estimated GFR, as well as tHcy—measuring either directly or after derivatization—may influence the final results, making comparisons of findings between studies difficult. In our study, we measured tHcy levels directly and photodocumented retinopathy status according to the EURODIAB protocol. In conclusion, we have shown that tHcy is associated with retinopathy in normoalbuminuric T1DM with normal renal function. The independent relationship between tHcy and retinopathy may have implications for the management of diabetes, such as dietary modulation of homocysteine level, because interventions with a combination of vitamins (B6, B9, and B12) have beneficial effects in subjects with nonproliferative diabetic retinopathy.43,44 Prospective studies are needed to confirm whether higher tHcy in normoalbuminuric T1DM patients has predictive value for the development of retinopathy.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Soedamah-Muthu SS, Chaturvedi N, Teerlink T, et al. Plasma homocysteine and microvascular and macrovascular complications in type 1 diabetes: A cross-sectional nested case-control study. J Intern Med 2005;258:450–459. 2. Wotherspoon F, Laight DW, Shaw KM, Cummings MH. Homocysteine, endothelial dysfunction and oxidative stress in type 1 diabetes mellitus. Br J Diabetes Vasc Dis 2003;3:334–340. 3. Hofmann MA, Kohl B, Zumbach MS, et al. Hyperhomocysteinemia and endothelial dysfunction in IDDM. Diabetes Care 1998;21:841–848. 4. Vaccaro O, Perna AF, Mancini FP, et al. Plasma homocysteine and microvascular complications in type 1 diabetes. Nutr Metab Cardiovasc Dis 2000;10:297–304. 5. Agardh E, Hultberg B, Agardh CD. Severe retinopathy in type 1 diabetic patients is not related to the level of plasma homocysteine. Scand J Clin Lab Invest 2000;60:169–174. Seminars in Ophthalmology
Semin Ophthalmol Downloaded from informahealthcare.com by Michigan University on 01/12/15 For personal use only.
Homocysteine and Retinopathy in Type 1 Diabetes 6. Saeed BO, Nixon SJ, White AJ, et al. Fasting homocysteine levels in adults with type 1 diabetes and retinopathy. Clin Chim Acta 2004;341:27–32. 7. Neugebauer S, Tarnow L, Stehouwer C, et al. Total plasma homocysteine is associated with hypertension in type 1 diabetic patients. Diabetologia 2002;45:1315–1324. 8. Buysschaert M, Jamart J, Dramais AS, et al. Micro- and macrovascular complications and hyperhomocysteinaemia in type 1 diabetic patients. Diabetes Metab 2001;27:655–659. 9. Veldman BAJ, Vervoort G, Blom H, Smits P. Reduced plasma total homocysteine concentrations in type 1 diabetes mellitus is determined by increased renal clearance. Diabet Med 2005;22:301–305. 10. Chen YF, Li PL, Zou AP. Effect of hyperhomocysteinemia on plasma or tissue adenosine levels and renal function. Circulation 2002;106:1275–1281. 11. Chico A, Perez A, Cordoba A, et al. Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: A new link between diabetic nephropathy and cardiovascular disease? Diabetologia 1998;41: 684–693. 12. Pavia C, Ferrer I, Valls C, et al. Total homocysteine in patients with type 1 diabetes. Diabetes Care 2000; 23:84–87. 13. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet 2010;376:124–136. 14. Grunwald JE, Alexander J, Ying GS, et al. Retinopathy and chronic kidney disease in the Chronic Renal Insufficiency Cohort (CRIC) study. Arch Ophthalmol 2012;130:1136–1144. 15. Brazionis L, Rowley K, Itsiopoulos C, et al. Homocysteine and diabetic retinopathy. Diabetes Care 2008;31:50–56. 16. Cho HC. The relationship among homocysteine, bilirubin, and diabetic retinopathy. Diabetes Metab J 2011;35:595–601. 17. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150:604–612. 18. Vucˇic´ LM, Radisˇic´ BV, Bozˇicˇevic´ S, et al. Estimating glomerular filtration rate (GFR) in diabetes: The performance of MDRD and CKD-EPI equations in patients with various degrees of albuminuria. Clin Biochem 2012;45: 1694–1696. 19. Aldington SJ, Kohner EM, Meuer S, et al. Methodology for retinal photography and assessment of diabetic retinopathy: The EURODIAB IDDM Complications Study. Diabetologia 1995;38:437–444. 