Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pKa determinations
BackgroundAqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIα, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure 13C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates.ResultsExtensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2.ConclusionsThe present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies.
Reassessment of the Unbound Concentrations of Unconjugated Bilirubin in Relation to Neurotoxicity In Vitro
Most studies of the cellular toxicity of unconjugated bilirubin (UCB) have been performed at concentrations of unbound UCB (BF) that exceed those in the plasma of neonates with bilirubin encephalopathy. We assessed whether UCB could be toxic to neurons and astrocytes at clinically relevant BF values (≤1.0 μM), a range in which spontaneous precipitation of UCB would be unlikely to occur, even though BF exceeded the aqueous saturation limit of 70 nM. A meta-analysis yielded twelve published studies that had determined the in vitro effects of UCB on the function of cultured neurons or astrocytes at calculable BF values ≤ 1.0 μM. BF values were recalculated from the stated UCB, albumin, and chloride concentrations by applying affinity constants derived from ultrafiltration of comparable solutions containing 14C-UCB and delipidated human serum albumin. At BF slightly above aqueous solubility, UCB impaired mitochondrial function and viability of astrocytes. Exposure of neuroblastoma and embryonic neuronal cell lines to BF above 250 nM impaired cellular proliferation and mitochondrial function and increased apoptosis. Purified UCB inhibited the uptake of glutamate into astrocytes at BF as low as 309 nM and induced apoptosis in brain neurons at BF as low as 85 nM. UCB can impair various cellular functions of astrocytes and neurons exposed to BF near or modestly above its aqueous solubility limit, at which UCB exists as soluble oligomers and metastable microaggregates. The results render doubtful the long-held concept that precipitation of UCB in or on cells is required to produce neurotoxicity.
Cross talk between redox signalling and metabolic activity of osteoblasts and fibroblasts in the presence of hydroxyapatite-based biomaterials influences bone regeneration
Regeneration of bone tissue defects that result from metabolic disorders, including periodontal diseases, can be supported by biomaterials based on hydroxyapatite. Despite of good biocompatibility of biomaterials they can cause oxidative stress and inflammatory processes as a result of mechanical interaction with surrounding tissues. Because osteoblasts are responsible for bone regeneration process in which gingival fibroblasts may also participate, the aim of the work was to investigate the influence of hydroxyapatite-based biomaterials (allogeneic and xenogeneic) and biomaterials combined with enamel matrix derivative (Emdogain) on osteoblast and fibroblast redox balance in the context of osteoblast proliferation and differentiation.
The results showed that examined substitutes were not cytotoxic in vitro, but affected redox balance of osteoblasts and fibroblasts (ROS level increase and GSH level decrease) which led to oxidative stress (MDA and protein carbonyl groups level increase) resulting in an increase of the Nrf2 and NFκB expression. The consequence of these changes was partial inhibition of proliferation and osteoblast differentiation. Emdogain alone and combined with biomaterials decreased ROS generation and increased GSH level in both osteoblasts and fibroblasts leading to reduction of transcription factors expression especially proinflammatory NFκB, which promoted osteoblast differentiation and mineralization process.