The significance of serum homocysteine levels in diabetic patients on haemodialysis

Kazuhiro Oishi, Yoshio Nagake, Hiroko Yamasaki, Shinji Fukuda, Haruo Ichikawa, Kosuke Ota and Hirofumi Makino

Department of Medicine III, Okayama University Medical School, Okayama, Japan



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Atherosclerotic diseases are the major cause of mortality and morbidity in patients on haemodialysis (HD). Furthermore, the prognosis of diabetic patients on HD is especially poor due to atherosclerotic complications. Because homocysteine (Hcy), a sulfur-containing amino acid, is emerging as an important risk factor for atherosclerosis in patients with end-stage renal disease, we examined the significance of serum Hcy levels in diabetic patients on HD.

Methods. We measured total serum Hcy levels (tHcy) in 31 patients with diabetes mellitus on HD (DM group) and 37 non-diabetic patients on HD (N group), adjusting for age and HD duration. Linear regression analysis was used to assess the correlation of multiple variables to tHcy.

Results. The proportion of atherosclerotic disease in the DM group was significantly higher than in the N group. However, serum tHcy, serum creatinine and per cent creatinine generation rate in the DM group were significantly lower than in the N group. In the DM group, serum tHcy was positively correlated with creatinine, albumin and per cent creatinine generation rate, respectively. This was not the case in the N group.

Conclusions. The demethylation pathway in methionine metabolism in the liver, which is linked directly to the creatinine generation system, may be disturbed in diabetic patients on HD. This may be the reason why serum tHcy and creatinine in diabetic patients on HD are lower than in non-diabetic patients on HD. Therefore, it is necessary to consider the possibility of an altered relation between serum tHcy and vessel disease when evaluating the atherogenic risk in diabetic patients on HD.

Keywords: atherosclerosis; creatinine; diabetes mellitus; haemodialysis; homocysteine



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Despite advanced dialysis techniques, atherosclerosis is the major cause of mortality and morbidity in patients with end-stage renal disease (ESRD) on maintenance haemodialysis (HD). Patients on HD have an annual mortality of 9.4% in Japan [1]. Cerebrovascular disease and cardiovascular disease, such as heart failure, cardiac arrhythmias and myocardial infarction, are responsible for 43.1% of these deaths [1]. Several factors, including the high prevalence of hypertension, diabetes mellitus (DM), dyslipidaemia, and perhaps an undefined predispositon toward accelerated atherosclerosis related to chronic uraemia, play an aetiologic role.

Homocysteine (Hcy), a sulfur-containing amino acid, is emerging as an important risk factor for atherosclerotic disease in patients with ESRD [2,3]. Hcy is an intermediate form generated during the metabolism of methionine and is not present in food. The transformation of methionine to Hcy involves the demethylation pathway, which provides a methyl group with glycocyamine in the creation of creatinine [4] (Figure 1Go). Notably, various studies show that the prevalence of increased serum Hcy levels is high in patients with ESRD [3,5–7]. In addition, DM is a recognized risk factor for atherosclerosis, and the morbidity of atherosclerosis in diabetic patients is two to three times higher than in the normal population [8]. However, little is known about the significance of Hcy in diabetic patients on HD. The purpose of this study was to elucidate the significance of Hcy in diabetic patients on HD.



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Fig. 1. Synthesis and metabolism of homocysteine. Synthesis and metabolism of Hcy involves three processes: demethylation, remethylation and trans-sulfuration. The demethylation pathway is associated with creatinine generation, i.e., S-adenosylmethionine provides a methyl group with glycocyamine on the generation of creatinine.

 



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
We evaluated 68 Japanese patients (38 men and 30 women; aged 66.7 ±1.8 years) with ESDR who received HD three times per week. The study population was restricted to patients whose urine volume was <100 ml between HD sessions with almost no residual renal function. Thirty-one patients (19 men and 12 women; aged 67.0±1.8 years) with ESRD caused by DM were assigned to the ‘DM group’ and 37 patients (19 men and 18 women; aged 66.3±1.8 years) with ESRD not caused by DM were placed in the ‘N group’. The characteristics of these two groups are given in Table 1Go.


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Table 1. Patient characteristics

 
Furthermore, we also classified the 68 patients according to three categories of vascular disease [9]. These categories were defined as follows. Cerebrovascular disease (CVD) was defined as a history of cerebrovascular accident not thought to be embolic in origin or an imaging study of the carotid arteries demonstrating significant occlusive disease (>50% stenosis). Coronary artery disease (CAD) was defined as a history of classic exertional angina, a previous angiogram showing significant occlusive disease (>50% stenosis), documentation of a previous myocardial infarction or a history of coronary artery bypass or angioplasty. Peripheral vascular disease (PVD) was defined as a history of intermittent claudication determined by patient interview, amputation of a limb due to obstructive arterial disease, or imaging studies documenting significant arterial disease. We assigned 47 patients with CAD, CVD or PVD to the ‘ASD(+) group’ (patients with atherosclerotic disease), and defined 21 patients with none of these diseases, the ‘ASD(-) group’.

