Serum Level of Myokines and Protein Energy Wasting, Inflammation and Atherosclerosis in Hemodialysis Patients
Serum Level of Myokines and Protein Energy Wasting, Inflammation and Atherosclerosis in Hemodialysis Patients
The aim is to test if serum level of myokines are associated with protein energy wasting,
 inflammation and atherosclerotic vascular disease among Taiwanese dialysis patients. Some
 novel myokines levels, anthropometric parameters, cardiovascular risk factors, and presence
 of endothelial dysfunction will be examined in 250 subjects. The distribution of some
 recently identified myokines such as irisin, myotatin etc levels will be studied and
 correlated markers of malnutrition, endothelial dysfunction, inflammation and insulin
 resistance. In the present study, the investigators will further follow and investigate
 whether serum myokines levels are associated with long-term mortality from all causes and
 from cardiovascular disease.
Study Design and Population

 In this study, patients who underwent stable hemodialysis at least 3 months at Tung Taichung
 Metroharbor Hospital will be enrolled. Baseline clinical data such as age, sex, height,
 bodyweight , clinical etiology of Chronic kidney disease(CKD) if possible, comorbidities,
 blood pressure, laboratory measures [such as serum creatinine, albumin, C-reactive protein
 (CRP), total cholesterol, complete blood counts, urine routines, anthropometric information
 and various potential indicators of protein energy wasting [such as body mass index,
 waist/hip ratio, mid-arm circumference (MAC), triceps skinfold thickness (TSF), mid-arm
 muscle circumference (MAMC), mid-arm muscle area (MAMA), SGA, BIS analyses, dual energy x-ray
 absorptiometry(DEXA),handgrip (HG)] will be collected. For the laboratory tests, fasting
 blood and urine samples are obtained from each patient and performed by means of routine
 methods in the investigators hospital. All patients received echocardiograms studies
 performed within 3 months after enrollment, which were used to estimate congestive heart
 failure(CHF, defined as an ejection fraction of <50%) or left ventricular hypertrophy (LVH,
 defined as left ventricle mass index >125 g/m2 in men and 100 g/m2 in women).Cardiovascular
 disease( CVD) was defined as a previous history of CHF, LVH, ischemic heart disease(including
 prior history of angina, myocardial infarction, coronary artery bypass grafting and
 percutaneous cardiac catheter intervention) or cerebrovascular disease (including prior
 history of transient ischemic attack and stroke). Information about conventional clinical
 risk factors, such as smoking, diabetes mellitus, hypertension, medication and other
 necessary data at baseline will be obtained from a standardized interview and examination.
 The diagnosis of type 2 diabetes is made according to the criteria of the American Diabetes
 Association.

 Patients with malignant disease, acute infectious disease, inflammatory disease (such as
 collagen disease), advanced liver disease, and those taking any type of immunosuppressive
 medication were also excluded. Patients with clinical manifestations of atherosclerosis,
 congestive heart failure, history of stroke, a previous history of coronary intervention or
 coronary artery bypass graft surgery, and presence peripheral vascular disease were recorded.

 Anthropometrics, and Handgrip Strength

 Body weight was measured with light clothing on up to 0.1 kg. Height was measured up to 0.1
 cm. Waist circumference was measured at the midpoint between upper iliac and lower costal
 level up to 0.1 cm at the end of normal expiration. Hip circumference was measured around the
 largest part of the hip. Anthropometric measurements were obtained immediately after the HD
 session by a trained researcher. Body mass index (BMI) was calculated as body mass divided by
 stature squared. The International Society of Renal Nutrition and Metabolism(ISRNM) panel
 recommends that a BMI lower than 23 kg/m2 is a marker of Protein Energy Wasting(PEW).
 Anthropometric measurements include TSF used skinfold calipers; MAC measured by a stretchable
 measuring tape, MAMC equals MAC(centimeter) - 3.14 x TSF (millimeter)/10 and MAMA equals
 (MAMC2/4π). Muscle mass area (MMA) was calculated using the following equation: MMA = ([MAC
 {cm} - π x triceps skinfold {cm}]2/4π) -n, where n = 10 for men and 6.5 for women . Muscle
 strength as hand grip strength (HGS) test was measured by Hand Dynamometer. HGS was measured
 3 times for both left and right hands with patients in a standing position using a
 dynamometer in units of kilograms. Patients held the dynamometer at thigh level and were
 encouraged to squeeze the instrument as hard as possible for 3 s. The maximum grip strength
 among all measurements was used for this study. Three sets of measurements at each site were
 averaged and used in the analyses.

