Protein adducts of hexahydrophthalic anhydride- chemical structures and biomarkers

Abstract: Organic acid anhydrides (OAAs) are low molecular weight chemicals used in the industry for manufacturing of plastics such as epoxy resins. Exposure to these chemicals may lead to symptoms from the eyes and the respiratory tract in form of conjunctivitis, rhinitis and asthma. In many workers production of specific IgE antibodies are induced suggesting that a type-1 allergy mediated mechanism is involved. The OAAs act as haptens and the binding to endogenous proteins is probably an important part of the pathophysiological mechanism. The aim of this thesis was to identify potentially allergenic chemical structures using hexahydrophthalic anhydride (HHPA), a highly sensitising OAA, as a model substance. The aim was also to develop a new method for biological monitoring of HHPA. The binding of HHPA to several nucleophilic amino acids and a model peptide was initially studied. Stable adducts to lysine and the N-terminal amino group were found. Unstable adducts to cysteine, histidine, tyrosine and tryptophan were also identified. The cysteine adduct could be transferred to lysine residues. Studies of potential allergenic structures were performed on hapten-protein conjugates between HHPA and hemoglobin (Hb) and human serum albumin (HSA), respectively, synthesised in vitro. Enzyme digestions of the conjugates were performed and the binding sites of HHPA were identified through analysis of the peptides using liquid chromatography coupled to tandem mass spectrometry. HHPA adducts were found with several lysine residues and the N-terminal amino group both in Hb and HSA. Further studies must be performed to determine the allergenic potential of these identified adducts. Based on the identifications of the binding sites of HHPA to HSA a method was developed to measure HHPA-adducted tryptic peptides of HSA in nasal lavage samples. The nasal lavages were obtained from five volunteers exposed to HHPA at different air concentrations of HHPA. The major binding sites of HHPA found in the in vitro studies were detected in the in vivo samples as well. The levels of these adducted peptides were highly correlated with the air levels of HHPA on an individual basis but there were large inter-individual differences. Thus, it should be investigated whether these peptides may be used as biomarkers of effective dose, which then would better reflect the risk than more traditional biomarkers.

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