Asymmetric dimethylarginine is associated with macrovascular disease and total homocysteine in patients with type 2 diabetes

Atherosclerosis 189 (2006) 236–240

Asymmetric dimethylarginine is associated with macrovascular disease and total homocysteine in patients with type 2 diabetes Katarzyna Krzyzanowska a,∗ , Friedrich Mittermayer b , Walter Krugluger c , Christoph Schnack a , Martin Hofer a , Michael Wolzt b , Guntram Schernthaner a a

Department of Internal Medicine I, Rudolfstiftung Hospital, Juchgasse 25, 1030 Vienna, Austria b Department of Clinical Pharmacology, Medical University Vienna, Vienna, Austria c Central Laboratory, Rudolfstiftung Hospital, Vienna, Austria

Received 23 November 2005; received in revised form 12 December 2005; accepted 13 December 2005 Available online 18 January 2006

Abstract Background: The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and total homocysteine (tHcy) are elevated in patients at increased cardiovascular risk. Patients with type 2 diabetes (T2DM) have higher incidence of macrovascular disease than the general population. Recent reports suggest a relationship between tHcy and ADMA. To evaluate the connection between ADMA and tHcy and macrovascular disease, we determined both risk factors in T2DM patients with and without macrovascular disease. Subjects and methods: Plasma concentrations of ADMA and tHcy were cross-sectionally determined in 136 T2DM patients. Fifty-five patients had macrovascular disease defined by history of stroke, myocardial infarction, coronary heart disease or peripheral arterial occlusive disease. Logistic regression analysis was performed to examine the relationship between macrovascular disease and these risk factors. Potential confounders were identified by significant Spearman rank correlation coefficients. Results: In unadjusted models ADMA (per 0.1 ␮mol/l) and tHcy (per 5 ␮mol/l) were both significantly related to macrovascular disease (OR = 1.63, 95% CI: 1.21–2.19 and OR = 1.49, 95% CI: 1.04–2.14). In multivariate models, ADMA was significantly associated with macrovascular disease independent of l-arginine, albumin excretion rate, tHcy and glomerular filtration rate (GFR; OR = 1.53, 95% CI: 1.04–2.26). The connection between tHcy and macrovascular disease was not independent of diastolic blood pressure, age, ADMA or GFR. Linear regression analyses revealed that ADMA, GFR and low-density lipoprotein cholesterol were independent predictors for tHcy. Conclusion: ADMA is associated with macrovascular disease independent of tHcy and traditional cardiovascular risk factors in patients with T2DM. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Asymmetric dimethylarginine; Homocysteine; Macrovascular disease; Type 2 diabetes mellitus

1. Introduction Elevated concentrations of the endogenous nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) or a decreased l-arginine/ADMA ratio have been described in patients at increased cardiovascular risk [1–4]. ADMA predicts future cardiovascular events in patients with ∗

Corresponding author. Tel.: +43 1 711 65 2107; fax: +43 1 711 65 2109. E-mail address: [email protected] (K. Krzyzanowska). 0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.12.007

coronary artery disease or chronic renal failure [5,6]. In healthy subjects ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase (DDAH) [7]. In patients with renal failure reduced urinary elimination contributes to elevated ADMA concentrations [3]. Patients with type 2 diabetes mellitus (T2DM) have a twoto three-fold higher incidence of macrovascular atherosclerotic disease compared to the general population [8]. Elevated total homocysteine (tHcy) plasma concentrations are associated with increased cardiovascular risk [9–11], in particular, in T2DM patients [12]. The mechanisms by which tHcy is

