Association of Serum Eotaxin Levels and Markers of Myocardiac Injury in Hemodialysis Patients

Study Design and Patient Population This cross-sectional trial, and will be carried out in accordance with the Declaration of Helsinki and applicable regulations. The protocol will be sent to the institutional ethics committee and patients' written informed consent will be obtained. To be included in the study, patients will be at least 20-years-old and on outpatient HD for at least 3 months. All the patients were dialyzed three times a week with a high-flux membrane and bicarbonate dialysate solutions. Patients will excluded if they had an acute infection or malignancy. A total of 400 long-term HD patients are randomly invited and enrolled in the study. The medical records will thoroughly reviewed for each subject by a collaborating physician in the study. Information such as underlying kidney disease, cardiovascular disease history, and other comorbid illnesses are abstracted. A pre-existing history of CVD was defined as a history of CAD ( including a history of myocardial infarction, coronary artery angioplasty/stenting/bypass surgery, carotid endarterectomy/stenting), cerebrovascular disease (CVD, e.g., stroke), non-traumatic lower extremity amputation, and lower limb artery bypass surgery/angioplasty/stenting

Measurements Predialysis blood samples are obtained on a mid-week day.Within 30 min after sampling, the remaining blood arecentrifuged at 3,000 g for 10 min, immediately aliquoted and frozen at-80 °C until further analysis. The total serum cholesterol is measured through the reaction of cholesterol esterase/cholesterol oxidase/peroxidase, using Hitachi 747 (Hitachi, Bohemia, NY, USA). HDL cholesterol is quantified after precipitation with polyethylene glycol at room temperature. Serum glucose concentrations are measured by the glucose oxidase method. Low-density lipoprotein cholesterol was calculated using the Friedewald formula. Total serum triglycerides were measured through the reaction of glycerol/ phosphate/oxidase and peroxidase. The serum levels of high-sensitivity C-reactive protein (hsCRP) are measured using a Behring Nephelometer II (Dade Behring, Tokyo, Japan). Serum levels of IL-6 and MCP-1 are measured by a commercially available enzyme-linked immunosorbent assay (Quantikine HS Immunoassaykit, R\&D System, Minneapolis, MN, USA). Serum 8-isoprostane concentrations were measured using the ELISA assay (Cayman Chemical, Ann Arbor, Michigan, USA) with a detection limit of 3 pg/mL, according to the manufacturer's instruction.

Serum eotaxin levels will be determined using a commercial ELISA kit (R\&D systems, Minneapolis, MN, USA)according to the manufacturer's protocol. In brief, mouse antihuman antibody against eotaxin is used to capture eotaxin from plasma. Captured eotaxin is detected with biotynilated goat antihuman eotaxin antibody. After washing, plates are incubated with streptavidin-HRP, developed with appropriate substrate, and OD450 is determined using an ELISA plate reader. Measurements are done in duplicate in an assay able to detect 10 pg of eotaxin/ml. The intraand inter-assay coefficients of variation values are <4% and <6%, respectively.

The Siemens Atellica IM High Sensitivity Troponin I assay (Siemens Healthineers) is a three-site sandwich immunoassay with a limit of detection of 1.6 ng/L and limit of quantification of 2.5 ng/L. The upper reference limit 99th centile was determined in 2007 samples from healthy individuals as 34 ng/L in women, and 53 ng/L in men, with a single threshold of 45 ng/L. In the reference range population, 75% of patients had values greater than the limit of detection. All measurements were undertaken at a central laboratory, who were blinded to all clinical information with results provided to the research team and linked to the trial database for analysis.

The redox state of serum albumin was determined by fractionating it into human mercaptalbumin (HMA), human nonmercaptalbumin-1 (HNA-1), and human non-mercaptalbumin-2 (HNA-2) with high-performance liquid chromatography (HPLC).