Impact of higher-order heme degradation products on hepatic function and hemodynamics

J Hepatol. 2017 Aug;67(2):272-281. doi: 10.1016/j.jhep.2017.03.037. Epub 2017 Apr 12.


Background & aims: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions.

Methods: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia.

Results: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton.

Conclusions: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia.

Lay summary: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.

Keywords: Bilirubin oxidation end products (BOXes); Bilirubin toxicity; Cholestasis; Cytoskeleton; Glutathione; Heme degradation; Hemodynamics; Pharmacokinetics; Reactive oxygen species; Rev-erb.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute-On-Chronic Liver Failure / metabolism
  • Animals
  • Bile / metabolism
  • Bilirubin / metabolism
  • Biliverdine / metabolism
  • Cholestasis / metabolism
  • Glutathione / metabolism
  • Heme / metabolism*
  • Hemodynamics
  • Hep G2 Cells
  • Humans
  • Hyperbilirubinemia / metabolism
  • In Vitro Techniques
  • Liver / metabolism*
  • Liver Circulation
  • Male
  • Oxidation-Reduction
  • Pyrroles / metabolism
  • Rats
  • Rats, Wistar


  • (Z)-2-(3-ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide
  • Pyrroles
  • Heme
  • Glutathione
  • Biliverdine
  • Bilirubin