K. Krzyzanowska et al. / Atherosclerosis 189 (2006) 236–240

involved in the development of atherosclerosis are not fully understood. In vitro and in vivo studies suggest that tHcy may cause endothelial damage, smooth muscle proliferation, platelet aggregation, enhanced coagulation and inflammation [13–16]. There is experimental evidence that tHcy and ADMA metabolism may be interrelated. S-adenosylmethionine serves as the methyldonor for arginine methylation and consequently, ADMA production and yields Sadenosylhomocysteine, which can be converted to homocysteine. In addition, elevated homocysteine can increase ADMA concentrations in cell culture experiments by inhibiting DDAH activity [17]. However, studies employing experimental hyperhomocysteinemia were inconsistent regarding ADMA plasma levels in healthy subjects [18,19]. Further, pharmacological reduction of tHcy concentrations by vitamin substitution in patients with hyperhomocysteinemia had no effect on ADMA levels [20]. Thus, the link between ADMA and tHcy in vivo is not clear. The aim of the present study was to evaluate if the cardiovascular risk factors ADMA and tHcy are associated with macrovascular disease in patients with T2DM on stable antidiabetic drug therapy.

2. Subjects and methods Clinical investigations were approved by the Institutional Review Board and were conducted in accordance with the guidelines of the Declaration of Helsinki. All subjects were carefully instructed about the aims of the study and written informed consent was obtained. In this cross-sectional study 136 patients with T2DM (mean age 65 years; 81 men) were included. All subjects were on stable antidiabetic therapy for at least 6 months with biguanides (n = 69), sulfonylurea (n = 53), glinides (n = 16), alpha-glucosidase inhibitors (n = 10) and insulin (n = 78). Antihypertensive treatment was as follows: angiotensin-converting enzyme inhibitors (n = 99), angiotensin II receptor blockers (n = 28), calcium channel blockers (n = 55), beta-blockers (n = 54) and diuretics (n = 73). Seventy-one patients were treated with statins and 10 patients received fibrates for hyperlipidaemia. Fifty-five patients (40%) had clinical evidence of macrovascular arteriosclerotic disease in their medical history defined as stroke, myocardial infarction, coronary heart disease or peripheral arterial occlusive disease. 2.1. Laboratory investigations Venous blood was drawn after an overnight fast to determine ADMA, l-arginine, tHcy and other laboratory parameters and 24 h urine collected. Blood levels of creatinine, HbA1c, blood glucose, total cholesterol, highdensity lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides and high-sensitivity C-reactive protein (hsCRP) as well as albumin excretion

237

rate (AER) were measured by standard laboratory methods. Glomerular filtration rate (GFR) was calculated with the Modification of Diet in Renal Disease Study Group formula (MDRD) [21]. For measurement of ADMA and l-arginine, plasma was subjected to cation exchange solid-phase extraction and analysed by high-performance liquid chromatography [22,23]. The coefficients of variation for inter- and intra-assay variations tested with a pooled plasma sample were below 3% for all analytes. The detection limit for dimethylarginines was 0.04 ␮mol/l. tHcy was measured using an IMX Homocysteine assay (Abbott Diagnostics, Wiesbaden, Germany). The coefficients of variation for inter- and intra-assay variations for tHcy are below 5% with this assay. 2.2. Statistical analysis The study population is described by medians and interquartile ranges for continuous variables and in terms of proportions (number of subjects) for categorical variables. Spearman rank correlations were applied to test for associations between ADMA and tHcy and continuous risk factors. The Mann–Whitney U-test was used to analyse ADMA and tHcy concentrations by sex and smoking status. To assess the relationship between ADMA or tHcy and macrovascular disease, logistic regression analysis was performed. Patients without macrovascular disease served as the reference group. Linear regression analysis was used to assess the relationship between ADMA and tHcy after adjustment for covariates. Confounding variables were chosen based on significant Spearman rank correlation coefficients. A P < 0.05 was considered the level of significance. SPSS Version 12.01 (SPSS Inc., Chicago, IL, USA) was used for all analyses.

