TOXICOLOGICAL SCIENCES 116(1), 23–31 (2010)
Advance Access publication April 15, 2010
Detection of Adduct on Tyrosine 411 of Albumin in Humans Poisoned by
Bin Li,* Ivan Ricordel,† Lawrence M. Schopfer,* Fre ´de ´ric Baud,‡ Bruno Me ´garbane,‡ Florian Nachon,§ Patrick Masson,*,§ and
*Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950; †Laboratoire de toxicologie de la pre ´fecture de police, Institut
National de Police Scientifique, 75012 Paris, France; ‡Department de Toxicologie, Service de Re ´animation Me ´dicale et Toxicologique and Universite ´ Paris-
Diderot, Ho ˆpital Lariboisie ´re, 75010 Paris, France; and §Institut de Recherche Biome ´dicale des Arme ´es—Centre de Recherches du Service de Sante ´ des Arme ´es,
38702 La Tronche, France
1To whom correspondence should be addressed at Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950. Fax: (402) 559-4651.
Received March 13, 2010; accepted April 12, 2010
Studies in mice and guinea pigs have shown that albumin is
a new biomarker of organophosphorus toxicant (OP) and nerve
agent exposure. Our goal was to determine whether OP-labeled
albumin could be detected in the blood of humans exposed
to OP. Blood from four OP-exposed patients was prepared for
mass spectrometry analysis by digesting 0.010 ml of serum
with pepsin and purifying the labeled albumin peptide by offline
high performance liquid chromatography. Dimethoxyphosphate-
VRY411TKKVPQVSTPTL and LVRY411TKKVPQVSTPTL from
two patients who had attempted suicide with dichlorvos. The
butyrylcholinesterase activity in these serum samples was inhibited
80%. A third patient whose serum BChE activity was inhibited 8%
by accidental inhalation of dichlorvos had undetectable levels of
adduct on albumin. A fourth patient whose BChE activity was
inhibited 60% by exposure to chlorpyrifos had no detectable adduct
on albumin. This is the first report to demonstrate the presence of
OP-labeled albumin in human patients. It is concluded that tyrosine
411 of human albumin is covalently modified in the serum of
humans poisoned by dichlorvos and that the modification is
detectable by mass spectrometry. The special reactivity of tyrosine
411 with OP suggests that other proteins may also be modified on
tyrosine. Identification of other OP-modified proteins may lead to
an understanding of neurotoxic symptoms that appear long after
the initial OP exposure.
butyrylcholinesterase; mass spectrometry.
Organophosphorus toxicants (OP) are toxic chemicals used
in agriculture, medicine, and warfare. Their acute toxicity is due
to inhibition of acetylcholinesterase in the cholinergic nervous
system by covalent modification of the active site serine.
OP are also highly effective inhibitors of butyrylcholinesterase
(BChE, accession number gi:116353), though inhibition of
BChE has no known clinical sequelae. Carbamates also inhibit
cholinesterases by covalently binding to the active site serine.
Acylpeptide hydrolase in red blood cells is 10 times more
reactive with dichlorvos, chlorpyrifos oxon, and diisopropyl-
fluorophosphate than acetylcholinesterase and has the potential
to serve as a biomarker of OP exposure (Quistad et al., 2005;
Richards et al., 2000).
We have successfully used mass spectrometry to identify
OP, dichlorvos and chlorpyrifos, and with the carbamates,
carbofuran and Aldicarb (Li et al., 2009, 2010). To find these
peptides, we had to develop methods for the purification of
carbamate-labeled and OP-labeled BChE from 2 ml plasma or
identify the mass of the adduct on serine 198, and to deduce the
type of pesticide to which the patient was exposed.
