S were provided with study instructions and were randomly assigned in
S were provided with study instructions and were randomly assigned in double-blind manner to one of following conditions: Acetyl L-carnitine arginate (ALCA; ArginoCarn? Sigma-tau HealthScience S.p.A, Rome, Italy; n = 20) or Placebo (cellulose; n = 20). The dosage of ALCA was 3 g ay-1 and was taken in two divided doses with meals (morning and evening ?3 capsules at each time). The composition of 3 grams of ALCA provided approximately 1350 mg of acetyl L-carnitine and 1200 mg of arginine. Arginine is a precursor to nitric oxide biosynthesis [34] and has been associated with positive effects related to vasodilatation when used in high dosages (e.g., 15 grams) via either intravenous infusion [39,40] or oral intake [41]. Capsules were identical in appearance and provided to subjects in unlabeled bottles every two weeks, for the duration of the eight week intervention. Capsule counts upon bottle return determined compliance to treatment. Meal Testing (pre-intervention and post-intervention) All subjects consumed two identical tests meals; one before and one following the eight week intervention. Subjects reported to the lab in the morning following a 10-hour overnight fast and a pre-meal blood sample was collected. Subjects then consumed a high-fat, high carbohydrate milkshake, made with whole milk, ice cream, and whipping cream. The caloric load of the test meal was based on subjects’ body mass and was equivalent to 1.2 grams of both fat and carbohydrate, and 0.25 grams of protein per kilogram. The test meal provided approximately 17 calories per kilogram of body weight. Blood samples were collected from subjects at 1, 2, 4, and 6 hours following meal intake. Subjects remained in the lab during the observation period and remained inactive. No additional meals or calorie containing beverages were allowed, although the intake of plain water was allowed ad libitum. These exact procedures, including the test meal, have been used in six recent investigations in our lab without incident [42-47] Blood Sampling Venous blood samples ( 10 mL per draw) were taken from subjects’ forearm via needle and collection tube. These blood samples were collected pre-meal (0 hour), and at 1, 2, 4, and 6 hour postprandial. The plasma/serum was immediately processed and stored PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27872238 at -80 until analyzed. Blood samples collected at all time points were assayed for malondialdehyde, xanthine oxidase activity, hydrogen peroxide, triglycerides, glucose, trolox equivalent antioxidant capacity (TEAC), and nitrate/nitrite (asurrogate marker of nitric oxide). Blood samples collected at the pre-meal time point only were assayed for insulin, HbA1c, total and HDL cholesterol, and C-reactive protein. Assays were performed on the first thaw. The procedures GW9662 supplement outlined above for meal testing and blood collection were used both pre- and post-intervention. Malondialdehyde was analyzed in plasma using a commercially available assay (Northwest Life Science Specialties, Vancouver, WA) using the method described by Jentzsch et al. [48] The intra-assay coefficient of variation (CV) was 4.7 . Xanthine oxidase activity and hydrogen peroxide were measured in plasma using the Amplex Red reagent method as described by the manufacturer (Molecular Probes, Invitrogen Detection Technologies, Eugene, OR; CV = 4.1 and 4.7 , respectively). Assays for glucose, triglycerides, and cholesterol were performed following standard enzymatic procedures as described by the reagent manufacturer (Thermo Electron Clini.