Acta Diabetol (2007) 44:6975 DOI 10.

1007/s00592-007-0245-7 ORIGINAL

T. Karabag A. Kaya A. Temizhan F. Ko S. Yavuz S. am

The influence of homocysteine levels on endothelial function and their relation with microvascular complications in T2DM patients without macrovascular disease
Received: 7 June 2006 / Accepted in revised form: 11 January 2007

Abstract The aim of this study was to investigate the influence of homocysteine (hcy) levels on endothelial function by the method of brachial artery ultrasonography and their relation with microvascular complications in type 2 diabetes mellitus (T2DM) patients without macrovascular disease. Fifty-nine T2DM patients with a mean age of 53.48.6 years and diabetes duration of 8.16.2 years and 16 healthy controls with a mean age of 4714.5 years were included in the study. Endothelialdependent and endothelium-independent flow-mediated dilatation (FMD) were evaluated via brachial artery ultrasonography. Fasting plasma glucose (FPG), glycosylated haemoglobin (A1c), lipid profile, hcy, B12 and folic acid

T. Karabag A. Temizhan F. Ko Department of Cardiology School of Medicine University of Seluk Konya, Turkey A. Kaya S. Yavuz S. am Department of Endocrinology and Metabolism School of Medicine University of Seluk Konya, Turkey T. Karabag ( ) Sezin Cardiology Center No:166, Meram-Konya, Turkey E-mail: turgutkarabag@yahoo.com

levels were measured. Diabetic patients and control group individuals were compared with regard to the laboratory values and brachial artery vascular reactivity. Factors influencing endothelium-dependent FMD were investigated with linear regression analysis. Age, gender, body mass index, lipid profiles and hcy levels were similar in both groups (p>0.05). Endothelium-dependent FMD percentages were significantly lower in diabetics than in the control group (7.75.9 vs. 11.77.1%, p<0.05). Endothelial-independent FMD percentage was similar for both groups (p>0.05). The upper limit of the reference hcy value was found to be 12.6 mol/l in the control group. In the diabetic group, hcy levels were high in 33 patients and normal in 26 patients. No difference was detected between the patients with high hcy levels and those with a normal level with regard to endotheliumdependent and endothelium-independent FMD values (p>0.05). Mean hcy levels were 161.7 and 13.34.3 mol/l in T2DM patients with microvascular complication and those with no microvascular complication, respectively (p<0.05). Regression analysis revealed that the main factors influencing the endothelial-dependent FMD were FPG, total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL-C) levels (p<0.05, p=0.05, p=0.05, p=0.02, respectively). Hcy, folic acid and B12 values did not influence endothelium-dependent FMD (p>0.05). Diabetes duration and A1c levels were close to being significant although they did not reach statistical significance (p=0.07 and p=0.08 respectively). Hcy levels have no effect on endothelium-dependent and endothelium-independent FMD in T2DM patients without macrovascular complications. The influence of classical atherogenic factors (such as FPG, TC, TG and HDL-C levels) on endothelium functions, detected with endothelium-dependent FMD, is greater. Key words Homocysteine Endothelial function Flowmediated dilatation

