Interaction between human serum albumin and the felbamate metabolites 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one and 2-phenylpropenal.
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.
Roller
Shane G
SG
Chemistry Department, University of Virginia, Charlottesville 22901, USA.
Dieckhaus
Christine M
CM
Santos
Webster L
WL
Sofia
R Duane
RD
Macdonald
Timothy L
TL
D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D003520:Cyclophosphamide / Q000378:metabolism; D000078328:Felbamate; D006207:Half-Life; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010078:Oxazines / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D048448:Phenylcarbamates; D011409:Propylene Glycols / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism
D000447:Aldehydes; D010078:Oxazines; D010446:Peptide Fragments; D048448:Phenylcarbamates; D011409:Propylene Glycols; D012709:Serum Albumin; D006639:Histidine; C013026:hydratropic aldehyde; D003520:Cyclophosphamide; D000078328:Felbamate