Hippokratia 2015, 19(2):131-135
Hemodialysis Unit, Ilion Medical Nursery, Medifil, Athens, Greece
Background: The aim of the study was to determine the effect of oral N-acetylcysteine (NAC) on levels of serum oxidative stress biomarkers in hemodialysis patients.
Methods: Forty eight hemodialysis patients were administered NAC orally for six months. Hematological, biochemical parameters and levels of asymmetric dimethylarginine (ADMA), malondialdehyde (MDA), myeloperoxidase (MPO) and nitrogen oxide (NO) were determined prior to and upon completion of the study period.
Results: At the end of the study period white blood cells, neutrophil percentage and C-reactive protein levels were significantly lower. Uric acid, albumin and hemoglobin were significantly higher compared to pre-treatment values. Statistically significant increase in NO, and decrease in MDA and ADMA levels were observed. Serum MPO demonstrated a measurable decrease trend, though not significant.
Conclusion It is suggested that treatment with NAC appears to be associated with restoration of important parameters of antioxidant defence and reduction in the levels of mediators of oxidative cellular damage.
Key words: N-acetylcysteine, oxidative stress, hemodialysis
The uremic milieu is an environment of high oxidative stress, in which increased generation of oxidant radicals is coupled to impaired antioxidant neutralization. This is correlated to major complications of the uremic syndrome namely inflammation, endothelial dysfunction, anaemia and accelerated atherosclerosis. The interruption in redox balance appears early in chronic kidney disease (CKD) and is progressively enhanced as it advances to end-stage (ESRD)1.
Studies concerning the effects of dialysis on oxidative stress have produced conflicting results, since dialysis-related factors promote the formation of free radicals. One of the principal parameters is biocompatibility of hemodialysis materials, while oxidant neutralization is impaired, due to intradialytic loss of water-soluble antioxidants exacerbating the antioxidant defence deficit.
The need to develop strategies to tackle the burden of oxidative stress in haemodialysis is highlighted. Administration of antioxidants is considered a promising approach. N-Acetylcysteine (NAC) is a thiol-molecule that demonstrated a positive effect on oxidative biomarkers2-6 and a reduction in the incidence of primary cardiovascular events7, in limited clinical trials involving dialysis patients.
Subjects and Methods
We examined 48 patients, aged 56.9 ± 9.3 years, under dialysis for 43 ± 31 months (Table 1). Inclusion criteria were: thrice-weekly, four-hour hemodialysis session, for a minimum of 12 months, efficacy of single-pool Kt/V (K is the dialyzer blood urea clearance in liters/hour, t is the dialysis session length in hours, V is the urea distribution volume in liters), (spKt/V) ≥1.2-1.4 and compliance with the prescribed treatment. Patients with: inflammatory diseases, peptic ulcer, vascular access failure at any time throughout the study or hypersensitivity to NAC, were excluded. The study protocol was approved by the Institution Scientific Committee, (Ilion Medical Nursery Scientific and Ethics Committe 12/05-10-2013), patients were informed and written consent was obtained.
The study period was set at 6 months, during which time patients received oral 600 mg of NAC, twice daily before meals. Alterations, involving patient therapeutic regimen or hemodialysis parameters, were not permitted during this period. Hematological and biochemical parameters, as well as levels of asymmetric dimethylarginine (ADMA), malondialdehyde (MDA), myeloperoxidase (MPO) and nitrogen oxide (NO), were determined prior to and upon completion of the study period, from samples obtained before the second dialysis session of the week.
Serum ADMA and MDA were quantified via competitive enzyme-linked immunosorbent assay (ELISA) (USNC Life Science Inc., Wuhan, China). Serum NO and MPO via sandwich ELISA, using the Human NO Kit (Cusabio, Wuhan, China) and MPO kit (USNC Life Science Inc., Wuhan, China). The delivered dose of hemodialysis was measured using Urea Reduction Ratio (URR) from predialysis and postdialysis serum urea samples [(predialysis Urea-postdialysis Urea)/predialysis Urea] and the single-pool urea kinetic volume model i.e. spKt/V [-ln(R-0.008×t)+(4-3.5×R)×UF/W, where R=1-URR, UF is the fluid volume removed during dialysis, t is session length and W is the postdialysis body weight].
