Glucose hk gen 3

Glucagon and alanine levels were determined using frozen (−80°C) plasma samples collected in proteinase-stabilised tubes (BD P800; BD Biosciences, San Jose, CA, USA) after participants had fasted overnight. Plasma glucagon was measured by sandwich ELISA (catalogue no. 10-1271-01; Mercodia, Uppsala, Sweden). Alanine, as well as 20 other amino acids (arginine, asparagine, aspartate, citrulline, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), were quantified by using targeted metabolomics (AbsoluteIDQ™ p180 Kit; Biocrates Life Sciences, Innsbruck, Austria) with LC-MS/MS. The measurement of plasma samples using this assay has been described in full detail previously [13 (link)]. Total amino acids represent the sum of all 21 l-amino acids, measured by the AbsoluteIDQ™ p180 Kit.
From the OGTT, plasma glucose was measured by a hexokinase method (Glucose HK Gen.3; Roche Diagnostics, Mannheim, Germany) and serum insulin by a chemiluminescent immunoassay (DiaSorin LIASON Systems, Saluggia, Italy). Fasting values of HDL-cholesterol and triacylglycerols were measured by enzymatic caloric test (Roche Diagnostics, Mannheim, Germany).

Glucose concentration in BAL samples was measured using the COBAS Integra 400 (Roche Diagnostics GmbH, Mannheim), with the Glucose HK Gen.3 assay (Roche Diagnosis)42 (link). Blood glucose concentration was measured by a glucometer (Precision QID, MediSense, Sao Paulo, SP, Brazil).

Blood samples were collected 2.5 h before transportation, IAT, and at 6, 24, and 48 h after transportation. The blood samples were collected by jugular venipuncture using both non-heparinized vacutainers (20 mL; BD Biosciences, San Jose, CA, USA) and ethylenediaminetetraacetic acid-treated vacutainers (20 mL). Blood samples were immediately placed in an icebox. The serum and plasma were separated by centrifugation at 1,500×g for 15-min at 4°C. The plasma and serum were subsequently stored at −80°C until analysis.
Serum glucose was analyzed with a fully automated Cobas 8000 C702 analyzer (Roche Diagnostics, Mannheim, Germany) using colorimetric methods with specific kits. A Roche GLUC2 kit was used for the analysis of serum glucose (Glucose HK Gen.3; Roche Diagnostics, Germany). Plasma cortisol was analyzed using a salivary cortisol enzyme immunoassay kit (Salimetrics, State College, PA, USA). The coefficient variances of the intra-assay and inter-assay of the cortisol kit for bovine plasma samples were 4.2% and 4.8%, respectively. The analytical method for cortisol assay was validated in previous report from our laboratory [1 (link)].

Blood samples were processed immediately after collection and analyzed directly or stored in aliquots at −80 °C. Standard methods were used to measure glucose (glucose oxidase method, Glucose HK Gen.3, Roche Diagnostics, Mannheim, Germany), hba1c (VARIANT™ II TURBO HbA1c Kit - 2.0, Bio-Rad Laboratories, Hercules, USA), gamma-GT (enzymatic caloric test, Roche Diagnostics), triglycerides and HDL (enzymatic caloric test, Roche Diagnostics). LDL was calculated by the Friedewald equation (all triglyceride levels were below 400 mg/dl). Stored serum samples were used to measure insulin by chemiluminescence technology (CLIA, DiaSorin LIAISON systems, Saluggia, Italy). High-sensitive CRP (hsCRP) and leptin were measured from stored plasma samples by ELISAs (hsCRP by wide-range CRP, Siemens AG, Erlangen, Germany and leptin by ELISA “Dual Range”, Merck Millipore, Darmstadt, Germany).

Blood biochemical analyses were performed after an 8-hour fast. FPG, insulin, lipid profile, hs-CRP, TNF-α, IL-6 and leptin levels were measured in all of the obese patients studied. The HOMA_IR was used to calculate the insulin resistance index^{35 (link)}. A 3-hour 75 g oral glucose tolerance test (OGTT) was performed for blood glucose and insulin plasma levels at baseline and 30, 90, 120, 150, 180 minutes after glucose loading (180 mL of syrup with 82.5 g glucose monohydrate equal to 75 g of glucose). Glucose and insulin AUC was calculated. OGTT was not performed in obese patients with a history of diabetes, and fasting insulin was not measured in patients receiving insulin treatment, meaning that HOMA_IR was not calculated in these patient categories. In patients with T2DM we also measured HbA1c (by high performance liquid chromatography) and microalbuminuria by the albumin/creatinine ratio (urinary albumin/creatinine ratio <30 mg/g creatinine was considered normal). Biochemical measurements were performed using diagnostic kits standardized according to the World Health Organization First International Reference Standard: glucose (Glucose HK Gen.3, Roche Diagnostic, USA), insulin, IL-6, TNF-α (IMMULITE 2000 Immunoassay, Siemens Healthcare GmbH, Germany), hs-CRP (CardioPhase High Sensitivity C-Reactive Protein, Siemens Healthcare) and leptin (Leptin-RIA-CT, Mediagnost, Germany).