3 research outputs found
LC–MS/MS Screening Strategy for Unknown Adducts to N‑Terminal Valine in Hemoglobin Applied to Smokers and Nonsmokers
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 <i>in vivo</i> 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, FI<i>R</i>E, 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 <i>a priori</i> 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 (<i>m</i>/<i>z</i> 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
Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples
Electrophiles
have the ability to form adducts to nucleophilic
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 <i>p</i>-nitroanilide
(Val-<i>p</i>NA), 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<sup>–1</sup>h<sup>–1</sup> at 37 °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 × 10<sup>3</sup> 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, <i>AUC</i>) of 7 nMh EVK was estimated. The <i>AUC</i> 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
Adductomic Screening of Hemoglobin Adducts and Monitoring of Micronuclei in School-Age Children
Electrophilic compounds/metabolites
present in humans, originating
from endogenous processes or exogenous exposure, pose a risk to health
effects through their reactions with nucleophilic sites in proteins
and DNA, forming adducts. Adductomic approaches are developed to screen
for adducts to biomacromolecules in vivo by mass spectrometry (MS),
with the aim to detect adducts corresponding to unknown exposures
from electrophiles. In the present study, adductomic screening was
performed using blood samples from healthy children about 12 years
old (<i>n</i> = 51). The frequencies of micronuclei (MN)
in erythrocytes in peripheral blood were monitored as a measure of
genotoxic effect/genotoxic exposure. The applied adductomic approach
has been reported earlier by us and is based on analysis of N-terminal
valine adducts in hemoglobin (Hb) by liquid chromatography tandem
mass spectrometry (LC-MS/MS). High resolution MS was introduced for
refined screening of previously unknown N-terminal Hb adducts. Measured
adduct levels were compared with MN frequencies using multivariate
data analysis. In the 51 individuals, a total of 24 adducts (whereof
12 were previously identified) were observed and their levels quantified.
Relatively large interindividual variations in adduct levels were
observed. The data analysis (with partial least-squares regression)
showed that as much as 60% of the MN variation could be explained
by the adduct levels. This study, for the first time, applies the
combination of these sensitive methods to measure the internal dose
of potentially genotoxic chemicals and genotoxic effects, respectively.
The results indicate that this is a valuable approach for the characterization
of exposure to chemical risk factors for the genotoxic effects present
in individuals of the general population