Administration of NAC was correlated with significant changes in haemoglobin levels (p=0.029), a decrease in leukocyte count (p=0.002), in particular, neutrophil percentage (p=0.001) while lymphocytes rose (p=0.008). Changes in serum iron (p=0.376) and ferritin (p=0.647) were not significant, while uric acid (p=0.008), phosphate (p=0.009) and calcium-phosphate product (p=0.014) differed significantly. Decrease in levels of C-reactive protein (CRP) (p=0.002) and increase in serum total protein (p=0.003), which was attributed to the increase in serum albumin (p=0.001), were also noted. The decrease in intact parathyroid hormone (iPTH) was statistically significant (p=0.001). Α comparison of the values for spKt/V (p=0.96) and URR (p=0.73) revealed no significant changes in adequacy markers, presuming thus that the state of oxidative stress was not affected by changes in hemodialysis efficacy (Table 2).
Significant increases in levels of NO (p=0.001) and decreases in MDA (p=0.001) and ADMA levels (p=0.032) were observed. Moreover, MPO levels demonstrated a decrease trend though not shown to be significant (p=0.385) (Table 3).
A correlation exists between indicators of antioxidant capacity, markers of inflammation and oxidative stress in uraemia. It seems that persistent inflammation depletes antioxidant defense mechanisms, disrupts redox potential, leading to and amplifying oxidative stress.
In our study, NAC appeared to have a positive effect on hemoglobin levels without modifications to either erythropoietin or iron dosage. Data suggests a correlation between erythrocytic lipid peroxidation products and resistance to erythropoietin, on one hand, and levels of oxidative stress biomarkers and hemoglobin, on the other. It seems that NAC acts as an adjuvant in the treatment of CKD anaemia8.
The relationship between oxidative stress, inflammation and endothelial dysfunction is observed early in CKD becoming prominent in ESRD. Oxidative stress biomarkers have been shown to be surrogate markers of cardiovascular morbidity and mortality of equal prognostic power to proinflammatory cytokines9. A positive correlation between CRP and lipid peroxidation by-products and a negative correlation between CRP and antioxidants has been described in ESRD10. In a recent study, treatment with NAC was associated with a significant reduction in inflammatory markers11.
Low albumin levels have been observed in CKD, being correlated to disease stage and are considered to be an independent risk factor of mortality12. The ratio of oxidized albumin is higher in ESRD and is attributed to high oxidative stress burden13, uremic inflammation and malnutrition, resulting in attenuation of total antioxidant plasma capacity14.
Considering the significant reduction in the number of leukocytes, neutrophils, CRP levels and the increase in serum albumin, it could be argued that the reduction of oxidative stress was likely associated with a decrease in uremic inflammation and restoration of key antioxidant mechanisms. Interpreting the increase in blood lymphocytes, it could be attributed to a potential decreased oxidative-mediated lymphocytic apoptotic rate, a collateral positive antioxidant effect of NAC therapy.
Uric acid is an essential component of plasma antioxidant capacity. A ‘J-shaped’ association is observed between urate and mortality in ESRD. Extremely high (>8.9mg/dL) and low (<6.3mg/dL) levels are associated with increased mortality15. The administration of NAC was possibly correlated to increase of urate to levels without negative outcome on end-points, attributed to reduction of oxidative stress accompanied by partial balancing of redox potential due to uric acid recovery.
Data analysis revealed a reduction in iPTH levels that could be attributed to NAC-mediated reduction in oxidative stress-induced osteoclastic activity and bone resorption. It has been reportedthat antioxidant treatment has had a beneficial effect on bone mass loss in experimental models. It is associated with osteoclastic inhibition, activation of transcription factor NF-κB and expression of TNF-a, inhibiting thus bone loss.
