Homocysteine, C-reactive protein, lipid peroxidation and mortality in haemodialysis patients

Beatriu Bayés1, M. Cruz Pastor2, Jordi Bonal1, Jordi Juncà3, José M. Hernandez2, Nadal Riutort2, Andreu Foraster4 and Ramón Romero1,

1 Nephrology Department, 2 Clinical Biochemistry Department, 3 Haematology Department and 4 Haemodialysis Unit, University Hospital ‘Germans Trias i Pujol’, Badalona, Universidad Autonoma de Barcelona, Barcelona, Spain



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cardiovascular disease (CVD) is common in haemodialysis patients with chronic renal insufficiency and is the leading cause of death. The accelerated state of atherosclerosis found in these patients is due to a combination of different mechanisms. Recent studies confirm that inflammation plays an important role in the development of atherosclerosis. However, the role of hyperhomocysteinaemia and the immune response to oxidation of low-density lipoproteins (LDL) remains unclear and studies show contradictory results. The objective of this study was to determine whether there is a relationship between inflammation, hyperhomocysteinaemia and oxidative stress and whether these CVD risk factors are predictors of mortality in haemodialysis patients.

Methods. A prospective follow-up study was carried out in 94 stable, chronic haemodialysis patients for 24 months (July 1999–July 2001). All the patients were given folic acid and vitamin B complex supplements. Homocysteine was determined by fluorescence polarization immunoassay. C-reactive protein (CRP) levels were determined by chemiluminescent enzyme-labelled immunometric assay. Plasma copper oxidized anti-LDL (oxLDL) antibodies were measured by ELISA using native LDL and oxLDL as antigens.

Results. Thirty-two patients died during the study and 59.3% of the deaths could be attributed to CVD (eight to acute myocardial infarction and 11 to non-coronary vascular disease). The patients had slight hyperhomocysteinaemia (25.8±7.82 µmol/l), evidence of inflammation (CRP 5.16 mg/l (0.35–88.7)) and oxidative stress (oxLDL antibodies=162±77 optical density at 495 nm x1000). Age (P<0.01), CRP (P=0.03) and the oxLDL antibody titre (P<0.01) were predictive of mortality. The patients who died from heart disease showed higher oxLDL antibody titres (P=0.03). No correlation was found between homocysteine, CRP and the oxLDL antibody titre, or between serum homocysteine levels and the different causes of mortality.

Conclusions. These results suggest that lipid peroxidation and inflammation, but not hyperhomocysteinaemia, are the main risk factors for mortality in haemodialysis patients receiving vitamin supplements. As the study was carried out in a relatively limited number of patients, our findings need to be confirmed in a larger patient population.

Keywords: C-reactive protein; folic acid; haemodialysis; homocysteine; mortality; oxidized LDL antibodies



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cardiovascular disease (CVD) is the leading cause of mortality in haemodialysis patients. The increased incidence of CVD is likely to be the result of a high prevalence of both traditional risk factors, such as hypertension, dyslipidaemia and smoking, and non-traditional risk factors, such as hyperhomocysteinaemia, oxidative stress and inflammation.

High total plasma homocysteine concentrations are often detected in patients with end-stage renal disease (ESRD) and are an independent risk factor for the development of CVD [1]. The hyperhomocysteinaemia found in these patients is refractory to different vitamin treatments [2] and, among other causes, may be attributed to a change in homocysteine metabolism as a result of uraemia. Experimental and clinical evidence suggests that moderate hyperhomocysteinaemia may predispose individuals to endothelial dysfunction through a mechanism that involves generation of reactive oxygen species [3].

The atherogenic effects of oxidative stress, especially those involving generation of oxidized low-density lipoproteins (LDL), have been reported in patients undergoing dialysis [4]. Target-specific oxidation processes in LDL generate an immunological response leading to the formation of anti-oxLDL (oxLDL) autoantibodies that may be involved in the overall process of atherogenesis [5].

Atherosclerosis is clearly multifactorial and it is well known that inflammation is an important factor in the onset and progression of lesions. C-reactive protein (CRP) is an excellent marker of systemic inflammation. Ridker et al. [6] have shown that systemic inflammation is a strong risk factor independent of CVD. A recent study shows that chronic inflammation is the main risk factor for CVD in ESRD [7]. It has also been suggested that chronic inflammation leads to lipid peroxidation, thereby promoting atherogenesis [8].