The purpose of the present work was to determine whether
albumin is labeled in people who have been exposed to OP. Mice
(Peeples et al., 2005) and guinea pigs (Read et al., 2010; Williams
et al., 2007) treated with OP in vivo have covalently bound OP on
albumin. Mass spectrometry has identified tyrosine 411 of human
albumin (accession number gi:122920512) as the residue that
is covalently modified by dichlorvos, chlorpyrifos oxon,
diisopropylfluorophosphate, soman, sarin, and fluorophosphinate-
biotin when human albumin or plasma is treated ex vivo with
these OP (Li et al., 2007).
the rate of reaction of BChE with OP (Li et al., 2008). However,
there is a 10,000-fold higher concentration of albumin
(0.6mM; 40,000 mg/l) compared with BChE (50nM; 4 mg/l);
in human serum. This difference in concentration compen-
sates for the slow reactivity, resulting in OP labeling of albumin
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in vivo. The most reactive residue in human albumin is
tyrosine 411, though other tyrosines are also labeled when
conditions are maximized in vitro (Ding et al., 2008; Means and
Wu, 1979). Tyrosine 411 can be found in peptic peptides
VRY411TKKVPQVSTPTL and LVRY411TKKVPQVSTPTL,
which are related by a missed cleavage. The most reactive
residue in BChE is serine 198 in the tryptic peptide
The adducts on albumin and BChE after reaction with
dichlorvos and chlorpyrifos oxon are shown in Figure 1. OP-
albumin adducts are stable, whereas OP-BChE adducts lose an
first to identify OP-albumin adducts in humans exposed to OP.
MATERIAL AND METHODS
Pepsin (Sigma, St Louis, MO; catalog number P6887 from porcine gastric
mucosa) 1 mg/ml in 10mM HCl was stored at ?80?C. Alpha-cyano
4-hydroxycinnamic acid (CHCA; Sigma; catalog number 70990) was recrystal-
lized before use. A 10 mg/ml solution in 50% acetonitrile, 0.1% trifluoroacetic
acid was stored at room temperature in the dark. Purified human serum
albumin, essentially fatty acid free (Fluka via Sigma; catalog number 05418),
butyrylthiocholine (catalog number B3253), and dithiobisnitrobenzoic acid
(catalog number D8130) were from Sigma. Modified trypsin (Promega,
Madison, WI; catalog number V5113) 0.4 lg/ll in 50mM acetic acid was
stored at ?80?C. Chlorpyrifos oxon 98% pure and dichlorvos 98% pure were
from ChemService Inc. (West Chester, PA; catalog numbers MET-674B and
Human serum. Serum samples from three attempted suicides and one
accidentally poisoned individual were provided by Dr Ivan Ricordel, Paris
Police in a protocol approved by the Institutional Review Board of the
University of Nebraska Medical Center. The samples were shipped on dry ice
and stored at ?80?C. The name of the poison in three cases was established
from the reports of family members who found bottles of dichlorvos pesticide
in the home. The name of the poison in one case was unknown but was
identified as chlorpyrifos (Li et al., 2010).
The two patients who attempted suicide with dichlorvos had severe
cholinergic symptoms, including bilateral myosis and hypersalivation. One
patient was unconscious when admitted to the hospital. The other was drowsy
without coma. The patient who accidentally inhaled dichlorvos had a brief period
of respiratory difficulty and paresthesis in four limbs but no other symptoms.
Nothing is known about the patient who attempted suicide with chlorpyrifos.
BChE activity assay. BChE activity was measured with 1mM butyrylth-
iocholine in 0.1M potassium phosphate pH 7.0 in the presence of 0.5mM
dithiobisnitrobenzoic acid by measuring the increase in absorbance at 412 nm
at 25?C. The reaction rate in delta absorbance per minute was converted to
micromoles per minute using the extinction coefficient E ¼ 13600/M?cm
(Ellman et al., 1961). One unit of activity is defined as 1 lmol of substrate
hydrolyzed per minute.
Pepsin digestion to release labeled albumin peptide. From previous
work, it was known that tyrosine 411 of human albumin is labeled by OP and
that the labeled peptide is easily released by digestion with pepsin (Li et al.,
2007, 2008). It was also known that the labeled albumin peptide could be
detected in the mass spectrometer if the peptide was isolated by offline high
performance liquid chromatography (HPLC) before
tandem mass spectrometry (LCMSMS). A 0.01 ml aliquot of human serum was
adjusted to pH 2.5 by addition of 0.01 ml of 1% trifluoroacetic and digested
with 2.5 lg of pepsin for 2 h at 37?C.
