Hemodialysis Raises Oxidative Stress Throughcarbon-centered Radicals Despite Improved Biocompatibility

Leukocyte activation and the resulting oxidative stress induced by bioincompatible materials during hemodialysis impact the prognosis of patients. Despite multiple advances in hemodialysis dialyzers, the prognosis of hemodialysis patients with complications deeply related to oxidative stress, such as diabetes mellitusremains poor. Thus, we re-evaluated the effects of hemodialysis on of multiple reactive oxygen species using electron spin resonance-based methods for further improvement of biocompatibility in hemodialysis. We enrolled 31 patients in stable condition undergoing hemodialysis using high-flux polysulfone dialyzers. The effects of hemodialysis on reactive oxygen species were evaluated by two methods: MULTiS, which evaluates serum scavenging activities against multiple hydrophilic reactive oxygen species, and i-STrap, which detects lipophilic carbon-center radicals. Similar to previous studies, we found that serum hydroxyl radical scavenging activity significantly improved after hemodialysis. Unlike previous studies, we discovered that scavenging activity against alkoxyl radicals was significantly reduced after hemodialysis. Moreover, patients with diabetes mellitus showed a decrease in serum scavenging activity against alkyl peroxyl radicals and an increase in lipophilic carbon-center radicals after hemodialysis. These results suggest that despite extensive improvements in dialyzer membranes, the forms of reactive oxygen species that can be eliminated during dialysis are limited, and multiple reactive oxygen species remain at increased levels during hemodialysis.

Key Words: hemodialysis, biocompatibility, alkyl peroxyl radical electron spin resonance, MULTiS, i-STrap

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Chronic kidney disease (CKD) is known to be associated with high oxidative stress. (-4)Accumulation of uremic toxins with pro-oxidative properties leading to microinflammation caused by the elevation of pro-inflammatory cytokines is the key feature of CKD pathophysiology. (s-, Hemodialysis (HD), a widely conducted renal replacement therapy, acts as a double-edged sword to reduce oxidative stress in patients with CKD. Activation of polymorphonuclear leukocytes, due to contact with the membrane or other artificial surfaces in HD circuits, is the main cause of oxidative stress induced by HD.s-) Conversely, the removal of pro-oxidative uremic toxins, especially low-molecular-weight hydrophilic compounds such as trimethylamine-Noxideor guanidino compounds, may improve the antioxidative nature of HD, leading to long-term improvement of survival rate and quality of life in patients with CKD. (2,13) In addition, correction of acidosis leads to the re-activation of antioxidative enzymes and reduction of volume overload leading to the prevention of cardio. vascular complications are antioxidative mechanisms. (4 Thus. is necessary for HD materials to minimize leukocyte activation and maximize antioxidative effects. To overcome this problemantioxidative and biocompatible HD material exemplified by the vitamin E-coated dialyzer have been developed. (si7) However despite the numerous advances in HD dialyzers, the prognosis of HD patients remains unsatisfactory, especially those with diabetes mellitus (DM), a disease that promotes a highly oxidative state. (8)These concerns are partly because the details of widely varied and complex in vivo oxidative stress-related reactions have not been sufficiently analyzed. Thus, this study aimed to clarify the effect of HD on reactive oxygen species(ROS) dynamics, as the upstream events of oxidative stress reactions, based on the concept that each target site of oxidative and antioxidant reactions in the body needs to be biochemically described in a specific time and space. One explanation for this is a lack of detailed analysis of the upstream side of oxidative stress-related reactions that stimulate these cellular reactions. The identification of ROS that act as stimulators of oxidative stress reactions or interactions among ROS to generate oxidative stimulators is difficult due to the high reaction rates and complex reaction chains. Since ROS are not uniform and individual ROS have specific characteristics during in vivo reactions, an analysis of multiple ROS is required. (9-2n Thus, to improve biocompatibility based on the use of antioxidative and biocompatible materials, one must understand the changes in ROS dynamics caused by HD.

To investigate ROS dynamics during HD, we employed two newly developed electron spin resonance (ESR)-based methods. the multiple hydrophilic free-radical scavenging assay(MULTIS)and the lipophilic radical detection assay on whole blood (i-STrap)Although research on ROS scavenging activity by ESR is not extensive, it remains the only method available for identifying type and examining the dynamics of ROS. The method combines a high-performance liquid chromatography type flow system with an ESR system and employs 5-(2,2-dimethyl-1.3-propoxycyclophosphoryl)-5-methyl-1-pyrrolineNoxide(CYPMPO) as the spin trap. MULTIS provides higher sensitivity and stability for the ROS scavenging measurement than conventional ESR assays.22-24) One of our co-authors has previously reported MULTIS-measured ROS scavenging activities in patients with stage 5 CKD. However, they did not evaluate the effect of HD.e2) Additionally, the i-STrap method is an ESRbased method that detects ROS in whole blood using 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxideDPhPMPO)as its spin trap and measures ESR after an organic extraction, thus reflecting lipophilic ROS.2s.26 In this study, we found that the levels of several ROS were increased and not eliminated by HD, indicating that radical chain reactions are still not adequately controlled following HD. This may affect the prognosis of patients with CKD.