 Malnutrition Inflammation Score

 To assess the malnutrition-inflammation complex syndrome(MICS), also known as the Kalantar
 score, developed, and validated by Kalantar- Zadeh et al. was measured at baseline. The
 Malnutrition-Inflammation Score(MIS) has ten components [change in end dialysis dry weight,
 dietary intake, gastrointestinal symptoms, functional capacity, comorbidities, decreased fat
 stores or loss of subcutaneous fat (according to SGA), signs of muscle wasting (according to
 SGA), BMI, serum albumin, and total iron binding capacity], each with four levels of
 severity, from 0 (normal) to 3 (severely abnormal). The sum of the MIS components ranges from
 0 (normal) to 30 (severely malnourished), in which a higher score reflects greater
 malnutrition and inflammation severity.

 The comorbidity component was scored as 0 if no comorbidities were present; 1 if there were
 mild comorbidities present and major comorbid conditions (MCCs) were absent (such as New York
 Heart Association Class III or IV congestive heart failure, severe coronary artery disease,
 acquired immunodeficiency syndrome, moderate- to-severe chronic obstructive pulmonary
 disease, and metastatic malignancies); 2 if moderate comorbidities were present, including
 one MCC; and 3 if multiple severe comorbidities were present, including two or more MCCs.
 Subjective global assessments (SGAs) were performed by an experienced physician according to
 conventional SGA guidelines.

 Dual-energy X-ray absorptiometry

 Whole-body scans are performed according to manufacturer's instructions, and body fat, lean
 tissue mass (LTM) and bone mineral content (BMC) are analysed using the manufacturer's
 software. DEXA estimate of fat-free mass (FFM) was calculated as a sum of LTM and BMC
 estimates. All patients were examined by the same observer.

 Bioelectric impedance spectroscopy (BIS)

 BIS measurement was performed in each of the participants enrolled in the study by a specific
 member of staff who had completed a training course in the BIS technique, using a portable
 whole body bioimpedance spectroscopy device, the body composition monitor. The body
 composition monitor(BCM) measures the impedance spectroscopy at 50 different frequencies
 between 5 kilohertz(kHz) and 1 megahertz (MHz). Measurements were taken on the midweek
 dialysis session before the start of the hemodialysis treatment with the patient calm,
 supine, and relaxed in the dialysis bed for 10 minutes. Specific exclusion criteria were
 dictated by the device and included history of a pacemaker, defibrillator, metallic sutures,
 or stent implantation and amputation of a major limb.

 Biochemical Analysis

 Blood samples were taken in a nonfasting state before a midweek hemodialysis session.
 Complete blood count, creatinine, urea, albumin, transferrin, total carbon dioxide, hsCRP ,
 triglycerides (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C),
 low-density lipoprotein-cholesterol (LDL-C), and fasting blood glucose are measured by
 routine laboratory methods. Another blood samples will be stored at -80 degree centigrade for
 subsequent assay of items such as insulin, interleukin-6, pentraxin, nitrotyrosine for
 markers of endothelial dysfunction such as Asymmetric dimethylarginine( ADMA), adropin and
 insulin resistance (adiponectin, resistin and leptin). Homeostatic Model Assessment Insulin
 Resistance(HOMA-IR) was calculated as fasting insulin (U/l) × fasting glucose (mg/dl)/405, as
 described by Matthews et al.

 Serum total p-cresol and indoxyl sulfate (i.e. combined free and protein-bound fractions)
 were analyzed with High-performance liquid chromatography (HPLC). Briefly, serum samples were
 deproteinized for the determination of indoxyl sulfate by the addition of three parts
 methanol to one part serum. Total p-cresol (i.e. the combined free and protein-bound
 fraction) was analysed after deproteinization (acid and heat) and extraction (ethyl acetate)
 of serum samples.

 Measurement of serum myostatin, irisin and myonectin

 Serum myostatin levels are measured with a competitive immunoassay kits according to the
 manufacturer's protocol. Serum irisin concentration was measured using the enzymelinked
 immunosorbent assay). The assay was proven to be highly sensitive to human irisin. Serum
 myonectin (Circulating Complement-C1q Tumor necrosis factor(TNF)-Related Protein 15) Serum
 myonectin was determined with a commercially available enzyme-linked immunosorbent assay kit.
Inclusion Criteria:

 -  Both sexes aged between 20-70 years.

 -  Received stable hemodialysis at least 3 months.

 -  Written informed consent.

 Exclusion Criteria:

 -  Patients with malignant disease, acute infectious disease, inflammatory disease (such
 as collagen disease), advanced liver disease, and those taking any type of
 immunosuppressive medication are excluded.