NDT Advance Access originally published online on February 22, 2007
Nephrology Dialysis Transplantation 2007 22(6):1685-1689; doi:10.1093/ndt/gfm068
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Autoantibodies against N-homocysteinylated proteins in patients on long-term haemodialysis
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Gowacki3awa Tracz1
1Institute of Cardiology, Jagiellonian University School of Medicine, Cracow, 2Haemodialysis Unit Miechow, Miechow, 3Department of Environmental Chemistry, University of 
odz, odz and 4Department of Endocrinology, Jagiellonian University School of Medicine, Cracow, Poland
Correspondence and offprint requests to: Anetta Undas, MD, PhD, Institute of Cardiology, Jagiellonian University School of Medicine, 80 Pradnicka St.31-202 Cracow, Poland. Email: mmundas{at}cyf-kr.edu.pl
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Abstract |
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Background. Autoantibodies against N
-homocysteinylated (N-Hcy) proteins at high titres have been demonstrated in patients with premature coronary artery disease (CAD) and stroke. Since recently, elevated N-Hcy-proteins levels have been reported in haemodialysis patients, we sought to investigate whether anti-N-Hcy-protein antibodies occur in such subjects and if they are associated with cardiovascular risk.
Methods. We studied 43 patients, aged 27–89 (mean 58.8) years, dialysed for, on average, 50 months and 31 age- and sex-matched healthy controls. IgG antibodies against N-Hcy-albumin and -haemoglobin were determined using an in-house enzyme-linked immunosorbent assay.
Results. Haemodialysis patients had higher plasma tHcy (23.18 ± 1.37 vs 13.51 ± 0.64; P < 0.0001), serum folate (29.7 ± 6.9 vs 9.9 ± 3.8 nmol/l; P < 0.0001) and anti-N-Hcy-albumin and -haemoglobin antibodies (absorbancy at 490 nm: 0.39 ± 0.22 vs 0.34 ± 0.12; P = 0.03 and 0.60 ± 0.31 vs 0.42 ± 0.09; P < 0.0001, respectively) than controls. Levels of anti-N-Hcy-albumin antibodies, but not those against haemoglobin, correlated negatively with the duration of haemodialysis (r = –0.39; P = 0.01). This correlation disappeared after a 6-month follow-up. Haemodialysis patients treated with folic acid on a long-term basis had similar levels of anti-N-Hcy-albumin and -haemoglobin antibodies compared with the minority which denied taking this vitamin (absorbancy A490: 0.35 ± 0.22 vs 0.38 ± 0.05; P = 0.5 and 0.63 ± 0.30 vs 0.51 ± 0.11; P = 0.4, respectively).
Conclusions. Our study shows that an autoimmune response to anti-N-Hcy-proteins occurs in patients on maintenance haemodialysis and is more pronounced than in healthy subjects.
Keywords: antibodies; haemodialysis; homocysteine; protein homocysteinylation
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Introduction |
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Elevated total homocysteine (tHcy) levels are found in 85–100% of patients with end-stage renal disease (ESRD) [1]. There is evidence that hyperhomocysteinaemia is involved in increased cardiovascular (CV) morbidity and mortality in patients with ESRD [2,3]. It has been estimated that 40% of ERSD patients developed CV disease, with an annual CV mortality of 10% [4].
Mechanisms by which homocysteine (Hcy) is involved in the pathogenesis of vascular disease remain unclear [5]. One of the potential mechanisms underlying harmful effects of Hcy is the protein modification induced by Hcy-thiolactone, a highly reactive cyclic thioester of Hcy [6], which is formed in the human body [7].
There is growing evidence that mild and moderate hyperhomocysteinaemia is associated with increased immune activation [8]. Elevated tHcy levels can result in the production of IgG autoantibodies directed against N-homocysteinylated (N-Hcy) protein adducts, in which a carboxyl group of Hcy is N-linked to lysine residues [9]. N-Hcy-proteins (e.g. human albumin and haemoglobin), which can be recognized as neoantigens, are formed in the presence of Hcy-thiolactone [9]. It has been documented that Hcy-thiolactone forms amide bonds with amino groups of lysine residues, thus generating N-Hcy-proteins that have an altered structure and biochemical properties [10]. Several investigators detected N-Hcy-proteins in human serum [11,12]. Increased levels of these antibodies have been demonstrated in patients with stroke [13] and early-onset coronary artery disease (CAD) [14].
Recently, Perna et al. [15] showed, using high-pressure liquid chromatography in 28 haemodialysis patients vs 14 controls, that N-homocysteinylated protein concentrations are markedly elevated in patients on haemodialysis. Therefore, we have hypothesized that also antibodies against these proteins are significantly increased in haemodialysis patients compared with healthy individuals.
