Serum Level of Lipopolysaccharide-binding Protein (LBP) and Atherosclerosis in Hemodialysis Patients

Overview

Background In hemodialysis (HD) patients, impaired gut barrier and alteration in microbiota in the gut is thought to increase the risk of bacterial translocation and chronic inflammation. Lipopolysaccharide-binding protein (LBP) is an acute-phase reactant that mediates immune responses triggered by microbial products. Our aim is to investigate the relationship between circulating levels of LBP, inflammatory markers and incident atherosclerotic vascular events in HD patients and follow-up this defined cohort for 3 years. Methods A total of 300 HD patients will be recruited. The LBP and inflammatory markers will be determined using immuoassay methods annually. A bioimpedance spectroscopy device will used for body fat composition measurement and measurements will be done annually. Arterial stiffness is evaluated by measuring PWV in the heart-femoral segment using an automatic waveform analyzer.

Study Design and Population Patient Population To be included in the study, patients have to be at least 20-years-old and on outpatient hemodialysis (HD) for at least 3 months. Patients are excluded if they had an acute infection or malignancy. A total of 360 long-term HD patients are randomly invited and agreed to participate in the study. The medical record is 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 will be abstracted. Study Parameters Predialysis blood samples are obtained on a mid-week day. Within 30 min after sampling, the remaining blood is centrifuged at 3,000 g for 10 min, immediately aliquoted and frozen at -70°C until further analysis. 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 is calculated using the Friedewald formula. Total serum triglycerides are 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). Plasma levels of Interleukin-6 (IL-6) and soluble CD14(sCD14) are measured by a commercially available enzyme linked immunosorbent assay (ELISA) (Quantikine HS Immunoassay kit, R\&D System, Minneapolis, MN). Concentrations Interleukin-6(IL-10), and Tumor necrosis factor-alpha(TNF-α) are simultaneously determined by the Multiplex® (R\&D Systems, Minnesota, MN, USA). Serum levels of LBP will be measured using a commercial enzyme-linked immunosorbent assay (HK315-02, HyCult Biotech Inc., Uden, the Netherlands) as per manufacturer's instructions. The intra- and inter-assay coefficients of LBP variation are \<5 and \<10%, respectively. Besides, serum from 40 normal control subjects will be used for interassay variation. Both the intra- and the interassay coefficients of variation were \<8.0%. All samples will be measured in duplicates and the mean value is reported in μg/mL Percent fat is measured with Bioelectric Impedance Spectroscopy Whole-body bioelectric impedance spectroscopy (BIS) measurement using a body composition monitor (BCM: Fresenius Medical Care, Bad Homburg, Germany) is performed on each of the participants enrolled in the study as described previously \[28\]. Briefly, measurements were taken on the day before dialysis with the patient calm, supine, and relaxed in the dialysis bed for 10 minutes. Four electrodes were placed on the patient's hand and foot on the side contra lateral to their arteriovenous fistula. The basic principle of this machine is the use of the bioimpedance spectroscopy which determines the fluid content of the body by measuring the serial values of electric impedance by applying small microAmp electric currents at 50 different frequencies between 5 and 1000 kHz. These impedance data are used to obtain the amount of total body water, intracellular water and extracellular water through a special fluid model and information about other body compositions, such as fat tissue or lean tissue, through a physiological body composition model \[29\]. The major estimates the body composition measured by BCM-BIS are as follows: (1) lean tissue mass (LTM -mainly muscle), fat mass (FAT), and adipose tissue mass (ATM-mainly fat with its relevant hydration) Measurement of arterial stiffness Arterial stiffness is evaluated by measuring PWV in the heart-femoral segment using an automatic waveform analyzer.