3. Results The clinical characteristics of the study population are presented in Table 1. ADMA correlated significantly with l-arginine, tHcy (Fig. 1), AER, creatinine and GFR. tHcy was associated with age, ADMA, creatinine, GFR, LDL cholesterol and diastolic blood pressure (Table 2). These correlation analyses were performed to identify the covariates for the regression analysis. For assessment of the contribution of renal function, GFR was used as a covariate. ADMA and tHcy were comparable between male and female T2DM patients (0.57 [0.50–0.64] versus 0.58 [0.51–0.69] ␮mol/1, P = 0.5; 11.6 [9.7–14.5] versus 11.1 [8.9–16.4] ␮mol/l, P = 0.9). No difference in ADMA or tHcy was detectable between smokers and nonsmokers (0.57 [0.52–0.63] versus 0.58 [0.49–0.69] ␮mol/1, P = 0.9; 11.1 [10.2–15.0] versus 11.5 [9.5–15.7] ␮mol/l, P = 0.8). In unadjusted regression models ADMA and tHcy were both positively related with macrovascular disease. ADMA remained significantly related to macrovascular disease after

238

K. Krzyzanowska et al. / Atherosclerosis 189 (2006) 236–240

Table 1 Characteristics of the study population by vascular disease status (n = 136) Patients with macrovascular disease (n = 55) Age (year) l-Arginine (␮mol/1) ADMA (␮mol/1) Total homocysteine (␮mol/1) Body mass index (kg/m2 ) Diabetes duration (year) Albumin excretion rate (mg/24 h) Glucose (mmol/l) HbA1c (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Creatinine (mmol/l) Glomerular filtration rate (ml/min/1.73 m2 ) Triglycerides (mmol/l) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) hsCRP (mg/l) Albuminuria, % (n) Men, % (n) Smokers, % (n)

70 (62–75) 64.7 (50.2–80.1) 0.63 (0.54–0.74) 12.3 (9.7–18.7) 29.1 (26.2–34.0) 15 (7–20) 75.9 (45.0–397.9) 8.2 (6.1–11.0) 7.8 (6.7–8.8) 148 (132–160) 80 (70–90) 97.4 (79.7–123.9) 63.8 (48.4–77.6) 1.9 (1.6–2.7) 4.4 (3.9–5.3) 2.5 (2.0–3.1) 1.1 (1.0–1.3) 5.5 (2.0–11.0) 82 (45) 65 (36) 18 (10)

Patients without macrovascular disease (n = 81) 62 (56–68) 61.6 (48.5–71.8) 0.55 (0.48–0.62) 11.1 (9.6–13.1) 29.1 (26.4–33.2) 9 (5–15) 44.6 (13.5–157.7) 8.5 (7.0–10.9) 7.5 (6.8–8.4) 155 (140–166) 80 (78–90) 79.7 (70.8–97.4) 77.7 (62.1–101.5) 2.0 (1.5–3.2) 5.1 (4.3–5.8) 2.6 (2.0–3.3) 1.2 (1.0–1.4) 3.3 (1.4–6.0) 64 (52) 56 (45) 25 (20)

Values are expressed as median (interquartile range); categorical variables are expressed as proportions (number of subjects); ADMA, asymmetric dimethylarginine; HbA1c, glycated haemoglobin; LDL, low-density lipoprotein; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; glomerular filtration rate was calculated with the MDRD formula.

adjustment for l-arginine, AER, homocysteine and GFR (Table 3). tHcy remained significantly related to macrovascular disease after adjustment for LDL cholesterol but this relationship was lost after adjustment for diastolic blood pressure, age, ADMA or GFR (Table 4). To determine if ADMA independently predicts tHcy concentrations, linear regression was applied with tHcy as dependent variable. The crude model showed a ␤-coefficient of 0.42 (P < 0.0001). After adjustment for age, l-arginine, AER, GFR, LDL cholesterol and diastolic blood pressure the ␤coefficient for ADMA was 0.30 (P = 0.001). Other independent predictors for tHcy were GFR (β = −0.30, P = 0.005) and LDL cholesterol (β = −0.19, P = 0.018).