Offline HPLC purification of the peptic albumin peptides from patient
samples. Peptides in the digested serum were purified by HPLC (Waters LC
625 system) on a Phenomenex Prodigy, 5 l C18 column 100 3 4.6mm eluted
with a 60-min gradient starting at 0.1% trifluoroacetic acid in water and ending
at 60% acetonitrile, 0.1% trifluoroacetic acid, at a flow rate of 1 ml/min. One-
milliliter fractions were collected. A 1 ll aliquot from each 1 ml fraction was
analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-
TOF) mass spectrometry to identify fractions containing the unlabeled albumin
LVRY411TKKVPQVSTPTL m/z 1830. When starting with 10 ll of serum
adding a mass of 108 amu, and, with chlorpyrifos oxon, adding a mass of 136 amu. Adducts on tyrosine do not age. Serine 198 of human BChE makes covalent
bonds with dichlorvos and chlorpyrifos oxon. The BChE adducts dealkylate during the aging process so that the added masses characteristically observed in
the mass spectrometer are 94 and 108 amu. The dimethoxyphosphate adduct on human BChE ages with a half-time of 3.9 h (Worek et al., 1999), whereas the
diethoxyphosphate adduct ages with a half-time of 11.6 h (Masson et al., 1997). The amino acid sequence of human BChE is in accession number gi:116353 in
the National Center for Biotechnology Information nonredundant database and that of human albumin in accession number gi:122920512.
Covalent binding of dichlorvos and chlorpyrifos oxon to albumin and BChE. Tyrosine 411 of human albumin makes a covalent bond with dichlorvos,
LI ET AL.
from a patient sample, the unlabeled peptides were detectable by MALDI-TOF
but the labeled peptides were present at too low a level to be detectable.
However, the elution position of the labeled peptides could be estimated from
previous studies. The unlabeled albumin tyrosine 411 peptides eluted between
19–24% acetonitrile, whereas labeled peptides eluted 1–8 min later. HPLC
fractions predicted to contain the labeled peptides were dried in a vacuum
centrifuge and dissolved in 50 ll of 5% of acetonitrile, 0.1% formic acid in
preparation for analysis on the QTRAP 4000 mass spectrometer.
Reaction of human plasma with chlorpyrifos oxon and dichlorvos. Human
plasma was treated with 0.1–1.5mM chlorpyrifos oxon or dichlorvos for 16 h
at 37?C. A 10 ll aliquot was mixed with 10 ll of 1% trifluoroacetic acid before
digestion with 1 ll of 1 mg/ml pepsin. After 2-h incubation at 37?C, the digest
was diluted 400-fold with 0.1% trifluoroacetic acid and 1 ll was spotted on
a matrix assisted laser desorption ionization (MALDI) plate.
The labeled and unlabeled albumin tyrosine 411 peptides were identified in
the same MALDI-TOF mass spectrum. Unlabeled peptides had masses of 1717
and 1830 amu. Chlorpyrifos oxon–labeled peptides had masses of 1853 and
1966 amu. Dichlorvos-labeled peptides had masses of 1825 and 1938 amu.
Relative quantities of labeled and unlabeled peptides were calculated by
comparison of cluster areas using the Data Explorer software. This method of
quantitation assumes that the labeled and unlabeled peptides ionize with similar
efficiencies. Each sample serves as its own internal control because the peptides
are in the same MALDI spot in the same MALDI spectrum.
MALDI-TOF mass spectrometry. A 1 ll aliquot of essentially salt-free
sample was spotted onto a 384 well Opti-TOF sample plate (#1016491; Applied
Biosystems, Foster City, CA). After the spot was dry, it was overlaid with 1
ll of CHCA matrix. Mass spectrometry spectra were acquired with a matrix
assisted laser desorption ionization tandem time of flight 4800 mass
spectrometer (Applied Biosystems), in positive reflector mode, with laser
intensity at 4000 V, using delayed extraction, and default calibration. Spectra
were saved to Data Explorer V4.9 software for analysis. Each spectrum was
the sum of 500 laser shots. The mass spectrometer was calibrated with Cal
Mix 5 (bradykinin, 2–9 clip; angiotensin I; Glu-fibrinopeptide B;
adrenocorticotropic hormone [ACTH], 1–17 clip; ACTH, 18–39 clip; and
ACTH, 7–38 clip from Applied Biosystems Inc., Framingham, MA).