Hemodialysis patients exhibit high levels of ADMA, a molecule with inhibitory action on nitric acid synthetase (NOS) and low levels of the endothelium-derived relaxant factor NO16. Reports concerning the effect of dialysis on ADMA and NO levels have been inconclusive17. A temporary reduction in ADMA, attributed to clearance by dialysis has been reported18. The administration of antioxidants appears to be associated with a persistent reduction in ADMA levels having a beneficial effect on outcome3,18. This is possibly due to restoration of the balance between enzymes involved in ADMA synthesis and catabolism, whose activity depends on oxidative stress magnitude, leading to partial restoration of the ADMA-NOS pathway, increase in the bioavailability of NO and oxidative stress reduction, via negative feedback.
Our study suggests a beneficial effect on the levels of NO and ADMA, which could be attributed to restoration of redox balance as a result of oxidative stress reduction. NAC has been shown to have a positive effect in the treatment of albuminuria in non-diabetic CKD patients19 and has been associatedwith increases in dialysis patients’ residual renal function20,21, attributed to reduction in ADMA and restoration of NO.
In interpreting the reduction in lipid peroxidation marker MDA, it could be argued that the administration of NAC contributed in limiting the extent of peroxidation reactions, due to oxidative stress decline. Available data concerning MDA levels in ESRD patients is limited and conflicting. Dialysis patients present with higher than normal MDA levels, which are negatively correlated with serum albumin and positively correlated with CRP levels22. In ESRD, the relationship between lipid peroxidation by-products (F2-isoprostanes), antioxidant mechanisms and inflammation is confirmed. Peroxidation products have been shown to be negatively correlated with albumin23 and positively correlated with MDA24, which appear higher in dialysis patients at high cardiovascular risk.
All the above highlight the negative consequences of oxidative stress (lipid peroxidation) on outcome25. The beneficial effect of NAC on MDA levels in hemodialysis patients have been demonstrated4, whereas in studies evaluating the effect of intradialytic iron therapy on MDA treatment with NAC was shown to have a protective effect5.
The value of MPO levels in predicting cardiovascular morbidity and endpoints for dialysis patients has been evaluated in recent clinical studies26. In the case of renal diseases, MPO appears to be primarily associated with oxidative stress27. It constitutes the link between inflammation, oxidative stress and endothelial dysfunction, acting as a NO oxidase, regulating NO availability27. In vitro studies28 have highlighted an inductive effect of advanced oxidation protein products on the MPO-dependent oxidative burst of leucocytes. Hemodialysis per se has been associated with an increase in MPO levels, attributed to leukocyte activation and related to the duration of dialysis, type of membrane and anticoagulant used26-29. Administration of heparin mobilizes vascular-bound MPO, suggesting that the observed increase in MPO levels could possibly be attributed, apart from the neutrophilic oxidative burst, to vascular release of MPO29.
Controlled in vitro studies involving dialysis patients found NAC to have a positive effect. According to certain in vivo studies, administering NAC to diabetic dialysis patients was associated with a significant persistent reduction in MPO levels30. In the present study, NAC administration was associated with a non significant reduction in MPO levels. The fact that the levels of advanced oxidation protein products, which are the prime indicators of MPO-dependent activation in ESRD, were not determined, prevents the establishment of conclusions. However, the response exhibited by other MPO-related oxidation markers has provided significant indirect evidence of an ultimately positive effect.
Our study is subject to limitations, such as the relatively small sample of patients, the absence of control group and randomized distribution. Moreover, markers of protein and DNA oxidation were not investigated. Measurement of NAC serum levels were not conducted while those concerning oxidative parameters were performed at two points in time and hence possibly do not reflect variations attributed to disease progression. Therefore, the generalization of the results is limited.
