56 - Excess circulating free heme triggers immense elevation in plasma xanthine oxidase activity

Abstract

Heme, an iron-containing compound and critical prosthetic group for metalloproteins, is found most commonly as an element of hemoglobin. Excess circulating heme is a key driver of hemolytic diseases including thalassemia, malaria, sickle cell disease, sepsis as well as a side effect of transplantation and cardiac bypass. Severe hemolysis (heme crisis) saturates heme degradation pathways such as hemopexin leading to abundant circulating free heme that can directly generate reactive oxygen species (ROS) and/or stimulate elevation in enzymatic sources of ROS, ultimately leading to overt tissue damage. A key enzymatic source of ROS reported to be upregulated in sickle cell disease is xanthine oxidase (XO). As such, we investigated the relationship between circulating XO activity and heme crisis in mice. Both 24 hour survival and plasma XO activity were assessed in C57BL6/J mice injected (tail-vein) with hemin (0-100 μmol/kg). Hemin dose-dependently reduced survival (100% (25 μmol/kg), 87% (50 μmol/kg), 12% (70 μmol/kg) and 0% (100 μmol/kg)). Hematological analysis revealed an increase in monocyte recruitment and diminished platelet counts with increasing doses of hemin. Doses of 50 and 70 μmol/kg hemin elevated plasma XO activity 20-fold and 39-fold, respectively compared to controls and correlated with substantial loss of hepatic XO. Histological analysis (H&E staining) also indicated that liver tissue from mice injected with 50 μmol/kg hemin demonstrated significant alterations in tissue organization. In conclusion, heme crisis induces extensive hepatocellular release of XO into the circulation where it may actively participate in the pathologic process. To validate this assumption, we have generated a liver-specific XO knockout and are currently investigating mechanisms underpinning heme crisis-associated hepatocellular release of XO as well as the impact of circulating XO on cardiovascular function. These ongoing experiments may serve to identify novel clinical strategies to address heme crisis.