Methods
Non-fasting serum total Hcy levels (tHcy) before and after HD were measured as the total amount of free and protein-bound Hcy by a modification of the fluorimetric high-performance liquid chromatography method originally described by Araki and Sako [10]. Kt/V (product of dialyser urea clearance and treatment time divided by body urea nitrogen) and protein catobolic rate (PCR) were calculated using the method described by Shinzato et al. [11]. Creatinine generation rate (CGR) was determined by the following equations of Shinzato et al. [12]: gint=gtotal-gext, where: gtotal=Cs[(7056/A)+({Delta}BW/IBW){240/(72-Td)}] and A=x-y, where x=3864+(7.8Td+411) ln(Cr/Cs)-1.5Td and y=1449/[(0.0190Td+0.999) ln(Cr/Cs)-(0.00367Td- 0.0219)], and where Cr=Ce[-81.622{ln(Ce/Cs)}/(60Td)+ 0.942]; and gext=7.79PCR2-7.91PCR+1.93.

The definitions of the symbols are as follows, gint (mg/kg/day) is the intrinsic creatinine generation rate, gext (mg/kg/day) is the extrinsic creatinine generation rate, gtotal (mg/kg/day) is the total creatinine generation rate, Cs, Ce and Cr (mg/dl) are the serum creatinine concentrations, before and after the first HD of the week, and the post rebound creatinine concentration of the same HD, respectively, {Delta}BW (kg) is the body weight loss during HD, IBW (kg) is the ideal body weight and Td (h) is the HD duration time. This method is not applicable to patients with residual renal function. We describe the intrinsic creatinine generation rate simply as the creatinine generation rate. Per cent creatinine generation rate (%CGR) is expressed as a percentage of the mean value of non-diabetic patients of the same gender and age.

Statistical analysis
Data are expressed as mean values±SE. Comparison between two groups was performed using the non-parametric Mann–Whitney U-test and the {chi}2 test. Linear regression analysis was used to assess the correlation of multiple variables on a single variable (serum tHcy). The variables included serum urea nitrogen (UN), serum creatinine (Cr), serum albumin (Alb), Kt/V, PCR, %CGR, total cholesterol (T Cho), low-density lipoprotein cholesterol (LDL Cho), high-density lipoprotein cholesterol (HDL Cho), triglyceride (TG) and systolic blood pressure. P<0.05 was accepted as statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Of the 68 patients, 64 had abnormally high serum tHcy (N, 100%; DM, 87.1%; Table 1Go). Serum tHcy after HD was significantly lower than before HD in both the N and the DM groups (pre-HD vs post-HD: N, 45.0±6.5 nmol/ml vs 27.0±3.8 nmol/ml, P<0.0001; DM, 24.6±1.7 nmol/ml vs 15.3± 1.1 nmol/ml, P<0.0001). Moreover, there was no significant difference in the rate of tHcy reduction by HD between the two groups (N vs DM, 39.2±1.7% vs 37.6±1.6%, NS; Table 1Go). The proportion of atherosclerotic diseases in the DM group was significantly higher than in the N group (N vs DM, 54.1% vs 87.1%, P=0.0075; Table 1Go). Serum tHcy in the DM group was significantly lower than in the N group (N vs DM, 45.0±6.5 nmol/ml vs 24.6±1.7 nmol/ml, P=0.0034; Table 1Go). In the N group, serum tHcy in the ASD(+) group was significantly higher than in the ASD(-) group (ASD(+) vs ASD(-), 57.7±11.1 nmol/ml vs 30.1±3.3 nmol/ml, P=0.0148), and serum tHcy was not correlated with any of the parameters evaluated (Table 2Go, Figure 2Go). In the DM group, there was no significant difference in serum tHcy between the ASD(+) group and the ASD(-) group (ASD(+) vs ASD(-), 24.3±1.9 nmol/ml vs 27.7±3.2 nmol/ml, NS), and serum tHcy was positively correlated with Cr, Alb and %CGR, respectively (r=0.551, P=0.0036; r=0.475, P=0.0155; r=0.462, P=0.0191, respectively; Table 2Go, Figure 2Go). %CGR was strongly correlated with Cr in both groups (N, r=0.701, P<0.0001; DM, r=0.852, P<0.0001).


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Table 2. Correlation between serum tHcy and parameters

 


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Fig. 2. Correlation between serum tHcy and Cr, Alb and %CGR. In the N group, serum tHcy was not correlated with Cr, Alb or %CGR, whereas in the DM group, serum tHcy was positively correlated with Cr, Alb and %CGR. (•) Patients with atherosclerotic disease. ({circ}) Patients without atherosclerotic disease.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
There were two major findings in this study: a significantly lower serum tHcy was found in diabetic patients compared with non-diabetic patients and there was an association between serum tHcy and %CGR in diabetic patients.