The objective of our preliminary study was to evaluate anti-N-Hcy-albumin and -haemoglobin antibodies in patients on maintenance haemodialysis.
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Subjects and methods |
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Forty-three patients [25 males and 18 females, aged 27–89 (mean 58.8) years] on maintenance haemodialysis for, on average, 50 months were recruited in southern Poland. Maintenance haemodialysis was carried out across Fresenius F6 to F8 polysulfone membranes. Exclusion criteria were cancer, any acute illness, acute vascular events or coronary intervention within the preceding 30 days, hypothyroidism and treatment affecting tHcy levels (e.g. carbamazepine). Patients were asked not to change their diet and abstain from vitamin supplements other than those prescribed by the physician (see further) throughout the study period. Thirty-one age- and sex-matched apparently healthy individuals who denied taking any medication, including vitamins, for at least 4 weeks prior to the enrolment were recruited from the hospital staff and served as controls. All study participants gave informed consent.
Laboratory investigations
Pre-dialysis blood samples were collected before heparin administration. In 24 patients, blood collection was repeated after 6 months to evaluate stability of the variables measured. Blood cell counts, lipid profile, glucose, creatinine, protein, ferritin and high-sensitivity C-reactive protein (CRP) were assessed by standard automated analysis. Plasma tHcy, i.e. the aggregate of all homocysteinyl moieties, whether in the sulfhydryl or disulfide form, but not those bound via the amide bond was determined by reverse-phase liquid chromatography with fluorescence detection [16]. Serum folate levels were measured using a commercial kit (Abbott Laboratoires, Abbott Park, IL, USA). Kt/V was calculated according to Daugirdas model [17].
Preparation of antigen
Human N-Hcy-albumin was prepared by incubation of 0.2 mM L-Hcy-thiolactone (Sigma) with 10 mg/ml human albumin (Sigma), 0.2 mM EDTA in 0.05 M potassium phosphate buffer, pH 7.4, at 37°C for 16 h. The extent of albumin modification, determined as previously described [18], amounted to 1 mol Hcy/1 mol albumin [10]. In a similar fashion, human N-Hcy-haemoglobin was prepared.
Detection of antibodies
Serum levels of IgG antibodies against N-Hcy-albumin and -haemoglobin were determined using an in-house enzyme-linked immunosorbent assay as described in detail previously [13,14], with a slight modification. Briefly, 96-well microtitre plates (Polysorp, Nunc) were coated with 100 µl of 10 µg/ml N-Hcy-albumin and N-Hcy-haemoglobin in carb/bicarb buffer, pH 9.6 and incubated at 37°C for 180 min. After washing with PBS, 0.05% Tween-20, plates were blocked using 2% bovine serum albumin (BSA) in PBS at 37°C for 60 min. After washing, serum samples were diluted 1:100 in 0.01 M PBS, pH 7.4, containing 1% BSA and 100 µl of the diluted sample was added to the well and incubated at 4°C for 24 h. After washing, the plates were incubated with horseradish peroxidase-conjugated anti-human IgG (Sigma) diluted 1:10 000 with PBS at 37°C for 60 min, and then following washing, 100 µl peroxidase substrate solution containing O-phenylenediamine (Sigma) was added to wells. After incubation of plates at room temperature for 30 min, absorbance at 490 nm was measured upon addition of 50 µl 2M sulfuric acid and results were corrected for a background absorbance and non-specific IgG binding. The intra- and interassay coefficients of variation were 5.5 and 7.9%, respectively. Seropositivity was defined as an ELISA reading above the value corresponding to the 90th percentile of the control samples.
Specificity of antibodies was confirmed by competition assays in which a fixed 1:100 dilution of serum was incubated with varying amounts of different competitors, including human albumin and haemoglobin in three forms: native, treated with 0.2 mM Hcy-thiolactone and also treated with 10 mM iodoacetic acid (IAA, Sigma) that blocks -SH groups, as well as Hcy, and Hcy-thiolactone, in antigen-coated wells at 4°C for 16 h.
Specificity of anti-N-Hcy-albumin and -haemoglobin antibodies was confirmed by competition assays, showing that N-Hcy-albumin and -haemoglobin, but not native proteins or IAA-treated proteins, were effective competitors as demonstrated in previous studies [13,14].
Statistical analysis
Data were given as mean ± SEM when normally distributed or median (IQR) otherwise. Intergroup differences were assessed by the Student's t-test for continuous variables with normal distribution or otherwise by the Mann–Whitney U-test. The chi-square test with appropriate corrections when required was used to assess intergroup differences of categorical variables. The Pearson's or Spearman's correlation coefficients were calculated to test the association between two variables with a normal or non-normal distribution, respectively. A P-value below 0.05 was considered to be statistically significant. Data analysis was done using the statistical package Statistica 6.0 for Windows.