Recent studies suggest that folic acid may play a role in the prevention of CVD [9]. Although the exact mechanisms underlying the ameliorative effects of folates on the endothelium remain to be elucidated, the beneficial effect of folates on endothelial function independent of changes in plasma homocysteine levels has been described recently. Potential mechanisms include antioxidants actions, effects on cofactor availability or direct interactions with the enzyme endothelial nitric oxide synthase.

Our study aimed to analyse, firstly, whether there is a relationship between the plasma concentration of homocysteine, systemic inflammation (measured according to CRP serum levels) and systemic lipid peroxidation (identified by oxLDL antibody titre) and, secondly, to determine whether hyperhomocysteinaemia, CRP and the oxLDL antibody titres are predictors of mortality in haemodialysis patients with ESRD.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
A prospective follow-up study of 94 stable, chronic haemodialysis patients was carried out during a 24 month period from July 1999 to July 2001, and the incidence and causes of mortality were determined. All patients received a minimum of 12 h per week maintenance haemodialysis therapy (three sessions of 4 h each) with an HCO3 bath on haemophane membranes.

The 94 haemodialysis patients studied included 50 men (53.2%) and 44 women (46.8%) aged 23–88 years (mean 66±14 years), 63.8% of whom were older than 65 years of age. The mean time from starting dialysis to joining the study was 46.90 (44.5) months. Eighty-two of the patients (87.2%) were undergoing haemodialysis due to arteriovenous fistula.

The underlying renal diseases were: diabetes mellitus (17%), nephroangiosclerosis (17%), chronic glomerular nephritis (13.6%), tubulointerstitial nephropathy (12.5%), polycystic liver and kidney disease (9.1%), and 30.7% were of unknown cause.

History of ischaemic cardiopathy (31.4%), peripheral vasculopathy (33.3%), cerebrovascular stroke (14.3%), smoking (34.1%), hypertension (76.1%) and diabetes mellitus (20.9%) were recorded.

Following dialysis, all patients were administered oral vitamin supplements: folic acid 5 mg (Acfol®, Italfarmaco), and hydroxycobalamin 1000 µg, pyridoxine 250 mg and thiamine chlorhydrate 250 mg (Benexol®, Roche B1-B6-B12).

Diagnostic criteria
The diagnostic criteria used to study previous disease activity are described below.

Coronary artery disease was diagnosed if the following were present: (i) a documented myocardial infarction; (ii) stenosis of >70% of at least one major epicardial coronary vessel recorded at the time of coronary angiography conducted according to standard procedure; or (iii) an abnormal cardiac effort test. Peripheral vascular disease was diagnosed if there were diminished pulses on clinical examination combined with measurements of peripheral vascular resistance and/or peripheral angiography. Cerebrovascular disease was suspected on clinical grounds and the diagnosis was confirmed by computerized tomography, magnetic resonance imaging and duplex carotic ultrasonography. Subjects were classified either as non-smokers (if they had never smoked) or ever-smokers (if they were current or ex-smokers).

Hypertension was defined as a blood pressure >140/90 mmHg or, in the presence of a history of hypertension, if the patient was also taking antihypertensive medications. Diabetes mellitus was diagnosed if patients were using insulin or if their fasting glucose concentration was >140 mg/dl.

Methods
Blood samples were drawn in the morning during fasting conditions before the start of the mid-week haemodialysis session. To determine the homocysteine plasma concentrations, blood was drawn into a vacutainer tube and EDTA was used as an anticoagulant. The samples were kept refrigerated before centrifuging (less than 1 h). To determine the other parameters, serum was collected from a tube containing blood with no anticoagulant. The samples were frozen at -80°C until examination.

Serum concentrations of vitamin B12 and serum folic acid were measured by radioimmunoassay (RIA) (Chiron, Emeryville, CA, USA). The level of vitamin B6 in plasma was determined using the tyrosine decarboxylase apoenzyme method. Total cholesterol, triglyceride and HDL cholesterol levels were all determined using enzymatic methods (Dax System, Bayer Diagnostic, Munich, Germany). The concentration of LDL cholesterol was calculated according to Friedewald's formula. Plasma albumin concentration was determined using Technicon Omnipack® bromocresol green method (Dax System, Bayer Diagnostic). Plasma homocysteine was assessed by fluorescence polarization immunoassay (Abbott IMX instrument, Chicago, IL, USA). Serum CRP levels were measured by chemiluminescent enzyme-labelled immunometric assay, based on ligand-labelled monoclonal antibody and separation by anti-ligand-coated solid phase (Immulite, Dipesa SA, Madrid, Spain).