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T. Karabag et al.: Influence of homocysteine levels on endothelial function and smokers were not included in the study. Subjects in the control group were non-smokers with normal lipid levels, no other cardiovascular risk factor and did not take medications. For both groups, caffeine-containing drinks were not allowed for 24 h before the scanning and fasting plasma glucose (FPG), glycosylated haemoglobin (A1c), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C) and triglyceride (TG) levels were measured. The venous blood samples obtained for hcy measurements were collected in EDTAcontaining tubes and measurement was performed with fluorescent immunoassay method on a Merck Hitachi L-6200A, Waters 470 (BioRad automatic sampling system AS100) device. Blood samples were not stored and were measured daily. The upper limit of the reference hcy values was obtained by calculation of the mean values of the control group and then adding 2SD. Endothelial function was evaluated non-invasively as endothelium-dependent flow-mediated dilatation (FMD) by means of brachial artery ultrasonography. The examination was performed in the morning. Patients who took oral antidiabetics were in a fasting state and did not receive medication before the examination; the control group was also asked not to have breakfast. Before the imaging process, patients were allowed to rest for 15 min in supine position in a silent room with its temperature adjusted to 24C. The examination was performed in the same room under the same conditions each time. Imaging was obtained with an ATL HDI 5000 (Philips Bothell, Washington) echocardiography device by using a 510-MHz linear transducer. Records were obtained with simultaneously accompanying electrocardiography. A sphygmomanometer was used, with its cuff placed in the proximal region of the right forearm. Right brachial artery images were obtained by using B mode imaging 23 cm below the cubital fossa, with the artery visualised in the longitudinal plane. This part of the artery was magnified and the initial baseline images were taken. Then, the cuff was inflated above a pressure of 100 mmHg and the artery was occluded for 5 min. The longitudinal artery image was obtained 1 min before releasing the cuff. Records were taken while the cuff was released. Arterial pulse signal was evaluated with a Doppler instrument in order to measure the hyperaemic velocity. Endothelium-dependent FMD was calculated by measuring the diameter of the brachial artery 60 s after the release of the cuff in the reactive hyperaemia phase. The patient was then asked to rest for 15 min. During the follow-up, further baseline images were obtained. Five milligrams of isosorbid dinitrate tablet was given sublingually and brachial artery response (endotheliumindependent response) was obtained 3 min later. For endothelium-independent (nitrate-dependent) vasodilatation, baseline diameter was compared to the diameter measured 3 min after the administration of sublingual nitrate, and the increase in the diameter in terms of percentage was obtained. In the diabetic group patients with A1c values greater than 7 were classified as having poor glycaemic control and those with A1c lower than 7 were classified as having good glycaemic control. The data were transferred to the computer using Statistical Package for Social Science (SPSS). The diabetic patients and the controls were compared with regard to their risk factors and brachial artery reactivity. The groups were compared with unpaired Students t-test. Correlation analysis was used to show the relation between the variables. All measurements were obtained as mean valueSD. Multiple linear regression analysis was used to evaluate the variables influencing endothelium-dependent FMD.

Introduction Type 2 diabetes mellitus (T2DM) patients are at increased risk for atherosclerotic diseases such as coronary heart disease, cerebrovascular disease and lower extremity occlusive vascular diseases that cause high mortality and morbidity [1, 2]. Micro- and macrovascular complications of diabetes have a complex pathogenesis, including dysfunction or damage of vascular endothelium [3, 4]. Several risk factors such as prolonged hyperglycaemia, hypertension, smoking, hyperlipidaemia, changes in the fibrinogen levels and decreased fibrinolytic activity may accelerate the atherosclerotic process [5]. It is known that hyperhomocysteinaemia can be associated with atherothrombotic vascular disease such as coronary artery disease, stroke and peripheral arterial disease [68]. Hyperhomocysteinaemia has been shown to be a stronger risk factor in T2DM patients than in non-diabetics, and has been thought to exert a synergistic effect with diabetes in accelerating the atherosclerosis process [9]. In our study we examined the influence of homocysteine (hcy) plasma levels on endothelial functions as detected by brachial artery ultrasound and the relation of hyperhomocysteinaemia with the microvascular complications of diabetes in T2DM patients.

Patients and methods

The study group consisted of 59 T2DM patients (29 male, 30 female) with a mean age of 53.48.6 years and diabetes duration of 8.16.2; 16 healthy individuals (8 male, 8 female) with a mean age of 4714.5 years served as the control group. Diabetic patients were required to have the diagnosis of diabetes for at least 1 year. Age, gender, diabetes duration and demographic characteristics of the patients and controls were recorded. A complete medical history, with an emphasis on the duration of diabetes, neuropathic symptoms and complications was taken and physical examinations were performed. The diagnosis of retinopathy was made by an ophthalmologist after the examination of the optic disc following the dilatation of pupils. For the diagnosis of neuropathy, patients were examined by a neurologist and patellar reflex, Achilles tendon reflex, sensitivity to heat and sensory stimuli were evaluated. The diagnosis was supported by electromyography when necessary. The mean urinary albumin excretion measured in three consecutive 24-h urine samples was used to diagnose nephropathy. Diagnosis of microalbuminuria was based on the detection of an albumin excretion of 30300 mg/day in a 24-h urine sample, or 30300 g/mg creatinine measured in the spot urine. All of our patients were on oral anti-diabetic medication. Medications that could positively affect the endothelial function, such as ACE inhibitors and statins, were stopped 2 days before test. Patients with hypertension, pregnant women, women in their menstruation period, patients diagnosed with coronary artery disease, patients with a history of myocardial infarction and/or cerebrovascular event

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Results There was no statistical difference between the diabetic group and the control group with regard to age, gender and body mass index (Table 1). FPG, TC, TG, LDL and hcy levels were higher in the diabetic group, but the difference was not statistically significant (p>0.05). Folic acid levels were also similar in both groups (p>0.05), whereas B12 levels were higher in the diabetic group compared to the control one (p<0.05) (Table 1). Baseline brachial artery diameters were 0.740.12 cm in the diabetic group and 0.760.11 cm in the control group. Brachial artery diameters in response to reactive hyperaemia (endothelium-dependent FMD) increased in both groups. The increase in the diabetic patients was significantly lower than the one in the control group (0.060.04 vs. 0.090.04; p<0.05, respectively). As a result, the percentage change in the hyperaemia phase with regard to baseline was significantly lower in diabetics (7.75.9 vs. 11.77.1%, p<0.05) (Table 2). Endothelium-independent FMD percentage, measured after nitroglycerin administration, was similar in both groups (p>0.05) (Table 2).

The upper limit of the reference hcy value was found to be 12.6 mol/l in our control group. Levels above 12.6 mol/l were conventionally accepted as hyperhomocysteinaemia, and those below this level were considered as normal. In the diabetic group, 33 patients were found to have high hcy levels, whereas 26 had normal values. When diabetic patients with high hcy levels were compared to those with normal values, the mean age of patients with higher hcy levels was significantly higher (p<0.05) (Table 3). No difference was detected with regard to diabetes duration, FPG and A1c levels. Folic acid and B12 levels were significantly lower in diabetic patients with high hcy levels (p=0.04 and p<0.01, respectively) (Table 3). With regard to endothelium-dependent and endothelium-independent FMD values, no statistical difference was detected between the diabetic patients with a high hcy level and those with normal hcy level. The relation between hcy levels and endothelium-dependent FMD can be seen in Figure 1. Furthermore there was a negative correlation between hcy, B12 and folic acid levels in the diabetic group (r=-38, p=0.001; r=-0.41, p<0.001). There was no correlation between FMD values and the other blood parameters we have studied.

Table 1 Baseline clinical and laboratory data in the diabetic patients and in the control group Diabetic patients (n=59) Age (years) Gender (male) Diabetes duration (year) BMI (kg/m2) FPG (mg/dl) A1c (%) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) hcy (mol/l) Folic acid (ng/ml) B12 (pg/ml) NS, non-significant Table 2 Endothelium-dependent and endothelium-independent FMD measurements in diabetic patients and in the control group Diabetics group (n=59) Endothelium-dependent FMD (%) Nitrate-dependent FMD (%) 7.75.9 13.56.3 Control group (n=16) 11.77.1 12.79.6 p <0.05 NS 53.48.6 27 27.18.1 29.44.1 18554 7.82.1 21447 216151 43.59.3 12435 14.15.4 9.53.7 35723.1 Control group (n=16) 52.910.2 8 26.85.1 9410 5.50.6 18442 16996 39.57.5 11929 12.62.8 11.76.4 274130.7 NS <0.001 <0.001 NS NS NS NS NS NS <0.05 p NS NS

NS, non-significant *Values are expressed as percentage increase with respect to baseline

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T. Karabag et al.: Influence of homocysteine levels on endothelial function

Table 3 Clinical and laboratory data and endothelium-dependent and endothelium-independent FMD measurements in T2DM patients with high and normal hcy levels High hcy (n=33) hcy Age FPG (mg/dl) A1c (%) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) Folic acid (ng/ml) B12 (pg/ml) Endothelium-dependent FMD (%) Endothelium-independent FMD (%) Retinopathy Neuropathy Nephropathy NS, non-significant 10.01.9 55.48.7 18157 7.52.2 21954 234164 4311 12541 8.43.5 321107 86.7 136.2 8 15 18 Normal hcy (n=26) 17.35.1 518 19151 8.22 20837 192131 446.9 12327 10.93.5 400129 7.35.1 14.16.5 4 8 10 p <0.001 <0.05 NS NS NS NS NS NS =0.04 <0.05 NS NS <0.001 NS NS

40,00

30,00

Homocysteine (m/dL)

20,00

1 10,00

12.6 mol/l

0,00

10,00 FMD (% change)

20,00 Fig. 1 The association between hcy levels and endothelium-dependent FMD in diabetic patients

When we applied regression analysis to the results of the diabetic group, the main factors influencing endothelium-dependent FMD were FPG, TC, TG and HDL-C levels (p<0.05, p=0.05, p=0.05, p=0.02 respectively). The same significance could not be observed for hcy, folic acid and B12 levels (p>0.05). Diabetes duration and A1c levels, although not reaching a statistically significant difference, were close to being significant (p=0.07, p=0.08 respectively).

In the diabetic group, retinopathy was diagnosed in 12 patients, neuropathy was detected in 23 patients and nephropathy was detected in 28 patients (Table 3). In 30 patients no complications were diagnosed. Hcy levels were significantly higher in diabetic subjects who had any microvascular complication than in those with no complication detected (167.1 vs. 13.34.3; p<0.05). This significance persisted after adjusting the analysis for age, diabetes duration and body mass index. Endothelium-

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Table 4 Clinical and laboratory data and endothelium-dependent and endothelium-independent FMD measurements in T2DM patients with poor and good glycaemic control Pts. with good glycaemic control (n=28) hcy Age FPG (mg/dl) A1c (%) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) Folic acid (ng/ml) B12 (pg/ml) Endothelium-dependent FMD (%) Endothelium-independent FMD (%) Retinopathy Neuropathy Nephropathy NS, non-significant 11.22.8 52.37.6 16944 6.10.6 21057 220148 437 11935 11.33.4 36624 6.76.4 13.76.1 4 6 8 Pts. with poor glycaemic control (n=31) 14.26.5 53.58.5 19954 91.8 22042 20195 447 12838 8.74.1 293106 8.35.7 13.46.6 8 13 16 p

<0.05 NS NS <0.001 NS NS NS NS <0.05 <0.05 NS NS <0.05 NS NS

dependent and endothelium-independent FMD values were similar in diabetic patients with microvascular complications and in those without (n=29, p>0.05). At linear regression analysis, both in diabetics with microvascular complication and in those without, hcy levels did not significantly influence endothelium-dependent FMD, but in diabetics with microvascular complication this was close to being significant (p=0.09). When diabetic patients were classified as having good glycaemic control (28 pts) and on those with poor glycaemic control (31 pts), by means of HbA1c levels lower and greater than 7%, a difference was found with respect to hcy, B12 and folic acid levels between groups (p<0.05) (Table 4). Both in patients with poor glycaemic control and in those with good glycaemic control there were significant correlations between hcy levels and B12 and folic acid levels (patients with poor glycaemic control r=-41, p<0.001 and r=38, p=0.001, respectively; patients with good glycaemic control, r=-52, p<0.001, and r=-36, p<0.01, respectively). Endothelium-dependent and -independent FMD values were similar between those groups (p>0.05).

Discussion Hcy is a sulphur-containing amino acid that is formed during the metabolism of methionin [10]. Plasma hcy concentrations are mainly affected by genetic and nutritional factors. Hyperhomocysteinaemia is common in the general population, and it is known to be a risk factor for atherosclerosis and thrombosis [1114]. Hcy levels are mainly regulated by methionin and folate intake in the

diet, and their levels are known to be affected by smoking, B12 intake and renal function [1517]. The inadequate activity of the enzymes in the trans-sulphuration and re-methylation pathways and mutations such as C677T may induce the formation of the methylenetetrahydrofolate reductase-thermolabile variant of the enzyme, and finally can cause hyperhomocysteinaemia. Vitamins that are effective in hcy metabolism, i.e., coenzyme B12 and B6, and folate, are important modulators that affect plasma hcy concentrations [18]. Hcy can affect atherosclerosis by causing injury to the vascular endothelium, suppressing the expression of thrombomodulin, increasing the platelet activity and platelet aggregation, increasing fibrinogen formation, and increasing smooth muscle cell proliferation and migration [1921]. In addition, hcy can activate protein kinase C [12, 18]. Furthermore, it can accelerate the expression of c-fos and c-myb, which are gene transcription factors found in the vascular smooth muscle cell [14, 2225]. In a study on children with T1DM, Pavia et al. found that hcy, folate and B12 levels were similar to those of the control group. A negative correlation between hcy levels and folate and B12 levels was detected. They found the hcy levels to be high in patients with microvascular disease and nephropathy [26]. We found that hcy and folate levels of the patients were similar to those of the control group, whereas B12 levels were higher. B12 and folate levels were lower in patients with microvascular complication than in those without, but this difference was not statistically significant. However, hcy levels were significantly higher in patients in whom complications were detected. In diabetic patients hcy levels were negatively correlated with B12 and folate levels.

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T. Karabag et al.: Influence of homocysteine levels on endothelial function 3. Vane JR, Angaard EE, Botting RM (1990) Regulatory functions of the vascular endothelium. N Engl J Med 323:2736 4. Stehouwer CD, Nauta JJ, Zeldenrust GC, Hacheng WH, Donker AJ, Ottolander GJ (1992) Urinary albumin excretion, cardiovascular disease, and endothelial dysfunction in noninsulin dependent diabetes mellitus. Lancet 340:319323 5. Colwell JA, Jokl RL (1996) Vascular thrombosis in diabetes. In: Porte D, Sherwin R, Rifkin H (eds) Diabetes mellitus: theory and practice, 5th edn. Appleton and Lange, Norwalk, CT, pp 207216 6. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH (1992) A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA 268:877881 7. Perry IJ, Refsum H, Morris RW, Ebrahim SB, Ueland PM, Shaper AG (1995) Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 346:13951398 8. van den Berg M, Stehouwer CDA, Bierdrager E, Rauwerda JA (1996) Plasma homocysteine and severity of atherosclerosis in young patients with lower-limb atherosclerotic disease. Arterioscler Thromb Vasc Biol 16:165171 9. Hoogeveen EK, Kostense PJ, Beks PJ, Mackaay AJ, Jakobs C, Bouter LM, Heine RJ, Stehouwer CD (1998) Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, especially in non-insulin dependent diabetes mellitus: a population-based study. Arterioscler Thromb Vasc Biol 18:133138 10. Mudd SH, Levy HL, Skovby F (1995) Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular basis of inherited disease. McGraw-Hill, New York, pp 12791327 11. Mayer EL, Jacobsen DW, Robinson K (1996) Homocysteine and coronary atherosclerosis. J Am Coll Cardiol 27:517527 12. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I (1991) Hyperhomocysteinemia: an independent risk factor for cardiovascular disease. N Engl J Med 324:11491155 13. Morita H, Kurihara H, Kuwaki T, Hamada C, Kitaoka M, Suzuki S, Yazaki Y, Nagai R (2000) Homocysteine as a risk factor for restenosis after coronary angioplasty. Thromb Haemost 84:2731 14. Morita H, Taguchi J, Kurihara H, Kitaoka M, Kaneda H, Kurihara Y, Maemura K, Shindo T, Minamino T, Ohno M, Yamaoki K, Ogasawara K, Aizawa T, Suzuki S, Yazaki Y (1997) Genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) as a risk factor for coronary artery disease. Circulation 95:20322036 15. Kaye JM, Stanton KG, McCann VJ, Vasikaran SD, Burke V, Taylor RR, van Bockxmeer FM (2002) Homocysteine, folate, methylene tetrahydrofolate reductase genotype and vascular morbidity in diabetic subjects. Clinical Sci (Lond) 102:631637 16. Bostom AG, Culleton BF (1999) Hyperhomocysteinemia in chronic renal disease. J Am Soc Nephrol 10:891900 17. Stein JH, Bushara M, Bushara K, McBride PE, Jorenby DE, Fiore MC (2002) Smoking cessation, but not smoking reduction, reduces plasma homocysteine levels. Clin Cardiol 25:2326 18. Ungvari Z, Pacher P, Rischak K, Szollar L, Koller A (1999) Dysfunction of nitric oxide mediation in isolated rat arterioles with methionine diet-induced hyperhomocysteinemia. Arterioscler Thromb Vasc Biol 19:18991904

Hofmann et al. measured soluble thrombomodulin levels as a marker of the impairment of endothelial functions in an early phase of T1DM patients. They observed that thrombodulin levels were significantly higher in T1DM patients, especially in those with high hcy levels and this finding correlated with hcy levels [27]. In our study, hcy levels were increased in T2DM patients with microvascular complication, but endothelium-dependent and endothelium-independent FMD were similar to that of the control group. Endothelium-dependent FMD was not correlated with hcy levels either in patients with complication or in those without. This finding suggested that hcy levels do not influence endothelial function of T2DM patients even when microvascular complications occur. In addition, the influence of factors such as FPG levels and lipid parameters is more prominent on endothelium-dependent FMD than that of hcy, B12 and folate levels. Soinio et al. [28] followed 830 T2DM patients for 7 years, and they detected a higher cardiovascular mortality and higher ratios of fatal and non-fatal myocardial infarction in patients with higher baseline hcy levels, in comparison to patients with normal or low hcy levels. They suggested that plasma hcy level is an independent risk factor for cardiovascular events and claimed that addition of folate to dietary intake can decrease cardiovascular event risk [28]. In our study, endothelial functions detected by FMD method were impaired both in diabetics with microvascular complication and in those without. This means that endothelial function can be impaired in the early stages of diabetes, before microvascular complications develop. As hcy levels were found to be higher in patients with microvascular complication, this could suggest that hcy levels may contribute to the development of the complications rather than initiating them. In summary, in patients with T2DM endothelial dysfunction seems to start in the early stages of the disease before microvascular complications occur. Hcy levels do not have an influence on the endothelial function evaluated with FMD by brachial artery ultrasonography in T2DM patients. The influence of classical atherogenic factors such as FPG, TG, TC and HDL on endothelial functions seems to be more prominent. In the presence of these risk factors hcy may have an accelerating effect on the atherosclerotic process, especially in patients in whom microvascular complications have developed.

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