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Results |
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Characteristics of the patient and control groups are given in Table 1. Haemodialysis patients had higher CRP, tHcy and folate levels and lower total cholesterol, LDL cholesterol, haematocrit, red cell count and platelets as compared with healthy subjects. Levels of both anti-N
-Hcy-albumin and -haemoglobin antibodies were significantly higher in the patient group than in healthy controls (Table 1). Levels of both anti-N-Hcy-albumin and -haemoglobin antibodies as well as plasma tHcy and serum folate were similar when repeated at 6-month intervals (data not shown).
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The cause of ERSD, age, sex, concomitant diseases and medications showed no associations with levels of both anti-N
-Hcy-albumin and -haemoglobin antibodies (data not shown). There were no associations between these antibodies and blood cell counts [with the exception of platelets (r = 0.27, P = 0.08 and r = 0.30, P = 0.05, respectively)], CRP, lipid profile, ferritin and Kt/V. Levels of anti-N-Hcy-albumin antibodies, but not those against haemoglobin, correlated negatively with the duration of haemodialysis (r = –0.39; P = 0.01). This correlation disappeared after a 6-month follow-up. In the control group, anti-N-Hcy-albumin and -haemoglobin antibodies showed no significant associations with any demographic, clinical or (other than tHcy or folate) laboratory variables (data not shown).
Haemodialysis patients treated with folic acid on a long-term basis had similar levels of anti-N-Hcy-albumin and -haemoglobin antibodies compared with the minority which denied taking this vitamin (absorbancy A490: 0.35 ± 0.22 vs 0.38 ± 0.05; P = 0.5 and 0.63 ± 0.30 vs 0.51 ± 0.11; P = 0.4, respectively).
As expected, there were significant associations between tHcy and levels of anti-N-Hcy-albumin antibodies (r = 0.64; P < 0.0001) and anti-N-Hcy-haemoglobin antibodies (r = 0.79; P < 0.0001) in haemodialysis patients. Even stronger associations among these variables were found in healthy controls (r = 0.91, P < 0.0001 and r = 0.83, P < 0.0001, respectively). In the control group, serum folate levels were negatively correlated with anti-N-Hcy-albumin (r = –0.41, P = 0.02) and anti-N-Hcy-haemoglobin antibodies (r = –0.54, P = 0.002). However, there were no significant associations between serum folate and anti-N-Hcy-protein antibodies levels in the haemodialysis group (r < 0.1; P > 0.05). Levels of both autoantibodies showed positive correlations in haemodialysis patients and controls (r = 0.51; P = 0.0001 and r = 0.87; P < 0.0001, respectively). There were negative correlations between tHcy and serum folate levels (r = –0.51, P = 0.003) in the control group, but not in haemodialysis patients (r = –0.07; P = 0.6).
Seropositivity based on the 90th percentile of absorbancy at 490 nm in controls, i.e 0.38 for anti-N-Hcy-albumin antibodies and 0.53 for anti-N-Hcy-haemoglobin antibodies, was found in 42 and 60% of haemodialysis subjects, respectively.
At baseline, 14 patients were diagnosed with CAD, all survivors of myocardial infarctions that developed on average, 61 (range, 4–38) months before enrolment, while on maintenance haemodialysis. Levels of both anti-N-Hcy-albumin and -haemoglobin antibodies determined at the time of enrolment were significantly higher in haemodialysis patients with a history of CAD as compared with those with a negative history of this disease (absorbancy A490: 0.48 ± 0.11 vs 0.33 ± 0.15; P = 0.0007 and 0.76 ± 0.37 vs 0.53 ± 0.19; P = 0.03, respectively). Among CAD patients on maintenance haemodialysis, the percentage of seropositive patients to anti-N-Hcy-albumin and anti-N-Hcy-haemoglobin was 78% (11 out of 14) for both antibodies, while in subjects without evidence for CAD the corresponding values were 24% (P = 0.002) and 51% (P = 0.1), respectively. Plasma tHcy levels were also elevated in CAD patients (P = 0.008), while serum folate levels did not differ with regard to the presence of CAD (P = 0.6).
