Cobas fara

Venous blood samples were collected and analysed for total cholesterol, very-low-density lipoprotein (VLDL), high-density lipoprotein (HDL) and triglycerides at the MRCG@LSHTM clinical laboratories using a centrifugal biochemical analyser (Cobas Fara, Roche, UK). The Friedewald formula was used in calculating the level of LDL.

During the individualized cooling protocol, blood was collected at thermoneutrality and during mild cold exposure. Plasma concentrations of glucose (ABX Glucose HK CP, Radiometer, Horiba ABX, Montpellier, France), free glycerol (Glycerol kit; R-Biopharm, Darmstadt, Germany), and total glycerol (ABX Triglycerides CP, Radiometer, Horiba ABX) were determined on a COBAS FARA centrifugal spectrophotometer (Roche Diagnostics, Woerden, the Netherlands). Triglyceride levels were calculated using the difference in total and free glycerol. Plasma catecholamines were determined using reagents from Recipe (Recipe Chemicals and Instruments, München, Germany) and analyzed on a HPLC and by electrochemical detection. Serum insulin was analyzed with a Human Insulin-Specific RIA Kit (Millipore) on a Gamma Counter (2470 Automatic Gamma Counter Wizard; Wallac, PerkinElmer, Waltham, MA). The plasma inflammatory marker C-reactive protein (CRP) was measured with a particle-enhanced immunoturbidimetric assay on a COBAS c311 system (Roche Diagnostics GmbH, Mannheim, Germany), and IL-6 and IL-8 were measured with a chemiluminescent immunometric assay on an IMMULITE 1000 system (Siemens, München, Germany).

The FRAP assay was conducted using Roche Diagnostic Systems, Inc. (NJ, USA) COBAS-FARA (Software version 8529) centrifugal fast analyzer. A total of 300 μl of freshly prepared FRAP reagent was heated to 37°C. Initially, a blank reading was taken at 593 nm using a spectrophotometer. Samples were added at various concentrations, along with water (H2O), resulting in a final dilution ratio of 1/34. Absorbance readings were recorded at 593 nm at regular 15-s intervals throughout the monitoring period. To determine the change in absorbance at 593 nm, the final reading was subtracted from the initial M1 reading for each sample. This alteration in absorbance at 593 nm offers insights into the antioxidant capacity of the samples, where a greater change indicates higher antioxidant activity. The FRAP assay assesses the ability of antioxidants to reduce ferric ions to ferrous ions, serving as an indicator of their antioxidant potential [43 ].