ABC | Volume 111, Nº1, July 2018

Original Article Kalkan et al Adropin and Irisin Arq Bras Cardiol. 2018; 111(1):39-47 cachectic patients with heart failure with reduced ejection fraction, and 2) to investigate the relationship between adropin and irisin levels and clinical and laboratory parameters in patients with heart failure with reduced ejection fraction. Methods Patient selection and study protocol To identify cachectic patients, the clinical records of patients admitted to the cardiology outpatient clinic of a training and research hospital either for the diagnosis or treatment of heart failure with reduced ejection fraction were screened. Subsequently, the patients were contacted by phone and asked to attend the clinic. Heart failure with reduced ejection fraction patients without weight loss were enrolled as a control group. The inclusion criteria were a diagnosis of heart failure with reduced ejection fraction according to the ‘2012 ESC Guidelines for the Diagnosis and Treatment of Acute and Chronic Heart Failure’ and treatment for heart failure with reduced ejection fraction for at least 6 months before enrolment in the study. 12 The following were exclusion criteria: acute decompensated heart failure, heart failure with preserved ejection fraction, hospitalization for acute coronary syndromes, primary valvular heart disease, chronic obstructive pulmonary disease, peripheral vascular disease, musculoskeletal disease, acute/chronic inflammatory or infectious diseases, connective tissue diseases, neoplastic diseases, congenital heart diseases, hepatic failure, acute or chronic end-stage kidney failure, recent trauma or major surgery and pregnancy. Demographic, clinical and laboratory data and the medical therapies administered to each patient during their index hospitalization were recorded by a systematic review of the patient files. To determine left ventricle ejection fraction values, all individuals underwent a transthoracic echocardiographic examination (Vivid S5; General Electric, Wisconsin, USA), which was performed by an experienced operator. The left ventricle ejection fraction was determined using Simpson’s method of discs and two-dimensional echocardiography. All patients were older than 18 years and able to provide written informed consent, whichwas a prerequisite for enrolment. The study compliedwith theDeclaration of Helsinki, and the trial protocol was approved by the Local Ethical Committee. Laboratory measurements Blood samples were drawn by venipuncture into tubes containing anticoagulant ethylenediaminetetraacetic acid (EDTA). The samples were collected after a 12-hour overnight fast from the antecubital vein, with the patient in a sitting position. The serum was obtained by centrifugation at 4000 rpm at 4°C for 20 min. The obtained sera were stored at -80°C until used in the analysis. All routine biochemical and hematological parameters were measured on the same day as the blood sampling. Biochemical parameters, including fasting blood glucose, creatinine, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and triglycerides (TG), were measured using an Abbott Diagnostics C8000i (Abbott, Germany) auto-analyzer with commercial kits. The LDL cholesterol was assayed by applying Friedewald’s formula to samples with TG ≤ 400 mg/dL. Hematological parameters were obtained using a Coulter LH 780 Hematology Analyzer (Beckman Coulter Ireland, Inc., Mervue, Galway, Ireland). Serum brain-natriuretic peptide (BNP) levels (pg/ml) were measured using commercially available kits (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). Serum adropin levels were measured with a commercially available kit using an enzyme-linked immunosorbent assay (ELISA) method (Human adropin ELISA kit, catalogue n°. ck-e90267, Hangzhou Eastbiopharm Co., Blue Ocean International Times Mansion, China), with a low sensitivity limit of 2.49 ng/L. All samples were measured in duplicate in a single experiment. The intra- and inter-assay coefficients of variance of this kit were < 10% and < 12%, respectively. The detection range of adropin was 5-1000 ng/L. Serum irisin levels were detected with a commercially available kit, using the ELISA method (Human irisin ELISA kit, catalogue n°. CK-E90905, Hangzhou Eastbiopharm Co., Blue Ocean International Times Mansion, China). The sensitivity limit was 0.023 μg/mL, and the intra- and inter-assay coefficients of variance were < 10% and < 12%, respectively. The detection range of irisin was 0.05-15 μg/mL. Definitions Cardiac cachexia can be defined as underlying disease and involuntary non-edematous weight loss ≥ 6% within the previous 6-12 month. 12,13 Hypertension was diagnosed if systolic arterial pressure exceeded 140 mm Hg, diastolic arterial pressure exceeded 90 mmHg, or the patient was taking antihypertensive drugs. Hyperlipidemia was defined as fasting total serum cholesterol > 200 mg/dL, LDL cholesterol > 130 mg/dL, serum TG > 180 mg/dL or the use of lipid-lowering drugs. Diabetes mellitus was defined as a previous history of the disease, the use of insulin or oral antidiabetic drugs, or a fasting venous blood glucose level ≥ 126 mg/dL on two occasions in previously untreated patients. 14 Anthropometric measurements were used to determine body mass index (BMI), triceps skinfold thickness (TST) and arm circumference (AC). The TST was measured using a Holtain skinfold caliper. The arm muscle area (AMA) was calculated by the formula (AC-TST × π) 2/4 × π and considered an indicator of body muscle mass. 15 The heights and weights of the study participants were measured, and the BMI was calculated as body weight in kilograms divided by the square of the height in meters (kg/m 2 ). Statistical Analysis Descriptive analyses are presented using means and standard deviations or the median and the interquartile range (IQR, range from the 25th to 75th percentile). The standard effect size of the current trial was determined 0.62 with power of 80% and error of 5% according to the equation reported by Pardo et al. 16 The sample size was established at a minimum of 41 volunteers per group to detect differences in irisin between cachectic and control patients. 40