Effect of bilirubin on erythrocyte shape and haemolysis, under hypotonic, aggregating or non-aggregating conditions, and correlation with cell age

Abstract

The effect of unconjugated bilirubin on the morphology and haemolysis of human erythrocytes was accomplished under distinct incubation conditions: (i) hypotonic medium, with bilirubin concentrations ranging from 1 x 10(-9) to 1 x 10(-4) mol l-1; (ii) isotonic medium, with 171 mumol l-1 bilirubin, in the absence of albumin (aggregating conditions), using non-separated and age-fractionated erythrocytes; (iii) isotonic medium, with 171 mumol l-1 bilirubin, in the presence of a surplus of human serum albumin (non-aggregating conditions), and using sulfisoxazole as a bilirubin displacer (bilirubin/albumin and sulfisoxazole/ albumin molar ratios of 0.5 and 4.0, respectively). Our data showed that low concentrations of bilirubin (1 x 10(-7) to 1 x 10(-5) mol l-1) protect against hypotonic haemolysis and induce crenation, while higher bilirubin concentrations induce haemolysis and lead to membrane disruption. When aggregating conditions were used, these phenomena were reproduced, the younger cells being significantly more susceptible to crenation while the older erythrocytes showed increased susceptibility to haemolysis. In non-aggregating conditions, haemolysis was virtually absent, though crenation was evident. Based on the above observations we conclude that the first step of erythrocyte bilirubin toxicity is crenation due to an expansion of the outer membrane leaflet by bilirubin mono-anion location. This effect is more evident in younger cells and explains the protection against the hypotonic haemolysis. Insertion of bilirubin deeper into the bilayer, facilitated by higher concentrations (> or = 1 x 10(-4) mol l-1) and cell age, produces an unstable situation, where bilirubin acid aggregation is apparently the main cause for haemolysis and cell destruction.

Rat Cultured Neuronal and Glial Cells Respond Differently to Toxicity of Unconjugated Bilirubin

“…This assumption is corroborated by alterations on cytoskeletal filaments, an intracellular target of both astrocytes and neurons, where disassembly of neuronal intermediate filaments and tubulin required much lower concentrations of UCB. Such type of cytotoxic phenomena is apparently due to the acid species of UCB (6) involving UCB aggregation and deposition into cell membranes or intracellularly (14,41,42). Indeed, if the anionic species of UCB were responsible for the cytoskeleton disassembly, then increased alterations would be expected as the pH rose to 8.0, which did not occur.…”

The Evolving Landscape of Neurotoxicity by Unconjugated Bilirubin: Role of Glial Cells and Inflammation

“…This finding, also observed in erythrocyte membranes (Brito et al, 2002) and synaptosomes (Brito et al, 2004) after exposure to UCB, was accompanied by reduced activities of Mg 2+ -ATPase aminophospholipid translocase (flippase) and Na + , K + -ATPase, together with enhanced intracellular levels of calcium (Ca 2+ ; Figure 2B). These effects can lead to cell destruction, as evidenced by the hemolysis of erythrocytes (Brites et al, 1997; Khan and Poduval, 2011). Ca 2+ influx or increased release of Ca 2+ from ER or other stores may contribute to the intracellular increase of Ca 2+ .…”

Bilirubin Toxicity to Human Erythrocytes: A More Sanguine View

“…[3][4][5][6][7] The bilirubin-induced morphologic changes were illustrated by striking photographs similar to those published some 30 years later, without reference to the earlier work, by others. 8,9 These changes were reversible by addition of serum albumin to the incubation medium and did not occur when albumin was present in the medium from the start. Subsequently, Brito et al 10 have confirmed and extended these observations and recently reviewed the toxicity of bilirubin to red cells (again without alluding to the seminal earlier work).…”

“…There is even evidence that bilirubin can strengthen the red cell membrane and protect red cells from oxidative damage that could lead to hemolysis. 8,15,16,39 Of course, it is hard to substantiate a negative. Documentation of mild hemolysis in newborns is difficult, and even a small degree of hemolysis, insufficient to have much effect on hematocrit measurements, could markedly augment the circulating bilirubin pool.…”

“…However, that lone observation is inconsistent with other observations on red cells in vitro, in which no hemolysis was observed at even higher bilirubin concentrations in the presence of serum albumin. 2,8 Red cells from just 1 adult were used in the studies, and the possible interference of bilirubin in the spectroscopic method used to estimate hemolysis, a phenomenon noted by Watson, 1 was not checked. 38 From the published clinical and in vitro evidence, with the exception of the article noted above, 38 it is hard to escape the conclusion that bilirubin-induced hemolysis is insignificant in vivo even at plasma bilirubin concentrations associated with kernicterus or at levels greater than those at which therapeutic intervention is considered mandatory.…”