4. Discussion This study shows that ADMA is independently related to the clinical diagnosis of macrovascular atherosclerotic disease in T2DM patients. The initial positive relationship of tHcy with macrovascular atherosclerosis in these patients depends on other cardiovascular risk factors such as age or reduced GFR and ADMA. Table 2 Spearman correlation coefficients (R) between asymmetric dimethylarginine (ADMA) and homocysteine and cardiovascular risk factors (n = 136) ADMA, R (P-value) Age l-Arginine ADMA Total homocysteine Body mass index Diabetes duration Glucose HbA1c Systolic blood pressure Diastolic blood pressure Creatinine Glomerular filtration rate Albumin excretion rate Triglycerides Total cholesterol LDL-cholesterol HDL-cholesterol hsCRP

Fig. 1. Correlation between ADMA and total homocysteine in patients with type 2 diabetes mellitus (R = 0.33, P < 0.0001).

0.14 (0.105) 0.31 (0.0006) 0.33 (<0.0001) −0.08 (0.382) 0.09 (0.311) 0.002 (0.979) −0.02 (0.796) −0.072 (0.411) −0.124 (0.154) 0.31 (0.0002) −0.33 (<0.0001) 0.21 (0.014) −0.09 (0.285) −0.04 (0.646) −0.02 (0.807) 0.09 (0.294) 0.17 (0.051)

Homocysteine, R (P-value) 0.28 (0.001) 0.05 (0.554) 0.33 (<0.0001) 0.03 (0.701) 0.15 (0.079) −0.0001 (0.999) 0.04 (0.616) −0.075 (0.389) −0.177 (0.041) 0.44 (<0.0001) −0.41 (<0.0001) 0.10 (0.251) −0.04 (0.638) −0.17 (0.050) −0.18 (0.041) 0.03 (0.728) −0.01 (0.919)

HbA1c, glycated haemoglobin; LDL, low-density lipoprotein; HDL, highdensity lipoprotein; hsCRP, high-sensitivity C-reactive protein; glomerular filtration rate was calculated with the MDRD formula.

K. Krzyzanowska et al. / Atherosclerosis 189 (2006) 236–240 Table 3 Odds ratios (95% CI) for the relationship between increasing asymmetric dimethylarginine (per 0.1 ␮mol/l) concentrations and macrovascular disease

ADMA Crude Model 1 Model 2 Model 3 Model 4

No. macrovascular disease (n = 81)

Macrovascular disease (n = 55)

P-value

1.00 1.00 1.00 1.00 1.00

1.63 (1.21–2.19) 1.84 (1.31–2.60) 1.83 (1.29–2.58) 1.64 (1.14–2.38) 1.53 (1.04–2.26)

0.001 <0.0001 0.001 0.008 0.032

Model 1, adjusted for l-arginine; Model 2, Model 1 + adjustment for albumin excretion rate; Model 3, Model 2 + adjustment for total homocysteine; Model 4, Model 3 + adjustment for glomerular filtration rate estimated with the MDRD formula.

This clinical observation is in good agreement with the experimental evidence on a direct connection between ADMA and tHcy metabolism. tHcy was strongly and independently associated with ADMA in our cohort of T2DM patients. ADMA was independently associated with macrovascular disease in our study population. Adjustment for tHcy concentrations only slightly reduced the odds ratio for ADMA, which suggests that the relation of ADMA with macrovascular disease is not mediated through effects of tHcy. Although ADMA concentrations are partly determined by renal function especially in patients with renal insufficiency, ADMA remained significantly predictive for macrovascular disease after adjusting for GFR. This implies that reduced degradation by DDAH and/or increased ADMA production by protein arginine methyltransferases play an important role for ADMA and consecutively, macrovascular disease in this cohort of T2DM patients. tHcy showed a definitely weaker association with macrovascular disease than ADMA. The relationship of tHcy and macrovascular disease remained significant after adjusting for LDL cholesterol but was lost after further adjustment for diastolic blood pressure and age. Additional correction for ADMA plasma concentrations clearly reduced the odds ratio for tHcy and macrovascular disease. Adjustment for GFR furTable 4 Odds ratios (95% CI) for the relationship between increasing total homocysteine (per 5 ␮mol/l) concentrations and macrovascular disease.

