Effects of Chronic Dietary Exposure to Branched Chain Amino Acids on Insulin Sensitivity Measures in Healthy Vegans and Omnivores

Subjects and methods Listed elsewhere Intervention Listed elsewhere

Clinical examination Each subject underwent a basic medical check-up with anthropometric examination (height (m), weight (kg), BMI (kg/m2), waist circumference (cm), waist-hip ratio). Body composition was measured using bioimpedance analysis (BIA, Nutriguard-M, Data Input GmbH, Germany). Each of above mentioned measurements was performed three times and mean was recorded.

Dietary assessment Each participant filled in a prospective questionnaire, where dietary data from 3 days were collected (2 working days, 1 weekend day). For dietary intake calculations Nutridan program was used. As nutritional data of some special vegan products are not available in the database, vegans were asked to collect packages from these products with producer declared nutritional content, and these data were used for calculations. Saccharides, lipid and protein intake were calculated separately. BCAA content of dietary proteins was estimated using nutritional database.

Physical activity assessment Physical activity was assessed in the whole sample using Baecke questionnaire for habitual physical activity [1], that correlates well with maximum oxygen consumption (VO2max)[2]. Maximal exercise test was performed in each subject on an electromagnetically braked bicycle ergometer (Ergoline 800, Bitz, Germany) to determine peak oxygen uptake (Vo2peak). An initial workload of 50W was increased by 25 W every minute continuously until fatigue despite the verbal encouragement. Oxygen uptake was measured using Vmax, Sensor Medics (Yorba Linda, CA). Heart rate was monitored continuously.

Laboratory analysis Peripheral venous blood was drawn from each subject after 12 hours of fasting. Parameters of glucose homeostasis were assessed in certified University hospital laboratory: plasma glucose using hexokinase reaction (kit KONELAB, Germany), HbA1c using high-pressure liquid boronate afinit chromatography (Primus corporation), insulin using solid phase competitive chemiluminescent enzyme immunoassay (Immulite 2000). Lipid profile: total cholesterol and triglycerides were measured using enzymatic method (kit KONELAB, Germany), HDL-cholesterol using PEG modified enzymatic measurement (kit ROCHE, Switzerland). Plasma levels of free fatty acids (FFA) were measured using method already described [12]. Briefly, FFA were extracted together with neutral lipids using isooctan and cleaned by reverse extraction. FA obtained this way were derivatized to methylesters and subsequently analyzed using gas chromatography (GC). Serum AA levels were determined using capillary electrophoresis (CE) with contactless conductivity detection, which has been already described in details [3, 4]. CE measurements were carried out using HP3DCE system (Agilent Technologies, Waldbronn, Germany) equipped with a built-in contactless conductivity detector.