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Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models
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).
Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
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2014 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 5, 1519-1530 p.Article in journal (Refereed) Published
Abstract [en]

Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

Place, publisher, year, edition, pages
2014. Vol. 406, no 5, 1519-1530 p.
Keyword [en]
Bulky serum albumin adducts, Polycyclic aromatic hydrocarbons, Extraction (SPE vertical bar HPLC vertical bar butanol), Diol epoxides, Mass Spectrometry, Hydrolysis (pronase enzymatic vertical bar alkaline)
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-102079DOI: 10.1007/s00216-013-7540-7ISI: 000331004700023OAI: oai:DiVA.org:su-102079DiVA: diva2:708425
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Note

AuthorCount:7;

Funding agencies:

European Commission, QLK4-CT-2002-02402; Swedish Research Council FORMAS;    Swedish Cancer and Allergy Foundation 

Available from: 2014-03-27 Created: 2014-03-26 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Biomarkers of internal exposure/dose: Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates
Open this publication in new window or tab >>Biomarkers of internal exposure/dose: Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation.

The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established.

In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation.

An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment. 

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry, Stockholm University, 2015. 66 p.
Keyword
biomarker, PAH, serum albumin, adduct, DNA, isocyanate
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-110490 (URN)978-91-7649-074-7 (ISBN)
Public defence
2015-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2015-01-07 Created: 2014-12-15 Last updated: 2015-08-17Bibliographically approved

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Westberg, EmelieAlsberg, TomasEriksson, JohanTörnqvist, Margareta
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