Relations between Concentrations of Asymmetric Dimethylarginine and Neopterin as Potential Risk Factors for Cardiovascular Diseases in Haemodialysis-Treated Patients

Abstract

Objectives. To investigate the correlation between concentrations of asymmetric dimethylarginine (ADMA) and neopterin (NP) as potential risk factors for cardiovascular diseases in chronic renal failure patients. Method. In this study, 33 patients with renal failure before and after haemodialysis were compared with healthy control subjects. Serum ADMA and NP levels were measured using high performance liquid chromatography (HPLC). Results. When ADMA and NP concentrations in renal failure patients were compared before and after dialysis, before dialysis ADMA and NP concentrations were higher than those in the control group. However, ADMA and NP levels showed a falling mean and clear after dialysis. While there is no correlation between ADMA and NP levels before dialysis, there is a mean and positive correlation between ADMA and NP levels after dialysis. Conclusion. Potential risk factors for cardiovascular diseases include high concentrations of both ADMA and NP levels in chronic renal failure patients. A correlation mean between ADMA and NP levels after dialysis was found, but no correlation between ADMA and NP levels before haemodialysis was discovered. These can be evaluated as two different risk factors independent from each other.

Keywords:

• asymmetric dimethylarginine
• neopterin
• chronic renal failure
• haemodialysis

INTRODUCTION

Atherosclerotic vascular complications are the major cause of morbidity and mortality in patients with different stages of chronic renal disease.[1] In recent years, there has been a growing body of evidence that even minor renal dysfunction is associated with high risks of cardiovascular events. Now, chronic kidney disease (CKD) is generally thought to be one of the risk factors for cardiovascular disease (CVD). Because endothelial dysfunction is an initial step to atherosclerosis in patients with hypertension, diabetes, and CKD, reduced generation and/or bioavailability of nitric oxide (NO) may link these risk factors to the events of CVD.[2] The association between conventional risk factors, together with malnutrition and chronic inflammation, seems to be a major source of oxidative stress, playing an important role in the pathogenesis of dependent-NO endothelial dysfunction.[3]

Nitric oxide (NO) is very active but short living, and has harmful effects on cell activity. NO is a critical modulator of blood flow and blood pressure. It is released by the endothelium in response to shear stress and plays an important role in flow-mediated vasodilatation.[4] The diminished bioavailability of NO impairs endothelium-dependent vasodilatation and activates other mechanisms that may play an important role in the pathogenesis of atherosclerosis.[5] NO, synthesized from L-arginine, accounts for the powerful vasodilator effects of endothelium-derived relaxing factor and consequently plays a decisive role in determining vasomotor tone.[6] Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase that can modulate NO production.[7–9] It is synthesized and metabolized by human endothelial cells.[10] ADMA and its regioisomer, symmetric dimethylarginine (SDMA), are generated by the degradation of methylated proteins due to protein arginine methyltransferase 1 (PRMT 1) activity[11] and subsequent physiological protein turnover.[8] ADMA is eliminated from the body either by renal excretion or by degradation to dimethylamine and citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH).[12] ADMA, but not SDMA, is an endogenous inhibitor of endothelial nitric oxide synthase (eNOS).[8] ADMA inhibits eNOS by competitive displacement of the physiological substrate, L-arginine, from the enzyme.[13],[14] The inhibition leads to decrease in NO production in the endothelium of vessel walls. Thus, ADMA levels are elevated, and endothelial dysfunction may result.[14] Further, ADMA level was recently found to be elevated in patients with CKD and atherosclerotic vascular complications.[15],[16] In addition, plasma ADMA level predicts future cardiovascular events and the progression of renal injury in patients with CKD.[17],[18]

Recently, the potential role of ADMA in CKD was comprehensively reviewed by Zoccali et al. (2006).[19] According to their theory, there are at least four possible mechanisms that may explain the accumulation of ADMA in CKD:

1. increased methylation of proteins;

2. increased protein turnover;

3. decreased metabolism by DDAH; and

4. impaired renal excretion.

As dimethylarginines are excreted in urine,[20] impaired renal clearance may, at least in part, account for the elevation of ADMA levels in patients with CKD.