Multiple reaction monitoring on the QTRAP 4000 mass spectrometer
(Applied Biosystems). Five microliter aliquots from selected fractions of
HPLC-purifiedpeptic peptides frompatientserawereinjected ontoa VydacC18
polymeric reverse-phase nanocolumn for a second phase of HPLC separation.
Peptides were separated on a HPLC nanocolumn (#218MS3.07515 Vydac C18
polymeric rev-phase, 75 lm i.d. 3 150 mm long; P.J. Cobert Assoc, St Louis,
MO) with a 90 min linear gradient from 0 to 60% acetonitrile, 0.1% formic acid,
at 300 nl/min, and electrosprayed through a fused silica emitter (360 lm o.d., 75
lm i.d., 15 lm taper, New Objective) directly into the QTRAP 4000, a hybrid
quadrupole linear ion trap mass spectrometer. The mass spectrometer was
calibrated on selected fragments from the tandem mass spectrometry (MSMS)
spectrum of Glu-Fibrinopeptide B. The MSMS data were collected and
processed using Analyst 1.4.1 software (Applied Biosystems).
The MRM algorithm screens ions entering the mass spectrometer for selected
parent ion masses, fragments the selected parent ions when they appear, and
examines the fragments for selected product ions. A signal is recorded when both
the correct parent and the product ions are observed. Preliminary experiments
showed that triply charged parent ions gave better signals in the QTRAP mass
a strong product ion fragment was observed at 748.8 amu (b12)þ2from parent
peptide VRY411TKKVPQVSTPTL, [M þ 3H]þ3¼ 609.4 amu. A strong product
ion wasobservedat805.3 amu
LVRY411TKKVPQVSTPTL, [M þ 3H]þ3¼ 647.4 amu. These parent/product
ion pairs were used in the MRM experiments on dichlorvos-labeled samples. The
dwell time for collecting the MRM signals was 40 ms, collision energy was 30 V,
and collision gas was pure nitrogen (40 lTorr). Data were collected using an
Information Directed Acquisition protocol that triggered the collection of an
from parent peptide
enhanced product ion spectrum (MSMS) following the detection of a peptide of
interest by the MRM algorithm. The enhanced product ion spectrum was taken
usingthetrap functionofthe QTRAP mass spectrometer. Collisionenergywas30
V, collision gas was pure nitrogen (40 lTorr), scan rate was 4000 Da/s, and 10
enhanced product ion scans were summed for each spectrum.
LCMSMS on the QTRAP 4000 mass spectrometer. Samples that gave
a positive result in the MRM method were also analyzed by LCMSMS. In
addition, the LCMSMS method was used to test unfractionated pepsin-digested
serum. The digested serum was diluted 2000-fold with 5% acetonitrile, 0.1%
formic acid to reduce the protein concentration to 0.5 lg/ll (~7 pmol albumin
per ll). Protein concentration was estimated from absorbance at 280 nm in the
NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE) relative to
albumin standards. Five microliter of the diluted digest were separated on an
HPLC nanocolumn and electrosprayed into the QTRAP 4000 mass
spectrometer as described for the MRM experiment.
An information directed acquisition protocol triggered the collection of an
enhanced resolution spectrum and an enhanced product ion spectrum on the
four most intense ions entering the mass spectrometer having an m/z between
200 and 1500, a charge state of þ2 to þ4 and an intensity greater than
500,000 counts per second. After an ion was analyzed twice, it was excluded
from analysis for 60 s. Collision energy was determined by the mass
spectrometer based on mass and charge state of the ion. Collision gas was pure
nitrogen (40 lTorr), and the scan rate was 4000 Da/s. Because of the time
required to obtain this series of scans, only one enhanced product ion scan was
taken for each ion. An ion spray voltage of 1900 V was maintained between the
emitter and the mass spectrometer. The mass spectrometer was calibrated using
MSMS fragments of Glu-Fibrinopeptide B.
