Effects Of Ultrapure Water On The Levels Of Oxidative Stress And Inflammation Parameters In Maintenance Hemodialysis Patients

Dialysis-related pathology(DRP) has been a major concern to nephrologists these years. DRP, which may be considered as one of the most frequent causes for morbidity and/or mortality after long-term hemodialysis. Among DRP manifestations, cardiovascular diseases (CVD) are the first cause of mortality in end-stage renal disease, and the majority of CVD results from complications of atherosclerosis. Hemoincompatibility of dialysis system, especially the microbiological contamination and the pyrogen content of the dialysate, may play a major role in the production of reactive oxygen species and chronic micro-inflammation.Oxidative stress resulting from an imbalance between prooxidative and antioxidative is responsible for increasing stress markers, such as malonylaldehyde (MDA), a lipid peroxidation product, and advanced oxidation protein products (AOPP), which can be formed in vitro by exposure of serum albumin to hypochlorous acid(HOCl).At the same time, oxygen radical scavenger system is impaired severely. That is to say, in the enzymatic pathway, the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) has reduced. During hemodialysis, dialysate pyrogens penetrating the dialyzer membranes is the most important inducer of cytokine production in mononuclear cells. Interleukin-1(IL-1), Tumor Necrosis Factor-α(TNF-α), Interleukin-6 (IL-6) are the critical mediators in acute or chronic inflammatory diseases of chronic hemodialysis. Interleukin-6 can increase production of acute-phase proteins(C- reactive protein, serum amyloid A) in the liver. Himmelfarb J considers oxidative stress and inflammation are linked in uremic patients through myeloperoxidase(MPO). Stimulated neutrophils and monocytes generate superoxide and its dismutation product, hydrogen peroxide, and simultaneously release the enzyme MPO. MPO has the unique property of converting chloride in the presence of hydrogen peroxide to hypochlorous acid.To a patient with hemodialysis, the contact between blood and water is through an un-selectivity semipermeable membrane. If the dialysis water is contaminanted, the patients will suffer from higher levels of oxidative stress and chronic inflammation. The quality standard that recommended by the Association for the Advancement of Medical Instrumentation(AAMI) for the water used to prepare dialysate was bacteria not exceeding 0.1 colony forming units(CFU)/ml and endotoxin not exceeding 0.03 endotoxin units(EU)/ml. Some investigations indicate that ultrapure water can reduce the production of cytokines, decrease the oxidative stress through non-enzyme systems, improve the nutritional status and reduce epoetin requirement. But there is still no report on the relationship between the improvement of water and the activation of monocytes. The productions of AOPP and antioxidant enzymatic systems during oxidative stress have not been investigated, either.Objective:To evaluate the effects of ultrapure water on the levels of oxidative stress and inflammation parameters in maintenance hemodialysis patients, and to approach the relationship between inflammation and oxidative stress, especially the relations among inflammation, MPO and AOPP, and their respective influential factors.Methods1.Cases selection 36 clinically stable maintenance hemodialysis patients(24 males, 12 females). Mean age 56.25±11.85 years (30-75 years) , median of dialysis time 19.50 months (3-192 months).They had no acute infection, vasculitis or disease activity and had undergone hemodialysis for at least three months before being changed to use ultrapure water. Each patient undergoes hemodialysis twice or three times every week, which lasts 4 hours each time, using heparin or low molecular heparin for anticoagulation.2.Experiment design The dialysis water and venous blood samples from patients were collected three times to detect the levels of oxidative stress and inflammation parameters, the first time was done before water processing equipment was changed to Aquaboss EcoROⅡand non-dead space circuit loop, the second was done six months after the change, and the last was done twelve months after the change. Serum was isolated at 4℃immediately and stored at -70℃until analysis.3. Detection of bacterial growth, endotoxin concentration and parameters The dialysis water from ingress of machines was collected for microbial counts and endotoxin concentration (Kinetic-Turbidimetric Assay, KTA).The level of AOPP was determined by spectral analysis with absorbance at 340nm in acidic condition. The level of MDA was measured by fluorometric assay. The activity of GSH-Px in serum was measured by spectrophotometry. The activity of MPO was detected by chromatometry. The inflammation parameters, including neopterin, TNF-α, IL-6, were measured by ELISA. Serum CRP and Alb were detected by immunoturbidimetry(ITM) and Albumin Kit(Bromcresol Green) in the clinical laboratory of Nan Fang hospital. 4. Statistical treatment Results are expressed as Mean±SD. Repeated measure ANOVA was used for each repeated measure parameter. Partial correlation analysis was applied to comparison of two parameters. Statistical significance was defined at P <0.05.All statistical calculations were performed with SPSS11.5.Result1. Before the water management system was improved, the microbial count of dialysis water was (85.05±13.35) CFU/ml. But 6 months and 12 months after the equipment was upgraded, there was no bacteria growth in the dialysis water (F=121.722, P=0.008). The concentrations of endotoxin at the above three time points were 0.30 EU/ml, <0.02 EU/ml, <0.02 EU/ml respectively.2. During study period, the levels of AOPP, MDA, GSH-Px, MPO differentiated significantly at each time point (P =0.034, P =0.006, P =0.049, P =0.001). After adopting to ultrapure water for six months, the levels of serum AOPP, MDA,GSH-Px, MPO have been improved significantly (P =0.01. P =0.015, P =0.027. P =0.001). After twelfth months, the level of serum MPO has achieved a further reduction (P =0.009) .3. During study period, the concentrations of CRP, neopterin, TNF-α, IL-6, Alb differentiated significantly at each time point(P=0.044, P =0.044, P =0.036, P =0.033, P =0.003). After switching to ultrapure water for six month, serum CRP, neopterin and TNF-αhas decreased markedly (P =0.005, P =0.044, P =0.033), while Alb has increased significantly (P=0.015). After twelfth months, each inflammation parameter has been improved (P=0.034, P =0.032, P =0.039, P =0.017. P =0.002).4.Before changing to ultrapure water, the level of serum CRP was positively correlated with TNF-α(r=0.5591, P =0.002), but negatively correlated with Alb (r=-0.5175, P =0.004); IL-6 was positively correlated with TNF-α(r=0.7712, P<0.001), but negatively correlated with Alb(r=-0.4474. P =0.015). After switching to ultrapure water for six months, serum AOPP was positively correlated with TNF-α(r=0.3881, P=0.037). After the past twelve months of study, serum AOPP was positively correlated with CRP(r=0.5028, P=0.005), CRP with MPO(r=0.4089, P=0.028), and MDA with neopterin (r=0.4572, P=0.013).Conclusions:1.When the microbial count and endotoxin concentration of dialysis water significantly decreased, the status of oxidative stress and that of inflammation in maintenance hemodialysis patients were improved after sixth month. Both statuses can be maintained or even become better after one year.2.Serum MPO activity may estimate the level of oxidative stress and that of inflammation at the same time.3.That MDA was correlated with neopterin shows that the activation of monocyte may also cause lipid peroxidation. MDA and neopterin would go hand in hand to promote and influence the development of the atherosclerosis.4.The reason that ultrapure water improved the level of Alb maybe relate with the improvement of the inflammation status.