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A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
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2010 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 23, no 3, 540-546 p.Article in journal (Refereed) Published
Abstract [en]

Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)-carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization tinder the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

Place, publisher, year, edition, pages
2010. Vol. 23, no 3, 540-546 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-52218DOI: 10.1021/tx900278pISI: 000275411700015OAI: oai:DiVA.org:su-52218DiVA: diva2:387119
Note

authorCount :6

Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2017-12-11Bibliographically approved
In thesis
1. New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane
Open this publication in new window or tab >>New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human exposure to harmful compounds in the environment, from intake via food, occupational exposures or other sources, could have health implications. Exposure to reactive compounds/metabolites can be identified and quantified as hemoglobin (Hb) adducts by mass spectrometry. This thesis aimed at improved synthetic pathways for reference standards, and improved analytical methods for adducts to N-terminal valine in Hb from a range of reactive compounds; isocyanates, aldehydes, methyl vinyl ketone (MVK), and diepoxybutane (DEB).

Isocyanates form urea adducts with N-terminal valine by carbamoylation, which are detachable as hydantoins by hydrolysis. A new synthetic pathway for reference standards of adducts from isocyanates and a method for their analysis by liquid chromatography/mass spectrometry (LC/MS) were developed.

Aldehydes form reversible imines (Schiff bases) with N-termini in Hb. After stabilisation by reduction and detachment by isothiocyanates using modified Edman methods, these adducts could be analysed by gas chromatography/mass spectrometry (GC/MS) or LC/MS. 5-Hydroxymethylfurfural, its metabolites, and other aldehydes related to exposure via food, were studied with regard to analysis by these methods with synthesised standard references. A considerably improved analytical method for imines was developed. Many of the studied adducts are too short-lived in vivo or in vitro to be used for long-term biomonitoring. However, different approaches for the analysis were evaluated.

Through synthesised reference standards, an observed unknown adduct in blood was verified as the adduct from MVK. There exist both natural and anthropogenic sources for MVK.

DEB, metabolite of butadiene, forms a cyclic adduct to valine-N. A new approach using hydrazinolysis of protein and enrichment by molecularly imprinted solid-phase extraction was tested on synthesised reference DEB-adduct and gave promising results.

Synthesised standards were characterized by NMR, LC/MS and GC/MS.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Chemistry, Stockholm University, 2009. 64 p.
Keyword
carbamoylation, modified-Edman procedure, 5-(hydroxymethyl)furfural, MISPE, biomarkers
National Category
Environmental Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-30138 (URN)978-91-7155-934-0 (ISBN)
Public defence
2009-10-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.

Available from: 2009-10-08 Created: 2009-10-05 Last updated: 2014-12-11Bibliographically approved
2. Biomarkers of internal exposure/dose: Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates
Open this publication in new window or tab >>Biomarkers of internal exposure/dose: Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation.

The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established.

In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation.

An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment. 

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry, Stockholm University, 2015. 66 p.
Keyword
biomarker, PAH, serum albumin, adduct, DNA, isocyanate
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-110490 (URN)978-91-7649-074-7 (ISBN)
Public defence
2015-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2015-01-07 Created: 2014-12-15 Last updated: 2015-08-17Bibliographically approved

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Rydberg, PerWestberg, EmelieMotwani, Hitesh V.Törnqvist, Margareta
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