Inhibitory action of bilirubin on superoxide production by polymorphonuclear leukocytes

To assess the interaction of bilirubin with albumin and to determine the site of bilirubin toxicity in cells, a study was made of the O2 production of neonatal neutrophils (PMNs) by two different stimulators: (1) concanavalin A (Con A) plus cytochalasin D (Cyt D), which acts on the cell surface, and (2) phorbol myristate acetate, which acts intracellularly. PMNs that had been separated from cord blood were incubated for 60 min at 37 °C in the solution with different molar ratios of bilirubin/albumin (unbound bilirubin, ranging from 0.35 to 3.92 μg/dl). The unbound bilirubin was determined by peroxidase oxidation method. A PMN viability of more than 96% was maintained after the incubation in each of solutions. The O2 production rate of PMNs stimulated by Con A plus Cyt D was inhibited in the presence of unbound bilirubin levels as low as 1.12 μg/dl, and the rate decreased as the levels of unbound bilirubin rose. The O2 production rate stimulated by Con A plus Cyt D was more remarkably inhibited than that by phorbol myristate acetate, which directly activates intracellular protein kinase C. These findings suggest that bilirubin toxicity to PMN can be shown at levels of unbound bilirubin as low as those in hyperbilirubinemic sera, and the critical site at which bilirubin exerts its toxicity is mainly in membrane level rather than on intracellular functions.