Glycerol kit

Blood was collected from the antecubital vein for analyses of several blood variables. Plasma concentrations of free fatty acids (NEFA-HR set; Wako Chemicals), free glycerol (Glycerol kit; R-Biopharm), total glycerol (ABX Triglyceriden CP; Horiba ABX), and glucose (ABX Glucose HK CP; Horiba ABX) were measured on a COBAS PENTRA centrifugal spectrophotometer (Horiba ABX). Plasma triglyceride concentrations were calculated by subtracting free glycerol from the total glycerol concentrations, and serum insulin was analyzed on a Gamma Counter (2470 Automatic Gamma Counter Wizard2 Wallac; Perkin-Elmer) with a Human Insulin specific RIA kit (Millipore). Plasma norepinephrine and adrenaline were analyzed by using reagents from Recipe Chemicals and Instruments with HPLC through electrochemical detection. Serum TSH was measured by an Electrochemiluminescence Immunoassay Kit on a COBAS 6000 system (Roche Diagnostica), and free thyroxine (FT4) was analyzed by a solid-phase time-resolved fluoroimmunoassay kit on an AutoDELFIA system (PerkinElmer). Plasma inflammatory marker C- reactive protein (CRP) was measured with a particle-enhanced immunoturbidimetric assay on a COBAS PENTRA system (Horiba ABX). For detailed description see S1 Text—Study Protocol.

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).