Oxidation of LDL was assessed by measuring immunoglobulin G (IgG) oxLDL antibody titres [10] using a solid-phase immunoassay method that involved isolating plasma LDL from blood of healthy subjects by sequential ultracentrifugation. Oxidation of LDL was performed by incubating LDL with Cu2+. Microtitre plates were coated with freshly isolated native LDL and Cu2+ oxidized LDL at a concentration of 8 µg/ml in PBS (pH 7.4).

Patient serum was assayed in duplicate at a 1/20 dilution and incubated overnight at 4°C. After the wells were washed three times, 100 µl of peroxidase-conjugated rabbit anti-human IgG was added at a dilution of 1:1500 in PBS and incubated for 3 h at 37°C. Phenylenediamine dihydrochloride and H2O2 was used as a chromogenic substrate. Plates were read at 495 nm using a Micro Plate Reader A4. Results were expressed as optical density (OD) x1000.

The levels of anti-oxLDL antibodies were calculated by subtracting the value obtained from binding to native LDL from binding to oxLDL [11]. All antibody determinations were conducted on the same day using the same batch of modified LDL and the same batch of reagents. The coefficient of variation was 6%.

The study received approval from the local ethics committee and all patients gave their informed consent to participate in the study.

Statistical analysis
For bivariate analysis between qualitative variables the chi-square test was used. However, when the conditions for application of this test were not met, Fisher's exact test was used. Student's t-test or the Mann–Whitney U-test were used to assess differences in continuous between-group variables. Pearson correlation was used to assess the association between continuous variables. In order to analyse the risk of death during the follow-up, Kaplan–Meier curves and Cox proportional risk models, including the variables showing statistical significance in the bivariate analysis, were conducted. All bivariate interactions were analysed. Statistically significant differences were defined as {alpha}<0.05.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Thirty-two patients died during the study. Ten underwent renal transplant and one changed the dialysis method to peritoneal dialysis. The analytical parameters studied are shown in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1.  Analytic parameters and age for the haemodialysis patients according to their vital status at the end of follow-up

 

Hyperhomocysteinaemia and vitamin concentration
The patients showed moderate hyperhomocysteinaemia, with a mean homocysteine plasma concentration of 25.8 (7.8) µmol/l. The median was 24.1 µmol/l. Normal homocysteine levels (<15 µmol/l) were recorded in 5.7% of the patients.

The mean concentrations of vitamin B12, vitamin B6 and serum folic acid were 664 (435) pg/ml, 79 (113) nmol/l and 8.21 (4.89) ng/ml, respectively. A total of 3.3% of the patients showed vitamin B12 deficiency (<200 pg/ml) and 40.4% vitamin B6 deficiency (<20 nmol/l). Serum folic acid deficiency (<2.2 mg/ml) was found in 6.6% of patients.

There was a significant inverse correlation between homocysteine and the main vitamins studied and co-factors that are involved in their metabolism: homocysteine–vitamin B12 (r=-0.29, P=0.005); homocysteine–vitamin B6 (r=-0.26, P=0.02); and serum homocysteine–folic acid (r=-0.37, P=0.0001).

C-reactive protein concentrations and serum albumin
The mean serum CRP level was 11 (16) mg/l. The median CRP level was 5.1 mg/l (0.35–88). A total of 45.3% of the patients had CRP >6 mg/l (normal CRP: <6 mg/l). A positive correlation was found between CRP and age (r=0.313, P=0.003).

The mean serum albumin concentration was 43 (4) g/l. No patients showed plasma albumin <30 g/l (normal range: 30–55 g/l). We found no correlation between the CRP and albumin level, or between CRP and vitamin B12, vitamin B6 or folic acid.

Oxidized LDL antibody titre
The mean antibody titre (IgG) was 162 (153) (OD at 495 nm x1000). The median oxLDL antibody titre was 153 (OD at 495 nm x1000). There was a correlation between oxLDL antibody titre and total cholesterol, LDL cholesterol and triglycerides but not with the other parameters studied.

