Immulite 1000 system

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).

Blood was collected at baseline and 30, 60, 120, 180, and 240 min from fasting through an indwelling catheter. Serum tubes were allowed to clot at room temp for at least 30 min, and all tubes were centrifuged at 3000× rpm at 4 °C for 10 min. Serum insulin concentration was determined using the IMMULITE 1000 System (Siemens Healthcare, Llanberis, Gwynedd, UK) with the insulin kit (cat# LKIN1). Serum ghrelin concentration was measured with an ELISA-based assay (Cat# EZGRA-88K; Waters-Millipore, Milford, MA, USA). Serum concentrations of glucose and triacylglycerol were measured by the COBAS INTEGRA 400 PLUS (Roche Diagnostics, Indianapolis, IN, USA) with the Glu HK Gen.3 kit (cat# 04404483190) for glucose and TRIGL kit (cat# 20767107322) for triacylglycerol.

Blood samples, drawn from all patients at admission, and at 24 ± 6, 48 ± 12, and 72 ± 12 h, were collected in glass chemistry test tubes, centrifuged at 3000 rpm during 10 min, and immediately frozen and stored at − 80 °C. Serum levels of IL-10 were measured using an immunodiagnostic IMMULITE 1000 System (Siemens Healthcare España, Madrid, Spain). Determinations were performed in an independent laboratory blinded to clinical and neuroimaging data.

Blood samples were obtained from each patient after an overnight fast upon admission. The blood samples were allowed to clot at room temperature for 30 mins and centrifuged for 10 minutes at 3000 rpm. The serum samples were separated as soon as possible from the clot of red cells after centrifugation to avoid TNF-α production by blood cells that falsely could increase its values. Separated sera were measured immediately. Levels of serum TNF-α were measured in the clinical laboratory at Peking Union Medical College Hospital using Tumor necrosis factor-alpha Assay Kit (chemiluminescent assay) through IMMULITE® 1000 system (Siemens Healthcare Diagnostics Inc., United Kingdom) according to the manufacturers’ instructions. Detection limit of the kits was obtained from the manufacturers, as follows: TNF-α (detection limit 1.7–1000 pg/mL). The recommended reference range was as follows: TNF-α (≤ 8.1 pg/mL).

PF and C-reactive protein (CRP) were measured from plasma samples collected on V0, V1 and V4 of the human study and were frozen until analysis which was conducted with an IMMULITE 1000 system (Siemens Healthcare) following the manufacturer’s instructions. Hb was measured in whole blood collected on V0, V4 and V7 on the day of collection by using either a Sysmex XE 5000 (Sysmex Corporation) or an Advia 2120 (Siemens Healthcare) hematology analyser.
Each blood sample was analysed in duplicate for its isotopic composition. Whole blood was mineralized by microwave digestion, and iron was separated by anion exchange chromatography and a subsequent precipitation step with ammonium hydroxide^{42 (link)}. Iron isotope composition was determined by a Multicollector-Inductively Coupled Plasma Mass Spectrometer (MC-ICP-MS) instrument (Neptune; Thermo Finnigan).
The PA concentration of the corn flour was analysed as earlier described^{43 (link)} and was used for calculation of the molar ratio of PA to iron.