Association Between Homocysteinemia and Renal Function in Patients with Type 2 Diabetes Mellitus

Transcription

1 Annals of Clinical & Laboratory Science, vol. 32, no. 3, Association Between Homocysteinemia and Renal Function in Patients with Type 2 Diabetes Mellitus Bilgin Ozmen, 1 Dilek Ozmen, 2 Nevbahar Turgan, 2 Sara Habif, 2 Isil Mutaf, 2, and Oya Bayindir 2 1 Department of Endocrinology, Celal Bayar University School of Medicine, Manisa, Turkey, and 2 Department of Clinical Biochemistry, Ege University School of Medicine, Izmir, Turkey Abstract. Homocysteinemia is an independent risk factor for cardiovascular disease, but information on its association with type 2 diabetes and mild renal dysfunction is limited. Plasma total homocysteine (thcy) concentration is partly determined by renal plasma clearance. Serum cystatin C (Cys C) concentration has been introduced as a marker of renal function, specifically as an indicator of glomerular filtration rate (GFR). The aim of this study was to explore the relationships among thcy, creatinine clearance (Ccr), serum Cys C, and microalbuminuria in a population with type 2 diabetes. Fasting plasma thcy, serum homocysteine-related vitamins (folate and vitamin B 12 ), serum Cys C, serum creatinine, urine microalbumin, and creatinine clearance were determined in 75 type 2 diabetic patients and 40 healthy control subjects. The patients were assigned to two groups based on urinary albumin excretion (UAE): normoalbuminuric (NAU, UAE <30 mg/24 hr, n = 35) and microalbuminuric (MAU, UAE mg/24 hr, n = 40). Ccr was calculated using the Cockroft-Gault formula. Plasma Hcy levels were determined by HPLC with fluorescence detection and serum Cys C by automated particle enhanced immunoturbidimetry. Plasma thcy levels were significantly higher in normoalbuminuric and microalbuminuric patients than in controls (10.64 ± 0.53, ± 0.78, 6.91 ± 0.37 mmol/l, respectively). Serum Cys C levels in microalbuminuric diabetics were higher than in normoalbuminurics and controls (1.36 ± 0.06, 1.12 ± 0.04, 1.10 ± 0.06 mg/ L, respectively). Positive correlations were noted between thcy and Cys C levels in normoalbuminuric and microalbuminuric diabetics (r = 0.72, r = 0.64, respectively ). Homocysteine and creatinine concentrations were correlated in both diabetic groups (r = 0.89, r = 0.93, NAU and MAU, respectively ). Elevated plasma total homocysteine concentrations in type 2 diabetics suggest an association between homocysteinemia and deterioration of renal function, evidenced by increased serum creatinine and Cys C, Ccr, and microalbuminuria. These findings implicate homocysteinemia in the relationship between diabetic nephropathy and cardiovascular complications of diabetes. (received 8 February 2002, accepted 25 March 2002) Keywords: diabetes mellitus type 2, homocysteine, cystatin C, microalbuminuria, renal function Introduction Homocysteine (Hcy) is the transmethylation product of the essential sulphur-containing amino acid methionine [1,2]. Experiments have shown that high concentrations of homocysteine may cause vascular damage [3]. Epidemiological research Address correspondence to Dilek Ozmen, M.D., Department of Clinical Biochemistry, Ege University School of Medicine, 35100, Bornova, Izmir, Turkey; tel and fax ; dozmen@med.ege.edu.tr. suggests an association between elevated total homocysteine (thcy) levels and cardiovascular disease (CVD), which is the most common cause of mortality in patients with type 2 diabetes mellitus [2]. The association between homocysteinemia and atherosclerotic vascular disease is especially strong in patients with type 2 diabetes, compared to nondiabetic subjects. Increased plasma thcy levels are reported to be associated with hypertension, hyperlipidemia, smoking, hyperuricemia, and impaired adrenal function [4-8]. Plasma thcy /02/0300/0279, $ by the Association of Clinical Scientists, Inc.

