Cobas c 501 autoanalyzer

The plasma levels of hApoA-I were determined using immunoturbidimetric commercial kits adapted to a COBAS c501 autoanalyzer (Roche Diagnostics). Mouse apoA-I levels were determined with an ELISA kit, and the wells were coated with a polyclonal rabbit antibody against mouse apoA-I, as previously reported [62 (link)]. Serum levels of arylesterase activity were determined using phenyl acetate as the substrate [60 (link)]. Ethylenediaminetetraacetic acid (EDTA)-sensitive serum arylesterase activity was calculated by subtracting the EDTA-resistant arylesterase activity in mixtures containing 1 mmol/L EDTA in the reaction buffer instead of calcium chloride (CaCl₂) and reported as paraoxonase (PON)1 activity. Plasma levels of Lp-PLA2 activity were determined using 2-thio-PAF (Cayman Chemical, Ann Arbor, MI, USA) as the substrate, according to the manufacturer’s instructions.

Height and weight were measured with the subject wearing light clothes and without shoes. Body mass index (BMI) was calculated and BMI categories were defined according to the World Health Organization (WHO) adult definition [24 ]. Complete blood count was carried out in a LH750 autoanalyzer (Beckman Coulter, Fullerton, CA, U.S.A.). Plasma Tf, apolipoprotein (apo) A-I and apo B concentrations were measured by nephelometry (IMMAGE®, Beckman Coulter, Fullerton, CA, U.S.A.). Ferritin concentration was assayed by an electrochemiluminescence assay (VITROS® ECiQ, Ortho-Clinical Diagnostics, Raritan City, NJ, U.S.A.). Serum levels of iron, glucose, triglycerides and total cholesterol as well as the activities of hepatic enzymes were quantified by standardized methods (Roche Diagnostics, Mannheim, Germany) in a COBAS C501 autoanalyzer (Roche Diagnostics, Mannheim, Germany). LDL-C and HDL-C concentrations were determined by selective precipitation methods.

Lean, male C57BL/6JRj mice (8–9 weeks, JanVier, Le Genest-Saint-Isle, France) were semi-fasted (∼50% of prior food intake) over-night and dosed orally with ulotaront (0.3, 1, 3 or 10 mg/kg), RO5166017 (0.1, 0.3, 1 mg/kg), RO5263397 (0.01, 0.1, 1 mg/kg) or the corresponding vehicle 30 min prior to acetaminophen administration (160 mg/kg, po suspended in 1.5% (w/v) HPMC/1.5% (w/v) HPCD in water). Semaglutide (10 nmol/kg, s.c. dissolved in 1× PBS with 0.1% BSA) was included as a positive control and administered 30 min prior to acetaminophen. Blood samples were collected at baseline (prior to test article dosing) and at 10, 30, 60 and 120 min post-acetaminophen administration. Serum was separated by centrifugation and acetaminophen concentrations determined using a commercially available kit (TDM Acetaminophen Gen.2, Roche Diagnostics) followed by quantification in the Cobas c 501 autoanalyzer. The ACET2 calibrator (Roche Diagnostics) was used to generate the standard curve.

All blood samples were collected in the morning after an overnight (8–10 h) fast and preferably on Days 2–5 of the spontaneous menstrual cycle in regularly menstruating women or during withdrawal bleeding in amenorrheic women. Blood samples were aliquoted for plasma insulin, TSH, total T4, LH, FSH, total T, plasma glucose, blood counts, and liver and kidney functions. Serum were separated at room temperature and aliquoted as per the requirements. The aliquots were stored at −70°C until the assay. All hormonal assays were carried out by chemiluminescence under Cobas e601 (Roche Diagnostics, Germany) using commercial kits. Plasma glucose and other biochemical parameters were assayed on Cobas c501 autoanalyzer (Roche Diagnostics, Germany). For all measurements, the inter- and intra-assay coefficient of variations were within the limits permitted by the manufacturers. To investigate insulin sensitivity, the quantitative insulin sensitivity check index (QUICKI) was calculated. QUICKI=1/[log(I₀)+log(G₀)], where I₀ is the fasting insulin, and G₀ is the fasting glucose. QUICKI is a validated surrogate marker for insulin resistance and has good agreement with gold standard hyperinsulinaemic euglycaemic clamp (11 (link)).

After 12 hours of fasting, peripheral blood samples from studied participants were obtained in the morning. Fasting plasma glucose (FPG), hemoglobin A1C (HbA1C), TC, TG, LDL-C, and HDL-C were assayed on Cobas C501 autoanalyzer (Roche Diagnostics, Germany).