20. Hankey GJ, Eikelboom JW, Ho WK, van Bockxmeer FM. Clinical usefulness of plasma homocysteine in vascular disease. Med J Aust 2004;181:314–318. 21. Lahiri KD, Dutta J, Datta H, Das HN. Hyperhomocysteinemia, as an independent risk factor for retinal venous occlusion in an Indian population. Indian J Clin Biochem 2013;28:61–64. 22. Sarici AM, Yetik H, Akar S, Arvas S. The association between serum homocysteine levels and retinopathy of prematurity. J Int Med Res 2012;40:1912–1918. 23. Gopinath B, Flood VM, Rochtchina E, et al. Homocysteine, folate, vitamin B-12, and 10-y incidence of age-related macular degeneration. Am J Clin Nutr 2013;98:129–135. 24. Aydin E, Demir HD, Ozyurt H, Etikan I. Association of plasma homocysteine and macular edema in type 2 diabetes mellitus. Eur J Ophthalmol 2008;18:226–232. 25. Lim CP, Loo AV, Khaw KW, et al. Plasma, aqueous and vitreous homocysteine levels in proliferative diabetic retinopathy. Br J Ophthalmol 2012;96:704–707. 26. Omae T, Nagaoka T, Tanano I, Yoshida A. Homocysteine inhibition of endothelium-dependent nitric oxide-mediated
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27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38. 39.
40.
41.
42.
43.
44.
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dilation of porcine retinal arterioles via enhanced superoxide production. Invest Ophthalmol Vis Sci 2013;54: 2288–2295. Chow K, Cheung F, Lao TT, O K. Effect of homocysteine on the production of nitric oxide in endothelial cells. Clin Exp Pharmacol Physiol 1999;26:817–818. Bellamy MF, McDowell IF, Ramsey MW, et al. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 1998;98:1848–1852. Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metab 2005;6:27–36. Maeda M, Fujio Y, Azuma J. MTHFR gene polymorphism and diabetic retinopathy. Curr Diabetes Rev 2006;2: 467–476. Poddar R, Sivasubramanian N, DiBello PM, et al. Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells. Circulation 2001;103:2717–2723. Berg K, Malinow MR, Kierulf P, Upson B. Population variation and genetic of plasma homocyst(e)ine level. Clin Genet 1992;41:315–321. Hoffman MA, Kohl B, Zumbach MS, et al. Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM. Diabetes Care 1998;21:841–848. Norlund L, Grubb A, Fex G, et al. The increase of plasma homocysteine concentrations with age is partly due to the deterioration of renal function as determined by plasma cystatin C. Clin Chem Lab Med 1998;36:175–178. Macisaac RJ, Premaratne E, Jerums G. Estimating glomerular filtration rate in diabetes using serum cystatin C. Clin Biochem Rev 2011;32:61–67. Wiesli P, Schwegler B, Spinas GA, Schmid C. Serum cystatin C is sensitive to small changes in thyroid function. Clin Chem Acta 2003;338:87–90. Wollesen F, Brattstrom L, Refsum H, et al. Plasma total homocysteine and cysteine in relation to glomerular filtration rate in diabetes mellitus. Kidney Int 1999;55: 1028–1035. Ubbink JB. Vitamin nutrition status and homocysteine: An atherogenic risk factor. Nutr Rev 1994;52:383–393. Kaye J, Stanton K, McCann V, Vasikaran S. Homocysteine, folate, MTHFR genotype and vascular morbidity in diabetic subjects. Clin Sci 2002;102:631–637. Shcherbak N, Shutsleaya Z, Sheridina A, et al. MTHFR gene polymorphism as a risk factor for diabetic nephropathy in IDDM patients. Mol Genet Metab 1999;68:375–378. Ukinc K, Ersoz HO, Karahan C, et al. Methyltetrahydrofolate reductase C677T gene mutation and hyperhomocysteinemia as a novel risk factor for diabetic nephropathy. Endocrine 2009;36:255–261. McGimpsey SJ, Woodside JV, Cardwell C, et al. Homocysteine, methylenetetrahydrofolate reductase C677T polymorphism, and risk of retinal vein occlusion: A meta-analysis. Ophthalmology 2009;116:1778–1787. Rowley KG, Lee AJ, Yarmirr D, O’Dea K. Homocysteine concentrations lowered following dietary intervention in an aboriginal community. Asia Pac J Clin Nutr 2003;12: 92–95. Smolek MK, Notaroberto NF, Jaramillo AG, Pradillo LR. Intervention with vitamins in patients with nonproliferative diabetic retinopathy: A pilot study. Clin Ophthalmol 2013;7:1451–1458.
