Homocysteine and lipid peroxidation in haemodialysis: role of folinic acid and vitamin E

Beatriz Bayés1, Mari Cruz Pastor2, Jordi Bonal1, Jordi Juncà3 and Ramon Romero1,

1 Servicio de Nefrologia, 2 Servicio de Bioquímica and 3 Servicio de Hematologia, Hospital Universitari ‘Germans Trias i Pujol’, Badalona, Spain



   Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Cardiovascular diseases are the leading cause of death in haemodialysis patients. Hyperhomocysteinaemia is an independent risk factor. Basic research has provided strong evidence that oxidation of low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis. Oxidative stress, lipid metabolism alterations, and hyperhomocysteinaemia observed in haemodialysis patients could induce increases in LDL oxidation. This study was designed to determine the effect of folinic acid on hyperhomocysteinaemia and to assess the antioxidant efficacy of folinic acid. The antioxidant effect of folinic acid was compared with that of vitamin E.

Methods. Sixteen stable patients (11 men, five women; mean age 54.3±6.32 years) on standard haemodialysis received 400 mg of vitamin E, orally, at the end of each haemodialysis session for 3 months. After a 1-month wash-out, they received 10 mg of folinic acid, intravenously, at the end of each haemodialysis session for an additional 3 months. Blood samples were drawn in the morning after an overnight fast and before dialysis. Plasma vitamin E was analysed by high-pressure liquid chromatography. Malondialdehyde (MDA) was determined using a fluorimetric method and plasma copper oxidized anti-LDL antibodies (Ab-LDLox) were measured with an ELISA method using native LDL and oxLDL as antigens. Plasma homocysteine was determined by an FPIA method.

Results. Folinic acid supplements significantly reduced hyperhomocysteinaemia (-44%), MDA concentrations (-40%), and IgG-LDLox titres (-13%).

Conclusions. Treatment with folinic acid lowers plasma homocysteine levels and, like vitamin E, affords antioxidant protection, which prevents lipid peroxidation. This lowering of lipid peroxidation may reduce the risk of atherosclerosis and prevent or delay cardiovascular complications in HD patients.

Keywords: folinic acid; haemodialysis; homocysteine; lipid peroxidation



   Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cardiovascular disease is the main cause of death in chronic renal failure (CRF) patients on haemodialysis [1]. The pathogenesis of arteriosclerosis involves classical cardiovascular risk factors and other factors, including hyperhomocysteinaemia [2] and increases in oxidative stress [3]. Homocysteine favours cardiovascular risk through a mechanism involving oxidative damage [4]. When released in plasma, homocysteine rapidly auto-oxidizes. During oxidation of the sulfhydryl group of the particle, free radicals, including the superoxide anion radical (O2) and hydrogen peroxide (H2O2) are generated [5,6]. Both O2 and H2O2 cause endothelial cytotoxicity and lipid peroxidation [7,8]. The oxidative changes in low-density lipoprotein (LDL) particles modify their composition and promote foam cell formation, fatty streak development, and endothelial lesion progression [9]. Recent studies showed that the concentration of malondialdehyde (MDA) and oxidized anti-LDL antibodies (IgG-LDLox), two lipid peroxidation markers, are increased in patients on haemodialysis [10,11].

Treatment with folinic acid lowers homocysteine levels [12,13] and improves endothelial dysfunction in patients with hyperhomocysteinaemia [14,15].

The aims of this study were to determine whether folinic acid reduces hyperhomocysteinaemia, and to assess whether folinic acid supplementation reduces oxidative stress. In addition, the antioxidant effect of folinic acid was compared with that of vitamin E.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Sixteen patients on haemodialysis (11 men, five women; mean age 54.3±6.32 years) participated in the study. All were stable and had no intercurrent phenomena 3 months before the study. Eighteen per cent were active smokers. Participants had no known history of malignancy, were not insulin-dependent, and had no active liver disease. All patients received a minimum of 12 h per week of maintenance haemodialysis therapy (three sessions of 4 h each) with an HCO3 bath on hemophane membranes. Mean haemodialysis treatment time was 42 months. Patients were treated with intravenous (i.v.) iron therapy and erythropoietin to control their anaemia. All received 400 mg of vitamin E orally at the end of each haemodialysis session for 3 months. After 1 month of wash-out, they received 10 mg of folinic acid intravenously at the end of each haemodialysis session for an additional 3 months. No patient had received vitamin supplements 3 months before the study. Blood samples were drawn in the morning during fasting conditions before the start of the mid-week haemodialysis session.

