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Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Antal upphovsmän: 42015 (Engelska)Ingår i: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 28, nr 11, s. 2120-2129Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Electrophiles have the ability to form adducts to nudeophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 +/- 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using L-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 degrees C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life <1 min. Parallel experiments with AA and MVK showed that the two vinyl ketones react approximately 2 x 10(3) times faster than AA. The EVK-Hb adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening.

Ort, förlag, år, upplaga, sidor
2015. Vol. 28, nr 11, s. 2120-2129
Nationell ämneskategori
Kemi Farmakologi och toxikologi
Forskningsämne
miljökemi
Identifikatorer
URN: urn:nbn:se:su:diva-125010DOI: 10.1021/acs.chemrestox.5b00287ISI: 000364980800008PubMedID: 26447499OAI: oai:DiVA.org:su-125010DiVA, id: diva2:893392
Tillgänglig från: 2016-01-12 Skapad: 2016-01-07 Senast uppdaterad: 2018-01-10Bibliografiskt 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, HenrikMotwani, Hitesh V.Osterman Golkar, SivTörnqvist, Margareta
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