Variant 2 turbo system

HbA1c was measured by ion-exchange high-performance liquid chromatography using BioRad VARIANT™ II Turbo system. Urine ketone was determined by Beckman Coulter iRICELL2000™ system. Serum albumin was measured by bromocresol purple method using Beckman Coulter UniCel DxC 800 Synchron™ Clinical Systems. Serum BHB was measured by Abbot Laboratories Optium Xceed™ system.

Measurements derived from the oral glucose tolerance test included glucose levels, insulin, and lipid profiles, determined using colorimetric enzymatic methods (Unicel DxC 600 Synchron Clinical System, Beckman Coulter). Insulin was measured with a chemiluminescence essay (Access 2, Beckman Coulter). As for HbA_1c, a 4-mL peripheral blood sample was drawn via venipuncture using the standardized technique and measured using a Variant II Turbo system (BIORAD); the method used was high pressure liquid chromatography.

Ambulatory blood pressure monitoring will be assessed throughout 24-h in intervals of 15 minutes in daytime and 20 minutes in nighttime periods. Daytime period starts at 7 AM and nighttime starts at 11 PM, except in case that the participant describes a different sleep time and wake-up time. Participants will receive a diary to note daily activities, symptoms, sleep time and wake-up time, and will be instructed to avoid strenuous physical exercises and alcohol ingestion 24-h prior to exam. Each exam is considered valid when at least 14 measurements during the day and seven measurements at night [26 (link)] were successfully performed. If any exam will not be considered valid, a new exam will be conduct within 48-h. Ambulatory blood pressure monitoring will be assessed using an automatic oscillometric device (ABP 2400, Mortara, Milwaukee, EUA).
Glycosylated hemoglobin will be assessed through a blood sample with high-performance liquid chromatography technique (Bio-Rad VARIANT II TURBO System, São Paulo, Brazil).

To measure the levels of glycated haemoglobin in blood, blood samples were collected at the 8th week of the experiment and injected into a glycated haemoglobin (HbA1c) analytical cartridge (Bio-Rad, Hercules, CA). The level of HbA1c was measured using the Bio-Rad Variant II Turbo system.
Levels of blood insulin were measured using a mouse insulin ELISA kit (ALPCo Diagnostics, Salem, NH) at the 8th week of the experiment with sera prepared from blood drawn from mice fasted for 12 h. The same sera samples were also used in the analysis of α-amylase concentrations using a QuantiChrom α-amylase assay kit (BioAssay Systems, Hayward, CA).

Fifty microliters of blood was collected from a small tail snip when mice were anesthetized for echocardiography and HbA1c level was determined using a Bio-Rad variant II turbo system (Bio-Rad Laboratories, Hercules, CA, United States).

Blood samples (10 mL) for plasma copeptin determination were centrifuged at 3000 rpm for 20 min at 8°C and stored at −80°C. For analysis, the samples were removed from the freezer and kept in room temperature to allow gradual thawing before measurements. Plasma copeptin levels were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Elabscience, Houston, TX, USA). This ELISA kit is based on the principle of competitive ELISA, with a sensitivity of 18.75 pg/mL and a detection range of 31.25–2000 pg/mL. The concentration of human copeptin was determined by comparing the optical density of the samples to a standard curve.
Blood samples for glucose determination were collected in tubes with sodium fluoride, centrifuged for 10 min at 3670 rpm, and analyzed by a hexokinase enzymatic method on the Roche COBAS c702 system (Roche Diagnostics, Mannheim, Germany). For HbA1c determination, samples were collected in EDTA tubes, homogenized and analyzed on the Bio-Rad Variant II Turbo system (Bio-Rad, Hercules, CA, USA), and processed by high-performance liquid chromatography. The concentration of glucose was expressed as mg/dL and of HbA1c as percentage.