Treatment of hemodialysis patients with NAC appears to be associated with restoration of important parameters of antioxidant defence and reduction in the levels of oxidative cellular damage mediators. The overall effect appears to affect both sides of the ESRD-associated redox imbalance. It appears to re-establish part of the antioxidant defence and to limit the magnitude of oxidation reactions and free radicals formation, leading to at least partial restoration of redox balance. Large-scale research and prospective controlled studies are required.
Conflict of interest
Author reports no conflict of interest.
1. Dounousi E, Papavasiliou E, Makedou A, Ioannou K, Katopodis KP, Tselepis A, et al. Oxidative Stress Is Progressively Enhanced With Advancing Stages of CKD. Am J Kidney Dis. 2006; 48: 752-760.
2. Coombes JS, Fassett RG. Antioxidant therapy in hemodialysis patients: a systematic review. Kidney Int. 2012; 81: 233-246.
3. Thaha M, Widodo, Pranawa W, Yogiantoro M, Tomino Y. Intravenous N-acetylcysteine during hemodialysis reduces asymmetric dimethylarginine level in end-stage renal disease patients. Clin Nephrol. 2008; 69: 24-32.
4. Trimarchi H, Mongitore MR, Baglioni P, Forrester M, Freixas EA, Schropp M, et al. N-acetylcysteine reduces malondialdehyde levels in chronic hemodialysis patients--a pilot study. Clin Nephrol. 2003; 59: 441-446.
5. Swarnalatha G, Ram R, Neela P, Naidu MU, Dakshina Murty KV. Oxidative stress in hemodialysis patients receiving intravenous iron therapy and the role of N-acetylcysteine in preventing oxidative stress. Saudi J Kidney Dis Transpl. 2010; 21: 852-858.
6. Garcia-Fernandez N, Echeverria A, Sanchez-Ibarrola A, Paramo JA, Coma-Canella I. Randomized clinical trial on acute effects of i.v. iron sucrose during haemodialysis. Nephrology (Carlton). 2010; 15: 178-183.
7. Tepel M, van der Giet M, Statz M, Jankowski J, Zidek W. The antioxidant acetylcysteine reduces cardiovascular events in patients with end-stage renal failure: a randomized, controlled trial. Circulation. 2003; 107: 992-995.
8. Hsu SP, Chiang CK, Yang SY, Chien CT. N-acetylcysteine for the management of anemia and oxidative stress in hemodialysis patients. Nephron Clin Pract. 2010; 116: C207-C216.
9. Stenvinkel P, Carrero JJ, Axelsson J, Lindholm B, Heimburger O, Massy Z. Emerging biomarkers for evaluating cardiovascular risk in the chronic kidney disease patient: how do new pieces fit into the uremic puzzle? Clin J Am Soc Nephrol. 2008; 3: 505-521.
10. Ikizler TA, Morrow JD, Roberts LJ, Evanson JA, Becker B, Hakim RM, et al. Plasma F2-isoprostane levels are elevated in chronic hemodialysis patients. Clin Nephrol. 2002; 58: 190-197.
11. Saddadi F, Alatab S, Pasha F, Ganji MR, Soleimanian T. The effect of treatment with N-acetylcysteine on the serum levels of C-reactive protein and interleukin-6 in patients on hemodialysis. Saudi J Kidney Dis Transpl. 2014; 25: 66-72.
12. Sitar ME, Aydin S, Cakatay U. Human serum albumin and its relation with oxidative stress. Clin Lab. 2013; 59: 945-952.
13. Menon V, Greene T, Wang X, Pereira AA, Marcovina SM, Beck GJ, et al. C-reactive protein and albumin as predictors of all-cause and cardiovascular mortality in chronic kidney disease. Kidney Int. 2005; 68: 766-772.
14. Danielski M, Ikizler TA, McMonagle E, Kane JC, Pupim L, Morrow J, et al. Linkage of hypoalbuminemia, inflammation, and oxidative stress in patients receiving maintenance hemodialysis therapy. Am J Kidney Dis. 2003; 42: 286-294.