No correlation was found between the homocysteine plasma concentration, CRP and anti-oxLDL antibody titre.

Causes of death, incidence and predictors of mortality
Thirty-two patients died during the 24 month follow-up period (July 1999–July 2001). Causes of death were divided into two large groups, CVD-related and non-CVD related. Nineteen deaths were CVD-related (eight from acute myocardial infarction (AMI) and 11 from non-coronary vascular disease). Thirteen patients died as a result of other disease processes: one from chronic lymphatic leukaemia; one from multiple myeloma; one from cancer of the pancreas; one from mesothelioma; one from sepsis; two from cirrhosis of the liver with concurrent hepatitis C infection; two from subdural haematoma due to head and brain injury; one following surgery for a fractured femur; and three from unknown causes.

The relation between analytical parameters studied and the survival of the patients during the follow-up period are shown in Table 1Go. The patients who died were often older (P<0.01) and had higher CRP levels (P=0.02) as well as a higher oxLDL antibody titre (P=0.01). There were no differences in the serum homocysteine levels or in the other parameters studied.

The clinical and analytical factors that were predictors of mortality are shown in Table 2Go. It was found that age (P<0.01), CRP (P=0.03) and oxLDL antibody titre (P<0.01) were predictors of mortality in this group of patients.


View this table:
[in this window]
[in a new window]
 
Table 2.  Unadjusted mortality-related risk factors

 
Figure 1Go shows the survival curve for the haemodialysis patients in relation to the CRP levels. The 30th percentile was used as a cut-off point (3 mg/l). The patients with a CRP <3 mg/l (a low inflammatory status) showed a better 24 month survival (P=0.01).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.  Kaplan–Meier estimate of survival in haemodialysis patients with serum CRP levels >=3 mg/l vs <3 mg/l ODx1000.

 
Figure 2Go shows the survival curve for the haemodialysis patients in relation to the oxLDL antibody titre. This parameter was categorized into two groups using the median oxLDL antibody titre (153 OD at 495 nm x1000) as the cut-off point. The patients with lower lipid peroxidation showed greater survival. Seventy-five per cent of the patients who died had oxLDL antibody levels >153 (OD at 495 nm x1000) compared with 33.3% of those who survived (P<0.01).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.  Kaplan–Meier estimate of survival in haemodialysis patients with plasma copper oxidized anti-LDL antibody levels >153 vs <=153.

 

Relation between cause of death, homocysteine level, CRP and oxidized LDL antibody titre
Table 3Go shows the relation between homocysteine, CRP level and oxLDL antibody titre and the cardiovascular mortality. The patients who died from non-coronary vascular disease showed higher CRP serum concentrations (P=0.02), while those who died from heart disease (AMI) showed higher levels of oxLDL antibody (P=0.03). The plasma concentration of homocysteine was not associated with the different causes of mortality.


View this table:
[in this window]
[in a new window]
 
Table 3.  Plasma homocysteine (Hcy), serum CRP, plasma copper oxidized LDL antibodies (IgG) (oxLDL Ab) and cardiovascular mortality

 
The multivariate analysis of mortality is shown in Table 4Go. When adjusted for age and anti-oxLDL antibody titre in the multivariate analysis, CRP continued to be a significant risk factor (OR: 3.1; CI 95%: 1.04–9.25; P=0.04). When adjusted for age and CRP level, oxLDL antibody titre remained a significant risk factor (OR: 1.006; CI 95%: 1.002–1.010; P<0.01).


View this table:
[in this window]
[in a new window]
 
Table 4.  Mortality study—multivariate analysis of the overall mortality

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The main finding of this study was that oxLDL antibody titres and CRP levels are predictors of mortality in haemodialysis patients on vitamin supplements, while homocysteine levels are not.

Hyperhomocysteinaemia is a new risk factor for atherosclerotic CVD in the general population and in patients with ESRD. Persistent, mild hyperhomocysteinaemia is common in patients with ESRD [1]. It is well known that vitamin supplements reduce the concentration of homocysteine in haemodialysis patients despite the fact that they do not normalize it [2].

