No patients were under treatment with glucocorticoids, bisphosphonates, or calcitonin, and all had normal serum calcium concentrations. The mean total serum bilirubin concentration measured by standard colorimetric assay in jaundiced patients was 330 μM (19.3 mg/dL). Sera was

separated from the blood samples and stored at −80°C until the assays were performed. Samples were mixed in equal volume condition of conventional culture medium. To avoid photodegradation, all bilirubin-containing serum samples were prepared in the dark, and all cell culture plates were wrapped in aluminum foil to avoid light exposure. The experiments were carried out with pooled samples. The protocol was reviewed and approved by the Hospital Clínic, and all subjects gave informed consent. The experiments were performed with human primary osteoblasts and SAOS-2 human osteosarcoma cells. Human primary osteoblasts were taken from trabecular learn more bone specimens using a modification of the procedure of Robey and Termine.15 Bone pieces were obtained from subjects without features of metabolic bone disease who were undergoing hip replacement for osteoarthritis, following the procedures approved by the Hospital mTOR inhibitor Clínic Ethics Committee. Trabecular bone pieces, processed according to previously

described protocol,16 were grown in DMEM/HAM F-12 (1:1) medium, supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin–streptomycin, and 10 μg/mL ascorbic acid. Cells were characterized as osteoblast phenotypes by determination of alkaline phosphatase activity4 and osteocalcin messenger RNA (mRNA) expression by histochemistry and reverse-transcription polymerase chain reaction (PCR), respectively. Only cells in the first passage were used in the experiments. Cell mineralization

was carried out with SAOS-2 human osteosarcoma cells from ATCC (American Type Culture Collection, Manassas, VA), which were cultured in DMEM supplemented with 10% FBS. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2, and the medium was changed twice a week. This cell line was used because it is the common model for mineralization assessment for calcium deposition capacity in osteogenic conditions.17-19 Cells were characterized as human osteoblastic cells by determination of the alkaline phosphatase activity, as measured only by histochemical technique. Cells were placed on 35-mm coverslips at a density of 4 × 105 cells/mL and incubated for 72 hours at 37°C with DMEM/HAM F-12 containing 10 μg/mL ascorbic acid and 1,25-dihydroxycholecalciferol at 10−7 M to stimulate the alkaline phosphatase synthesis, and the enzyme activity was assessed in cells grown to confluence. Cells were rinsed twice with HBSS and fixed in cold 95% ethanol, then were incubated at room temperature with a solution of α-naphthylphosphate and 0.1% Tris-buffered HCl at pH 10 containing 0.1% fast blue RR. Stained cells were identified by optical microscopy.