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Methodology for hemoglobin adduct measurement: Fetal exposures to acrylamide and other genotoxic agents
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
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: urn:nbn:se:su:diva-63026ISBN: 978-91-7447-376-6 (print)OAI: oai:DiVA.org:su-63026DiVA: diva2:446413
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
List of papers
1. LC/MS/MS analysis of N-terminal protein adducts with improved sensitivity: A comparison of selected Edman isothiocyanate reagents.
Open this publication in new window or tab >>LC/MS/MS analysis of N-terminal protein adducts with improved sensitivity: A comparison of selected Edman isothiocyanate reagents.
2009 (English)In: International Journal of Analytical Chemistry, ISSN 1687-8779Article in journal (Refereed) Published
Abstract [en]

This study provides a basis for a new and straightforward method for LC/MS/MS-based screening of N-terminal protein adducts. This procedure is denoted the “FIRE procedure” as fluorescein isothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts (R) of electrophilic compounds with a modified Edman procedure. The principles of the FIRE-procedure are that adducts to N-terminal amino acids selectively are detached and measured from of proteins after derivatisation by isothiocyanate Edman reagents. In this study, FITC, 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesize thiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhanced by up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared to pentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low background adduct levels. Synthesized analytes were characterised with, for example, 1H NMR, 13C NMR, LC/MS/MS, and UV.

National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-35829 (URN)10.1155/2009/153472 (DOI)
Note
Article ID 153472.Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2011-10-10Bibliographically approved
2. A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC-MS/MS
Open this publication in new window or tab >>A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC-MS/MS
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.

Keyword
Hemoglobin adducts, Acrylamide, Ethylene oxide, Modified Edman, Adduct FIRE procedure, LC-MS/MS
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-51442 (URN)10.1016/j.jchromb.2010.03.034 (DOI)000283410200004 ()
Note
authorCount :3Available from: 2011-01-12 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
3. Analysis of hemoglobin adducts from acrylamide, glycidamide and ethylene oxide in paired mother/cordblood samples from Denmark
Open this publication in new window or tab >>Analysis of hemoglobin adducts from acrylamide, glycidamide and ethylene oxide in paired mother/cordblood samples from Denmark
Show others...
2011 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 24, no 11, 1957-1965 p.Article in journal (Refereed) Published
Abstract [en]

The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography–mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27–0.86) for acrylamide, 0.38 (range 0.20–0.73) for glycidamide, and 0.43 (range 0.17–1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38–0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large population studies.

National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-62978 (URN)10.1021/tx200284u (DOI)000297082500020 ()
Available from: 2011-10-07 Created: 2011-10-06 Last updated: 2017-12-08Bibliographically approved
4. 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: 2017-12-13Bibliographically approved

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