15. Hsu SP, Pai MF, Peng YS, Chiang CK, Ho TI, Hung KY. Serum uric acid levels show a ‘J-shaped’ association with all-cause mortality in haemodialysis patients. Nephrol Dial Transplant. 2004; 19: 457-462.
16. Uzun H, Konukoglu D, Besler M, Erdenen F, Sezgin C, Muderrisoglu C. The effects of renal replacement therapy on plasma, asymmetric dimethylarginine, nitric oxide and C-reactive protein levels. Clin Invest Med. 2008; 31: E1-E7.
17. Eiselt J, Rajdl D, Racek J, Široká R, Trefil L, Opatrná S. Asymmetric dimethylarginine in hemodialysis, hemodiafiltration, and peritoneal dialysis. Artif Organs. 2010; 34: 420-425.
18. Chang JW, Lee EK, Kim TH, Min WK, Chun S, Lee KU, et al. Effects of alpha-lipoic acid on the plasma levels of asymmetric dimethylarginine in diabetic end-stage renal disease patients on hemodialysis: a pilot study. Am J Nephrol. 2007; 27: 70-74.
19. Feldman L, Shani M, Sinuani I, Beberashvili I, Weissgarten J. N-acetylcysteine may improve residual renal function in hemodialysis patients: A pilot study. Hemodial Int. 2012; 16: 512-516.
20. De Vecchi AF, Bamonti F, Novembrino C, Ippolito S, Guerra L, Lonati S, et al. Free and total plasma malondialdehyde in chronic renal insufficiency and in dialysis patients. Nephrol Dial Transplant. 2009; 24: 2524-2529.
21.Lim PS, Chang YM, Thien LM, Wang NP, Yang CC, Chen TT, et al. 8-iso-prostaglandin F2alpha as a useful clinical biomarker of oxidative stress in ESRD patients. Blood Purif. 2002; 20: 537-542.
22. Cavalca V, Sisillo E, Veglia F, Tremoli E, Cighetti G, Salvi L, et al. Isoprostanes and oxidative stress in off-pump and on-pump coronary bypass surgery. Ann Thorac Surg. 2006; 81: 562-567.
23. Pecoits-Filho R, Stenvinkel P, Marchlewska A, Heimburger O, Barany P, Hoff CM, et al. A functional variant of the myeloperoxidase gene is associated with cardiovascular disease in end-stage renal disease patients. Kidney Int Suppl. 2003; 84: S172-S176.
24. Eiserich JP, Baldus S, Brennan ML, Ma W, Zhang C, Tousson A, et al. Myeloperoxidase, a leukocyte-derived vascular NO oxidase. Science. 2002; 296: 2391-2394.
25. Witko-Sarsat V, Gausson V, Nguyen AT, Touam M, Drüeke T, Santangelo F, et al. AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target for N-acetylcysteine treatment in dialysis patients. Kidney Int. 2003; 64: 82–91.
26. Tang WH, Wu Y, Nicholls SJ, Hazen SL. Plasma myeloperoxidase predicts incident cardiovascular risks in stable patients undergoing medical management for coronary artery disease. Clin Chem. 2011; 57: 33-39.
27. Strobel NA, Fassett RG, Marsh SA, Coombes JS. Oxidative stress biomarkers as predictors of cardiovascular disease. Int J Cardiol. 2011; 147: 191-201.
28. Tang WHW, Wu Y, Nicholls SJ, Hazen SL. Plasma myeloperoxidase predicts incident cardiovascular risks in stable patients undergoing medical management for coronary artery disease. Clin Chem. 2011; 57: 33–39.
29. Borawski J. Myeloperoxidase as a marker of hemodialysis biocompatibility and oxidative stress: The underestimated modifying effects of heparin. Am J Kidney Dis. 2006; 47: 37-41.
30. Amore A, Formica M, Giacchino F, Gigliola G, Bonello F, Conti G, et al. N-Acetylcysteine in hemodialysis diabetic patients resets the activation of NF-kB in lymphomonocytes to normal values. J Nephrol. 2013; 26: 778-786.