An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase

Abstract

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.

Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometry

“…Rough estimations of BOXes concentrations in CSF were obtained with high performance liquid chromatography coupled to ultra violet spectroscopy (HPLC-UV). That method delivered appraised values of 1.2-1.4 M BOXes in CSF after subarachnoid hemorrhage [10,15,16].…”

Recombinant human brain-derived neurotrophic factor prevents neuronal apoptosis in a novel in vitro model of subarachnoid hemorrhage

“…However, in vitro SAH models, such as stimulating glial cells with hemoglobin (Hb)6,21 or mimicking SAH with cytochrome oxidase,22 are still under investigation. In this present study, we established an in vitro model of SAH by application of hemolysate, which sufficiently simulated the clinical pathophysiological processes of SAH observed in vivo 18,19.…”

Bilirubin and its Oxidation Products Damage Brain White Matter

“…22 They can be quantified by assessing the absorbance at a wavelength of 320 nm. 6 The 7-day stock solution of bilirubin either stored ot oxidized at 4°C or 37°C had significantly higher absorbance at 320 nm compared with freshly-made solution (Figuress 5A and 5B, P o0.05 to 0.001 across different concentrations).…”