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LC–MS/MS Screening Strategy for Unknown Adducts to N-Terminal Valine in Hemoglobin Applied to Smokers and Nonsmokers
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
2014 (Engelska)Ingår i: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 27, nr 12, s. 2062-2070Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

Ort, förlag, år, upplaga, sidor
2014. Vol. 27, nr 12, s. 2062-2070
Nationell ämneskategori
Kemi
Forskningsämne
miljökemi
Identifikatorer
URN: urn:nbn:se:su:diva-112092DOI: 10.1021/tx5002749ISI: 000346543900008OAI: oai:DiVA.org:su-112092DiVA, id: diva2:778086
Tillgänglig från: 2015-01-09 Skapad: 2015-01-09 Senast uppdaterad: 2017-12-05Bibliografiskt granskad
Ingår i avhandling
1. Development of an adductomic approach to identify electrophiles in vivo through their hemoglobin adducts
Öppna denna publikation i ny flik eller fönster >>Development of an adductomic approach to identify electrophiles in vivo through their hemoglobin adducts
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Humans are exposed to electrophilically reactive compounds, both formed endogenously and from exogenous exposure. Such compounds could react and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA. The formation of adducts constitutes a risk for effects, such as cancer and contact allergy, and plays a role in ageing processes. Adducts to proteins offer a possibility to measure electrophilic compounds in vivo.

Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This thesis describes the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC/MS/MS).

The adductomic approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC/MS/MS. The adduct screening was performed by stepwise scanning of precursor ions in small mass increments and monitoring four fragments common for derivatives of detached Val adducts, in the multiple reaction monitoring mode. Samples from 12 smokers/nonsmokers were screened with the adductomic approach, and seven previously identified adducts and 19 unknown adducts were detected. A semiquantitative approach was applied for approximate quantification of adduct levels.

A strategy for identifying unknown Hb adducts using adductome LC/MS/MS data was formulated and applied for the identification of unknown adducts. Identifications were based on the observed m/z of precursor ions and retention times combined with databases and Log P calculations. Hypothesized adducts were generated in vitro for comparison and matching with the corresponding unknown adducts. Five identified adducts correspond to the precursor electrophiles ethyl vinyl ketone (EVK), glyoxal, methylglyoxal, acrylic acid, and 1-octen-3-one. These adducts, except the adducts corresponding to glyoxal and methylglyoxal, have not been observed as protein adducts before.  Probable exposure sources to these electrophiles are diet and/or endogenous formation. The observation of these adducts motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.

The adduct from EVK was quantitatively assessed through different experiments to estimate the daily internal dose (area under the concentration-time-curve, AUC). EVK is about 2 × 103 more reactive than the reference compound acrylamide. The EVK adduct was shown to be unstable, with a relatively short half-life. The daily AUC in humans of EVK was estimated to be about 20 times lower than the corresponding AUC of acrylamide from intake via food.

To confirm the observation of the detected unknown adducts and obtain a statistical foundation, analysis of unknown adducts were performed in large sets of blood samples (n = 50–120) from human cohorts. The majority of the previously detected unknown adducts were found in all analyzed samples, and the levels of many adducts showed large variations between individuals. The cause and significance of these observed variations are not yet clarified, but are of importance for the directions of future studies.

In conclusion, a new approach for identification of unknown human exposure to electrophiles was developed and successfully applied. 

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Environmental Science and Analytical Chemistry, 2016. s. 87
Nyckelord
Adductomics, Hemoglobin adducts, LC/MS/MS, Ethyl vinyl ketone, Glyoxal, Methylglyoxal, Acrylic acid, 1-Octen-3-one
Nationell ämneskategori
Annan kemi Analytisk kemi
Forskningsämne
miljökemi
Identifikatorer
urn:nbn:se:su:diva-129248 (URN)978-91-7649-348-9 (ISBN)
Disputation
2016-06-10, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Cancer- och AllergifondenVetenskapsrådet
Anmärkning

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.

Tillgänglig från: 2016-05-18 Skapad: 2016-04-18 Senast uppdaterad: 2017-02-17Bibliografiskt granskad

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Carlsson, Henrikvon Stedingk, HansNilsson, UlrikaTörnqvist, Margareta
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Institutionen för material- och miljökemi (MMK)Institutionen för analytisk kemi
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