2 280 Annals of Clinical & Laboratory Science concentration is strongly related to renal function [9]. A study in rats identified the kidney as a major site for removal and metabolism of Hcy [10]. Two mechanisms appear to be involved. The main source of Hcy is adenosylmethionine-dependent methylation of guanidoacetate to form creatine and its anhydride creatinine [11]. Second, renal function plays a central role for clearance of both creatinine and Hcy [12,13]. Plasma thcy concentrations in diabetic patients can be affected by both glomerular hyper- and hypofiltration, which can respectively decrease and increase the thcy concentrations [14,15]. However, reduced glomerular filtration rate (GFR) accompanies microalbuminuria (MAU) in the late phase of diabetic nephropathy, and reduction of GFR causes elevation of plasma Hcy levels [16,17]. These data provide a potential link between microalbuminuria, diabetic nephropathy and cardiovascular disease [18-20]. Despite some disadvantages, in clinical practice serum creatinine and creatinine clearance determinations are routinely used for the estimation of GFR in diabetic patients. Recently, serum cystatin C (Cys C) concentration has been introduced as a new marker of renal function, specifically as an indicator of the GFR. Cys C comprises one non-glycosylated polypeptide chain with 120 amino acid residues (mol wt 13 kda) [21]. Investigations have indicated that serum Cys C is superior to serum creatinine for the detection of early decreases of GFR [21-24]. In contrast to serum creatinine, Cys C is not related to muscle mass or the formation of creatinine; it only reflects the GFR [22,25]. GFR is proposed as an independent determinant of plasma thcy and a rate limiting parameter for renal clearance of homocysteine and cysteine in diabetic patients without overt nephropathy [13,14]. Based on these considerations, this study was designed to assess the association between plasma thcy and serum Cys C for the evaluation of renal function in type 2 diabetic patients. Materials and Methods This study included 75 patients (30 men, 45 women, age yr) with type 2 diabetes mellitus, with or without microalbuminuria, who received care at the Department of Endocrinology of Celal Bayar University Hospital. Their diagnosis of diabetes mellitus was based on a previous history of diabetes or the American Diabetes Association (ADA) criteria [26] questionnaire, which includes age, duration of diabetes, use of medication for diabetes, vitamin supplementation, smoking habits, and ethanol intake. The control group consisted of 40 healthy subjects (18 men, 22 women, age yr) who were screened by a physician for signs of diabetes mellitus and who did not show any clinical evidence of the disease. The subjects were all informed about the protocol and their written consent was obtained. The diabetic patients were divided into two groups based on their urinary albumin excretion (UAE). Group 1 included 35 patients with normoalbuminuria (NAU), (UAE <30 mg/24 hr); Group 2 included 40 patients with microalbuminuria (MAU), (UAE mg/24 hr). HbA ıc, creatinine, homocysteine, folate, vitamin B 12 and cystatin C concentrations were determined in blood or serum samples after an overnight fast. Urine albumin excretion was measured in 24 hr urine specimens. Among the diabetic patients, 42 were treated with sulphonylurea drugs, 10 with combinations of sulphonylurea and alpha-glucosidase inhibitors, and 23 with insulin. Serum creatinine concentrations were analyzed by standard laboratory methods with an automatic analyzer (Technicon Dax-48, Bayer Diagnostics, Toshiba, Japan). HbA ıc and urine albumin excretion were measured by immunoturbidimetric assay kits (Roche Diagnostic GMBH, Germany) with a Hitachi 704 analyzer (Tokyo, Japan). Creatinine clearance in males was calculated by the Cockroft-Gault formula [27]: (140-age) x weight kg / 72 x serum creatinine mg/100ml. For females, the value obtained by this equation was multiplied by 0.85.

