2.1. Human Study

This study was conducted by the Declaration of Helsinki (1964) and was approved by the Ethics Committee of the Department of Internal Medicine at State University of Campinas (UNICAMP), Campinas, SP, Brazil. All the subjects gave written informed consent.

All volunteers were employees with a majority of nurses at the Sumare State Hospital in Sumare city, São Paulo state, Brazil. A total of 150 volunteers were randomly recruited via advertisements placed around the hospital. In this double-blind study, 39 volunteers completed the intervention period. The inclusion criteria to participate were as follows: men or women adults aged between 20 and 60 years old and diagnosed as overweight or obese. Subjects who reported renal or thyroid disease, pregnancy, taking an antidepressant, and anorectic or laxative drugs were excluded from the study. Before beginning the study, body weight and height were measured using a Filizola scale with a calibrated digital scale and stadiometer, respectively (PL 200 model). The Body Mass Index (BMI) (kg/m2) was calculated as weight (kg) divided by height (m2) squared. Only overweight (BMI ≥ 25 kg/m2) and obese (BMI ≥ 30 kg/m2) volunteers were included in the study (WHO, 2000).

On the first day, volunteers were random divided into four groups according to the BMI and the supplement that they would receive: Overweight Alanine (Ala), Obese Ala, Overweight Glutamine, and Obese Gln. Afterward, blood samples were collected and waist circumference was measured. The volunteers received a kit containing small packs with 15 g of amino acid (Gln or Ala) each. They were instructed to take two packs per day, taking a total of 30 g of amino acids per day. The supplementation lasted for 14 days. The volunteers were instructed to mix the pack content in a cup of water (200 mL) before drinking and maintain the same levels of physical activity and the same diet during the 14 days of supplementation. The second measurement was done 15 days after the supplementation started. Fasting volunteers came to the hospital for the second time for BW and WC measurements and blood sample collections.

®, St. Charles, Missouri, United States. Human TNF-α (DTA00C), human IL-1β (DLB50), and human IL-6 (D6050) kits were from R&D Systems Inc., Minneapolis, MN, USA. To determine serum lipopolysaccharides (LPS) levels, the Limulus Amebocyte Assay from Cambrex (LAL kit endpoint-QCL-1000, Lonza, Walkersville, MD, United States) was used. Analyses were performed by following the specific instructions for each manufactory. HPLC system/SCL-10avp (Shimadzu Scientific Instruments, Columbia, MD, USA) and the CLASS-VP 6.12 software Class VP were used to measure serum glutamine and alanine [ Biochemical Analysis was conducted by blood samples, which were obtained before and after the supplementation from the same volunteer. Overnight fasted volunteers had blood samples collected into tubes placed on ice. After collection, blood samples were immediately centrifuged at 1500 rpm for 15 min at 18 °C using a Centrifuge Biofuge Stratos (Hereaus, Dijkstra Vereenigde, Lelystad, Netherlands). The serum obtained was separated and transferred into 2 mL Eppendorf and stored at −80 °C until analysis. Glucose concentration was determined using the Glucose Liquiform Test (Labtest, Brazil) that applied the glucose oxidase method. All the other assays were quantified by the specific commercial enzyme-linked immunosorbent assay (ELISA). The human insulin (EZHI-14K) kit was from Millipore, St. Charles, Missouri, United States. Human TNF-α (DTA00C), human IL-1β (DLB50), and human IL-6 (D6050) kits were from R&D Systems Inc., Minneapolis, MN, USA. To determine serum lipopolysaccharides (LPS) levels, the Limulus Amebocyte Assay from Cambrex (LAL kit endpoint-QCL-1000, Lonza, Walkersville, MD, United States) was used. Analyses were performed by following the specific instructions for each manufactory. HPLC system/SCL-10avp (Shimadzu Scientific Instruments, Columbia, MD, USA) and the CLASS-VP 6.12 software Class VP were used to measure serum glutamine and alanine [ 30 ] levels in order to assess the adherence to glutamine or alanine supplementation. The serum was collected before and after supplementation.

To assess the caloric intake of the individuals, we applied a 24-h food record before and after the supplementation. For analysis of the 24-h food record, we used the software Diet Pro 4.0 (Viçosa, Brazil). To assess physical activity, subjects were informed of their physical activity before and after supplementation during an interview.