ORIGINAL ARTICLE
Plasma Homocysteine is Associated with Retinopathy in Type 1 Diabetic Patients in the Absence of Nephropathy
Semin Ophthalmol Downloaded from informahealthcare.com by Michigan University on 01/12/15 For personal use only.
Tomislav Bulum, Kristina Blaslov, and Lea Duvnjak Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic Diseases, University Hospital Merkur, Medical School, University of Zagreb, Zagreb, Croatia
ABSTRACT Introduction: Previous cross-sectional studies suggested that plasma total homocysteine (tHcy) is associated with retinopathy in patients with type 1 diabetes (T1DM) only in cases of impaired renal function. The objective of this study was to examine whether there is an independent relationship between tHcy and retinopathy in normoalbuminuric T1DM patients with normal estimated glomerular filtration rate (eGFR). Methods: The study included 163 normoalbuminuric patients with T1DM and normal renal function (eGFR 460 125 ml min 1 1.73 m 2). Urinary albumin excretion rate (UAE) was measured from at least two 24 h urine samples. Photodocumented retinopathy status was made according to the EURODIAB protocol. tHcy level was measured with the chemiluminescent immunoassay. Results: Retinopathy was present in 48% of normoalbuminuric patients. Patients with retinopathy were older (49 vs 42 years, p = 0.001), had higher systolic blood pressure (130 vs 120 mmHg, p = 0.001), triglycerides (0.89 vs 0.77 mmol/L, p = 0.01), tHcy (9.8 vs 9.1 mmol/L, p = 0.04), and lower eGFR (100 vs 106 ml min 1 1.73 m 2, p = 0.03). In multivariate logistic regression analysis, after adjustment for variables that reached statistical significance in the univariate analysis, only tHcy was significantly associated with a risk of retinopathy in our subjects (p = 0.02), with odds ratios of 1.02 to 1.43. Conclusion: These data suggest that tHcy is independently associated with retinopathy in normoalbuminuric T1DM with normal eGFR. The mechanisms relating tHcy and retinopathy in T1DM are not clear. Prospectives studies are needed to confirm whether higher tHcy in normoalbuminuric T1DM patients has predictive value for development of retinopathy. Keywords: Homocysteine, microvascular complications, retinopathy, type 1 diabetes
INTRODUCTION
increased levels of tHcy and cardiovascular disease was reported in T1DM.7,8 tHcy is independently associated with glomerular filtration rate (GFR) and albuminuria.1,2 The independent relationship between GFR and tHcy is based on a relationship between declining GFR and increasing tHcy levels in T1DM.9 In addition, experimental findings suggested that Hcy may directly impair glomerular filtration.10 Diabetic patients with microalbuminuria and normal GFR also have mild hyperhomocysteinaemia.6,11 On the other hand, it is assumed that relative hyperfiltration in younger normoalbuminuric T1DM is responsible for similar
Moderate hyperhomocysteinaemia has been observed in studies of patients with type 1 diabetes (T1DM) in which the plasma total homocysteine (tHcy) levels are independent of vitamin status and reflect the heterogeneous nature of the patients, including a variety of microvascular and macrovascular complications.1,2 Most previous cross-sectional studies exploring the relationship between tHcy and microvascular complications reported positive associations between tHcy and nephropathy,1,3,4 but not for retinopathy.5,6 In addition, no independent relationship between
Received 9 January 2014; accepted 1 April 2014; published online 2 May 2014 Correspondence: Tomislav Bulum, MD, PhD, Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic Diseases, University Hospital Merkur, School of Medicine, Dugi dol 4a, University of Zagreb, Zagreb, Croatia. E-mail: [email protected]
1
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tHcy levels in those with and without microvascular complications.12 Identification of the determinants of the onset of diabetic retinopathy is essential for reducing the morbidity and mortality associated with diabetes because diabetic retinopathy is one of the leading causes of visual impairment and blindness in T1DM.13 It is assumed that retinopathy and nephropathy, as the most important microvascular complications in diabetes, occur at the same time and that retinovascular pathology reflects renal disease.14 However, it is suggested that higher tHcy is associated with retinopathy in T1DM only in cases of impaired renal function measured with GFR and/or albuminuria,1,2,5,6,12 although recent studies including type 2 diabetic patients found higher tHcy in individuals with retinopathy, independent of renal dysfunction and other major risk factors.15,16 The objective of this study was to examine whether there is an independent relationship between tHcy and retinopathy in normoalbuminuric T1DM patients with normal GFR.
