A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes

Maria Alexandra Brito, Dora Brites, D. Allan Butterfield

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93 Scopus citations


Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (≤1 μM), for 4 h at 37°C. Intracellular concentrations of reactive oxygen species (ROS) and calcium were determined by dichlorofluorescin and BAPTA fluorescent probes, respectively. Membrane protein and lipid oxidation were evaluated by immunocytochemistry and phosphatidylserine exposure by annexin V binding. Levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as activities of Mg 2+-ATPase aminophospholipid translocase (flippase) and Na +,K+-ATPase, were also measured. Our results showed that bilirubin induced oxidative stress, due to a rise in lipid (≥10%, P<0.05) and protein oxidation (≥20%, P<0.01), ROS content (∼17%, P<0.01), and a decrease in GSH/GSSG ratio (>30%, P<0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (∼10%, P<0.05), together with decreased flippase and NA +,K+-ATPase (≥15%, P<0.05) activities, events that were accompanied by enhanced intracellular calcium levels (∼20%, P<0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.

Original languageEnglish
Pages (from-to)33-43
Number of pages11
JournalBrain Research
Issue number1
StatePublished - Nov 5 2004

Bibliographical note

Funding Information:
The authors greatly acknowledge Dr. Jennifer Drake and Dr. Alessandra Castagna for their expertise in all the techniques performed, as well as all the other members of Butterfield's laboratory for their help and encouragement during the course of this work. They also thank Dr. Rui Silva for the revision of the present manuscript. This work was supported by grants from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal (POCTI/39906/FCB/2001 to D.B.), NIH (AG-10836; AG-05119 to D.A.B.), and by a fellowship to M.A.B. (FLA-288/02).


  • ATPase
  • Bilirubin neurotoxicity
  • Calcium
  • Oxidative stress
  • Phospholipid asymmetry
  • Synaptosomal membrane

ASJC Scopus subject areas

  • General Neuroscience
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology


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