Identification of modification sites on human serum albumin and human hemoglobin adducts with houttuynin using liquid chromatography coupled with mass spectrometry

Houttuynin, a β‐keto aldehyde compound, is the major active ingredient in herba houttuyniae injection. The injection was once used as an anti‐inflammatory drug associated with occasional serious hypersensitivity reactions in the clinic, which were proposed to be related to the formation of protein adducts. N^α‐Boc‐lysine, FEEM and IVTNTT were used as model amino acids or peptides containing one nucleophilic residue to investigate adduct types by liquid chromatography coupled with ion trap mass spectrometry (LC/MSⁿ) and high‐resolution quadrupole time‐of‐flight mass spectrometry (Q‐TOF MS). These adducts were respectively characterized as Schiff bases formed by 1:1 reaction of houttuynin with lysine or N‐terminal residue and pyridinium adducts by 2:1 reaction. LC/MSⁿ analysis of trypsin digests of HSA/Hb incubations with houttuynin revealed that houttuynin‐modified HSA adducts were formed mainly at N‐terminal amino acid and lysine residues, specifically at Lys‐212, Lys‐414 and Lys‐525 for Schiff base adducts, and at Lys‐414 and Lys‐432 for pyridinium adducts, and houttuynin adducted more readily with N‐terminal valine of the α‐ and β‐chains in Hb and lysine amine (Lys‐62) of the β‐chain for Schiff base adducts. The results showed the direct modification of houttuynin to HSA/Hb in vitro, which was speculated to be responsible for the adverse reactions induced by houttuyniae injection. Copyright © 2012 John Wiley & Sons, Ltd.