Homocysteine Crude Model 1 Model 2 Model 3 Model 4 Model 5

No. macrovascular disease (n = 81)

Macrovascular disease (n = 55)

P-value

1.00 1.00 1.00 1.00 1.00 1.00

1.49 (1.04–2.14) 1.49 (1.02–2.18) 1.40 (0.94–2.06) 1.30 (0.87–1.94) 1.02 (0.65–1.60) 0.95 (0.60–1.50)

0.031 0.039 0.094 0.196 0.929 0.812

Model 1, adjusted for low-density lipoprotein cholesterol; Model 2, Model 1 + adjustment for diastolic blood pressure; Model 3, Model 2 + adjustment for age; Model 4, Model 3 + adjustment for asymmetric dimethylarginine; Model 5, Model 4 + adjustment for glomerular filtration rate estimated with the MDRD formula.

239

ther reduced the odds ratio, which implies that tHcy concentrations are related to ADMA and renal function. This finding is supported by the strong correlation between tHcy and ADMA as well as GFR. However, the molar tHcy concentrations were much higher than the molar ADMA concentrations, which attenuates the hypothesis on a direct connection between ADMA and tHcy formation. It is possible that tHcy reduces DDAH expression and activity [17]. But only ADMA was independently related to macrovascular disease in our cohort. If tHcy had been the determining factor for ADMA, tHcy should also have been more clearly related to macrovascular disease. Regarding these findings increased ADMA and tHcy might also just be an epiphenomenon. T2DM patients are at high risk for cardiovascular disease [8]. ADMA and tHcy might both contribute to this adverse risk profile. ADMA was shown to be related to carotid intima media thickness [24] and induces development of arteriosclerotic lesions in mice [25]. Hyperhomocysteinemia is also linked to increased carotid intima media thickness [26] and the development of arteriosclerosis [27]. However, the finding that the relationship of tHcy with macrovascular disease is mainly due to other cardiovascular risk factors including reduced renal function, and ADMA diminishes the importance of tHcy in our population of T2DM patients. Our study cohort also included patients with different stages of diabetic nephropathy as indicated by the presence of albuminuria. This may contribute to the missing independent relationship between tHcy and macrovascular disease because of GFR, which is an important determinant of tHcy serum concentrations [28]. As development of macrovascular disease is a process of long duration, recent changes in tHcy concentrations by reduced renal excretion might only have a small impact on the development of vascular disease. Due to the cross-sectional design the predictive value of elevated ADMA cannot be assessed from our study. But evidence from prospective studies in other patient cohorts [5,6] and animal experiments [25] suggests a causative role of ADMA for vascular lesions. The median ADMA concentrations were 15% higher in patients with macrovascular disease than in those without the disease. Although this difference might be too low to induce measurable haemodynamic effects [7], it has been shown that an ADMA increase of 11% predicts cardiovascular events in patients with coronary artery disease [5]. Longitudinal studies in T2DM patients are needed to confirm the role of ADMA for cardiovascular disease in T2DM patients. In conclusion, ADMA is associated with macrovascular disease independent of tHcy and traditional cardiovascular risk factors in patients with T2DM. The relationship between tHcy and macrovascular disease is strongly influenced by ADMA and other risk factors. ADMA predicts tHcy concentrations in T2DM patients.

