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A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC-MS/MS
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, 2483-2490 p.Article in journal (Refereed) Published
Abstract [en]

A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the "adduct FIRE procedure" as the FITC reagent is used for measurement of adducts ((R) under bar) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamicle, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC-MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure (TM), resulted in a procedure in which low background levels of the studied adducts could be measured from 250 mu L lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC-MS/MS with LOQ clown to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.

Place, publisher, year, edition, pages
2010. Vol. 878, no 27, 2483-2490 p.
Keyword [en]
Hemoglobin adducts, Acrylamide, Ethylene oxide, Modified Edman, Adduct FIRE procedure, LC-MS/MS
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
URN: urn:nbn:se:su:diva-51442DOI: 10.1016/j.jchromb.2010.03.034ISI: 000283410200004OAI: oai:DiVA.org:su-51442DiVA: diva2:385679
Note
authorCount :3Available from: 2011-01-12 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
In thesis
1. 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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