Interaction between human serum albumin and the felbamate metabolites 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one and 2-phenylpropenal

Chem Res Toxicol. 2002 Jun;15(6):815-24. doi: 10.1021/tx025509h.


Felbamate is an anti-epileptic drug associated with hepatotoxicity and aplastic anemia. These toxicities are believed to be mediated by the formation of the reactive species 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one is a metabolic precursor for 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one exists in equilibrium with 3-oxo-2-phenylpropyl carbamate, which can undergo beta-elimination to form 2-phenylpropenal. The work presented here investigates the interaction between 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one and human serum albumin (HSA). HSA (40 mg/mL) was found to decrease the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one from 4.57 +/- 0.44 h to 1.07 +/- 0.10 h at pH 7.4. This decrease in the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was due to increased beta-elimination of 3-oxo-2-phenylpropyl carbamate, presumably through HSA-mediated general base catalysis. The k(cat) for HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was determined to be 12.04 min(-)(1) M(-)(1). Competitive binding assays using warfarin and ibuprofen showed that HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one is dependent on the subdomain IIA binding site of HSA. LC/MS/MS analyses of trypsin digests of HSA incubations with either 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one or 2-phenylpropenal identified HSA-2-phenylpropenal adducts formed specifically at residues His-242 and His-247. These HSA-2-phenylpropenal adducts were found to be slowly reversible, with a decrease in alkylation of 74.0 +/- 0.6% after extensive dialysis. Interestingly, only the bis-adduct (His-242 and His-247) could be identified after dialysis. These results demonstrate the first direct example of 2-phenylpropenal conjugation to a human protein in vitro and suggest the possibility that HSA may be involved in the development of felbamate toxicity either by antigen formation or as a route of detoxification of 2-phenylpropenal.

MeSH terms

  • Aldehydes / chemistry*
  • Aldehydes / metabolism
  • Aldehydes / toxicity
  • Alkylation
  • Amino Acid Sequence
  • Binding Sites
  • Binding, Competitive
  • Catalysis
  • Chromatography / methods
  • Cyclophosphamide / metabolism
  • Felbamate
  • Half-Life
  • Histidine / chemistry
  • Humans
  • Kinetics
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Oxazines / chemistry*
  • Oxazines / metabolism
  • Peptide Fragments / chemistry
  • Phenylcarbamates
  • Propylene Glycols / chemistry*
  • Propylene Glycols / metabolism
  • Propylene Glycols / toxicity
  • Protein Binding
  • Sequence Analysis, Protein
  • Serum Albumin / chemistry*
  • Serum Albumin / metabolism


  • Aldehydes
  • Oxazines
  • Peptide Fragments
  • Phenylcarbamates
  • Propylene Glycols
  • Serum Albumin
  • Histidine
  • hydratropic aldehyde
  • Cyclophosphamide
  • Felbamate