Although it is accepted that hyperhomocysteinaemia is a CVD risk factor, recent studies analysing the role of homocysteine as a predictor of cardiovascular morbidity and mortality show controversial results both in haemodialysis patients and in the general population. Bostom et al. [1] and Moustapha et al. [12] showed that hyperhomocysteinaemia entailed an increased risk for CVD. Recently, Mallamaci et al. [13] have confirmed that there is an association between hyperhomocysteinaemia and cardiovascular mortality in haemodialysis patients not receiving vitamin supplements, with a 20% increased risk of fatal cardiovascular events for every 10 µmol/l increase in homocysteine concentration. However, other studies [14,15] found no association or only a small association between plasma homocysteine levels and cardiovascular disease. Furthermore, Suliman et al. [16] suggested that patients with lower homocysteine levels have a worse chance of survival than those with high plasma homocysteine concentrations as a result of malnutrition. In our study, homocysteine was not a good predictor of overall or cardiovascular mortality. Our patients showed very homogeneous moderate hyperhomocysteinaemia after taking oral vitamin supplements and there were no statistically significant differences (P=0.51) in the plasma homocysteine concentrations in the different groups with regard to causes of mortality. The lack of correlation between hyperhomocysteinaemia and CVD mortality could be due to the beneficial effect of the vitamin supplements.

There are various mechanisms by which homocysteine leads to endothelial lesions, but recent studies have proposed that increased oxidant stress is the main one [3]. Voutilainen et al. [17] found a strong association between F2-isoprostane and homocysteine and suggested that elevated levels of homocysteine are associated with an increase in in vivo lipid peroxidation. In a previous study [10], we analysed the relation between homocysteine and lipid peroxidation in haemodialysis patients and found that treatment with folic acid lowered plasma homocysteine levels and improved lipid peroxidation while decreasing oxLDL antibody titre, although it did not reach the normal level. Despite the fact that previous studies were not able to show an improvement in endothelial function in uraemic patients treated with folic acid [18], it has been demonstrated recently [19] that folic acid, in addition to reducing homocysteine concentration, improves endothelial function in haemodialysis patients. In the present study we found no correlation between plasma homocysteine levels and oxLDL antibody titre. This may be attributed to the fact that since the beginning of renal replacement treatment, all the patients had been receiving vitamin supplements, which had been reported previously to reduce homocysteine plasma concentration and oxLDL antibody titre [10], although not proportionately. In addition, oxLDL is influenced by factors other than hyperhomocysteinaemia (hypertriglyceridaemia, smoking, hypertension, diabetes, low levels of HDL cholesterol and predominance of small and dense LDL cholesterol) independent of homocysteine metabolism.

In haemodialysis patients receiving vitamin supplements, Wrone et al. [15] found no relation between homocysteine levels and CVD and suggested that inflammation may play a role in the development of this disease. The relation between homocysteine and CRP has also been a topic of research. Although an experimental study [20] has suggested that there is a relation between the two, our study confirms the observations of Mezzano et al. [7] in finding no association between the increase in homocysteine levels and markers of inflammation.

CRP is a good marker of systemic inflammation and is higher in haemodialysis patients. Like other studies, we showed elevated CRP levels (45.3% of the patients had CRP >6 mg/l (normal CRP: <6 mg/l)). Many studies describe CRP as an independent CVD risk factor and a predictor of cardiovascular mortality [6,21]. However, the stimulus that generates moderate increases in CRP and its role as a predictor of coronary events is unknown. Our study confirms the results of previous trials in demonstrating that CRP is a predictor of overall and cardiovascular mortality, despite there being no correlation between inflammation and the other cardiovascular risk factors studied. Patients who die as a result of CVD (non-coronary vascular disease and AMI) show a greater inflammatory response than those who die from other causes (P<0.01).

Various studies in the literature have analysed the relation between inflammation and oxidative stress. A recent study has shown that the host response to infection and inflammation increases oxidized lipids in serum and induces LDL oxidation in vivo [8]. It also suggested that the increase in the oxidation of LDL during the inflammatory process can promote atherogenesis and may be responsible for the increase in the incidence of CVD in patients with chronic inflammatory disease. In our study, unlike other previously published trials [22], we found no correlation between inflammatory state and lipid peroxidation despite them being two independent risk factors for mortality. The role of folic acid in the markers of inflammation remains unclear, but an increasing number of studies have shown that it is related to a beneficial effect which is additional to that caused by the reduction of homocysteine [9]. As mentioned above, our previous study demonstrated that in haemodialysis patients, folic acid acted as an antioxidant, since it led to a reduction in the markers of lipid peroxidation [10]. A recent study has observed that folic acid treatment reduces LDL oxidation and regulates the inflammatory response of peripheral blood mononuclear cells in hyperhomocysteinaemic subjects [23]. It is possible that no correlation will be found between inflammation and lipid peroxidation due to the additional effects of folic acid [9].