3 Plasma homocysteine and renal function in diabetics 281 Serum cystatin C levels were determined by a latex particle enhanced immunoturbidimetric assay (DAKO, Glostrop, Denmark). Folate and vitamin B 12 in serum were measured by a chemiluminescent immunoassay (Access, Sanofi Diagnostics Pasteur, Marnes La Coquette, France); the reference intervals were >6.8 nmol/l for folate and 133 to 675 pmol/ L for vitamin B 12. For homocysteine analyses, blood was collected in EDTA-containing tubes; the plasma was separated within min and stored at -20 C. Total homocysteine (thcy) in plasma was measured by HPLC and fluorescence detection [28]. A Shimadzu LC 10A HPLC system (Shimadzu Corp., Kyoto, Japan) was used, consisting of a Shimadzu LC-10AD pump, a Shimadzu SIL-10AXL autoinjector with a 20 µl loop, and a Shimadzu RF-10AXL fluorescence detector. The system was controlled through a Shimadzu CBM-10A communication module and a personal computer. The column was EC 150/4.6 Nucleosil C18 5 mm (Macherey-Nagel, Duren, Germany). Tri-n-butyl-phosphine (TBP) in dimethly formamide (DMF) was applied for reduction of disulfide-bound homocysteine and ammonium 7-fluorobenzo-2-oxa-1,3-diazide-4- sulphonate (SBDF) served as a derivatization agent. Cysteamine hydrochloride was added as an internal standard. Results are reported as mean ± SE. Differences in mean values of the three groups were evaluated by the t-test (p <0.05 was considered significant). Correlations were tested by the Pearson rank correlation test. The Statistical Package for Social Sciences (SPSS Version 7.5, SPSS Inc., Chicago, IL, USA) was used. Results Laboratory findings of patients and control subjects are listed in Table 1. There were no significant differences in age or sex between the two diabetic groups and the controls. Plasma homocysteine, serum cystatin C, and serum creatinine concentrations showed significant differences between the two diabetic groups. Creatinine clearance values were lower in both diabetic groups (p < 0.01 for both values), compared to controls. Serum folate and vitamin B 12 concentrations are not included in Table 1, since these parameters were used only to exclude deficiencies of these vitamins that could affect thcy levels. All subjects had folate concentrations >6.8 nmol/l and vitamin B 12 concentrations between 133 and 675 pmol/l. Mean plasma homocysteine concentrations were significantly higher in normo- and microalbuminuric diabetic patients than in the controls (10.64 ± 0.53 and ± 0.78 vs 6.91 ± 0.37 µmol/l, respectively, p < 0.01) The mean serum cystatin C concentration in microalbuminuric patients was higher than in normoalbuminuric patients and controls (1.36 ± 0.06, 1.12 ± 0.04, and 1.10 ± 0.06 mg/l, respectively, p <0.01) The difference between normoalbuminuric diabetics and controls was insignificant. Serum creatinine levels in microalbuminuric patients were higher than in normoalbuminuric patients and controls ( ± 4.42, ± 3.54, and ± 2.86 µmol/l, respectively, p <0,01) There were no significant differences in serum creatinine levels between normoalbuminuric diabetics and control subjects. Mean creatinine clearance values in normo- and microalbuminuric groups were higher than in controls (60.50 ± 5.19 and ± 3.49, versus ± 0.06 ml/min, respectively, p < 0,01). In normoalbuminuric diabetics, positive correlations were observed between plasma homocysteine and serum cystatin C concentrations (r = 0.72, p <0.01, Fig. 1a), between plasma homocysteine and serum creatinine levels (r = 0.89, p <0.01) (Fig. 1b), and between plasma homocysteine and age (r = 0.48, p <0.01) (Table 2). In microalbuminuric diabetic patients, there were positive correlations between plasma homocysteine and serum cystatin C levels (r = 0.64, p <0.01) (Fig. 2a), homocysteine and serum creatinine levels (r = 0.93, p <0.01) (Fig. 2b), and homocysteine and age (r = 0.58, p <0.01) (Table 2).

