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New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
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 [en]
carbamoylation, modified-Edman procedure, 5-(hydroxymethyl)furfural, MISPE, biomarkers
National Category
Environmental Sciences
Research subject
Environmental Chemistry
Identifiers
URN: urn:nbn:se:su:diva-30138ISBN: 978-91-7155-934-0 (print)OAI: oai:DiVA.org:su-30138DiVA: diva2:241663
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
List of papers
1. A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts
Open this publication in new window or tab >>A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts
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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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-52218 (URN)10.1021/tx900278p (DOI)000275411700015 ()
Note

authorCount :6

Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2017-12-11Bibliographically approved
2. Improved method to measure aldehyde adducts to N-terminal valine in hemoglobin using 5-hydroxymethylfurfural and 2,5-furandialdehyde as model compounds
Open this publication in new window or tab >>Improved method to measure aldehyde adducts to N-terminal valine in hemoglobin using 5-hydroxymethylfurfural and 2,5-furandialdehyde as model compounds
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2009 (English)In: Food and Chemical Toxicology, ISSN 0278-6915, Vol. 47, no 8, 1950-1957 p.Article in journal (Refereed) Published
Abstract [en]

Hemoglobin (Hb) adducts are used to measure reactive compounds/metabolites in vivo. Schiff base adducts from aldehydes to N-termini in Hb have been measured by GC-MS/MS after stabilisation through reduction, and detachment by a modified Edman procedure. This paper describes a further development using 5-hydroxymethylfurfural (HMF) and its probable metabolite, 2,5-furandialdehyde (FDA), as model compounds. Reference compounds were synthesized and characterized. The conditions for the reduction of the Schiff bases were optimized using NaBH(3)CN as a mild reducing agent, and steps used in the earlier method could be deleted. The adduct from FDA could not be specifically analysed, as selective reduction of the imine could not be achieved. In a few samples of human blood, background levels of 10-35 pmol/g globin of the HMF adduct were observed. Half-lifes of the reversible Schiff base adduct from HMF were determined to 3.4h at 37 degrees C and 10.9h at 25 degrees C. The developed method showed good sensitivity and reproducibility for the analysis of the Schiff base from HMF, with improvements regarding simplicity of work-up procedures due to mild conditions. The developed method could be explored for application to adducts from other aldehydes bound as Schiff bases to N-termini in Hb.

Identifiers
urn:nbn:se:su:diva-30142 (URN)10.1016/j.fct.2009.05.015 (DOI)000269100700034 ()19457441 (PubMedID)
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2010-01-11Bibliographically approved
3. Methyl vinyl ketone – identification and quantification of adduct to N-terminal valine in human haemoglobin
Open this publication in new window or tab >>Methyl vinyl ketone – identification and quantification of adduct to N-terminal valine in human haemoglobin
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.

Keyword
Hemoglobin adducts, Methyl vinyl ketone, Adduct FIRE procedure, LC–MS/MS
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-30126 (URN)10.1016/j.jchromb.2010.03.037 (DOI)000283410200005 ()
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2011-11-22Bibliographically approved
4. Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine in haemoglobin
Open this publication in new window or tab >>Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine in haemoglobin
Show others...
2010 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 27, 2497-2501 p.Article in journal (Refereed) Published
Abstract [en]

A molecularly imprinted polymer, MIP, was prepared and evaluated as SPE sorbent for a cyclicized adduct formed to N-terminal valine (Pyr-Val) in hemoglobin from 1,2:3,4-diepoxybutane (DEB). This metabolite plays an important role in the carcinogenesis of 1,3-butadiene. The hydrazide of Pyr-Val, formed after hydrazinolysis of hemoglobin, as well as necessary standards was synthesized. The MIP was prepared from methacrylic acid with a structure analogue to the investigated adduct as template and the method was developed for aqueous conditions. Selective desorption was achieved when the sample was washed with water after loading in 10% acetonitrile. The primary interaction with the binding sites in the imprints was most likely of ionic character. Quantification of the Pyr-Val adduct was performed with LC/ESI-MS/MS, yielding an instrumental LOD of 150 pg injected amount.

Keyword
Hemoglobin adducts, Diepoxybutane, Molecularly imprinted polymers, Solid-phase extraction
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-30127 (URN)10.1016/j.jchromb.2010.02.012 (DOI)000283410200006 ()
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2011-11-22Bibliographically approved

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