Neopterin (NP), belonging to the pteridine family, is synthesized primarily by human monocytes and macrophages after stimulation by interferon-gamma (IFN-γ) produced by activated helper T-cells.[21],[22] The biosynthesis of neopterin starts from purinic guanosine triphosphate base that is converted to 7,8-dihydroneopterintriphosphate by GTP ciclohydrolase I.[23] NP levels rise in many pathologic conditions: renal failure[24]; diabetic nephropathy[25]; glomerulonephritis[26],[27]; renal transplant rejection[28]; insulin resistance[29]; autoimmune diseases[30]; viral,[31] bacterial,[32] and protozoic infections[33]; coronary artery disease [34]; and psoriasis.[35] In humans, there is a correlation between serum NP concentration and the extent of atherosclerosis.[36] In patients undergoing haemodialysis, serum NP levels decrease as a consequence of the procedure.[37] In renal diseases, two mechanisms are responsible for increased NP concentrations: impaired excretion of the compound and its production in the course of inflammation.

Studies have reported an association between ADMA and NP levels and the grade of atherosclerosis.[38] These findings suggest that the elevation of ADMA and NP may be a missing link between CVD and CKD. The purpose of the present study was to investigate any relationship between concentrations of asymmetric dimethylarginine and neopterin as potential risk factors for cardiovascular diseases in chronic renal failure patients, with control subject.

METHODS

Patients

Thirty-three patients with haemodialysis treatment at Gazi University, Faculty of Medicine, (Ankara, Turkey) were examined in this study. Dialyzer membrane type was polysulfone semisynthetic and dialyzer machine was Fresenius 4008B. We studied three groups of individuals: 33 healthy subjects, 33 subjects with renal failure disease that were being prepared before dialysis, and 33 subjects with renal failure disease after dialysis. Then, the samples were stored at −80°C.

Biochemical Analysis

Measurement of ADMA by HPLC Methods

Measurement of ADMA was accomplished by HPLC using a method described by Chen et al. [39] In brief, 20 mg 5-sulfosalisilic acid was added to 1 ml serum, and the mixture was left in an ice bath for 10 min. The precipitated protein was removed by centrifugation at 2000 × g for 10 min. Ten microlitres of the supernatant, which was filtered through a 0.2 μm-pore size filter, was mixed with 100 μl derivatization reagent (prepared by dissolving 10 mg ο-phtaldialdehyde in 0.5 ml methanol, 2 ml 0.4 M borate buffer (pH10.0), and 30 μl 2-mercartoethanol were added) and then injected into the chromatographic system. Separation of ADMA was achieved with a 150 × 4 mm-interior diameter Nova-Pak C₁₈ column with a particle size of 5 μm (Waters, Millipore Corp., Milford, Massachusetts, USA) using 50 mM sodium acetate (pH 6.8), methanol, and tetrahydrofurane as mobile phase (A; 82/17/1, B; 22/77/1 [v/v/v], respectively) at a flow rate of 1.0 ml/min. The areas of peaks detected by fluorescent detector (excitations, 338 nm; emission, 425 nm) were used for quantification. The intra- and inter-assay coefficients of variation for ADMA were 2.8% and 4.5%, respectively.

Measurement of NP by HPLC Methods

The measurement of NP was accomplishment by HPLC. In this study, measurements were made using a C₁₈/5 μm reverse-phase column 4.6 × 250 mm, and the most appropriate mobile phase has been determinate to be 0.015 mol/L phosphate buffer with pH 6.4 as a consequence of various optimization studies. For the neopterin analysis, 100 μL of TCA (2 mol/L) were added to 100 μL of standard or serum sample and vortexed for 10 s. The samples were centrifuged at 10,000 g for 10 min. Then, 50 μL of supernatant was diluted with 200 μL of bidistilled water, and 20 μL aliquot was used for HPLC analysis. The areas of peaks detected by fluorescent detector (excitations, 353 nm; emission, 438 nm) were used for quantification. The intra- and inter-assay coefficients of variation for NP were 4.7% and 6.9%.

Statistical Analysis

All data were expressed in terms of mean ± standard deviation. Statistics were done using the software Statistical Package of Social Science (SPSS 11.0.0, 2001; SPSS Inc., Chicago, Illinois, USA). The Mann-Whitney U-test was used to compare differences between two independent groups, and the Spearman rank correlation test was used for estimating relationships between variables. A p value of ≤0.05 was considered statistically significant.

RESULTS

The characteristics of patients; BUN, creatinine, ADMA, and NP levels; and other parameters both before dialysis and after dialysis in patients with chronic renal failure and control subjects are shown in Table 1. ADMA and NP levels were not affected by age, sex, HD vintage, original CKD, or HD membrane in our study group.

Table 1 Characteristics of patients. Comparison of ADMA and Neopterin levels between before dialysis and after dialysis in patients with chronic renal failure and control subjects

Patients (n = 33: male, 22; female, 11)	Before dialysis	After dialysis	Control
Age	51 ±14	51 ±14	48 ± 11
Age at haemodialysis (in years)	9 ± 3	9 ± 3	NA
Hemoglobin (g/dL)	11.3 ± 1.5	11.3 ± 1.5	13.7 ± 1.8
Total protein (g/dL)	6.5 ± 0.6	6.5 ± 0.6	7.9 ± 1.1
Albumin (g/dL)	4.1 ± 0.6	4.1 ± 0.6	4.5 ± 1
BUN (mg/dL)	64.9 ± 14.1	23.0 ± 6.7	14.2 ± 5.2
Creatinine (mg/dL)	9.2 ± 2.7	3.8 ± 1.3	0.8 ± 0.3
ADMA (μmol/L)	3.14 ± 2.50*^†	1.53 ± 1.18	0.89 ± 0.26
NP (nmol/L)	291 ± 135*^†	182 ± 98	19 ± 7

*p < 0.05 versus control group; ^†p < 0.05 versus after dialysis.

Serum ADMA Concentrations in Renal Failure

The serum concentrations of ADMA are low in healthy subjects (ADMA: 0.89 ± 0.26 μmol/L). This study was compared with control subjects; HD-treated patients exhibited significantly (p < 0.05) higher serum ADMA concentrations. However, serum ADMA concentrations were determined before dialysis and after dialysis in patients with chronic renal failure. In our results, ADMA levels were found to be significantly (p < 0.05) higher before dialysis (3.14 ± 2.50 μmol/L) than after dialysis (1.53 ± 1.18 μmol/L). Nevertheless, high ADMA levels are not enough to eliminate by dialysis.

Serum NP Concentrations in Renal Failure

The serum concentrations of NP are very low in healthy subjects (NP: 19 ± 7 nmol/L). In our study, serum NP concentrations were measured both before dialysis and after dialysis in patients with chronic renal failure. Serum NP concentrations were greater before dialysis (291 ± 135 nmol/L) than after dialysis (182 ± 98 nmol/L). Nevertheless, this reduction does not reach normal serum NP concentration levels. Also, compared with control subjects, HD-treated patients exhibited significantly (p < 0.05) higher serum NP concentrations.

We also analyzed any association between levels of ADMA, NP, and uremic molecules. The NP levels correlated positively with ADMA levels in both HD-treated patients and control groups (r = 0.381, p < 0.05; r = 0.497; p < 0.05, respectively; see Figures 1 and 2). There were no correlations between uremic molecules and ADMA or NP.

Figure 1. A positive correlation was found between concentrations of ADMA and neopterin in a studied HD-treated patients (n = 33, r = 0.497, p < 0.05).

Figure 2. A positive correlation was found between concentrations of ADMA and neopterin in a studied controls (n = 30, r = 0.381, p < 0.05).

DISCUSSION

Recently, biochemical factors including ADMA and NP showed in most diseases, such as renal failure, diabetic disease, cardiovascular disease, and hepatitis.[40] Several investigators have proposed ADMA as a potentially useful biomarker in cardiovascular disease. Until recently, there was no compelling evidence that ADMA plays a causal role in the pathophysiology of vascular disease. Thus, although elevated ADMA levels have been frequently associated with various cardiovascular risk factors, it remained unclear as to whether this molecule is simply a marker or also a “maker” of vascular disease.[41] Despite the wide range of reported ADMA levels in patients with renal disease, there is some evidence that ADMA may be a useful marker to assess severity of renal disease. Most studies are of limited value, as they were cross-sectional in nature and simply show an association of elevated ADMA levels with renal disease.[42],[43]

Our finding that ADMA levels are significantly higher in patients with chronic renal failure than in healthy control subjects is consistent with the first description by Vallance and colleagues[8] of nine HD-treated patients. They reported a mean total ADMA level of 8.7 ± 0.7 μmol/L. This concentration was six times higher than that measured for control subjects (1.2 ± 0.1 μmol/L).[8] In our study, ADMA level before dialysis in patients was found to be three times higher than in healthy subjects, a statistically significant finding (p < 0.05). However, ADMA level after dialysis was found to be lower than before dialysis. MacAllister and colleagues found elevated ADMA level (0.9 ± 0.1 μmol/L) in six HD-treated patients.[12] Kielstein and colleagues observed that the plasma concentration of ADMA was higher in patients with end-stage renal disease and atherosclerotic vascular disease than in those without vascular complications.[44] Miyazaki and colleagues measured dimethylarginine levels in the serum of 116 human subjects who had no signs of coronary or peripheral arterial disease, and found that ADMA levels were positively correlated with age, mean arterial pressure, and glucose tolerance. Most intriguingly, ADMA levels were significantly correlated with carotid artery intima-media thickness in a stepwise regression analysis of this population.[45] As the relationship between carotid intima-media thickness and major cardiovascular events had also recently been established,[46],[47] these authors proposed that ADMA is a marker of cardiovascular disease.

Irrespective of its absolute concentration, serum ADMA levels correlate significantly with established risk factors of atherosclerosis in both renal and nonrenal patients.[15],[44],[45],[47–51] Thus, ADMA is thought to be not only a novel biochemical marker of atherosclerosis, but it may even be causally involved in the pathogenesis of atherosclerotic disease.[4]

Neopterin, which is a biochemical marker of cellular immune response, belongs to the group of compounds known as pteridines. The first pteridines were initially identified in 1889, and in 1979, Wachter et al. reported that urine neopterin levels were elevated in cases of viral infections and tumors.[53] Recently, NP has been reported as the indicator of local macrophage activity in different body fluids.[54] Neopterin is biologically stable, and increased concentrations in serum and urine have been used as a diagnostic and prognostic criterion for cell-mediated immunity in some clinical conditions. Because neopterin excretion takes place before clinical symptoms appear, biochemical follow-up of neopterin levels has been accepted as a strong indicator for the clinical severity of some diseases.[55] Further, a marked abnormality in pteridine metabolism has been noted in cases of chronic renal disease.[56] Pecoits-Filho et al. also reported that neopterin levels were significantly higher in the subgroup of patients with a low glomerular filtration rate, suggesting either impaired renal elimination, increased generation in uremia, or an adverse effect of inflammation on renal function.[57] In our study, there was a trend toward increased serum neopterin levels in patients with chronic renal failure compared to the control group that was statistically significant (p < 0.05).

A possible explanation for the prognostic value of neopterin, as observed separately by Gupta[58] and Schumacher,[59] is that neopterin is a marker of coronary disease activity. Neopterin affects intracellular redox state and has been shown to be involved in the activation of both constitutive and inducible NO synthase.[60],[61]

Previously, neopterin has been used as a marker of the stimulation of the cellular immune system. Our finding that neopterin is higher in patients with chronic renal failure than in healthy control subjects is consistent with previous reports, which have shown that coronary syndromes are associated inflammatory mechanisms.[62],[63]

CONCLUSION

Our study researched the relationship between ADMA and NP concentrations as potential risks factors for cardiovascular disease in patients with chronic renal failure. We have found elevated ADMA and NP levels in patients with chronic renal failure compared with controls. There found a correlation mean between ADMA and NP levels after dialysis, but there was not found any correlation between ADMA and NP levels before haemodialysis. However, a correlation between ADMA and NP levels after haemodilaysis may be evaluated in terms of dialysis effect. These can be evaluated as two different risk factors independent from each other. In addition, in examining this decrease, it is reasonable to assume that hemodialysis is not more effective with respect to ADMA and NP elimination than the kidney.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.