4 282 Annals of Clinical & Laboratory Science Table 1. Summary of the data (mean ± SE) for diabetic patients and healthy controls Parameter Control Normoalbuminuric Microalbuminuric p subjects diabetic patients diabetic patients (n = 40) (NAU) (n = 35) (MAU) (n = 40) Plasma homocysteine (µmol/l) 6.91 ± ± ± 0.78 a,b,c Serum cystatin C (mg/l) 1.10 ± ± ± 0.06 b,c Serum creatinine (µmol/l) 82.21± ± ± 5.65 b,c Creatinine clearance (ml/min) ± ± ± 3.49 a,b Blood HbA ıc (%) 5.54 ± ± ± 0.45 ns Urine microalbumin (mg/24 hr) 13.25± ± ± b,c Age (yr) ± ± ± 1.96 ns Duration of diabetes (yr) 9.7 ± ± 1.2 ns a p <0.01; NAU versus controls b p <0.01; MAU versus controls c p <0.01; MAU versus NAU ns = not significant Table 2. Pearson s correlation coefficient (r) and p values for determinants of plasma homocysteine concentrations in diabetic patients Parameter Plasma homocysteine concentration (µmol/l) Normoalbuminuric Microalbuminuric diabetic patients diabetic patients (n = 35) (n = 40) r p r p Serum cystatin C (mg/l) 0.72 < <0.01 Serum creatinine (µmol/l) 0.89 < <0.01 Age (yr) 0.48 < <0.01

5 Plasma homocysteine and renal function in diabetics 283 Fig. 1a (left panel) and Fig. 1b (right panel) show the graphs and regression equations for comparisons between plasma homocysteine and serum cystatin C (1a) and plasma homocysteine and serum creatinine (1b) concentrations in normoalbuminuric diabetic patients. Fig. 2a (left panel) and Fig. 2b (right panel show the graphs and regression equations for the comparisons between plasma homocysteine and serum cystatin C (2a) and plasma homocysteine and serum creatinine (2b) concentrations in microalbuminuric diabetic patients

6 284 Annals of Clinical & Laboratory Science No significant correlations were observed between any of the parameters in healthy controls. Discussion Growing interest is being focused on the association of homocysteinemia with diabetes mellitus. However, consensus on how type 2 diabetes affects plasma homocysteine concentrations has not been achieved [2]. Homocysteinemia has been established as a risk factor for cardiovascular disease and occurs with high prevalence in patients with type 2 diabetes: 31% of type 2 diabetic patients have homocysteine concentrations above >15 mmol/l [29]. Only a few studies found no significant differences in fasting plasma thcy levels between type 2 diabetic patients without microalbuminuria and healthy control subjects [30]. A large amount of evidence supports increased plasma thcy levels in type 2 diabetes [2,4, 15,18,20,29,31,32]. Buysschaert et al [29] reported increased plasma thcy levels in type 2 diabetics with advanced nephropathy, compared to control subjects and diabetics without nephropathy. The relationship between homocysteinemia and microalbuminuria is more problematic. Some authors found an association between plasma thcy and microalbuminuria in diabetes [2,18,30,33,34], while others did not [32,35]. In our study, plasma homocysteine concentrations were significantly higher in microalbuminuric patients than in normoalbuminuric patients or controls. Chico et al [18], Emoto et al [2], and Lanfredini et al [30] found strong association between homocysteinemia and deteriorated renal function. According to previous reports, homocysteine is ultrafiltrated through the glomeruli, almost completely reabsorbed in the tubuli, and degraded in kidney tissue by transmethylation and transsulfuration [36]. Decreased renal clearance of homocysteine results in homocysteinemia [10-12], but the exact mechanism is unknown [37]. Recently, serum cystatin C has been introduced as a more sensitive marker for mildly