SUBJECTS, MATERIALS, AND METHODS This cross-sectional study included 163 patients with T1DM, defined as an onset of diabetes before the age of 35 years, positive autoantibodies, and permanent insulin treatment initiated within one year of diagnosis. The study included patients with the following characteristics: age of 18–65 years, minimum duration of type 1 diabetes of one year, no medical history of cardiovascular diseases or electrocardiogram (ECG) evidence of ischemic heart disease, absence of any systemic disease, and absence of any infections in the previous month. Those with microalbuminuria (urinary albumin excretion rate (UAE) 305300 mg/24h), macroalbuminuria (UAE 300 mg/24h), impaired estimated GFR (less than 60 ml min 1 1.73 m 2), and hyperfiltration (estimated GFR over 125 ml min 1 1.73 m 2) were also excluded from the study. All subjects were studied in the morning after an overnight fast. Basic anthropometric measurements were performed on all study subjects, including body mass index (BMI) and waist to hip ratio (WHR). Blood pressure was measured on both arms twice in the sitting position with a mercury sphygmomanometer, after a resting period of 10 minutes, and was expressed in mmHg. UAE was measured from at least two 24 h urine samples and determined as the mean of 24 h urine collections. Patients performed collections on two consecutive days to minimize variability. Normoalbuminuria was defined as a UAE530 mg/24h. Serum creatinine was measured in a fasting blood sample. Data on serum creatinine levels, age, sex, and race were used to calculate the
estimated GFR using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula, which has been shown to be accurate in determining renal function in diabetic patients with normal renal function.17,18 All subjects were confirmed to be free of urinary tract infections. Retinopathy was diagnosed by binocular indirect slit lamp fundoscopy and fundus photography after mydriasis with eye drops containing 0.5% tropicamide and 5% phenylephrine. Color fundus photographs of two fields of both eyes were taken with a suitable 45 fundus camera (VISUCAM, Zeiss) according to the EURODIAB retinal photography methodology.19 In each patient, the ‘‘worse’’ eye was graded for retinopathy using fundus photographs. Fasting venous blood samples were collected in the morning between 08:00 and 09:30 hours after an overnight fast for the determination of HbA1c, total LDL, HDL cholesterol, triglycerides, and tHcy. Microalbumin and HbA1c were measured spectrophotometrically by turbidimetric immuno-inhibition (Olympus AU600, Beckman-Coulter, USA). Results of HbA1c (%) are expressed in the DCCT-equivalent. Serum tHcy level was measured with the Chemiluminescent Immunoassay (CLIA). Glucose, cholesterol, and triglycerides in serum were measured by an enzymatic colorimetric method (Olympus AU600, Beckman-Coulter, USA). Complete blood count was determined on an automatic blood counter (Advia 120, Siemens Diagnostic Solutions, USA). The study protocol complies with the Declaration of Helsinki as well as with local institutional guidelines, and was approved by the local ethics committees. Data are expressed as means ± SD for normally distributed values, as median with range for nonnormally distributed values, and percentage. Differences between groups were examined, depending on the nature of the data, parametric (t-test), or nonparametric tests (Mann-Whitney). All of the variables that reached statistical significance in the univariate analysis were included in multivariate logistic regression in order to assess associations of tHcy and risk of retinopathy. Level of statistical significance was chosen to be = 0.05. Statistical analysis was performed by statistical package SPSS, version 17.0 for Windows.
RESULTS The characteristics of the study subjects are listed in Table 1. The average age was approximately 47 years, most were not overweight, and 51% of subjects were male. Mean/median values of BMI, WHR, systolic blood pressure, diastolic blood pressure, LDL, HDL cholesterol, and triglycerides were within the normal range for patients with T1DM, with slightly elevated Seminars in Ophthalmology
Homocysteine and Retinopathy in Type 1 Diabetes TABLE 1. Clinical and metabolic characteristics of all patients.
