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| 2. |
- Kåredal, Monica, et al.
(författare)
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Determination of hexahydrophthalic anhydride adducts to human serum albumin
- 2003
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Ingår i: Biomarkers. - : Informa UK Limited. - 1366-5804 .- 1354-750X. ; 8:5, s. 343-359
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Tidskriftsartikel (refereegranskat)abstract
- Hexahydrophthalic anhydride (HHPA) is a highly sensitizing industrial chemical that is known to covalently bind to endogenous proteins. The aim of this study was to determine the binding sites of HHPA to human serum albumin (HSA). Conjugates between HSA and HHPA, at two different molar ratios, were synthesized under physiological conditions. The conjugates were digested with trypsin and Pronase E to obtain specific peptides and amino acids, which were separated by liquid chromatography (LC). Fractions containing modified peptides were detected through quantification of hydrolysable HHPA using LC coupled to a triple quadrupole mass spectrometer with electrospray ionization. Modified residues in albumin were identified by sequence analyses using nanoelectrospray quadrupole time-of-flight mass spectrometry. A total of 36 HHPA adducts were found in the HSA - HHPA conjugate with 10 times molar excess of added HHPA. In the conjugate with a molar ratio of 1: 0.1 of added HHPA, seven HHPA adducts were found bound to Lys(137) (domain IB), Lys(190), Lys(199) and Lys(212) (domain IIA), Lys(351) (domain IIB), and Lys(432) and Lys(436) (domain IIIA). Moreover, several of these adducted albumin peptides were detected in nasal lavage fluid from one volunteer exposed to HHPA. The binding sites of HHPA to HSA have been determined, thus identifying potential allergenic chemical structures. This knowledge generates the possibility of developing methods for the biological monitoring of HHPA exposure by analysing tryptic peptides including these binding sites.
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| 3. |
- Kåredal, Monica, et al.
(författare)
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Mass spectrometric characterization of human hemoglobin adducts formed in vitro by hexahydrophthalic anhydride.
- 2002
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Ingår i: Chemical Research in Toxicology. - : American Chemical Society (ACS). - 1520-5010 .- 0893-228X. ; 15:4, s. 562-569
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Tidskriftsartikel (refereegranskat)abstract
- Primary structural information of anhydride binding to endogenous proteins is of interest in order to determine the mechanism causing the type-I allergy seen in many anhydride-exposed workers. In addition, studies on specific protein adducts may generate new methods for biological monitoring. In this study, the binding of hexahydrophthalic anhydride (HHPA) to human hemoglobin (Hb) in vitro was investigated. The in vitro synthesized conjugates were analyzed using a hybrid quadrupole-time-of-flight mass spectrometer (Q-TOF) with electrospray ionization (ESI) to determine the number of HHPA adducts per Hb molecule. Structural information on the locations of the adducts was obtained through nanospray Q-TOF, liquid chromatography-ESI mass spectrometric analysis, and gas chromatography/mass spectrometric analysis of Pronase E and tryptic digests. Up to six adducts were found on the alpha-chain and five on the beta-chain. The HHPA-adducts were localized to the N-terminal valine of the alpha- and beta-chains of Hb and to lysine residues at positions 7, 11, 16, and 40 of the alpha-chain and 8, 17, 59, 66, and 144 of the beta-chain. These results will constitute a basis for studies on structure-activity relationships as well as for development of methods for biological monitoring of acid anhydrides.
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| 4. |
- Kåredal, Monica, et al.
(författare)
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Mass spectrometric monitoring of in vitro formed ad ducts between hexahydrophthalic anhydride (HHPA) and cysteine and lysine
- 2002
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Ingår i: Proceedings 50th ASMS Conference on Mass Spectrmetry and Allied Topics. ; , s. 325-326
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Konferensbidrag (refereegranskat)abstract
- The in vitro formed adducts between hexahydrophthalic anhydride (HHPA) and cysteine and lysine were monitored by mass spectrometry. Freshly synthesized N-acetyl-S-hexahydrophthaloyl-L-cysteine was dissolved in dry acetonitrile and added to a solution of N-acetyl-L-lysine in 0.1 M sodium phosphate buffer (pH 7.4) an incubated at 37°C. The reactions were monitored by LC/MS on a triple quadrupole mass spectrometer with electrospray ionization coupled to a HPLC system from Perkin Elmer. The results show that there was a rapid rearrangement of HHPA from most of the cysteines to the lysines, and that HHPA-adducts of cysteine will be transferred to lysine when encountered.
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| 5. |
- Kåredal, Monica
(författare)
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Protein adducts of hexahydrophthalic anhydride- chemical structures and biomarkers
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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