Haemodialysis patients show an increase in oxidative stress that leads to lipid peroxidation. Numerous factors affect oxidized levels, such as the production of oxygen-free radicals, LDL lipid content, LDL concentrations, antioxidant concentrations, the condition of the vascular wall, blood pressure and clearance efficiency. The in vitro and in vivo increase in LDL oxidation seen in uraemic patients may play a significant role in the development of atherosclerosis [22]. Oxidation of LDL transforms low-density lipoprotein into more atherogenic particles with vasomotor and thrombogenic properties, thereby favouring the onset and progression of atheromatous plaques. Autoantibodies against oxidized LDL have been considered a sensitive marker for the detection of LDL oxidation in vivo; haemodialysis patients show elevated oxLDL antibody titres [4]. Evidence that oxLDL is present in the circulation is strongly supported by the fact that autoantibodies against oxLDL exist in patients with atherosclerosis. A study by Toshima et al. [24] suggests that plasma oxLDL levels may be a biochemical risk marker for CVD. The results of our study demonstrate that the IgG oxLDL antibody titre is a predictor of mortality in these patients and that the levels of oxLDL antibodies are significantly higher statistically in patients who die from AMI. Despite the fact that oxLDL antibody titre is a predictor of mortality from AMI, that other studies support the hypothesis that a high oxLDL titre predisposes patients to atherosclerosis and CVD, and that one study even suggests that oxLDL titre is also a marker of coronary atheromatous plaque instability [25], this finding should be taken with caution because the sample of patients who die from AMI is small.

Our study suggests that the increase in CRP and lipid peroxidation is not associated with hyperhomocysteinaemia, and that cardiovascular mortality is not a reflection of the vascular damage produced by homocysteine, but rather a synergism between oxidative stress and systemic inflammatory response.

In conclusion, stable haemodialysis patients who took vitamin B complex and folic acid supplements showed moderate hyperhomocysteinaemia, increase in systemic inflammation (CRP) and marked moderate lipid peroxidation (oxLDL antibody titre), without correlation between the parameters studied. In these patients, CRP, oxLDL antibody titres and age, but not homocysteine, were predictors of mortality. As the study was carried out in a relatively limited number of patients, the results need to be confirmed in a larger patient population.