Materials and Methods

Subjects and informed consent. All procedures conducted on human subjects were performed after obtaining individual written consent. The protocol used was approved by the Tsukuba University of Technology Committee (AuthorizationNo. 201809). This is an observational study; therefore, interventions were performed on the patients. A total of 31 stable HD patients were included in the study. We obtained and recorded clinical and demographic information for each patient. All patients were treated with high-flux polysulfone membrane dialyzers (Toray Medical Co., Ltd., Tokyo, Japan) with various membrane surface areas, adapted to the body constitution of each individual. The patients were divided into two groups depending on the cause of their end-stage renal disease: the non-diabetic group (non-DM group, n=17)and the diabetic group, n = 14).

Sample collection. Blood samples were obtained at the onset and end of the dialysis treatment from the sampling located on the arterial side of the HD circuit. For MULTIS measurements, sera were separated and stored in a freezer at -80°(until the measurement was performed, for i-STrap measurements. whole-blood samples were collected in heparin-coated tubes that were allowed to stand for one hour before measurements. Measurements of multiple free radical scavenging activity in serum. Hydrophilic ROS scavenging activities were measured with the MULTIS method using previously described protocols with minor modifications.2227) Scavenging activities against five ROS, namely hydroxyl radical(OH), superoxide(0,"), alkoxyl radical (RO"), alkyl peroxyl radical (ROO"), and singlet oxygen ('0,), were measured. An X-band ESR spectrometer(RR-Xl ESR: Radical Research Inc., Tokyo, Japan) employing 100 kHz field modulation and WN-RAD operation software(Radical Research Inc.) was used. The ESR spin trapping reagents used were CYPMPO for "OH, O, RO', and ROO' and 4-hydroxy-2,2,6,6-tetramethylpiperidine (TEMP)for 'O. The typical spectrometer settings were as follows: field modulation width.0.1 mT; microwave power, 10 mW; field scan width and rate+7.5 mT/2 min; and time constant, 0.1 s.Each ROS was generated via in situ illumination with UV/visible light from an illuminatorRUVF-203SR UV illuminator, Radical Research Inc.) equipped with a 200W medium-pressure mercury/xenon arc lamp and quartz light guide, connected to the resonator cavity. The light sources, illumination times, precursors, and photosensitizers used to produce ROS are summarized in Table 1. The ROS scavenging activities were calculated according to a previously describedmethod22) and converted into the unit equivalent to known pure scavengers: glutathione(GSH)for OH and 'O,  superoxide dismutase (SOD)for O, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)for RO', and a-lipoic acid for ROO'MULTISmeasurements for each ROS were performed in triplicate.

Measurement of lipophilic 

Ros scavenging activity in whole blood using i-Strap. Lipophilic ROsscavenging activity was measured using the i-STrap ESR measurement ki(Dojin-Glocal/Dojindo, Kumamoto, Japan) based on the manufacturer's protocol and a previous report.2s This method is based on the competitive reaction between antioxidants in whole blood and DPhPMPO with the tert-butyl hydroperoxide (BuOOHradical. Whole blood samples were incubated with DPhPMPO(10 mM) and test-BuOOH (10 mM) for 30 min at room temperature. After incubation, the spin adducts in the organic phase were extracted using chloroform and methanol solutions; ESR measurements were conducted using these organic samples. The measuring conditions were the same as for the MULTISmeasurement, except for the field scan width and rate, set at at+5.0 mT/2 min, There were no specific antioxidants for i-STrapmeasurement; therefore, scavenging activity was expressed as an10/-1 value, where "0" denotes the signal intensity without a sample and "' denotes the signal intensity with a sample. (28)Reagents. CYPMPO was obtained from RR INC.(Japan); riboflavin, 2,2'-azobis (2-amidinopropane) dihydrochloride(AAPH), text-BuOOH, dimethyl sulfoxide(DMSO), and rose Bengal were purchased from Sigma-Aldrich Japan (Tokyo, Japan)and TEMP was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydrogen peroxide and buffers were obtained from WakoChemical Co.(Osaka, Japan).Statistical analysis."Statistical analysis was performed using Prism 6 for Mac OS X computer software (GraphPad SoftwareInc., La Jolla, CA). Data were tested using the Student's paired t-test. Data were expressed as mean values with a 95% confidence interval(95% CI.