Cobas c system

First morning urine samples were collected at a symptoms-free stage, at least after 6 months of an acute attack. Samples were available only in 45 patients. Urinary creatinine was analysed with a commercial kit (CREJ2) based on the Jaffé method in a Roche Cobas c system (Roche, Mannheim, Germany). ALA and PBG were measured by spectrophotometry after column chromatography with a commercial kit (code 11017, BioSystems S.A., Barcelona, Spain) following manufacturer’s instructions. The results were normalized to urinary creatinine.

Hemopexin was measured in a sandwich ELISA. A microtiter plate (Greiner Bio-One™) was coated with 500 ng/ml anti-human hemopexin capture antibody (Bioporto) and incubated at 4°C overnight. Subsequently, standard dilutions of hemopexin from human plasma (0–50 ng/ml, Sigma-Aldrich) as well as diluted patient samples were added to the plate. Next 100 ng/ml of the detection antibody biotinylated anti-human hemopexin (Bioporto) was added to the plate followed by 1:1,000 dilution of streptavidin-HRP (RPN1231V GE healthcare). Subsequently, TMB Substrate (Sigma-Aldrich) was added and the absorbance was measured at wavelength of 450 nanometer (nm) with Victor 3 V multilabel plate reader (PerkinElmer).
Plasma HO-1 levels were determined by a sandwich ELISA using the human HO-1 matched antibody pair kit (Abcam) according to the manufacturer’s instructions.
Hemolysis and icterus index (respectively H-index and I-index) were determined using Serum Index Gen. 2 assay according manufacturer’s instructions (Cobas c system, Roche Diagnostics). H-index was used as marker for free hemoglobin. I-index was used as marker for bilirubin (both conjugated and unconjugated). H-index and I-index were determined based on absorbance measurements (570 and 480 nm primary wavelength and 700 and 505 nm secondary wavelength respectively) and expressed as µmol/L.

Lactate and glucose content of both cell lines was analysed on day 10 of cultivation. Therefore, apical and basolateral supernatants were collected of both cell lines. Medium was changed 72 h before and medium without cells was used as blank. All samples were stored on ice and immediately measured with a Roche/Hitachi Cobas c system (Roche, Basel, Switzerland). The difference between blank and sample represents the glucose consumption respectively lactate production of the cell.

Blood samples were collected at the schools after an overnight fast to analyse serum BDNF, TC, HDL-C, TG, glucose and insulin. Blood samples used for serum BDNF analyses were put directly on ice. Subsequently, all blood samples were transported to the laboratory. The blood samples were centrifuged with 1000g for 15 minutes at room temperature. Time between blood drawing and centrifugation was <4 hours (time was noted). Blood samples were stored 4–6 months at -80°C. Sandwich ELISA kits (Quantikine, R&D Systems, Minneapolis, MN, USA) were used to measure levels of serum BDNF. Samples were analyzed in duplicates, and the mean concentrations were used in the statistical analyses. Analyses were performed at the Centre for Physical Activity Research, Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, Denmark.
TC, TG, glucose and HDL-C were analysed by quantitative determination using enzymatic, colorimetric method on Roche/Hitachi cobas c system (Roche, Mannheim, Germany). Insulin was analysed using solid phase enzyme labeled chemiluminescent immunometric assay. Insulin resistance was estimated by the homeostasis model assessment, HOMA-IR; blood glucose (mmol/L) x insulin (μU/mL) divided by 22.5 [21 (link)].

Measurements of a panel of cytokines and chemokines in serum were performed using Luminex technology on a Bioplex Suspension Array System (Bio-Rad Laboratories Inc, Hercules, CA, USA) with a Milliplex Map kit (Millipore, Billerica, MA, USA). C- reactive protein (CRP) levels were determined using high sensitive immunoturbidimetric assay (CRPL3, Cobas C system, Roche Diagnostics, IN, USA).