240

K. Krzyzanowska et al. / Atherosclerosis 189 (2006) 236–240

References [1] B¨oger RH, Bode-B¨oger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98: 1842–7. [2] Surdacki A, Nowicki M, Sandmann J, et al. Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension. J Cardiovasc Pharmacol 1999;33:652–8. [3] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–5. [4] H¨orl WH. Atherosclerosis and uremia: significance of non-traditional risk factors. Wien Klin Wochenschr 2003;115:220–34. [5] Schnabel R, Blankenberg S, Lubos E, et al. Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the AtheroGene Study. Circ Res 2005;97:e53–9. [6] Zoccali C, Bode-Boger S, Mallamaci F, et al. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 2001;358: 2113–7. [7] Achan V, Broadhead M, Malaki M, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23:1455–9. [8] Kannel WB, McGee DL. Diabetes and cardiovascular disease. The Framingham study. JAMA 1979;241:2035–8. [9] Clarke R, Daly L, Robinson K, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149–55. [10] Coull BM, Malinow MR, Beamer N, et al. Elevated plasma homocyst(e)ine concentration as a possible independent risk factor for stroke. Stroke 1990;21:572–6. [11] Perry IJ, Refsum H, Morris RW, et al. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 1995;346:1395–8. [12] Hoogeveen EK, Kostense PJ, Jager A, et al. Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study. Kidney Int 1998;54:203–9. [13] De Bree A, Verschuren WM, Kromhout D, Kluijtmans LA, Blom HJ. Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease. Pharmacol Rev 2002;54:599–618. [14] Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem 1998;44:1833–43. [15] D’Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1–11.

[16] Araki A, Hosoi T, Orimo H, Ito H. Association of plasma homocysteine with serum interleukin-6 and C-peptide levels in patients with type 2 diabetes. Metabolism 2005;54:809–14. [17] St¨uhlinger MC, Tsao PS, Her JH, et al. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 2001;104:2569–75. [18] B¨oger RH, Lentz SR, Bode-B¨oger SM, Knapp HR, Haynes WG. Elevation of asymmetrical dimethylarginine may mediate endothelial dysfunction during experimental hyperhomocyst(e)inaemia in humans. Clin Sci (Lond) 2001;100:161–7. [19] Wanby P, Brattstrom L, Brudin L, Hultberg B, Teerlink T. Asymmetric dimethylarginine and total homocysteine in plasma after oral methionine loading. Scand J Clin Lab Invest 2003;63:347–53. [20] Ziegler S, Mittermayer F, Plank C, et al. Homocyst(e)ine-lowering therapy does not affect plasma asymmetrical dimethylarginine concentrations in patients with peripheral artery disease. J Clin Endocrinol Metab 2005;90:2175–8. [21] Levey AS, Bosch JP, Lewis JB, et al. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461–70. [22] Teerlink T, Nijveldt RJ, de Jong S, van Leeuwen PA. Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by highperformance liquid chromatography. Anal Biochem 2002;303:131–7. [23] Mittermayer F, Namiranian K, Pleiner J, Schaller G, Wolzt M. Acute Escherichia coli endotoxaemia decreases the plasma larginine/asymmetrical dimethylarginine ratio in humans. Clin Sci (Lond) 2004;106:577–81. [24] Nanayakkara PW, Teerlink T, Stehouwer CD, et al. Plasma asymmetric dimethylarginine (ADMA) concentration is independently associated with carotid intima-media thickness and plasma soluble vascular cell adhesion molecule-1 (sVCAM-1) concentration in patients with mildto-moderate renal failure. Kidney Int 2005;68:2230–6. [25] Suda O, Tsutsui M, Morishita T, et al. Asymmetric dimethylarginine produces vascular lesions in endothelial nitric oxide synthase-deficient mice: involvement of rennin–angiotensin system and oxidative stress. Arterioscler Thromb Vasc Biol 2004;24:1682–8. [26] McQuillan BM, Beilby JP, Nidorf M, Thompson PL, Hung J. Hyperhomocysteinemia but not the C677T mutation of methylenetetrahydrofolate reductase is an independent risk determinant of carotid wall thickening The Perth Carotid Ultrasound Disease Assessment Study (CUDAS). Circulation 1999;99:2383–8. [27] Wilson KM, Lentz SR. Mechanisms of the atherogenic effects of elevated homocysteine in experimental models. Semin Vasc Med 2005;5:163–71. [28] 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;55:1028–35.