Most patients had abnormally high tHcy levels (reference values; 3–14 nmol/ml), which agrees with the results of previous reports [2]. Serum tHcy in the DM group was significantly lower than in the N group and was not associated with atherosclerotic disease. In contrast, Chico et al. [13] demonstrated that non-insulin-dependent diabetes mellitus (NIDDM) patients with microalbuminuria had increased Hcy levels compared with patients with normoalbuminuria; however, the causality between Hcy and albuminuria remains equivocal [14] and diabetic patients in previous studies may have included patients with various degrees of renal function and/or with nephropathy. Okada et al. [15] reported a clear relationship between hyperhomocysteinaemia and an increased risk of coronary arteriosclerosis in Japanese patients with type 2 diabetes. In our study, most of the diabetic patients had atherosclerotic disease at the beginning of HD therapy. The low number of patients without atherosclerotic disease (N=4) in the DM group (N=31) may have influenced the results of our statistical analysis, or factors other than tHcy may have contributed to atherosclerotic disease in the diabetic patients on HD. However, Hoogeveen et al. [16] found that serum tHcy >14 nmol/ml was a significant risk factor for ischaemic heart disease in NIDDM patients independent of classical risk factors. In addition, Kark et al. [17] reported that despite lower serum tHcy in diabetes, values >10 nmol/ml were a risk marker for death in diabetic patients. Therefore, we should pay adequate attention to evaluating serum tHcy although the relationship between serum tHcy and atherosclerosis could be altered by the presence of DM in patients on HD.

In the metabolism of Hcy, trans-sulfuration and remethylation are the main pathways in both the liver and kidney. The kidney plays a role in Hcy metabolism [19,18] although Hcy clearance is not affected to the same extent as creatinine clearance in patients with renal failure. In addition to reduced glomerular filtration, depressed tubular uptake of Hcy may occur in chronic renal failure, which then counteracts the reduced glomerular filtration rate (GFR) [7,18,20]. In contrast, Wollesen et al. [21] reported that GFR is a rate-limiting factor for renal clearance of Hcy and an independent determinant of Hcy in diabetes. They also suggested that reduced serum tHcy in diabetic patients is not the result of metabolic abnormalities. However, since all of the subjects in our study had almost no residual renal function, decreased GFR does not explain our results. Insulin is also involved in the regulation of Hcy by affecting the trans-sulfuration pathways in the liver [22]. Guldener et al. [23] measured serum tHcy and free Hcy in arterial and renal venous blood from the aorta and right renal vein in fasting patients with normal renal function, and demonstrated that no significant net renal uptake of Hcy occurs in fasting humans, suggesting that hyperhomocysteinaemia in renal failure is more likely to be caused by an impairment in extrarenal metabolism. Therefore, the major candidate site for extrarenal Hcy metabolism is the liver, where the metabolism of Hcy may be impaired by factors related to uraemia [23] and/or to DM.

We speculate that the significantly lower serum tHcy in the DM group is due to the metabolic process of methionine in the liver. Lower serum tHcy in the DM group may result from depression of the demethylation pathway and enhancement of the remethylation or trans-sulfuration pathway. Interestingly, Cr and %CGR, as well as serum tHcy, were significantly lower in the DM group than in the N group, and serum tHcy was positively correlated with Cr and %CGR in the DM group. No significant differences in other parameters, such as UN, Alb, PCR, Kt/V, T Cho, LDL Cho, TG, HDL Cho, vitamin B12 or folic acid, were found between the two groups (Table 1Go). Therefore, the nutritional status was similar between the two groups. As reported previously [18], in both groups HD significantly lowered serum tHcy by about 40% but failed to normalize it, although the rates of Hcy reduction by HD were similar between the two groups. Furthermore, %CGR was correlated strongly with Cr in both groups. The rates of Cr reduction by HD were similar in the two groups. CGR reflects the muscle mass, a possible indicator of protein nutritional status [12]. However, since some enzymatic reactions are associated with the intrinsic creatinine generation system, CGR alone does not reflect the muscle mass in patients with a disorder in creatinine synthesis. Therefore, our study suggests that the lower serum tHcy in the DM group may have been associated with impairment of the demethylation pathway of methionine metabolism in the liver. The demethylation pathway in methionine metabolism is linked directly to the creatinine generation system, i.e. transmethylation and demethylation are co-reactions: S-adenosylmethionine+glycocyamine (receptor) -> (S-adenosylhomocysteine+creatinphosphate (CH3-receptor) (Figure 1Go). In addition, serum tHcy in the DM group was also correlated with Alb and showed a weak positive correlation with PCR.

In conclusion, the demethylation pathway in methionine metabolism, which is linked directly to the creatinine generation system, may be disturbed in diabetic patients on HD, whereas serum tHcy in diabetic patients tends to be lower than those in non-diabetic patients. Therefore, the possibility of an altered relation between serum tHcy concentrations and the occurrence of vascular lesions needs to be considered when evaluating the atherogenic risk in diabetic patients on HD.



   Notes
 
Correspondence and offprint requests to: Kazuhiro Oishi, MD, Department of Medicine III, Okayama University Medical School, 2–5-1 Shikata-cho, Okayama 700–8558, Japan. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 25. 5.99
Revision received 18. 1.00.