Vacutainer plus

Vein blood samples were collected by BD Vacutainer Plus and centrifuged at 3000 rpm for 15 min within 4 h. The supernatant plasmas were immediately recovered and stored at −80 ^oC. Plasma miRNAs were extracted using Total RNA Isolation Kit (QuantoBio). Synthetic Caenorhabditis elegans miRNA (cel-miR-67) was used as a exogenous control and added to the plasma lysate before extraction. Quantities of miRNAs were determined by SYBR-based qRT-PCR according to manufactures’ instructions (Quantobio). Briefly, Escherichia coli polyA polymerase was used to add a polyA tail at the 3′ end of RNA molecules. With oligo(dT) annealing, a universal tag was attached to the 3′ end of cDNAs during the cDNA synthesis using reverse transcriptase (Quantobio). Quantitative PCR was performed with miRNA-specific forward primers and a universal reverse primer mix according to the universal tag.

Arterial blood samples were collected at rest before each CPET and every 30th second during exercise. Test tubes contained an antiglycolytic agent to prevent continued glycolysis in the blood samples (2.0 mL sodium fluoride, potassium oxalate BD Vacutainer^® Plus, Franklin Lakes, NJ). The samples were immediately placed in iced water and centrifuged at 1500g for 15 min at 21°C within 30 min (Sigma 2‐7, Osterode am Harz, Germany). Plasma was separated and put on dry ice before storing at −80°C until analyzed. Arterial Lactate concentration ([La_a]) was determined by spectrophotometer (MaxMat PL, Montpellier, France). We added 4‐chlorophenol, Lactate oxidase, peroxidase and 4‐aminophenazone (Lactate, Spinreact, Girona, Spain) to plasma and the tubes were mixed at 37°C for 10 min. The absorbance of samples and standards was read at 505 nm against the blank, with a detection limit of 0.044 mmol/L and a linearity limit of 16.85 mmol/L. Control sera (SPINTROL H Normal and Pathologic, Spinreact, Girona, Spain) were used to monitor the performance of assay procedures, as recommended by the manufacturer. Arterial blood samples for analyses of hemoglobin concentration were collected at rest before each CPET and within 90 sec after peak exercise had been achieved, and analyzed within 1 minute after sampling (ABL835 version 6.16 Flex; Radiometer, Copenhagen, Denmark).

Blood samples were collected from healthy donors under approved IRB #IRCM-2019-203 (Protocol #CB-IR-002) using BD Vacutainer Plus plastic serum tube (BD Biosciences, San Jose, CA, USA, Cat# 367820) (10 mL/tube, 2 tubes for each donor). Blood samples were incubated for 30–45 min at room temperature (RT) in an upright position, then were centrifuged at 2000× g for 15 min at RT. Supernatant (serum) was collected, aliquoted, and stored at −80 °C. Each sample was assigned an identifying number (ex.: CBD01, CBD02…). The Human AB Serum was obtained from Valley Biomedical (Winchester, VA, USA, Cat# H1022).

Plasma samples were collected at 56 d of feeding trial and at slaughter (80 d) by jugular venipuncture using vacutainer tubes containing sodium heparin as anticoagulant (BD Vacutainer Plus, BD, Brazil). All blood samples were centrifuged at 3,500×g for 15 min at 4 °C, and plasma was collected and stored at −20 °C until assayed for leptin. The concentration was determined in duplicate 100 μL aliquots of plasma samples, using the leptin RIA kit (Multi-species leptin RIA kit, Cat. # XL-85 K, Millipore, St. Charles, MO, USA) and following the manufacturer’s instructions. Intra and interassay CVs for the leptin assay were less than 10% as described by Delavaud et al. [20 (link)].

Venus blood was collected (BD Vacutainer Plus) and centrifuged at 3000 rpm for 15 min within 4 h. The supernatant serum was immediately collected and stored at −80 °C. Serum miRNA was extracted using Total RNA Isolation Kit (QuantoBio). Synthetic Caenorhabditis elegans miRNA (cel-miR-67) was used as a exogenous control and added to the plasma lysate before extraction. Quantities of miRNAs were determined by SYBR-based qRT-PCR according to manufactures’ instructions (Quantobio). Briefly, Escherichia coli polyA polymerase was used to add a polyA tail at the 3′ end of RNA molecules. With oligo(dT) annealing, a universal tag was attached to the 3′ end of cDNAs during the cDNA synthesis using reverse transcriptase (Quantobio). Quantitative PCR was performed with miRNA-specific forward primers and a universal reverse primer mix according to the universal tag.

All blood samples were collected before initial treatment. To avoid the effects of diet on the level of serum CCL18, all participants were told to fast more than 8 hours, and their blood samples were collected in the morning between 7 a.m. and 10 a.m. by venipuncture (BD Vacutainer Plus). Blood samples were allowed to clot at room temperature followed by centrifuging at 3000 rpm for 15 min, and then tested within 2 hours. Samples with hemolysis were excluded from the study. Serum CCL18 was determined by an ELISA commercial kit (R&D Systems, MN, USA) according to the manufacturer's instructions. Each experiment was performed in triplicate.