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Variable
Value
Age (years) Sex (m/f, %) Body mass index (kg/m2) Waist to hip ratio HbA1c (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L) Triglycerides (mmol/L) Serum creatinine (mmol/L) eGFR (ml min 11.73 m 2) Urinary albumin excretion (mg/24 h) Retinopathy (no/yes, %) Homocysteine (mmol/L)
47 (23–65) 54/46 25 (18–38) 0.82 ± 0.07 6.7 (4.2–10.6) 120 (90–180) 80 (55–105) 5.1 ± 0.9 2.8 ± 0.8 1.7 (0.6–3.4) 0.84 (0.3–4.3) 73 (42–120) 104 (60–124) 12.2 (1.2–29.8) 52/48 9.5 (1.7–19.7)
eGFR, estimated glomerular filtration rate.
No (n = 85)
Yes (n = 78)
Age (years) 42 (23–65) 49 (26–65) Sex (m/f) 45/40 43/35 24 (18–38) 25 (18–34) Body mass index (kg/m2) Waist to hip ratio 0.81 ± 0.07 0.83 ± 0.07 HbA1c (%) 6.5 (4.6–10.6) 6.8 (4.2–10.2) SBP (mmHg) 120 (90–180) 130 (105–160) DBP (mmHg) 80 (60–105) 80 (55–100) Total cholesterol 5.0 ± 0.8 5.2 ± 1.0 (mmol/L) LDL cholesterol 2.7 ± 0.7 3.0 ± 0.9 (mmol/L) HDL cholesterol 1.8 (0.6–3.4) 1.7 (0.8–2.7) (mmol/L) Triglycerides (mmol/L) 0.77 (0.2–4.3) 0.89 (0.4–2.8) Serum creatinine 73 (56–105) 73 (42–120) (mmol/L) 106 (71–125) 100 (60–123) eGFR (ml min 1 1.73 m 2) UAE (mg/24 h) 11.5 (2.1–29.8) 13.2 (1.2–29.6) Homocysteine (mmol/L) 9.1 (1.7–19.7) 9.8 (6.0–19.0)
p 0.001 0.7 0.6 0.1 0.05 0.001 0.6 0.2 0.5 0.2 0.01 0.7 0.03 0.2 0.04
SBP, systolic blood pressure; DBP, diastolic blood pressure; UAE, urinary albumin excretion rate.
levels of total cholesterol. The majority of patients had no retinopathy (48% with retinopathy). The mean GFR estimated by the CKD-EPI was 104 ml min 1 1.73 m 2. Clinical and metabolic characteristics of patients with and without retinopathy are presented in Table 2. Patients with retinopathy were older, had higher systolic blood pressure, triglycerides, tHcy, and lower estimated GFR (p50.05 for all). BMI, WHR, diastolic blood pressure, total LDL, HDL cholesterol, serum creatinine, and UAE levels did not differ between the two groups. !
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TABLE 3. Multivariate logistic regression analysis of risk factors for development of retinopathy in normoalbuminuric type 1 diabetic patients. Independent variable
Odds ratio (95% CI)
Age Homocysteine eGFR Systolic blood pressure Triglycerides
0.96 1.21 1.00 1.01 1.05
(0.91–1.03) (1.02–1.43) (0.7–1.03) (0.98–1.05) (0.99–3.3)
p 0.3 0.02 0.8 0.2 0.5
eGFR, estimated glomerular filtration rate.
In multivariate logistic regression analysis, after adjustment for variables that reached statistical significance in the univariate analysis, only tHcy was significantly associated with risk of retinopathy in our subjects (p = 0.02), with odds ratios of 1.02 to 1.43 (Table 3).
