Rag018r

The samples were collected twice: during the 1^st week of life, and then 4 weeks later (5^th week of life). We used test tubes with aprotinin, a protease inhibitor (Becton Dickinson Vacutainer) to collect blood and milk samples. Blood samples were transferred immediately after drawing to the Department of Clinical Biochemistry, Institute of Pediatrics, Jagiellonian University Medical College, Krakow, Poland and centrifuged. The supernatant plasma was separated and immediately stored in a deep freezer (-80°C) until assay. Milk samples were transferred to the above laboratory and immediately stored in a deep freezer (-80°C) until assay.
Levels of irisin were measured by an ELISA commercial kit (RAG018R, BioVendor, Brno, Czech Republic). The minimum detectable concentration, intraassay and interassay coefficients of variation were 0.001μg/ml, 5–8%, 8–10%, respectively.
Levels of visfatin were measured by an ELISA commercial kit (RAG004R, BioVendor, Brno, Czech Republic). The minimum detectable concentration, intraassay and interassay coefficients of variation were 0.003μg/ml, 2.5–9%, 4.66–7.24%, respectively.

Blood samples were obtained from patients with TBI. The anticoagulated blood was centrifuged (1500 rpm, 15 min) to obtain the serum for ELISA analysis. Commercial ELISA Kits (ml037372, mlbio, RAG018R, BioVendor R&D, and NBP3-08117, Novus) were used to test and validate serum irisin levels, and the irisin-specific Luminex bead-based multiplex detection system (Merck Millipore) was further employed to detect human serum irisin. Detailed irisin assays were performed according to the kit instructions.

The plasma concentration of TNFα, interleukin (IL)-6, IL-10, leptin, adiponectin, angiotensinogen, and irisin was measured using an enzyme-linked immunosorbent assay kit (eBioscience BMS223HS, BMS213HS, BMS215HS, Thermo Fisher Scientific, Waltham, MA, USA; Biovendor RD191001100, Brno, Czech Republic; Assaypro EA3501-1, St Charles, MO, USA; IBL International 27412, Hamburg, Germany; Biovendor RAG018R, Brno, Czech Republic). To determine malondialdehyde (MDA) levels, plasma samples were measured using the thiobarbituric acid-reactive substance (TBARS) method [17 (link)]. Briefly, we mixed samples with 20% acetic acid (pH 3.5), 0.8% thiobarbituric acid, and 8.1% sodium dodecyl sulfate and incubated the mixture at 95 °C for 1 h. After the mixture had cooled down to room temperature, trichloroacetic acid was added and detected spectrophotometrically at 532 nm.