impaired GFR, compared to creatinine and GFR, and also as an independent determinant of plasma thcy concentrations [21,22]. Norlund et al [17] suggested that the increase of plasma homocysteine concentrations with advancing age may be due to age-related decline in renal function. It has also been shown that cystatin C shows a higher predictive value for thcy concentrations than age or serum creatinine [17]. Our data indicate that serum cystatin C levels in patients with microalbuminuria were significantly higher than in patients with normoalbuminuria or control subjects. Additionally, we found that plasma homocysteine concentrations correlated better with serum creatinine than with serum cystatin C. Our data are in agreement with the study of Abdella et al [31], who also showed that plasma homocysteine concentrations correlated better with serum creatinine than with cystatin C, presumably because of the relationship between creatine-creatinine synthesis and homocysteine production. Contradicting these data, Bostom et al [38] and Aras et al [39] reported that serum cystatin C was a much better predictor of fasting total homocysteine concentrations in renal transplant recipients with normal renal function. In patients with coronary artery disease and normal renal function (serum creatinine concentrations < µmol/l), serum cystatin C was reported to be an independent predictor of fasting thcy concentrations [40]. Although kidney function plays an important role in the metabolism of homocysteine, it is not clear whether homocysteinemia is associated with or can precede microalbuminuria [5,41]. Homocysteinemia is believed to increase the risk of atherothrombic disease directly by impairing endothelial function, stimulating vascular smooth muscle cell proliferation, and altering extracellular matrix properties [42]. High plasma homocysteine levels may also exert an atherothrombic effect by inducing oxidative stress, which may impair endothelial function. Oxidative stress is thought to be increased in type 2 diabetes [43]. Thus, homocysteinemia may contribute to the development of renal impairment and subsequent microalbuminuria in diabetic patients through increased oxidative stress [5]. In summary, the present study shows that plasma homocysteine concentrations are elevated in type 2 diabetic patients with and without microalbuminuria. Microalbuminuric type 2 diabetic patients have higher plasma homocysteine levels than normoalbuminuric patients and controls.

7 Plasma homocysteine and renal function in diabetics 285 Elevated plasma homocysteine concentrations in type 2 diabetic patients suggest an association between homocysteinemia and impaired renal function, as evidenced by increased serum creatinine and cystatin C levels. These findings suggest that homocysteinemia may partly explain the link between diabetic nephropathy and cardiovascular complications of diabetes. References 1. Medina MA, Amores-Sanchez MI. Homocysteine : an emergent cardiovascular risk factor? Eur J Clin Invest 2000;30: Emoto M, Kanda H, Shoji T, Kawagishi T, Komatsu M, Mori K, Tahara H, Ishimura E, Inaba M, Okuno Y, Nishizawa Y. Impact of ınsulin resistance and nephropathy on homocysteine in type 2 diabetes. Diabetes Care 2001;24: Stamler JS, Osborne JA, Jaraki O, Rabbanı LE, Mullins M, Sıngel D, Loscalzo J. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing oxides of nitrogen. J Clin Invest 1993;91: Fiorina P, Lanfredini M, Montanari A, Peca MG, Verpnelli A, Mello A, Astorri E, Craveri A. Plasma homocysteine and folate are related to arterial blood pressure in type 2 diabetes mellitus. Am J Hypertens 1998;11: Hoogeveen EK, Kostense PJ, Jager A, Heine RJ, Jakobs C, Bouter LM, Donker AJ, Stehouwer CD. Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study. Kidney Int 1998;54: Munshi MN, Stone A, Fink L, Fonseca V. Homocysteinemia following a methionine load in patients with noninsulın dependent diabetes mellitus and macrovascular disease. Metabolism 1996;45: Okada E, Oida K, Tada H, Asazuma K, Eguchi K, Tohda G, Kosaka S, Takahashi S, Miyamori I. Homocysteinemia is a risk factor for coronary arteriosclerosis in Japanese patients with type 2 diabetes. Diabetes Care 1999;22: Graham IM, Daly LE, Refsum HM, et al. Plasma homocysteine as a risk factor for vascular disease. The European Concerned Action Project. JAMA 1997;277: Bostom AG, Lathrop L. Homocysteinemia in end stage renal disease : prevalence, etiology and potential relationship to arteriosclerotic outcomes. Kidney Int 1997;52: House JD, Brosnan ME, Brosnan JT. Characterization of homocysteine metabolism in the rat kidney. Biochem J 1997;328: Brattström L, Lindgren A, Israelsson B, Andersson A, Hultberg B. Homocysteine and cysteine: determinants of plasma levels in middle-aged and elderly subjects. J Intern Med 1994;236: Bostom A, Brosnan JT, Hall B, Nadeau MR, Selhub J. Net uptake of plasma homocysteine by the rat kidney in vivo. Atherosclerosis 1995;116: Arnadottir M, Hultberg B, Nilsson-Ehle P, Thysell H. The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scand J Clin Lab Invest 1996;56: Wollesen F, Brattström L, Refsum H, Ueland PM, Berglund L Berne C. Plasma total homocysteine and cysteine in relation to glomerular filtration rate in diabetes mellitus. Kidney Int 1999;55: Hultberg B, Agardh E, Andersson A, Brattström L, Isaksson A, Israelsson B, Agardh CD. Increased levels of plasma homocysteine are associated with nephropathy, but not severe retinopathy in type 1 diabetes mellitus. Scand J Clin Lab Invest 1991;51: Hultberg B, Andersson A, Sterner G. Plasma homocysteine in renal failure. Clin Nephrol 1993; 40: Norlund L, Grubb A, Fex G, Lksell H, Nilsson JE, Schenck H, Hultberg B. The increase of plasma homocysteine with age is partly due to the deterioration of renal function as determined by plasma cystatin C. Clin Chem Lab Med 1998;36: Chico A, Perez A, Cordoba A, Arcelus R, Carreras G, de Leiva A, Gonzales-Sastre F, Blanco-Vaca F. 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: Hoogeveen EK, Kostense PJ, Beks PJ, Mackay AJ, Jakobs C, Bouter LM, Heine RJ, Stehouwer CD. Homocysteinemia is associated with an increased risk of cardivascular disease, especially in non-insulin dependent diabetes mellitus. a population-based study. Arterioscler Thromb Vasc Biol 1998;18: Stabler SP,. Estacio R,.Jeffers B W,. Cohen JA, Allen R H,.Schrier RW, Total homocysteine is associated with nephropathy in non-insulin-dependent diabetes mellitus. Metabolism 1999;48: Randers E, Erlandsen EJ, Serum cystatin C as an endogenous marker of the renal function: a review. Clin Chem Lab Med 1999; 7:

8 286 Annals of Clinical & Laboratory Science 22. Newman DJ, Thakkar H, Edwards RG,. Wilkie M, White T, Grubb AO. Price Serum cystatin C measured by automated immunassay: a more sensitive marker of changes in GFR than serum creatinine, Kidney Int. 1995;47: Filler G, Witt I, Priem F, Ehrich JH, Jung K, Are cystatin C and beta 2 -microglobulin better markers than serum creatinine for prediction of a normal GFR in pediatric subjects? Clin Chem 1997; 43: Mojiminiyi OA, Shayji IA, George S. Serum cystatin could replace serum creatinine as an indicator of GFR in routine practice. Eur J Lab Med 1998;6: Kyhse-Andersson J, Schmidt C, Nordin G, Andersson