DISCUSSION
TABLE 2. Clinical and metabolic characteristics of patients without and with retinopathy. Retinopathy
3
Previous cross-sectional studies suggested that higher tHcy is associated with retinopathy in T1DM only in cases of impaired renal function measured with GFR and/or albuminuria.1,2,5,6,12 Our patients with retinopathy had significantly higher metabolic risk factors including age, systolic blood pressure, triglycerides, tHcy, and lower estimated GFR. The difference in the mean tHcy concentration between subjects with and without retinopathy was relatively small but significant, and the median tHcy concentration for both subjects was below the upper limit for adults, as previously documented in diabetic and nondiabetic subjects with retinopathy.15,20 In multiple logistic regression analysis, only tHcy was independently related to retinopathy in our normoalbuminuric T1DM with normal renal function. In support of these data, recent studies, including a similar number of type 2 diabetic patients, found higher tHcy in individuals with retinopathy, independent of renal dysfunction and other major risk factors.15,16 Furthermore, hyperhomocysteinemia is an independent risk factor for retinal venous occlusion, retinopathy of prematurity, age-related macular degeneration, and macular edema.21 24 Higher homocysteine levels were observed not only in blood plasma, but also in aqueous of eyes with proliferative diabetic retinopathy.25 The mechanisms relating tHcy and retinopathy in T1DM are not clear, although proposals include oxidative stress, endothelial damage, and decreased nitric oxide bioavailability.2 Homocysteine impairs retinal endothelial function regarding nitric oxidemediated vasodilatation in the retinal arterioles and reduces vasodilation of the retinal arterioles in response to the calcium, indicating that hyperhomocysteinemia is implicated in the pathogenesis of
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ocular vascular disorders.26 Moreover, it has been shown that the nitric oxide concentration can decrease after acute and chronic exposure of homocysteine to human vein endothelial cells.27 Homocysteine is a sulfur-containing amino acid that forms during metabolism of methionine, an essential amino acid, and hyperhomocysteinemia induced by oral administration of methionine impaired vascular endothelial function in human brachial arteries.28 Homocysteine can also induce endothelial toxicity by reactive oxygen species (ROS) generated via both enzymatic and nonenzymatic mechanisms.29 Furthermore, accumulating evidence suggests that homocysteine activates vascular inflammation through inflammatory cytokines.30,31 Renal failure is an important cause of hyperhomocysteinaemia because the kidneys play a major role in the metabolism of Hcy.1,2,9,10,32 In our study, although it included normoalbuminuric patients with normal estimated GFR, those with retinopathy had slightly higher UAE and significantly lower estimated GFR. Higher tHcy in patients with retinopathy may be due to (although non-significant) higher UAE because previous studies demonstrated a strong correlation between UAE and retinopathy, even in the absence of chronic kidney disease.6,11,33 On the other hand, lower estimated GFR probably results from the synthesis of the precursor of creatinine and donation of methyl groups formed in the transformation of methionine to Hcy.34 It can be argued that we should measure kidney function with gold standard methods like cystatin C in this specific population (with special regard to hyperfiltration). However, cystatin C is impractical in studies with large numbers of subjects; it should not be used for assessment of GFR in patients with thyroid disorders, which are common in patients with T1DM, and it has yet to be shown whether estimating GFR from cystatin C compared with creatinine-based methods improves risk stratification for adverse vascular and renal outcomes in subjects with diabetes.35,36 In the early stages of T1DM and in those with normoalbuminuria and without retinopathy, hyperfiltration may cause lower plasma tHcy, but we excluded patients with hyperfiltration from the study.37 Vitamins B12 and B6 and especially folate are important determinants of plasma tHcy.38 Determinations of these factors are needed in evaluating hyperhomocysteinemia and its possible etiology. However, the majority of our patients had tHcy in the reference range, and previous studies documented that, in most studies of plasma tHcy levels and T1DM serum, B12 and folate levels were in the normal range and those with poor folate status did not account for the tHcy-retinopathy relationship.2,15 In addition, the most common genetic cause of elevated tHcy is the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene, and a previous study on a