   Notes
 
Correspondence and offprint requests to: R. Romero, Nephrology Department, University Hospital ‘Germans Trias i Pujol’, Badalona, Universidad Autonoma de Barcelona, Barcelona, Spain. Email: rromero{at}ns.hugtip.scs.es Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Bostom AG, Shemin D, Verhoef P et al. Elevated fasting total plasma homocysteine levels and cardiovascular disease outcomes in maintenance dialysis patients. A prospective study. Arterioscler Thromb Vasc Biol1997; 17:2554–2558[Abstract/Free Full Text]
  2. Shemin D, Bostom A, Selhub J. Treatment of hyperhomocysteinemia in end-stage renal disease. Am J Kidney Dis2001; 38:S91–S94[Medline]
  3. Kanani P, Sinkey C, Browning R, Allaman M, Knapp H, Haynes W. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation1999; 100:1161–1168[Abstract/Free Full Text]
  4. Maggi E, Bellazzi R, Falaschi F et al. Enhanced LDL oxidation in uremic patients: an additional mechanism for accelerated atherosclerosis? Kidney Int1994; 45:876–883[ISI][Medline]
  5. Witztum JL, Steinberg D. Role of oxidised low density lipoprotein in atherogenesis. J Clin Invest1991; 88:1785–1792[ISI][Medline]
  6. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med1997; 336:973–979[Abstract/Free Full Text]
  7. Mezzano D, Pais EO, Aranda E et al. Inflammation, not hyperhomocysteinemia, is related to oxidative stress and hemostatic and endothelial dysfunction in uremia. Kidney Int2001; 60:1844–1850[CrossRef][ISI][Medline]
  8. Memon RA, Staprans I, Noor M et al. Infection and inflammation induce LDL oxidation in vivo. Arterioscler Thromb Vasc Biol2000; 20:1536–1542[Abstract/Free Full Text]
  9. Verhaar MC, Stroes E, Rabelink TJ. Folates and cardiovascular disease. Arterioscler Thromb Vasc Biol2002; 22:6–13[Abstract/Free Full Text]
  10. Bayés B, Pastor MC, Bonal J, Juncà J, Romero R. Homocysteine and lipid peroxidation in haemodialysis: role of folic acid and vitamin E. Nephrol Dial Transplant2001; 16:2172–2175[Abstract/Free Full Text]
  11. Sherer Y, Tenenbaum A, Blank M et al. Autoantibodies to oxidised low-density lipoprotein in coronary artery disease. Am J Hypertens2001; 14:149–154[CrossRef][ISI][Medline]
  12. Moustapha A, Naso A, Nahlawi M et al. Prospective study of hyperhomocysteinemia as an adverse cardiovascular risk factor in end-stage renal disease. Circulation1998; 97:138–141[Abstract/Free Full Text]
  13. Mallamaci F, Zoccali C, Tripepi G et al. on behalf of the CRREED Investigators. Hyperhomocysteinemia predicts cardiovascular outcomes in hemodialysis patients. Kidney Int2002; 61:609–614[CrossRef][ISI][Medline]
  14. Eikelboom JW, Lonn E, Genest J Jr, Hankey G, Yusuf S. Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence. Ann Intern Med1999; 131:363–375[Abstract/Free Full Text]
  15. Wrone E, Zehnder J, Hornberger J, McCann L, Coplon N, Fortmann S. An MTHFR variant, homocysteine, and cardiovascular comorbidity in renal disease. Kidney Int2001; 60:1106–1113[CrossRef][ISI][Medline]
  16. Suliman ME, Qureshi AR, Barany P et al. Hyperhomocysteinemia, nutritional status, and cardiovascular disease in hemodialysis patients. Kidney Int2000; 57:1727–1735[CrossRef][ISI][Medline]
  17. Voutilainen S, Morrow J, Roberts J et al. Enhanced in vivo lipid peroxidation at elevated plasma total homocysteine levels. Arterioscler Thromb Vasc Biol1999; 19:1263–1266[Abstract/Free Full Text]
  18. Thambyrajah J, Landray MJ, McGlynn FJ, Jones HJ, Wheeler DC, Townend JN. Does folic acid decrease plasma homocysteine and improve endothelial function in patients with predialysis renal failure? Circulation2000; 102:871–875[Abstract/Free Full Text]
  19. Buccianti G, Raselli S, Baragetti I et al. 5-Methyltetrahydrofolate restores endothelial function in uraemic patients on convective haemodialysis. Nephrol Dial Transplant2002; 17:857–864[Abstract/Free Full Text]
  20. Hofmann MA, Lalla E, Lu Y et al. Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model. J Clin Invest2001; 107:675–683[Abstract/Free Full Text]
  21. Wanner C, Zimmermann J, Schwedler S, Metzger T. Inflammation and cardiovascular risk in dialysis patients. Kidney Int2002; 61:99
  22. Stenvinkel P, Heimbürger O, Paultre F et al. Strong association between malnutrition, inflammation, and atherosclerosis in chronic renal failure. Kidney Int1999; 55:1899–1911[CrossRef][ISI][Medline]
  23. Holven K, Aukrust P, Holm T, Ose L, Nenseter M. Folic acid treatment reduces chemokine release from peripheral blood mononuclear cells in hyperhomocysteinemic subjects. Arterioscler Thromb Vasc Biol2002; 22:699–703[Abstract/Free Full Text]
  24. Toshima S, Hasegawa A, Kurabayashi M et al. Circulating oxidised low density lipoprotein levels. A biochemical risk marker for coronary heart disease. Arterioscler Thromb Vasc Biol2000; 20:2243–2247[Abstract/Free Full Text]
  25. Ehara S, Ueda M, Naruko T et al. Elevated levels of oxidised low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation2001; 103:1955–1960[Abstract/Free Full Text]
Received for publication: 17. 2.02
Accepted in revised form: 21. 8.02