B, Nilsson-Ehle P, Lindström V, et al. Serum cystatin C, determined by a rapid automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for GFR. Clin Chem 1994;40: Expert Committee on Diabetes Mellitus. The Expert Committee s report on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997;20: Cockroft DW, Gault DA. Prediction of creatinine clearance from creatinine. Nephron 1976;16: Turgan N, Boydak B, Habif S, Apakkan S, Özmen D, Mutaf I, Bayındır O. Plasma homocysteine levels in acute coronary syndromes. Jpn Heart J 1999;40: Buysschaert M, Dramaıs AS, Wallemaco PE, Hermansa MP. Homocysteinemia in type 2 diabetes. Diabetes Care 2000;23: Lanfredini M, Fiorina P, Peca MG, Veronelli A, Mello A, Astorri E, Dall Aglio P, Craveri A. Fasting and post-methionine load homocyst(e)ine values are correlated with microalbuminuria and could contribute to worsening vascular damage in noninsulin-dependent diabetes mellitus. Metabolism 1998;47: Abdella N, Mojiminiyi OA, Akanji AO., Homocysteine and endogenous markers of renal function in type 2 diabetic patients without coronary heart disease. Diabetes Res Clin Pr 2000;50: Smulders YM, Rakie M, Slaats EH, Treskes M, Sijbrands EJ, Oderkerken DA, Stehouwer CD, Silberbusch J. Fasting and post-methionine homocysteine levels in NIDDM. Determinants and correlations with retinopathy, albuminuria and cardiovascular disease. Diabetes Care 1999;22: Hofmann MA, Kohl B, Zumbach MS,Borcea V, Bierhaus A, Henkels M, Amiral J,Schmidt AM, Fiehn W, Ziegler R, Nawroth PP. Homocyst(e)ine and endothelial dysfunction in IDDM. Diabetes Care 1998;21: Agardh CD, Agardh E, Andersson A, Hultberg B. Lack of association between plasma homocysteine levels and microangiopathy in type I diabetes mellitus. Scand J Clin Lab Invest 1994;54: Baliga BS, Reynolds T, Fink LM, Fonseca VA. Homocysteinemia in type 2 diabetes mellitus: cardiovascular risk factors and effect of treatment with folic acid and pyridoxine. Endocr Pract 2000; 6: Dudman NP, Guo XV, Gordon RB, Dawson PA, Wılcken DE. Human homocysteine catabolism: Three major pathways and their relevance to develoment of arterial occlusive disease. J Nutr 1996;126 : 1295S-3000S. 37. Guttormsen AB, Ueland PM, Svarstad E, Refsum H. Kinetic basis of homocysteinemia in patients with chronic renal failure. Kidney Int 1997;52: Bostom AG, Gohh RY, Bausserman L, Hakas D, Jacques PF, Selhub J, Dworkin L, Rosenberg IH. Serum cystatin C as a determinant of fasting total homocysteine levels in renal recipients with a normal serum creatinine. J Am Soc Neph 1999;10: Aras Ö, Tsai MY, Hanson NQ, Bailey R, Rao G, Hunnınghake DB. Cystatin C is an independent predictor of fasting and post-methionine load total homocysteine concentrations among stable transplant recipients.clin Chem 2001;47: Bostom AG, Bausserman L, Jacques PF, Liaugaudas G, Selhub J, Rosenberg IH. Cystatin C as a determinant of fasting plasma total homocysteine levels in coronary artery disease patients with normal serum creatinine. Arterioscler Thromb Vasc Biol 1999; 19: Jager A, Kostense PJ, Nijpels G, Dekker JM, Heine RJ, Bouter LM, Donker AJ, Stehouwer CD. Serum homocysteine levels are associated with the development of (micro)albuminuria: the Hoorn Study. Arterioscler Thromb Vasc Biol 2001;21: Stehouwer CDA, Gall MA, Hougaard P, Jakobs C, Parving HH. Plasma homocysteine concentration predicts mortality in non-insulin dependent diabetic patients with and without microalbuminuria. Kidney Int 1999;55: Hoogeveen EK, Kontense PJ, Jakobs C, Dekker JM, Nijpels G, Heine RJ, Bouter LM, Stehouwer CDA. Homocysteinemia increases risk of death, especially in type 2 diabetes. Circulation 2000;101: