Hemodialysis International 2014; 18:680–685
Predictive value of serum myeloperoxidase activity for thrombosis of arteriovenous fistulas Radojica V. STOLIC,1 Goran Z. TRAJKOVIC,2 Mirjana KOSTIC,3 Dragica Z. STOLIC,4 Dijana J. MIRIC,5 Bojana M. KISIC,5 Slavica D. PAJOVIC,1 Vladan M. PERIC1 1
Internal Clinic, Faculty of Medicine Pristina/Kosovska Mitrovica, University of Pristina, Kosovska Mitrovica, Serbia, 2Faculty of Medicine, Institute of Medical Statistics and Informatics, University of Belgrade, Serbia, 3Faculty of Medicine Pristina/Kosovska Mitrovica, Institute of Medical Statistics and Informatics, University of Pristina, Kosovska Mitrovica, Serbia, 4Health Center Pristina, Gracanica, Serbia, 5Faculty of Medicine Pristina/Kosovska Mitrovica, Institute of Biochemistry, University of Pristina, Kosovska Mitrovica, Serbia
Abstract Myeloperoxidase is a proinflammatory protein that appears as a result of increased oxidative stress. It plays an important role in the promotion and progression of atherosclerosis. The aim of this study was to determine the importance of MPO as a predictive parameter for thrombosis of arteriovenous fistula (AVF). The study involved monitoring patients with AVFs for hemodialysis over a period of 2 years. There were 41 patients, 19 (46%) men and 22 (54%) women, with mean age of 65 ± 12.7 years. Routine laboratory analyses were carried out in all respondents, including determination of MPO concentration. Gender, demographic and anthropometrical characteristics, smoking, alcohol consumption, as well as the presence of diabetic nephropathy, as an etiological factor of kidney disease, were recorded. The group of patients who developed initial thrombosis of the AVFs had significantly different values for leukocytes (8.5 ± 3.8 vs. 7.3 ± 2.1, P = 0.024), erythrocytes (2.8 ± 0.27 vs. 3.2 ± 0.65; P = 0.019), hemoglobin (88.5 ± 81 vs. 99.1 ± 6.02; P = 0.041), and myeloperoxidase (19.3 ± 4.67 vs. 11.1 ± 4.43; P = 0.007) when compared with the group without fistula thrombosis. Diabetic nephropathy (P = 0.02) characterized the group of patients with thrombosis of the fistula. Diabetic nephropathy (B = 2.53, P = 0.049) and MPO (B = 0.03, P = 0.029) were statistically significant predictors of fistula thrombosis. In our study, MPO and diabetic nephropathy were predictors of thrombosis of the AVF. Key words: Oxidative stress, myeloperoxidase, atherosclerosis, thrombosis, arteriovenous fistula, hemodialysis
INTRODUCTION Oxidative stress is defined as tissue damage caused by an imbalance between synthesis of oxidative compounds and Correspondence to: R. V. Stolic, MD, PhD, Faculty of Medicine, Anri Dinan bb, 38220 Kosovska Mitrovica, Serbia. E-mail: [email protected]
antioxidant defense mechanisms. In end-stage renal disease, increased oxidative stress can occur due to loss of antioxidants during hemodialysis (HD), interaction between the blood and bioincompatible dialysis membranes, and bacterial contamination of the dialysate, leading to activation and recruitment of polymorphonuclear leukocytes.1,2 However, increased oxidative stress was also found in patients without end-stage renal disease,
© 2014 International Society for Hemodialysis DOI:10.1111/hdi.12155
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suggesting that the nature of the pathological process itself largely contributes to this situation.3 In fact, reduced glomerular filtration rate and chronic inflammation in chronic kidney disease can be important determinants of oxidative stress.4 Inflammatory events are known to be incorporated into all evolution phases of atherosclerotic plaque development, from the early stage of endothelial dysfunction through the formation of atheroma to the final damage.5 Myeloperoxidase (MPO) is a proinflammatory enzyme implicated in the pathophysiology of atherosclerosis, particularly in endothelial dysfunction, where its predictive significance seems to be much higher than that of C-reactive protein. Moreover, experimental and clinical studies now suggest that MPO may serve as a general marker of inflammation.6 However, data regarding the importance of MPO in predicting vascular access thrombosis in HD patients are scarce. The aim of the current study was to determine the predictive value of serum MPO in the development of thrombosis of arteriovenous fistula (AVF) for HD.
MATERIALS AND METHODS Patients The study was organized within the framework of junior project number 17/11 (oxidative stress and markers of inflammation in patients with chronic kidney disease), Medical Faculty Pristina/Kosovska Mitrovica, Serbia, and conducted at the Center for Hemodialysis, Medical Center, Kosovska Mitrovica, Serbia, as a nonrandomized clinical investigation. It involved monitoring patients with AVF for HD created over a period of 2 years. The protocol was approved by the Ethics Committee of the Medical Center, Kosovska Mitrovica, Serbia, according to the Declaration of Helsinki. All participants verbally consented to be included and were patients with the initial form of native AVF for HD. Fistulas were created when creatinine clearance was between 25 and 30 mL/min. The project involved 41 patients, 19 (46%) men and 22 (54%) women, with mean age of 65 ± 12.7 years. Patients were divided into a group of 14 (34%; 7 men and 7 women) with thrombosis of the AVF (mean age 64.9 ± 12 years) and 27 (66%; 12 men and 15 women) without fistula thrombosis (mean age 63.1 ± 14.7 years). In this study, the following were not included: 1. Patients on chronic HD, in whom AVF thrombosis had occurred earlier. 2. Patients younger than 18 years.
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3. Patients who were being treated for cardiovascular disease. 4. Patients with manifested forms of vasculitis. 5. Patients who had had any form of actual infection.
Biochemical methods Before the scheduled surgical interventions, 10 mL of blood was withdrawn into tubes with or without ethylenediaminetetraacetic acid (EDTA), as an anticoagulant. Serum samples were obtained after centrifuging clotted blood at 3000 × g for 15 minutes. Total and differential blood cell counts were determined in the EDTA samples by flow cytometry (Beckman Coulter Inc., Fullerton, CA, USA). Blood hemoglobin level was determined colorimetrically by the cyanmethemoglobin method. Serum concentrations of urea, creatinine, glucose, total calcium, inorganic phosphorus, uric acid, and iron were measured on an ILAB-600 chemistry analyzer (Diamond Diagnostics, Holliston, MA, USA), according to the manufacturer’s instructions, using original test reagents. Since MPO activity was not routinely measured, serum samples (1 mL) from the patients were frozen and kept at −20°C until a sufficient number accumulated but for no longer than 2 weeks. Serum MPO activity was determined kinetically in the 4-aminoantipyrine/phenol system with hydrogen peroxide as the substrate, by monitoring the formation of quinoneimine at λ = 505 nm, as described.7 Serum MPO activity was calculated using the molar absorbance: ε = 1.3 × 104 L × M−1 × cm−1. One unit of MPO activity was defined as the amount of enzyme degrading 1 μmol of hydrogen peroxide per minute, at 25°C.
Clinical, demographic, and anthropometrical parameters Age and sex were registered for all patients. Data regarding abuse of alcohol, diabetic nephropathy, and body mass index (BMI) were also recorded for all participants. Their BMI was calculated with respect to preoperative body weight by the formula: BMI (kg/m2) = present body weight/height2.
Statistical methods Data were summarized as frequencies or percentages for categorical variables, and as means ± SD or medians and range for numerical variables, respectively. To test the differences between cases and controls, Pearson’s chisquare test or Fisher’s exact test was used for categorical variables and, depending on the distribution of the data,
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the independent samples t test or Mann-Whitney U test were used for numerical variables. All variables that were significant at the probability level, P ≤ 0.05, were entered into a multiple logistic regression model with AVF thrombosis as the dependent variable. Statistical analysis was performed using SPSS version 15.0 (SPSS, Chicago, IL, USA) and INSTAT (Prism 4 GraphPad InStat, San Diego, CA, USA) software programs.
We performed multiple logistic regression analysis in order to evaluate the relationship between BMI, diabetes mellitus, and serum MPO activity, with the occurrence of AVF thrombosis. Of all variables included, only diabetic nephropathy (B = 2.53; P = 0.049) and MPO activity (B = 0.03; P = 0.029) showed statistically significant relationships as independent predictors of AVF thrombosis (Table 3).
RESULTS
DISCUSSION
Basic demographic, anthropometric, and clinical characteristics of patients with and without AVF thrombosis are presented in Table 1. There was a significant difference between the groups concerning BMI (19.9 ± 1.12 kg/m2 vs. 24.7 ± 3.2 kg/m2; P ≤ 0.0001). Among patients with diabetic nephropathy, five (18%) were without AVF thrombosis, whereas eight (57%) patients were with AVF thrombosis. In the group without diabetic nephropathy, six (43%) patients were with AVF thrombosis, whereas 22 (82%) patients were without AVF thrombosis. Thus, among patients with diabetic nephropathy, the frequency of AVF thrombosis was significantly higher (P = 0.02). Patients with AVF thrombosis had a significantly elevated number of leukocytes (8.55 ± 3.82) in comparison to those without fistula thrombosis (7.3 ± 2.13); P = 0.024. However, patients with AVF thrombosis had markedly fewer erythrocytes (2.8 ± 0.65 vs. 3.2 ± 0.27; P = 0.019), as well as a lower mean hemoglobin concentration (88.5 ± 81 vs. 99.5 ± 6.02; P = 0.041), compared to the group without AVF thrombosis. On the contrary, serum MPO activity was significantly higher in the group with thrombosis of the AVF (19.3 ± 4.67 vs. 11.1 ± 4.43; P = 0.007) (Table 2).
The principal finding of the current study is that higher extracellular MPO activity was an independent predictor of AVF thrombosis in patients undergoing HD treatment. These results are consistent with the recently reported benefits of low expression of leukocytic MPO on the vascular tone of resistance and conductance vessels,8 suggesting that extracellular MPO could play a role in oxidative damage and the development of vascular complications in HD patients. We observed that erythrocyte cell count and hemoglobin levels were far below the reference values in almost all of our patients. It is well known that erythrocytes are under constant challenge to maintain hemoglobin in its reduced functional state and to maintain the integrity of the cell membrane. Once hemoglobin is denatured it aggregates. In addition, oxidative damage of membranebound polyunsaturated fatty acids may result in delipidation and increased stiffness of the erythrocyte cell membrane.9,10 These perturbations usually result in premature removal of erythrocytes from the circulation,11,12 and our finding that anemia was more severe in patients with AVF thrombosis may therefore suggest more extensive oxidative damage in that group.
Table 1 Demographic, anthropometric, and clinical characteristics of the examined patients Investigated parameters Age (y), mean ± SD Gender, n (%) Males Females BMI ([kg/m2], [mean ± SD]) Diabetic nephropathy, n (%) Yes No Alcohol consumption, n (%) Yes No
AVF with thrombosis (n = 14)
AVF without thrombosis (n = 27)
64.9 ± 12.0
63.1 ± 14.7
7 (50) 7 (50) 23 ± 2.7
12 (44) 15 (56) 25.2 ± 3.4
8 (57) 6 (43)
5 (18) 22 (82)
2 (14) 12 (86)
4 (15) 23 (85)
P 0.706 0.735
0.046* 0.02*
1.00
*Statistically significant difference. BMI = body mass index.
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Myeloperoxidase and fistulas thrombosis
Table 2 Comparison of biochemical parameters between the examined groups of patients Biochemical parameters
AVF with thrombosis (n = 14)
AVF without thrombosis (n = 27)
P
8.55 ± 3.82 2.8 ± 0.65 81 ± 88.5 12.5 ± 8.4 26.4 ± 4.8 731.9 ± 230.1 2.1 ± 0.1 1.7 ± 0.6 170.6 ± 50.9 19.3 ± 4.67
7.3 ± 2.13 3.2 ± 0.27 99.5 ± 6.02 11.1 ± 6.3 25.9 ± 9.4 743.5 ± 347.7 2.1 ± 0.3 1.6 ± 0.4 196.5 ± 98.2 11.1 ± 4.43
0.024* 0.019* 0.041* 0.558 0.810 0.911 0.630 0.373 0.293 0.007*
Leukocytes (×10 /L), mean ± SD Erythrocytes (×1012/L), mean ± SD Hemoglobin (g/L), mean ± SD Serum iron (μM), mean ± SD Urea (mM), mean ± SD Creatinine (μM), mean ± SD Total calcium (mM) mean ± SD Inorganic phosphorus (mM) mean ± SD Uric acid (μM), mean ± SD MPO (U/L), mean ± SD 9
*Statistically significant difference. AVF = arteriovenous fistula; MPO = myeloperoxidase; SD = standard deviation.
In the past decade, much effort has been made to elucidate the role of MPO and oxidative stress in the promotion and progression of atherosclerosis. It has been shown, e.g., that higher extracellular MPO levels could predict an abnormal coronary angiogram in patients undergoing diagnostic cardiac catheterization, whereas in acute coronary syndrome and heart failure, serum MPO activity was predictive for an adverse clinical outcome.8 We have previously reported that atherosclerosis can significantly reduce the functionality of the AVF,13 and the results of our current study suggest that high serum MPO activity may predict the occurrence of AVF thrombosis in HD patients. Although these findings may help planning and creation of vascular access, the relevance of extracellular MPO levels in predicting AVF thrombosis is still to be explained. Myeloperoxidase is an important microbiocidal agent present in polymorphonuclear leukocytes and, to lesser extent, in other professional phagocytes. However, MPO can be released into the vascular compartment and is present either as a soluble form in circulation or bound to the endothelial cell surface. This extracellular MPO was suggested to impose oxidative damage on circulating lipoproteins, proteins, nitric oxide, and other biomolecules,
thus being implicated in the generation of a proinflammatory state, vascular dysfunction, and accelerated atherosclerosis. Uncontrolled production of free radicals and other oxidants may affect the structural integrity of the atherosclerotic plaque, i.e., accelerated activation of matrix metalloproteinases in the fibrous cap can induce plaque rupture, thereby creating conditions for blood clot formation. Such a sequence of events suggests that oxidative stress also plays a significant role in thrombogenesis and platelet activity. Oxidants generated by polymorphonuclear leukocytes have been shown to facilitate aggregation of monocytes to the endothelium, transformation of macrophages into foam cells, eventually leading to endothelial injury.14 In the current study, there was a higher leukocyte cell count among the group with initial AVF thrombosis than in patients in whom AVF thrombosis did not occur. This finding indicates that neutrophil leukocytes are a significant source of MPO in the circulation, and a role for them in AVF thrombosis. Another finding was that after exclusion of morbidly obese patients, the respondents with AVF thrombosis had significantly lower BMI than those without AVF thrombo-
Table 3 Multiple logistic regression model with thrombosed arteriovenous fistula as the dependent variable Independent variables
B
P
OR (95% CI)
BMI Diabetic nephropathy MPO
−0.16 2.53 0.03
0.250 0.049* 0.029*
0.85 (0.64–1.12) 12.61 (1.01–157.1) 1.032 (1.033–1.062)
*Statistically significant difference. BMI = body mass index; CI = confidence interval; MPO = myeloperoxidase; OR = odds ratio.
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sis. These results are in agreement with earlier reports that elevated levels of MPO can be associated with poor nutritional status, thereby connecting increased oxidative stress, malnutrition, and inflammation. Moreover, in malnourished end-stage renal disease patients, oxidative stress biomarkers were reported to be much higher than in well-nourished patients, in agreement with our previous study.15 If there is no ongoing infection, leukocytes are at rest, and release of oxidants into the vascular compartment occurs at a slow rate. However, in chronic kidney disease, there is permanent inflammation, in which polymorphonuclear leukocytes seem to be the common denominator of other clinical conditions, such as hypertension, diabetes mellitus, and other risk factors. These conditions are associated with endothelial dysfunction, accelerated atherosclerosis, and increased prevalence of cardiovascular mortality. The state of low-grade systemic inflammation causes oxidative stress, which greatly contributes to increased concentrations of extracellular MPO.16 Previous studies have shown that parameters indicating increased oxidative stress in predialysis patients are closely correlated with reduced glomerular filtration rate, chronic inflammation, and oxidative modifications of high-density lipoproteins. Thus, if excessive formation of oxidants plays a role in the development and progression of chronic kidney disease, there is a pertinent question of therapy to limit the impact of these harmful species. Due to the complexity of oxidative stress, it is almost impossible to accept and adopt a wide enough range of biomarkers to include all types of oxidative damage. Earlier findings indicate the importance of MPO activity in cardiovascular disease, although serum levels of MPO may not always reflect the state of inflammation. In uremic patients, however, an increased concentration of extracellular MPO has been shown to be a relevant marker of inflammation and an independent predictor of atherosclerosis.17,18 This was also confirmed in the current study by the finding that serum MPO activity was independently associated with the development of AVF thrombosis. Taken together, the results of our study have shown that serum MPO activity has predictive significance for the development of AVF thrombosis. Although it is limited by the small sample size and exclusion of morbidly overweight patients, this investigation raises the question of a preventive strategy for reduction of high proinflammatory, and obviously high prothrombotic levels of extracellular MPO in patients undergoing HD treatment. In that regard, there may be a promising impact of vitamin E and some other naturally occurring antioxidants on biomarkers of oxidative stress,17 which requires further research.
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As a limitation of the study, we must emphasize the small group of participants, and the single measurement of MPO that could not reflect changes over time.
CONCLUSION In our study, we found that MPO and diabetic nephropathy are major predictive factors of fistula thrombosis. Our patients with fistula thrombosis had a lower BMI, probably as a consequence of malnutrition, inflammation, and atherosclerosis. They also had a significantly lower erythrocyte count and hemoglobin concentration, while the number of leukocytes was larger.
ACKNOWLEDGMENTS Part of this research is supported by the Ministry of Education and Science of Serbia (Grant No. III41010) and the Medical Faculty Pristina/Kosovska Mitrovica, Serbia ( Junior Project No. 07/09). Conflict of interest: The authors declare no financial or other conflict of interests regarding this paper.
Manuscript received December 2013; revised January 2014.
REFERENCES 1 Crawford A, Fassett RG, Coombes JS, et al. Relationship between antioxidant enzyme genotype and activity and kidney function: A case-control study. Clin Nephrol. 2012; 78:135–144. 2 Capeillère-Blandin C, Gausson V, Nguyen AT, Descamps-Latscha B, Drüeke T, Witko-Sarsat V. Respective role of uraemic toxins and myeloperoxidase in the uraemic state. Nephrol Dial Transplant. 2006; 21:1555– 1563. 3 Krieter DH, Lemke HD, Wanner C. Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes. Nephrol Dial Transplant. 2006; 21:546. 4 Wu CC, Chen JS, Wu WM, et al. Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes. Nephrol Dial Transplant. 2005; 20:1134– 1139. 5 Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol. 2005; 25:1102–1111.
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6 Vita JA, Keaney JF Jr, Larson MG, et al. Brachial artery vasodilator function and systemic inflammation in the Framingham Offspring Study. Circulation. 2004; 110:3604–3609. 7 Metcalf JA, Gallin JI, Nauseef WM, Root RK. Myeloperoxidase functional assays. In: Laboratory Manual of Neutrophil Function. New York, New York, Raven Press. 1986; 150–151. 8 Rudolph TK, Wipper S, Reiter B, et al. Myeloperoxidase deficiency preserves vasomotor function in humans. Eur Heart J. 2012; 33:1625–1634. 9 Lipinski B. Is it oxidative or free radical stress and why does it matter? Oxid Antioxid Med Sci. 2012; 1:5–9. 10 Bergeron F, Auvre F, Radicella JP, Ravanat JL. HO• radicals induce an unexpected high proportion of tandem base lesions refractory to repair by DNA glycosylases. Proc Natl Acad Sci U S A. 2010; 107:5528–5533. 11 Edwards CJ, Fuller J. Oxidative stress in erythrocytes. Comp Haematol Int. 1996; 6:24–31. 12 Pandey KB, Rizvi SI. Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxid Med Cell Longev. 2010; 3:2–12. 13 Stolic R, Trajkovic G, Peric V, Jovanovic A, Subaric-Gorgieva G. Impact of arteriosclerosis on the
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14
15
16
17
18
functioning of arteriovenous fistula for hemodialysis. Vojnosanit Pregl. 2007; 64:13–18 [Serbian]. Hsu SP, Wu MS, Yang CC, et al. Chronic green tea extract supplementation reduces hemodialysis-enhanced production of hydrogen peroxide and hypochlorous acid, atherosclerotic factors, and proinflammatory cytokines. Oxid Antioxid Med Sci. 2012; 35–39. Miric D, Kisic B, Stolic R, Miric B, Mitic R, Janicijevic-Hudomal S. The role of xanthine oxidase in hemodialysis-induced oxidative injury: Relationship with nutritional status. Oxid Med Cell Longev. 2013; 2013:Article ID 245253. Sela S, Shurtz-Swirski R, Cohen-Mazor M, et al. Primed peripheral polymorphonuclear leukocyte: A culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease. J Am Soc Nephrol. 2005; 16:2431–2438. Coombes JS, Fassett RG. Antioxidant therapy in hemodialysis patients: A systematic review. Kidney Int. 2012; 81:233–236. Honda H, Ueda M, Kojima S, et al. Assessment of myeloperoxidase and oxidative α1-antitrypsin in patients on hemodialysis. Clin J Am Soc Nephrol. 2009; 4:142– 145.
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Predictive value of serum myeloperoxidase activity for thrombosis of arteriovenous fistulas Radojica V. STOLIC,1 Goran Z. TRAJKOVIC,2 Mirjana KOSTIC,3 Dragica Z. STOLIC,4 Dijana J. MIRIC,5 Bojana M. KISIC,5 Slavica D. PAJOVIC,1 Vladan M. PERIC1 1
Internal Clinic, Faculty of Medicine Pristina/Kosovska Mitrovica, University of Pristina, Kosovska Mitrovica, Serbia, 2Faculty of Medicine, Institute of Medical Statistics and Informatics, University of Belgrade, Serbia, 3Faculty of Medicine Pristina/Kosovska Mitrovica, Institute of Medical Statistics and Informatics, University of Pristina, Kosovska Mitrovica, Serbia, 4Health Center Pristina, Gracanica, Serbia, 5Faculty of Medicine Pristina/Kosovska Mitrovica, Institute of Biochemistry, University of Pristina, Kosovska Mitrovica, Serbia
Abstract Myeloperoxidase is a proinflammatory protein that appears as a result of increased oxidative stress. It plays an important role in the promotion and progression of atherosclerosis. The aim of this study was to determine the importance of MPO as a predictive parameter for thrombosis of arteriovenous fistula (AVF). The study involved monitoring patients with AVFs for hemodialysis over a period of 2 years. There were 41 patients, 19 (46%) men and 22 (54%) women, with mean age of 65 ± 12.7 years. Routine laboratory analyses were carried out in all respondents, including determination of MPO concentration. Gender, demographic and anthropometrical characteristics, smoking, alcohol consumption, as well as the presence of diabetic nephropathy, as an etiological factor of kidney disease, were recorded. The group of patients who developed initial thrombosis of the AVFs had significantly different values for leukocytes (8.5 ± 3.8 vs. 7.3 ± 2.1, P = 0.024), erythrocytes (2.8 ± 0.27 vs. 3.2 ± 0.65; P = 0.019), hemoglobin (88.5 ± 81 vs. 99.1 ± 6.02; P = 0.041), and myeloperoxidase (19.3 ± 4.67 vs. 11.1 ± 4.43; P = 0.007) when compared with the group without fistula thrombosis. Diabetic nephropathy (P = 0.02) characterized the group of patients with thrombosis of the fistula. Diabetic nephropathy (B = 2.53, P = 0.049) and MPO (B = 0.03, P = 0.029) were statistically significant predictors of fistula thrombosis. In our study, MPO and diabetic nephropathy were predictors of thrombosis of the AVF. Key words: Oxidative stress, myeloperoxidase, atherosclerosis, thrombosis, arteriovenous fistula, hemodialysis
INTRODUCTION Oxidative stress is defined as tissue damage caused by an imbalance between synthesis of oxidative compounds and Correspondence to: R. V. Stolic, MD, PhD, Faculty of Medicine, Anri Dinan bb, 38220 Kosovska Mitrovica, Serbia. E-mail: [email protected]
antioxidant defense mechanisms. In end-stage renal disease, increased oxidative stress can occur due to loss of antioxidants during hemodialysis (HD), interaction between the blood and bioincompatible dialysis membranes, and bacterial contamination of the dialysate, leading to activation and recruitment of polymorphonuclear leukocytes.1,2 However, increased oxidative stress was also found in patients without end-stage renal disease,
© 2014 International Society for Hemodialysis DOI:10.1111/hdi.12155
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Myeloperoxidase and fistulas thrombosis
suggesting that the nature of the pathological process itself largely contributes to this situation.3 In fact, reduced glomerular filtration rate and chronic inflammation in chronic kidney disease can be important determinants of oxidative stress.4 Inflammatory events are known to be incorporated into all evolution phases of atherosclerotic plaque development, from the early stage of endothelial dysfunction through the formation of atheroma to the final damage.5 Myeloperoxidase (MPO) is a proinflammatory enzyme implicated in the pathophysiology of atherosclerosis, particularly in endothelial dysfunction, where its predictive significance seems to be much higher than that of C-reactive protein. Moreover, experimental and clinical studies now suggest that MPO may serve as a general marker of inflammation.6 However, data regarding the importance of MPO in predicting vascular access thrombosis in HD patients are scarce. The aim of the current study was to determine the predictive value of serum MPO in the development of thrombosis of arteriovenous fistula (AVF) for HD.
MATERIALS AND METHODS Patients The study was organized within the framework of junior project number 17/11 (oxidative stress and markers of inflammation in patients with chronic kidney disease), Medical Faculty Pristina/Kosovska Mitrovica, Serbia, and conducted at the Center for Hemodialysis, Medical Center, Kosovska Mitrovica, Serbia, as a nonrandomized clinical investigation. It involved monitoring patients with AVF for HD created over a period of 2 years. The protocol was approved by the Ethics Committee of the Medical Center, Kosovska Mitrovica, Serbia, according to the Declaration of Helsinki. All participants verbally consented to be included and were patients with the initial form of native AVF for HD. Fistulas were created when creatinine clearance was between 25 and 30 mL/min. The project involved 41 patients, 19 (46%) men and 22 (54%) women, with mean age of 65 ± 12.7 years. Patients were divided into a group of 14 (34%; 7 men and 7 women) with thrombosis of the AVF (mean age 64.9 ± 12 years) and 27 (66%; 12 men and 15 women) without fistula thrombosis (mean age 63.1 ± 14.7 years). In this study, the following were not included: 1. Patients on chronic HD, in whom AVF thrombosis had occurred earlier. 2. Patients younger than 18 years.
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3. Patients who were being treated for cardiovascular disease. 4. Patients with manifested forms of vasculitis. 5. Patients who had had any form of actual infection.
Biochemical methods Before the scheduled surgical interventions, 10 mL of blood was withdrawn into tubes with or without ethylenediaminetetraacetic acid (EDTA), as an anticoagulant. Serum samples were obtained after centrifuging clotted blood at 3000 × g for 15 minutes. Total and differential blood cell counts were determined in the EDTA samples by flow cytometry (Beckman Coulter Inc., Fullerton, CA, USA). Blood hemoglobin level was determined colorimetrically by the cyanmethemoglobin method. Serum concentrations of urea, creatinine, glucose, total calcium, inorganic phosphorus, uric acid, and iron were measured on an ILAB-600 chemistry analyzer (Diamond Diagnostics, Holliston, MA, USA), according to the manufacturer’s instructions, using original test reagents. Since MPO activity was not routinely measured, serum samples (1 mL) from the patients were frozen and kept at −20°C until a sufficient number accumulated but for no longer than 2 weeks. Serum MPO activity was determined kinetically in the 4-aminoantipyrine/phenol system with hydrogen peroxide as the substrate, by monitoring the formation of quinoneimine at λ = 505 nm, as described.7 Serum MPO activity was calculated using the molar absorbance: ε = 1.3 × 104 L × M−1 × cm−1. One unit of MPO activity was defined as the amount of enzyme degrading 1 μmol of hydrogen peroxide per minute, at 25°C.
Clinical, demographic, and anthropometrical parameters Age and sex were registered for all patients. Data regarding abuse of alcohol, diabetic nephropathy, and body mass index (BMI) were also recorded for all participants. Their BMI was calculated with respect to preoperative body weight by the formula: BMI (kg/m2) = present body weight/height2.
Statistical methods Data were summarized as frequencies or percentages for categorical variables, and as means ± SD or medians and range for numerical variables, respectively. To test the differences between cases and controls, Pearson’s chisquare test or Fisher’s exact test was used for categorical variables and, depending on the distribution of the data,
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Stolic et al.
the independent samples t test or Mann-Whitney U test were used for numerical variables. All variables that were significant at the probability level, P ≤ 0.05, were entered into a multiple logistic regression model with AVF thrombosis as the dependent variable. Statistical analysis was performed using SPSS version 15.0 (SPSS, Chicago, IL, USA) and INSTAT (Prism 4 GraphPad InStat, San Diego, CA, USA) software programs.
We performed multiple logistic regression analysis in order to evaluate the relationship between BMI, diabetes mellitus, and serum MPO activity, with the occurrence of AVF thrombosis. Of all variables included, only diabetic nephropathy (B = 2.53; P = 0.049) and MPO activity (B = 0.03; P = 0.029) showed statistically significant relationships as independent predictors of AVF thrombosis (Table 3).
RESULTS
DISCUSSION
Basic demographic, anthropometric, and clinical characteristics of patients with and without AVF thrombosis are presented in Table 1. There was a significant difference between the groups concerning BMI (19.9 ± 1.12 kg/m2 vs. 24.7 ± 3.2 kg/m2; P ≤ 0.0001). Among patients with diabetic nephropathy, five (18%) were without AVF thrombosis, whereas eight (57%) patients were with AVF thrombosis. In the group without diabetic nephropathy, six (43%) patients were with AVF thrombosis, whereas 22 (82%) patients were without AVF thrombosis. Thus, among patients with diabetic nephropathy, the frequency of AVF thrombosis was significantly higher (P = 0.02). Patients with AVF thrombosis had a significantly elevated number of leukocytes (8.55 ± 3.82) in comparison to those without fistula thrombosis (7.3 ± 2.13); P = 0.024. However, patients with AVF thrombosis had markedly fewer erythrocytes (2.8 ± 0.65 vs. 3.2 ± 0.27; P = 0.019), as well as a lower mean hemoglobin concentration (88.5 ± 81 vs. 99.5 ± 6.02; P = 0.041), compared to the group without AVF thrombosis. On the contrary, serum MPO activity was significantly higher in the group with thrombosis of the AVF (19.3 ± 4.67 vs. 11.1 ± 4.43; P = 0.007) (Table 2).
The principal finding of the current study is that higher extracellular MPO activity was an independent predictor of AVF thrombosis in patients undergoing HD treatment. These results are consistent with the recently reported benefits of low expression of leukocytic MPO on the vascular tone of resistance and conductance vessels,8 suggesting that extracellular MPO could play a role in oxidative damage and the development of vascular complications in HD patients. We observed that erythrocyte cell count and hemoglobin levels were far below the reference values in almost all of our patients. It is well known that erythrocytes are under constant challenge to maintain hemoglobin in its reduced functional state and to maintain the integrity of the cell membrane. Once hemoglobin is denatured it aggregates. In addition, oxidative damage of membranebound polyunsaturated fatty acids may result in delipidation and increased stiffness of the erythrocyte cell membrane.9,10 These perturbations usually result in premature removal of erythrocytes from the circulation,11,12 and our finding that anemia was more severe in patients with AVF thrombosis may therefore suggest more extensive oxidative damage in that group.
Table 1 Demographic, anthropometric, and clinical characteristics of the examined patients Investigated parameters Age (y), mean ± SD Gender, n (%) Males Females BMI ([kg/m2], [mean ± SD]) Diabetic nephropathy, n (%) Yes No Alcohol consumption, n (%) Yes No
AVF with thrombosis (n = 14)
AVF without thrombosis (n = 27)
64.9 ± 12.0
63.1 ± 14.7
7 (50) 7 (50) 23 ± 2.7
12 (44) 15 (56) 25.2 ± 3.4
8 (57) 6 (43)
5 (18) 22 (82)
2 (14) 12 (86)
4 (15) 23 (85)
P 0.706 0.735
0.046* 0.02*
1.00
*Statistically significant difference. BMI = body mass index.
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Table 2 Comparison of biochemical parameters between the examined groups of patients Biochemical parameters
AVF with thrombosis (n = 14)
AVF without thrombosis (n = 27)
P
8.55 ± 3.82 2.8 ± 0.65 81 ± 88.5 12.5 ± 8.4 26.4 ± 4.8 731.9 ± 230.1 2.1 ± 0.1 1.7 ± 0.6 170.6 ± 50.9 19.3 ± 4.67
7.3 ± 2.13 3.2 ± 0.27 99.5 ± 6.02 11.1 ± 6.3 25.9 ± 9.4 743.5 ± 347.7 2.1 ± 0.3 1.6 ± 0.4 196.5 ± 98.2 11.1 ± 4.43
0.024* 0.019* 0.041* 0.558 0.810 0.911 0.630 0.373 0.293 0.007*
Leukocytes (×10 /L), mean ± SD Erythrocytes (×1012/L), mean ± SD Hemoglobin (g/L), mean ± SD Serum iron (μM), mean ± SD Urea (mM), mean ± SD Creatinine (μM), mean ± SD Total calcium (mM) mean ± SD Inorganic phosphorus (mM) mean ± SD Uric acid (μM), mean ± SD MPO (U/L), mean ± SD 9
*Statistically significant difference. AVF = arteriovenous fistula; MPO = myeloperoxidase; SD = standard deviation.
In the past decade, much effort has been made to elucidate the role of MPO and oxidative stress in the promotion and progression of atherosclerosis. It has been shown, e.g., that higher extracellular MPO levels could predict an abnormal coronary angiogram in patients undergoing diagnostic cardiac catheterization, whereas in acute coronary syndrome and heart failure, serum MPO activity was predictive for an adverse clinical outcome.8 We have previously reported that atherosclerosis can significantly reduce the functionality of the AVF,13 and the results of our current study suggest that high serum MPO activity may predict the occurrence of AVF thrombosis in HD patients. Although these findings may help planning and creation of vascular access, the relevance of extracellular MPO levels in predicting AVF thrombosis is still to be explained. Myeloperoxidase is an important microbiocidal agent present in polymorphonuclear leukocytes and, to lesser extent, in other professional phagocytes. However, MPO can be released into the vascular compartment and is present either as a soluble form in circulation or bound to the endothelial cell surface. This extracellular MPO was suggested to impose oxidative damage on circulating lipoproteins, proteins, nitric oxide, and other biomolecules,
thus being implicated in the generation of a proinflammatory state, vascular dysfunction, and accelerated atherosclerosis. Uncontrolled production of free radicals and other oxidants may affect the structural integrity of the atherosclerotic plaque, i.e., accelerated activation of matrix metalloproteinases in the fibrous cap can induce plaque rupture, thereby creating conditions for blood clot formation. Such a sequence of events suggests that oxidative stress also plays a significant role in thrombogenesis and platelet activity. Oxidants generated by polymorphonuclear leukocytes have been shown to facilitate aggregation of monocytes to the endothelium, transformation of macrophages into foam cells, eventually leading to endothelial injury.14 In the current study, there was a higher leukocyte cell count among the group with initial AVF thrombosis than in patients in whom AVF thrombosis did not occur. This finding indicates that neutrophil leukocytes are a significant source of MPO in the circulation, and a role for them in AVF thrombosis. Another finding was that after exclusion of morbidly obese patients, the respondents with AVF thrombosis had significantly lower BMI than those without AVF thrombo-
Table 3 Multiple logistic regression model with thrombosed arteriovenous fistula as the dependent variable Independent variables
B
P
OR (95% CI)
BMI Diabetic nephropathy MPO
−0.16 2.53 0.03
0.250 0.049* 0.029*
0.85 (0.64–1.12) 12.61 (1.01–157.1) 1.032 (1.033–1.062)
*Statistically significant difference. BMI = body mass index; CI = confidence interval; MPO = myeloperoxidase; OR = odds ratio.
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sis. These results are in agreement with earlier reports that elevated levels of MPO can be associated with poor nutritional status, thereby connecting increased oxidative stress, malnutrition, and inflammation. Moreover, in malnourished end-stage renal disease patients, oxidative stress biomarkers were reported to be much higher than in well-nourished patients, in agreement with our previous study.15 If there is no ongoing infection, leukocytes are at rest, and release of oxidants into the vascular compartment occurs at a slow rate. However, in chronic kidney disease, there is permanent inflammation, in which polymorphonuclear leukocytes seem to be the common denominator of other clinical conditions, such as hypertension, diabetes mellitus, and other risk factors. These conditions are associated with endothelial dysfunction, accelerated atherosclerosis, and increased prevalence of cardiovascular mortality. The state of low-grade systemic inflammation causes oxidative stress, which greatly contributes to increased concentrations of extracellular MPO.16 Previous studies have shown that parameters indicating increased oxidative stress in predialysis patients are closely correlated with reduced glomerular filtration rate, chronic inflammation, and oxidative modifications of high-density lipoproteins. Thus, if excessive formation of oxidants plays a role in the development and progression of chronic kidney disease, there is a pertinent question of therapy to limit the impact of these harmful species. Due to the complexity of oxidative stress, it is almost impossible to accept and adopt a wide enough range of biomarkers to include all types of oxidative damage. Earlier findings indicate the importance of MPO activity in cardiovascular disease, although serum levels of MPO may not always reflect the state of inflammation. In uremic patients, however, an increased concentration of extracellular MPO has been shown to be a relevant marker of inflammation and an independent predictor of atherosclerosis.17,18 This was also confirmed in the current study by the finding that serum MPO activity was independently associated with the development of AVF thrombosis. Taken together, the results of our study have shown that serum MPO activity has predictive significance for the development of AVF thrombosis. Although it is limited by the small sample size and exclusion of morbidly overweight patients, this investigation raises the question of a preventive strategy for reduction of high proinflammatory, and obviously high prothrombotic levels of extracellular MPO in patients undergoing HD treatment. In that regard, there may be a promising impact of vitamin E and some other naturally occurring antioxidants on biomarkers of oxidative stress,17 which requires further research.
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As a limitation of the study, we must emphasize the small group of participants, and the single measurement of MPO that could not reflect changes over time.
CONCLUSION In our study, we found that MPO and diabetic nephropathy are major predictive factors of fistula thrombosis. Our patients with fistula thrombosis had a lower BMI, probably as a consequence of malnutrition, inflammation, and atherosclerosis. They also had a significantly lower erythrocyte count and hemoglobin concentration, while the number of leukocytes was larger.
ACKNOWLEDGMENTS Part of this research is supported by the Ministry of Education and Science of Serbia (Grant No. III41010) and the Medical Faculty Pristina/Kosovska Mitrovica, Serbia ( Junior Project No. 07/09). Conflict of interest: The authors declare no financial or other conflict of interests regarding this paper.
Manuscript received December 2013; revised January 2014.
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