large cohort of T1DM found that C677T mutation did not significantly affect plasma tHcy levels.39 Furthermore, the MTHFR polymorphism has been observed more frequently in diabetic patients with nephropathy and it is not associated with retinal vein occlusion, an important cause of vision loss.4,40–42 The present study and the results have a number of potential limitations, in addition to those previously mentioned. First, our study was cross-sectional, which limited our ability to infer a causal relation between tHcy and risk for the development of retinopathy. Second, our analyses were based on a single measurement of tHcy and serum creatinine that may not reflect the relationship over time. Third, the methods used for diagnosis of retinopathy, estimated GFR, as well as tHcy—measuring either directly or after derivatization—may influence the final results, making comparisons of findings between studies difficult. In our study, we measured tHcy levels directly and photodocumented retinopathy status according to the EURODIAB protocol. In conclusion, we have shown that tHcy is associated with retinopathy in normoalbuminuric T1DM with normal renal function. The independent relationship between tHcy and retinopathy may have implications for the management of diabetes, such as dietary modulation of homocysteine level, because interventions with a combination of vitamins (B6, B9, and B12) have beneficial effects in subjects with nonproliferative diabetic retinopathy.43,44 Prospective studies are needed to confirm whether higher tHcy in normoalbuminuric T1DM patients has predictive value for the development of retinopathy.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Soedamah-Muthu SS, Chaturvedi N, Teerlink T, et al. Plasma homocysteine and microvascular and macrovascular complications in type 1 diabetes: A cross-sectional nested case-control study. J Intern Med 2005;258:450–459. 2. Wotherspoon F, Laight DW, Shaw KM, Cummings MH. Homocysteine, endothelial dysfunction and oxidative stress in type 1 diabetes mellitus. Br J Diabetes Vasc Dis 2003;3:334–340. 3. Hofmann MA, Kohl B, Zumbach MS, et al. Hyperhomocysteinemia and endothelial dysfunction in IDDM. Diabetes Care 1998;21:841–848. 4. Vaccaro O, Perna AF, Mancini FP, et al. Plasma homocysteine and microvascular complications in type 1 diabetes. Nutr Metab Cardiovasc Dis 2000;10:297–304. 5. Agardh E, Hultberg B, Agardh CD. Severe retinopathy in type 1 diabetic patients is not related to the level of plasma homocysteine. Scand J Clin Lab Invest 2000;60:169–174. Seminars in Ophthalmology
Semin Ophthalmol Downloaded from informahealthcare.com by Michigan University on 01/12/15 For personal use only.
Homocysteine and Retinopathy in Type 1 Diabetes 6. Saeed BO, Nixon SJ, White AJ, et al. Fasting homocysteine levels in adults with type 1 diabetes and retinopathy. Clin Chim Acta 2004;341:27–32. 7. Neugebauer S, Tarnow L, Stehouwer C, et al. Total plasma homocysteine is associated with hypertension in type 1 diabetic patients. Diabetologia 2002;45:1315–1324. 8. Buysschaert M, Jamart J, Dramais AS, et al. Micro- and macrovascular complications and hyperhomocysteinaemia in type 1 diabetic patients. Diabetes Metab 2001;27:655–659. 9. Veldman BAJ, Vervoort G, Blom H, Smits P. Reduced plasma total homocysteine concentrations in type 1 diabetes mellitus is determined by increased renal clearance. Diabet Med 2005;22:301–305. 10. Chen YF, Li PL, Zou AP. Effect of hyperhomocysteinemia on plasma or tissue adenosine levels and renal function. Circulation 2002;106:1275–1281. 11. Chico A, Perez A, Cordoba A, et al. Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: A new link between diabetic nephropathy and cardiovascular disease? Diabetologia 1998;41: 684–693. 12. Pavia C, Ferrer I, Valls C, et al. Total homocysteine in patients with type 1 diabetes. Diabetes Care 2000; 23:84–87. 13. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet 2010;376:124–136. 14. Grunwald JE, Alexander J, Ying GS, et al. Retinopathy and chronic kidney disease in the Chronic Renal Insufficiency Cohort (CRIC) study. Arch Ophthalmol 2012;130:1136–1144. 15. Brazionis L, Rowley K, Itsiopoulos C, et al. Homocysteine and diabetic retinopathy. Diabetes Care 2008;31:50–56. 16. Cho HC. The relationship among homocysteine, bilirubin, and diabetic retinopathy. Diabetes Metab J 2011;35:595–601. 17. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150:604–612. 18. Vucˇic´ LM, Radisˇic´ BV, Bozˇicˇevic´ S, et al. Estimating glomerular filtration rate (GFR) in diabetes: The performance of MDRD and CKD-EPI equations in patients with various degrees of albuminuria. Clin Biochem 2012;45: 1694–1696. 19. Aldington SJ, Kohner EM, Meuer S, et al. Methodology for retinal photography and assessment of diabetic retinopathy: The EURODIAB IDDM Complications Study. Diabetologia 1995;38:437–444. 20. Hankey GJ, Eikelboom JW, Ho WK, van Bockxmeer FM. Clinical usefulness of plasma homocysteine in vascular disease. Med J Aust 2004;181:314–318. 21. Lahiri KD, Dutta J, Datta H, Das HN. Hyperhomocysteinemia, as an independent risk factor for retinal venous occlusion in an Indian population. Indian J Clin Biochem 2013;28:61–64. 22. Sarici AM, Yetik H, Akar S, Arvas S. The association between serum homocysteine levels and retinopathy of prematurity. J Int Med Res 2012;40:1912–1918. 23. Gopinath B, Flood VM, Rochtchina E, et al. Homocysteine, folate, vitamin B-12, and 10-y incidence of age-related macular degeneration. Am J Clin Nutr 2013;98:129–135. 24. Aydin E, Demir HD, Ozyurt H, Etikan I. Association of plasma homocysteine and macular edema in type 2 diabetes mellitus. Eur J Ophthalmol 2008;18:226–232. 25. Lim CP, Loo AV, Khaw KW, et al. Plasma, aqueous and vitreous homocysteine levels in proliferative diabetic retinopathy. Br J Ophthalmol 2012;96:704–707. 26. Omae T, Nagaoka T, Tanano I, Yoshida A. Homocysteine inhibition of endothelium-dependent nitric oxide-mediated
!