The control group consisted of 30 healthy age-matched individuals extracted from the blood bank of our hospital.

Plasma vitamin E concentrations were measured by high-pressure liquid chromotography (HPLC) following methods described previously [16]. Within-day and between-day methodological errors were 4.65 and 5.2%, respectively. Concentrations of vitamin B12 and serum and erythrocytic folic acid were measured by radioimmunoassay (RIA) (Chiron, Emeryville, CA, USA): intra-assay coefficients of variation were 8.5% at 144.6 pmol/l and 7.3% at 604.9 pmol/l; inter-assay coefficients of variation were 4.3 and 4.5%, respectively, at the same concentrations. The detection limit of the assay was 60 pmol/l. Plasma homocysteine was assessed by fluorescence polarization immunoassay (Abbott), with within-day and between-day coefficients of variations of 2.45 and 5.6%, respectively. MDA concentrations were determined following the fluorimetric method of Yagi [17]. Within-day and between-day methodological errors were 5 and 7%, respectively [18]. LDL oxidation was assessed by the measurement of IgG-LDLox antibody titre. IgG-LDLox titre was measured using a solid base ELISA method that involved isolating plasma LDL from blood of healthy individuals by sequential ultra-centrifugation at a density range of 1025–1063 g/ml. LDL oxidation was performed by incubation of LDL (1–2 mg/ml protein) with 5 µmol/l Cu2+ for 16 h at 4°C. LDL was dialysed overnight in phosphate-buffered saline (PBS) to remove copper. Microtitre plates were coated with 100 µl of freshly isolated native LDL (nLDL) or 100 µl of Cu2+ oxidized LDL at a concentration of 8 µg/ml in PBS (pH 7.4). Following 16 h of incubation at 4°C, each well was washed three times with PBS containing 0.05% Tween 20. The remaining binding sites were blocked by incubating with a blocking reagent (1% human serum albumin) for 2 h at room temperature, and the wells were then washed three times as described above.

Patient serum (50 µl) was added in duplicate at a dilution 1/20 and incubated overnight at 4°C, and wells were washed three times. Following washing of the wells, 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. Plates were washed three times and 100 µl of chromogenic substrate (phenylenediamine dihydrochloride and H2O2) was added to each well. Following 30 min of incubation in the dark at room temperature, the reaction was stopped with 50 µl of 3 M HCl. Plates were read at 495 nm using a Micro Plate Reader A4.

Antibody titre was expressed as the ratio of antibodies against oxidized vs nLDL (absorbance with Cu-oxLDL/nLDL antibodies) [19]. All antibody determinations were made on a single day using the same batch of modified LDL and the same batch of reagents. The coefficient of variation was 6%.

Results are expressed as means±SD. Statistical analyses were performed using the non-parametric Wilcoxon test for paired data. The level of statistical significance was defined as P<0.05. All patients were informed of the purpose of the study and gave informed consent.



   Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The concentrations of vitamin E and serum and erythrocytic folic acid before and after treatment are shown in Table 1Go. Before treatment, all patients had vitamin B12 concentrations within the normal range. Treatment with folinic acid significantly reduced (P<0.05) plasma homocysteine, while vitamin E administration produced no modifications in plasma homocysteine concentration. Vitamin E significantly decreased MDA concentrations and IgG-LDLox antibody titres, and these values returned to basal values after 1 month of wash-out. Folinic acid significantly decreased MDA and IgG-LDLox antibody titres although the reduction in IgG-LDLox titre was greater with vitamin E than with folinic acid. Plasma levels of homocysteine, MDA, and IgG-LDLox before and after vitamin supplementation are shown in Table 1Go. No patient had adverse effects to the treatment during the study.


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Table 1. Plasma homocystein, MDA, and IgG-LDLox before and after vitamin supplementation

 



   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our results show that folinic acid reduced homocysteine by 44%, but vitamin E produced no effect on homocysteine levels. However, and more importantly, folinic acid reduced lipid peroxidation. Cardiovascular disease is the main cause of morbidity and mortality in haemodialysis [1]. Thus, while control and reduction of classical risk factors are important, other risk factors must be considered, including oxidative stress and hyperhomocysteinaemia [4,20]. The study by Stenvinkel et al. [21] indicated that the rapidly developing atherosclerosis in uraemic patients appeared to be a synergism of malnutrition, inflammation, and oxidative stress. In fact, uraemic patients possess numerous factors that increase oxidative stress and favour LDL oxidation. These factors include increases in free radical production, decreases in primary antioxidant mechanisms (vitamins and enzymes), increases in the time LDL remains in the vascular wall, reductions in the antioxidant capacity of HDL, and accumulation of homocysteine [22].

