Bilirubin Degradation by Hypochlorous Acid: Identification of Products and Their Formation Ex Vivo and In Vivo

Abstract Bilirubin (BR) is the breakdown product of heme, and has antioxidative and anti‐inflammatory activities. BR has been known to be degraded by H 2 O 2 ; however, its degradation by HOCl, an oxidant specific to activated neutrophils, has not been investigated. We herein reported that at pH = 7.4 and 37 °C, BR was rapidly degraded by HOCl into many products. Three major products were characterized: hematinic acid (HA), 2,5‐diformyl‐4‐methyl‐1 H ‐pyrrole‐3‐propanoic acid (BHP2), and 4‐methyl‐3‐(1‐hydroxy‐2‐chloroethyl) maleimide (MHCM), which is a novel compound. Acidic pH favored the MHCM formation, whereas neutral and alkaline pH and high HOCl:BR ratios favored production of HA and particularly BHP2. In murine whole blood and bone marrow suspensions, the levels of HA and BHP2 increased significantly after neutrophil activation with MHCM being undetected, and inhibition of HOCl generation abolished the increases. In the lipopolysaccharide (LPS)‐induced murine inflammatory model, the BHP2 concentration in plasma, but not HA, increased significantly at 6 h and 24 h post‐LPS administration, and returned to the pre‐LPS stimulation level at 48 h. The results show that BR is readily degraded by HOCl, which can occur ex vivo and in vivo upon neutrophil activation, with BHP2 being a potential biomarker for HOCl production.