Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy S. R. AHLFORS 1 , M. H. KRISTIANSSON 2 , C. H. LINDH 2 , B. A. G. JO ¨ NSSON 2 , & C. HANSSON 1 1 Department of Dermatology and 2 Department of Occupational and Environmental Medicine, Institute of Laboratory Medicine, University Hospital, Lund, Sweden Abstract Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC- UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/ MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N a - acetylated lysine and cysteine and the non-acetylated a-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine  /HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine  /HHPA adducts. The results will be useful for understanding the formation of HHPA  /protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy. Keywords: Adduct, allergy, amino acids, antigen, hapten, hexahydrophthalic anhydride (Received 18 October 2004; accepted 12 April 2005) Introduction Allergy is a common and increasing cause of human health problems today. Two of the most common types of allergy are type I allergy, with symptoms such as asthma, rhinitis, conjunctivitis and urticaria; and type IV allergy, with allergic contact dermatitis. Type IV allergy is induced by small reactive organic chemicals or metal salts (haptens) found in consumer goods, industrial chemicals and materials. Correspondence: S. Ahlfors, Department of Dermatology, University Hospital, SE-221 85 Lund, Sweden. Fax: 46-46-17-39-47. E-mail: Stefan.Ahlfors@med.lu.se ISSN 1354-750X print/ISSN 1366-5804 online # 2005 Taylor & Francis DOI: 10.1080/13547500500283488 Biomarkers, September  /October 2005; 10(5): 321  /335