2014 Informa Healthcare USA, Inc.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38. 39.
40.
41.
42.
43.
44.
5
dilation of porcine retinal arterioles via enhanced superoxide production. Invest Ophthalmol Vis Sci 2013;54: 2288–2295. Chow K, Cheung F, Lao TT, O K. Effect of homocysteine on the production of nitric oxide in endothelial cells. Clin Exp Pharmacol Physiol 1999;26:817–818. Bellamy MF, McDowell IF, Ramsey MW, et al. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 1998;98:1848–1852. Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metab 2005;6:27–36. Maeda M, Fujio Y, Azuma J. MTHFR gene polymorphism and diabetic retinopathy. Curr Diabetes Rev 2006;2: 467–476. Poddar R, Sivasubramanian N, DiBello PM, et al. Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells. Circulation 2001;103:2717–2723. Berg K, Malinow MR, Kierulf P, Upson B. Population variation and genetic of plasma homocyst(e)ine level. Clin Genet 1992;41:315–321. Hoffman MA, Kohl B, Zumbach MS, et al. Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM. Diabetes Care 1998;21:841–848. Norlund L, Grubb A, Fex G, et al. The increase of plasma homocysteine concentrations with age is partly due to the deterioration of renal function as determined by plasma cystatin C. Clin Chem Lab Med 1998;36:175–178. Macisaac RJ, Premaratne E, Jerums G. Estimating glomerular filtration rate in diabetes using serum cystatin C. Clin Biochem Rev 2011;32:61–67. Wiesli P, Schwegler B, Spinas GA, Schmid C. Serum cystatin C is sensitive to small changes in thyroid function. Clin Chem Acta 2003;338:87–90. Wollesen F, Brattstrom L, Refsum H, et al. Plasma total homocysteine and cysteine in relation to glomerular filtration rate in diabetes mellitus. Kidney Int 1999;55: 1028–1035. Ubbink JB. Vitamin nutrition status and homocysteine: An atherogenic risk factor. Nutr Rev 1994;52:383–393. Kaye J, Stanton K, McCann V, Vasikaran S. Homocysteine, folate, MTHFR genotype and vascular morbidity in diabetic subjects. Clin Sci 2002;102:631–637. Shcherbak N, Shutsleaya Z, Sheridina A, et al. MTHFR gene polymorphism as a risk factor for diabetic nephropathy in IDDM patients. Mol Genet Metab 1999;68:375–378. Ukinc K, Ersoz HO, Karahan C, et al. Methyltetrahydrofolate reductase C677T gene mutation and hyperhomocysteinemia as a novel risk factor for diabetic nephropathy. Endocrine 2009;36:255–261. McGimpsey SJ, Woodside JV, Cardwell C, et al. Homocysteine, methylenetetrahydrofolate reductase C677T polymorphism, and risk of retinal vein occlusion: A meta-analysis. Ophthalmology 2009;116:1778–1787. Rowley KG, Lee AJ, Yarmirr D, O’Dea K. Homocysteine concentrations lowered following dietary intervention in an aboriginal community. Asia Pac J Clin Nutr 2003;12: 92–95. Smolek MK, Notaroberto NF, Jaramillo AG, Pradillo LR. Intervention with vitamins in patients with nonproliferative diabetic retinopathy: A pilot study. Clin Ophthalmol 2013;7:1451–1458.
