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Methyl vinyl ketone – identification and quantification of adduct to N-terminal valine in human haemoglobin
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).
2010 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 27, 2491-2496 p.Article in journal (Refereed) Published
Abstract [en]

Adducts to N-terminal valines in Hb have been shown useful as biomarkers of exposure to electrophilic compounds. Adducts from many compounds have earlier been measured with a modified Edman degradation method using a GC–MS/MS method. A recently developed method, the adduct FIRE procedure™, adopted for analysis by LC–MS/MS, has been applied in this study. With this method a fluorescein isothiocyanate (FITC) reagent is used to measure adducts (R) from electrophiles with a modified Edman procedure. By using LC–MS/MS in product ion scan mode, a new peak was identified and the obtained MS data indicated that this adduct could originate from methyl vinyl ketone (MVK). Incubation of human-, sheep- and bovine blood with MVK increased the signal of the identified peak. By comparing the LC–MS/MS data from the unknown background peak with data obtained from synthesized fluorescein thiohydantoin (FTH) standards of the MVK adduct to valine and d8-valine, the identity of this adduct was confirmed. The MVK adduct was shown present in human blood (35 pmol/g globin, n = 3) and only just above LOD in bovine blood, n = 1 (LOD = 2 pmol/g globin). MVK reacts, in similarity with acrylamide, via Michael addition. MVK is known to occur in the environment and has earlier been observed in biological samples, which means that there are possible natural and anthropogenic exposure sources. Analysis of an Hb adduct from MVK in humans has to our knowledge not been described before.

Place, publisher, year, edition, pages
2010. Vol. 878, no 27, 2491-2496 p.
Keyword [en]
Hemoglobin adducts, Methyl vinyl ketone, Adduct FIRE procedure, LC–MS/MS
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
URN: urn:nbn:se:su:diva-30126DOI: 10.1016/j.jchromb.2010.03.037ISI: 000283410200005OAI: oai:DiVA.org:su-30126DiVA: diva2:241637
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2017-12-13Bibliographically 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. Methodology for hemoglobin adduct measurement: Fetal exposures to acrylamide and other genotoxic agents
Open this publication in new window or tab >>Methodology for hemoglobin adduct measurement: Fetal exposures to acrylamide and other genotoxic agents
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is increasing evidence that exposure to toxic chemicals during the prenatal period constitute a higher health risk than exposure during adulthood. To characterize exposure and identify risk factors, sensitive methods for analysis of chemicals in vivo with biomarker methods are needed. Adducts to hemoglobin (Hb) have been shown useful as biomarkers of dose in blood of reactive compounds/metabolites, which are toxic due to reactions with biomacromolecules.

The aim of this thesis was to develop a new method for the analysis of Hb adducts suitable for analysis of large sample series, and then to apply the method for measurements of Hb adducts from exposure to acrylamide, glycidamide and ethylene oxide in mother/cord blood samples from five European countries.

A new method for measurements of N-terminal Hb adducts, denoted the adduct FIRE procedure, was developed using the fluorescein isothiocyanate Edman reagent. With the new procedure, optimized for LC/MS analysis, a high sensitivity and reproducibility was achieved. The new method made it possible to perform measurements of low exposures to the studied genotoxic compounds in approximately 1350 maternal and cord blood samples.

The results show that the fetus is exposed to a similar in vivo dose of the studied compounds as the mother. The measured Hb adduct levels show that acrylamide exposure from food intake is higher for the participating mothers fromUK compared to the mothers from the other countries. The measured Hb adduct levels form a basis for evaluations of relationships between exposure and health risks, and ongoing studies indicate associations between acrylamide Hb adduct levels and birth weight.

The developed method was also used for identification of an unknown Hb adduct, which was shown to originate from methyl vinyl ketone (MVK), a highly reactive and toxic compound. The identity of the adduct was confirmed with synthesized standards. There exist both natural and anthropogenic sources to MVK, and to what extent the MVK-adduct reflects exogenous exposure is yet not clarified.

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry (MMK), Stockholm University, 2011. 54 p.
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-63026 (URN)978-91-7447-376-6 (ISBN)
Public defence
2011-11-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print.Available from: 2011-10-20 Created: 2011-10-07 Last updated: 2011-10-10Bibliographically approved

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