We analysed the role of lipid peroxidation and hyperhomocysteinaemia as non-traditional cardiovascular risk factors in patients on haemodialysis.

As in the majority of studies [23], our patients on haemodialysis presented moderate hyperhomocysteinaemia (mean plasma Hcy 36±25 µmol/l). In two recent prospective studies by Bostom et al. [24] and Moustapha et al. [25], hyperhomocysteinaemia was associated with atherosclerotic disease and with the increase in relative risk of cardiovascular morbidity-mortality in patients with CRF. Recent studies have identified the epithelium as the site where hyperhomocysteinaemia produces the greatest damage. Although the exact mechanism of homocysteine-induced endothelial dysfunction remains unknown, there is growing evidence that it facilitates oxidative injury. When homocysteine is added to plasma, it readily auto-oxidizes, leading to the formation of metabolites and the generation of oxygen free radicals (O2 and H2O2) which are implicated in vascular toxicity and initiate lipid peroxidation [26]. Oxidation of LDL transforms these lipoproteins into more atherogenic particles with vasomotor and thrombogenic properties, thereby favouring the onset and progression of atheromatous plaques [27]. Thus, we wished to determine whether folinic acid decreases homocysteine and assess its effects on LDL oxidation.

LDL oxidation is increased in haemodialysis patients. In agreement, we found increases in MDA and IgG-LDLox, both markers of lipid peroxidation. Whereas MDA is an end product of non-specific lipid peroxidation, IgG-LDLox antibodies result from an autoimmune response against LDL oxidation. Previous studies have demonstrated the usefulness of these markers for assessing cardiovascular risk [10,19,28]. In a cross-sectional study, Boaz et al. [10] found that serum MDA was the single strongest predictor of cardiovascular disease prevalence in a haemodialysis population, whereas traditional risk factors, including LDL cholesterol, were not predictive. Salonen et al. [19] showed that Ab-LDLox antibody titres were a predictive factor of carotid atheromatosis progression.

Several studies have used antioxidant treatments to reduce and prevent atherosclerosis. In the present study, we observed a decrease in both MDA and Ab-LDLox titre after treatment with vitamin E, which indicates an improvement in lipid peroxidation parameters. Although conflicting results have been published, several studies have shown that vitamin E administration may decrease the atherogenic potential of LDL. Mune et al. [29] showed that patients dialysed with vitamin E-coated membrane for 2 years had lower levels of oxidized LDL and slower atherosclerosis progression compared with controls.

Treatment with folinic acid has proved to be highly effective for reducing plasma homocysteine concentrations [12], except for renal failure where this and other treatments achieve only moderate decreases in homocysteine levels. A recent study by Bostom et al. [30] confirmed that moderate hyperhomocysteinaemia in haemodialysis patients is refractory to different vitamin treatments. In our study, homocysteine decreased by 44% after a total dose of 30 mg of i.v. folinic acid per week. Furthermore, we observed statistically significant decreases in both MDA and IgG-LDLox titres. This suggests that folinic acid exerts an indirect antioxidant effect by reducing the pro-oxidant action of homocysteine and, consequently, lowering oxidative stress [5]. A recent study in hypercholesterolaemic patients showed that folinic acid also exerts direct antioxidant effects during restoration of endothelial function, probably by affecting cellular oxidative metabolism [31].

In conclusion, treatment with folinic acid lowers plasma homocysteine levels and exerts adequate antioxidant protection by decreasing lipid peroxidation. These results suggest that folinic acid may be a safe and efficacious treatment for improving cardiovascular prognosis in patients on haemodialysis.



   Notes
 
Correspondence and offprint requests to: Ramon Romero, Department of Nephrology, Hospital Universitari ‘Germans Trias i Pujol’, Carretera del Canyet s/n., E-08916 Badalona, Spain. Email: bbayes{at}teleline.es Back



   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 14. 3.00
Revision received 29. 1.01.