Food intake was assessed by 7-day food diaries. This method consists of the listing of foods and beverages consumed during 7 consecutive days. Energy and macronutrients were

analyzed by the Dietpro® 5i GSK3326595 molecular weight software (Sao Paulo, Brazil). Creatine supplementation protocol and blinding procedure The creatine group received creatine monohydrate (20 g/d for 5 d followed by 5 g/d throughout the trial). The placebo group received the same dosage of dextrose. The participants were advised to consume their supplements preferably along with meals selleckchem (e.g., breakfast, lunch, afternoon snack, and dinner). The supplement packages were coded so that neither the investigators nor the participants were aware of the contents until the completion of the analyses. In order to verify the purity of the creatine used, a sample was analyzed by high-performance

liquid chromatography (HPLC). This established 99.9% of purity, with no other peaks detected (creatinine, dicyandiamide, and cyclocreatine < 0.01%). 51Cr-EDTA clearance After a 24h-protein-restricted diet and a 12-h overnight fasting, the participants were admitted to the clinical research center at 7:00 a.m., where they rested in a supine position with an indwelling polyethylene catheter inserted into a cubital vein in both arms. A single dose of 3.7 MBq (100 μCi) of the 51Cr-EDTA tracer, in a volume of 1 ml was injected intravenously in the right arm. The catheter was flushed through with 10 ml of saline. Accurately timed 10-ml blood-samples selleck products were drawn

into a heparinized tube from the opposite arm Sulfite dehydrogenase at 4 and 6 h after the injection. The plasma disappearance curve was designed using the results of these time-points. To measure the radioisotope activity, the blood samples were centrifuged at 1500 g for 10 min and 3 ml of plasma was measured in a well-calibrated counter (Genesys Genii™, LabLogic Systems Inc, Brandon, Florida, USA) for the energy of chromium-51 (320 keV). Each sample, including 3 ml of standard solution taken as an aliquot from 3.7 MBq (100 μCi) 51Cr-EDTA diluted to 500 mL in saline, was counted for 5 min. The plasma clearance rate was calculated by the slope-intercept method with a single-compartment model, which assumes that the tracer spreads out immediately after injection in its volume of distribution. The Brochner–Mortensen method was used for correcting systematic errors of the slope-intercept technique according to the following equation: where Clc is the clearance corrected for the first exponential and Clnc is the non-corrected clearance. Systematic errors caused by an abnormal radioisotope distribution were corrected using the Groth method. 51Cr-EDTA clearance was also corrected for 1.73 m2 body surface area. The coefficient of variation (CV) for 51Cr-EDTA clearance was 9.7%. Blood and urinary analyses Blood samples were obtained from an antecubital vein, following a 12-h overnight fasting.