Access ultrasensitive insulin assay

Blood samples were collected in Vacutainer EDTA tubes and used to measure clinical lab tests, as well as for long-term storage at −80 °C freezers until ready for assay. At the date of the visit, the collected blood was used to measure lipid and glycemic profiles using the Siemens Dimension RXL chemistry analyzer (Diamond Diagnostics, Holliston, MA, USA). The blood samples were used to measure lipid and glycemic profiles, including fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), fasting insulin, triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). HbA1c levels were measured using the VariantTM device (BioRad, Hercules, CA, USA). Insulin resistance was calculated using the homeostatic model assessment for insulin resistance (HOMA-IR) formula: (FBG in mmol/L) × (fasting insulin in mU/L)/22.5. The WHO criteria for the diagnosis of diabetes were used: FPG ≥ 7 mmol/L or HbA1c ≥ 6.5% (48 mmol/mol) [39 ]. Insulin levels were quantified using the Access Ultrasensitive Insulin Assay (Beckman Coulter, Brea, CA, USA), with both intra- and inter-assay coefficients of variation not exceeding 6%. HOMA-β was determined using the following formula: (20 × fasting insulin in mU/L)/(FBG in mmol/L − 3.5) multiplied by 100%.

Whole blood samples were collected into designated tubes from mice at different time points following treatment. Immediately after collection serum was extracted from the samples and stored at −20 °C until processing. To determine serum fasting glucose levels (mg dl⁻¹), we used the hexokinase G-6-PDH method assay (Beckman Coulter). To determine serum insulin levels (μIU/ ml), we used the Access Ultrasensitive Insulin assay (Beckman Coulter).

The method used for the measurement of biochemical parameters in this survey has been published previously [17 (link),20 (link)]. For brevity, study participants fasted for at least 10 h prior to the collection of fasting blood samples. The Siemens Dimension RXL chemistry analyser (Diamond Diagnostics, Holliston, MA, USA) was used to measure the blood glucose and lipids profile. The participants were measured for HbA1c using a Variant device (Bio-Rad Laboratories, Hercules, GA, USA). The Access Ultrasensitive Insulin Assay (Beckman Coulter, Brea, CA, USA) was used in measuring insulin. Insulin resistance was calculated using the HOMA-IR formula: FBG (mmol/L) × fasting insulin (mU/L)/22.5 [23 (link)]. Hs-CRP concentrations were determined using ELISA (Hycult Biotech, Cat. no. HK369, Uden, The Netherlands), while ALT values in blood were analysed using the Dimension EXL Chemistry Analyser (Berlin, Germany) following manufacturer’s instructions. All blood analyses were conducted at the Dasman Diabetes Institute clinical laboratories in Kuwait.

Fasting blood samples (overnight) were taken from the anterior cubital fossa, and serum, plasma and fluoride oxalate aliquots were prepared in 1 ml tubes and frozen at − 80℃ until further analyses. Fasting insulin (serum), glucose (plasma) and HbA1c (whole blood) concentrations were measured using Beckman Coulter Access Ultrasensitive Insulin assay (paramagnetic particle, chemiluminescent immunoassay), Beckman Coulter enzymatic UV test (hexokinase method) and Sebia CAPILLARYS HbA1c (separation and quantification of the HbA1c glycated fraction of haemoglobin by capillary electrophoresis), respectively. HOMA2-IR was calculated using the HOMA2 Calculator (v.2.23, University of Oxford) available online at https://www.dtu.ox.ac.uk/homacalculator/. After an overnight fast, percutaneous vastus lateralis muscle biopsies (Weil-Blakesley conchotome) containing between 10 and 60 mg of the tissue were conducted under local anesthetic, using a standardised protocol [28 ], and muscle cell cultures were isolated and frozen down.

To determine insulin resistance, the homeostasis model assessment of insulin resistance (HOMA) was utilized. Upon arrival at the hospital laboratory following a 12-hour fast, blood was taken from the antecubital vein of each participant, centrifuged at 4°C for 15 min at 2000 g, and stored in aliquots at −20°C. Fasting insulin levels (μU/L) were determined using the Access Ultrasensitive Insulin assay (Beckman Coulter, Inc., Brea, CA). Fasting glucose levels were determined using the Dimension Vista System and the Flex reagent cartridge (Siemens, Deerfield, IL). Using these fasting insulin and glucose levels, HOMA was computed as follows: fasting insulin (μU/mL) × fasting glucose (mg/dL)/405. HOMA is frequently used as a valid measure of insulin resistance as it has been shown to be comparable to the euglycemic clamp method [21 (link)]. More than 500 studies using HOMA have been published [22 (link)].