Bilirubin oxidation products, oxidative stress, and intracerebral hemorrhage

Abstract

Hematoma and perihematomal regions after intracerebral hemorrhage (ICH) are biochemically active environments known to undergo potent oxidizing reactions. We report facile production of bilirubin oxidation products (BOXes) via hemoglobin/Fenton reaction under conditions approximating putative in vivo conditions seen following ICH. Using a mixture of human hemoglobin, physiological buffers, unconjugated solubilized bilirubin, and molecular oxygen and/or hydrogen peroxide, we generated BOXes, confirmed by spectral signature consistent with known BOXes mixtures produced by independent chemical synthesis, as well as HPLC-MS of BOX A and BOX B. Kinetics are straightforward and uncomplicated, having initial rates around 0.002 microM bilirubin per microM hemoglobin per second under normal experimental conditions. In hematomas from porcine ICH model, we observed significant production of BOXes, malondialdehyde, and superoxide dismutase, indicating a potent oxidizing environment. BOX concentrations increased from 0.084 +/- 0.01 in fresh blood to 22.24 +/- 4.28 in hematoma at 72h, and were 11.22 +/- 1.90 in adjacent white matter (nmol/g). Similar chemical and analytical results are seen in ICH in vivo, indicating the hematoma is undergoing similar potent oxidations. This is the first report of BOXes production using a well-defined biological reaction and in vivo model of same. Following ICH, amounts of unconjugated bilirubin in hematoma can be substantial, as can levels of iron and hemoglobin. Oxidation of unconjugated bilirubin to yield bioactive molecules, such as BOXes, is an important discovery, expanding the role of bilirubin in pathological processes seen after ICH.

Fig. 1

Simple oxidization reaction vessel showing in vitro system for modeling the oxidizing environment in porcine hematoma

Fig. 2

Fig. 2A. A solution of 1 g sodium carbonate, 105 μM solubilized bilirubin, saturating levels of carbon dioxide gas and compressed air, 1 mg human hemoglobin, and 42 mM hydrogen peroxide were allowed to react with constant stirring in an open system. Aliquots of reaction were taken at 0 min (square), 5 min (triangle), 10 min (X), and 30 min (diamond). Optical density was followed in the visible region. The pH of this reaction is 6.8 Fig. 2B. The apparent kinetics of bilirubin degradation and BOX production using visible spectroscopy and extinction coefficients for bilirubin and BOXes. The rise in BOXes is apparent within 10 min and plateaus at about 10 min, reaching a steady state. Bilirubin falls at 426 nm, mirroring the production of BOXes

Fig. 2

Fig. 2A. A solution of 1 g sodium carbonate, 105 μM solubilized bilirubin, saturating levels of carbon dioxide gas and compressed air, 1 mg human hemoglobin, and 42 mM hydrogen peroxide were allowed to react with constant stirring in an open system. Aliquots of reaction were taken at 0 min (square), 5 min (triangle), 10 min (X), and 30 min (diamond). Optical density was followed in the visible region. The pH of this reaction is 6.8 Fig. 2B. The apparent kinetics of bilirubin degradation and BOX production using visible spectroscopy and extinction coefficients for bilirubin and BOXes. The rise in BOXes is apparent within 10 min and plateaus at about 10 min, reaching a steady state. Bilirubin falls at 426 nm, mirroring the production of BOXes

Fig. 3

Relative amounts of BOX production normalized to control condition A. A = hemoglobin (0.64 μM), bilirubin (105 μM), peroxide (383 mM); B = incubation A without hemoglobin; C = incubation without hemoglobin and peroxide; D = incubation A without peroxide; E = incubation A with added deferoxamine (65 mM); F = incubation A with carbon monoxide; G = incubation A with benzoic acid (l.2 mM); H = incubation A with added potassium cyanide (6.5 mM); I = incubation A with added arachidonic acid (4.3 mM); J = incubation A with added oxalic acid (550 mM); K = incubation A with added thiourea (110 mM). Error bars are standard deviation. N=3 or more for all conditions. Reactions were performed in phosphate-buffered saline at pH 7.5

Fig. 4

Representative cross-section of porcine brain 16 h after ICH. Breakdown of blood-brain barrier is indicated by Evans blue perfusion. Arrows indicate relative areas of sampling for use in generating data presented in Table 1

Fig. 5

Edema in the pig brain after ICH is evident in 8 h after hemorrhage and appears to peak at 2 days