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

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.