Oxidative degradation of bilirubin produces vasoactive compounds

Abstract

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C 9 H 11 N 2 O 2 ) derivatives, 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.

BilirubinOxidation Products (BOXes) and Their Role in Cerebral Vasospasm after Subarachnoid Hemorrhage

“…This culminated in the identification of BOXes, compounds that are metabolites of bilirubin (Kranc et al, 2000). Bilirubin oxidation product was coined as a term for the compounds because they were formed by bilirubin oxidation.…”

“…The BOX designation is also fitting since the BOXes were first identified in the Department of Biochemistry, OXford University in collaboration with the Department of Chemistry. Because there are isomers of the BOXes formed from the two ends of the bilirubin molecule (differing by the position of the methyl and vinyl groups; see also Figure 6), we have adopted the term BOXes as the plural form of BOX A and BOX B (Kranc et al, 2000). To date, we have observed no significant difference in the biologic activity of BOX A versus BOX B and consistently observe them to be present in equimolar concentrations.…”

“…Hydrogen peroxide, in the presence of iron, forms hydroxyl ions (Fenton reaction) that would be expected to oxidize bilirubin, biliverdin, and heme-all of which would be found in high concentration in clots surrounding cerebral blood vessels after SAH. The initial studies showed that treatment of pure bilirubin with H 2 O 2 (BOX) produced compounds that increased oxygen consumption and increased contractile tension of porcine carotid artery rings to a similar degree or even greater than CSF from SAH patients with vasospasm (Kranc et al, 2000). In addition, the oxidized bilirubin compounds were extractable with chloroform and they were light sensitive-just as the vasoactive compounds in CSF were (Kranc et al, 2000).…”

“…Mass spectrometry identified one peak as MVM, which is 4-methyl-3-vinylmaleimide-a previously isolated photodegradation product of biliverdin. The other two molecules were novel, and were named BOX A and BOX B (Kranc et al, 2000), as are shown in Figure 6. Bilirubin oxidation product A and BOX B were vasoactive in vitro, and lost all biologic activity when exposed to sunlight (Kranc et al, 2000).…”

“…The other two molecules were novel, and were named BOX A and BOX B (Kranc et al, 2000), as are shown in Figure 6. Bilirubin oxidation product A and BOX B were vasoactive in vitro, and lost all biologic activity when exposed to sunlight (Kranc et al, 2000). Importantly, CSF from SAH patients with vasospasm was extracted with chloroform and applied to the HPLC.…”