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Discussion |
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The current study is the first to demonstrate that in haemodialysis patients elevated tHcy levels are associated with significantly increased levels of IgG anti-N
-Hcy-albumin and -haemoglobin antibodies as compared with healthy individuals matched for age and sex. Moreover, both these autoantibodies displayed higher titers in haemodialysis subjects diagnosed with CAD. Our findings supplement the study by Perna et al. [15] showing that both S-Hcy-protein and N-Hcy-proteins occur in significant amounts in patients on maintenance haemodialysis. We confirmed these observations in an indirect way by providing evidence for the presence of an autoimmune response to these N-Hcy proteins in haemodialysis patients. Moreover, measurement of antibodies against homocysteinylated proteins is more feasible and useful in clinical practice than assessment of the N-Hcy proteins by HPLC. One might suspect that this form of autoimmune response related to hyperhomocysteinaemia is initiated by haemodialysis. However, based on previous observations [9], it can be concluded that elevated tHcy levels also support the formation of anti-N-Hcy-protein antibodies most likely in the majority of clinical settings, including patients with renal failure, who are not treated with haemodialysis. A negative correlation between duration of heamodialysis treatment and levels of anti-N-Hcy-albumin antibodies, which disappeared over time, might indicate that chronic haemodialysis can blunt the Hcy-mediated autoimmune response.
As expected, levels of anti-N-Hcy-albumin and -haemoglobin antibodies were positively associated with tHcy both in controls and haemodialysis patients. Given higher (by 42%) tHcy levels in haemodialysis patients compared with controls, it could have been expected that levels of both antibodies will be higher than those found in the current study. However, in uraemia other competing processes such as carbamyolation, affecting among others, N-amino groups [19] and decreased paraoxonase/thiolactonase activity [20], are able to mask the effects of homocysteinylation, as evidenced by a 2-fold increase in N-Hcy-proteins vs 5-fold increase in tHcy in haemodialysis patients [15].
We did not observe lower levels of anti-N-Hcy protein antibodies in patients treated with folic acid compared with those without such a supplementation. Perna et al. [15] reported that folic acid reduces N-Hcy-protein levels in patients on haemodialysis. Therefore, it might have been speculated that folic acid administration will decrease levels of anti-N-Hcy-albumin and -haemoglobin antibodies in haemodialysis patients. The lack of folate-induced reduction in serum levels of these antibodies may be associated with similar plasma tHcy levels in subjects treated or untreated with folic acid in our study. It has already been reported that if baseline tHcy is between 20–30 µmol/l in haemodialysis patients, vitamin therapy is commonly ineffective because the greatest reduction in tHcy can be seen in subjects with high tHcy above 40 µmol/l [21]. Like in our recent study [22], we also found a significant negative correlation between both antibodies and serum folate in apparently healthy individuals. The lack of such associations in haemodialysis patients, of whom the majority were taking folic acid, combined with similar levels of antibodies against N-Hcy-proteins regardless of the treatment with folic acid or not, indicates that long-term haemodialysis may result in the resistance to homocysteine-lowering effects of this vitamin, which leads to a persistently enhanced autoimmune response to N-Hcy-proteins. Moreover, our findings suggest that although folic acid administration for 2 months can reduce N-Hcy-protein levels almost to normal values in haemodialysis patients [15], an immune response to them is prone to persist, as shown in patients with CAD and normal renal function [22]. Interestingly, the association between anti-N-Hcy-albumin antibodies and haemodialysis was stronger than that for anti-N-Hcy-haemoglobin antibodies, which is in line with our recent report showing that anti-N-Hcy-albumin antibodies are a more sensitive marker of protein N-homocysteinylation compared with those against haemoglobin in subjects receiving folic acid for 6 months or more [22]. It might be hypothesized that N-Hcy-albumin at lysine 525, which has been found to display altered susceptibility to oxidation [12], typical of ESRD [23], actually reflects the combined effect of enhanced homocysteine and reactive oxygen species and until now such properties have not been demonstrated in vitro for N-Hcy-haemoglobin.
A difference in levels of autoantibodies against homocysteinylated proteins between patients with a positive history of CAD and those with a negative history might indicate that an autoimmune response to Hcy contributes to the development of CAD. However, lack of randomization and the small size of the population studied make it impossible to draw firm conclusions on the relationship between CAD and the Hcy-mediated autoimmune response. Further prospective studies are needed to validate our preliminary observations.
Our findings indicate that anti-N-Hcy-albumin and -haemoglobin antibodies circulate in blood of haemodialysis patients in greater amounts than in healthy individuals. Moreover, these antibodies might constitute a useful indicator of Hcy-related activation of the immune system in haemodialysis patients. Larger prospective studies are needed to elucidate a role of anti-N-Hcy-protein antibodies in atherosclerotic vascular disease in haemodialysis patients.
Conflict of interest statement. None declared.
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Acknowledgements |
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The authors would like to thank Agnieszka Padjas and Magdalena Zeglin for technical assistance.
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References |
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Accepted in revised form: 19. 1.07
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