IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Unbound, Not Total Bilirubin, Concentration Predicts Cytotoxicity In Vitro
Autor/es:
SEBASTIAN D. CALLIGARIS, CRISTINA BELLAROSA, PABLO GIRAUDI, RICHARD P. WENNBERG, J. DONALD OSTROW, CLAUDIO TIREBELLI.
Lugar:
Toronto, Canada
Reunión:
Congreso; 2007 Pediatric Academic Societies' Meeting; 2007
Resumen:
BACKGROUND: Theoretical considerations and limited clinical data indicate that the unbound bilirubin level (Bf) should predict the risk for bilirubin toxicity better than the total serum bilirubin concentration (BT).OBJECTIVE: We hypothesized that Bf, measured using the peroxidase method, would predict bilirubin-induced cytotoxicity in vitro independent of BT, the albumin reservoir source, and/or the presence of binding competitors or photoisomers.DESIGN/METHODS: Four cell types, human neuroblastoma (SHSY5Y), primary mouse embryo fibroblast, HeLa cells, and striatal precursor cell line 2a1, were grown in multiwell plates for 1 day to 70-80% confluence, then washed and incubated for 2 hours with medium containing either 15% fetal calf serum (FCS), 30 or 60 M human (HSA) or 30 M bovine (BSA) serum albumin and varying BT. Cell viability was then determined by tetrazolium dye reduction. The effect of photoisomers and photo-degradation products on Bf determination and toxicity was evaluated by exposing culture medium containing FCS and a toxic level of bilirubin to intense light for 60 min prior to incubation.RESULTS: Susceptibility to bilirubin cytotoxicity varied with cell type, being greatest with SHSY5Y cells (30% and 46% reduction in cell viability at Bf of 80 and 160 nM respectively). The magnitude of toxicity was invariably related to Bf and not to BT. By using albumin preparations with different binding affinities (BSA, FCS, HSA), varying the albumin concentration, and/or adding a bilirubin displacer (sulfadimethoxine), a constant Bf could be obtained at widely different BT. Thus, a Bf of 80 nM (and associated cell toxicity) was achieved with BT ranging 9-45 M. Intense light exposure reduced BT, Bf and toxicity by 18-20%, but the calculated binding affinity, Kf, did not change and the decrease in toxicity was consistent with the decrease in Bf.CONCLUSIONS: Within a given cell line, cytotoxicity was predicted by Bf, not BT, and was independent of albumin source. The presence of photo-products did not alter the relationship between Bf and cytotoxicity in vitro.