The MSMS data files were searched for spectra that included a singly
charged mass of 330 amu. The 330 amu mass is the y3 ion in both labeled and
unlabeled albumin peptides VRYTKKVPQVSTPTL and LVRYTKKVPQVST
PTL. The extracted ion chromatogram feature of the Analyst software (version
1.4) was used for the search. MSMS spectra were accepted if the other masses
in the spectra matched the fragment masses for the albumin peptides. Predicted
fragment masses, for comparison with the observed fragment masses, were
calculated with the aid of the fragment ion calculator in the Proteomics Toolkit
Infusion into the QTRAP 4000 mass spectrometer. The MRM and
LCMSMSprotocols proved that
LVRYTKKVPQVSTPTL peptides had a mass 108 amu higher than the mass of
that proved the adduct was on tyrosine 411. To obtain proof that the reaction of
dichlorvos with albumin resulted in covalent modification of tyrosine 411, we
pH 8.5 with 1.5mM dichlorvos overnight. The albumin was digested with pepsin
and the peptides purified by offline HPLC. Purified peptides were dried and
infusion. Samples were infused into the QTRAP mass spectrometer because
infusion allows one to sum hundreds of MSMS spectra into one final spectrum,
whereas the MRM method sums ten spectra and the LCMSMS method only one
MSMS spectrum. The improved signal-to-noise ratio after summing 500 MSMS
spectra in the infusion method reveals low-intensity ions.
consistent with those expected for dichlorvos-labeled peptic peptides were then
fragmented in the mass spectrometer. Collision gas was nitrogen (40 lTorr),
rate was 4000 Da/s. The final spectrum was the sum of 500 MSMS spectra.
MRM for Detection of Dichlorvos-Albumin Adducts
Use of the MRM feature of the mass spectrometer requires
one to know the masses of the parent and product ions that best
DICHLORVOS ADDUCT ON ALBUMIN IN HUMANS
indicate the presence of the desired peptide. This information
was obtained from MSMS spectra of dichlorvos-labeled pure
human albumin peptic peptides. The MSMS spectra acquired
in the QTRAP 4000 mass spectrometer showed that the parent
ions were most readily detectable in the triply charged state.
The b12 product ion (also called transition and daughter ion) in
charge state þ 2 was intense for the OP-labeled VRYT
KKVPQVSTPTL peptide. The b13 product ion in charge state
þ 2 was intense for the OP-labeled LVRYTKKVPQVSTPTL
peptide. Therefore, the triply charged parent ion masses and the
doubly charged product ion masses listed in Table 1 were
selected for MRM.
Sera from Dichlorvos-Poisoned Patients Contain Dichlorvos
Adducts on Tyrosine 411 of Albumin
Albumin peptides partially purified from 0.010 ml patient
serum were subjected to reverse-phase liquid chromatography
followed by electrospray ionization and fragmentation in
the triple quadrupole linear ion trap mass spectrometer. The
mass spectrometer was programmed to search for the ion
partners listed in Table 1. An example of an MRM hit is given
in Figure 2, where two parent ions coeluted from the
nanocolumn at the same time. Parent ion 609.4 m/z had
product ion 748.8 m/z. Parent ion 647.4 had product ion 805.3.
A positive MRM signal does not prove that the ion represents
the peptide of interest. Proof comes from the MSMS spectrum,
which is automatically acquired following the appearance of an
The MSMS spectrum for the triply charged parent ion at
609.4 m/z is given in Figure 3A. The mass of the parent ion is
consistent with the peptide VRYTKKVPQVSTPTL plus an
added mass from dichlorvos of 108 amu. The y and b ion series
fit the predicted peptide sequence and appear with approxi-
mately the same relative intensities as the ions from a control
sample of dichlorvos-labeled peptide (Figure 3B). These
observations provide strong evidence that the 609.4 amu mass
is dichlorvos-labeled albumin peptide VRYTKKVPQVSTPTL.
There is no direct proof in this spectrum that the
dimethoxyphosphate is attached to tyrosine because there are
no signals for the b2 and b3 ions that define tyrosine 411.
However, the masses of the remainder of the b ions in the
spectrum (b7), (b10), (b11)þ2, (b12)þ2, and (b14)þ2are all
consistent with the presence of the label. Therefore, the labeled
residue must reside on the VRYTKKVP portion of the peptide.
There are four nucleophilic residues in that portion of the
peptide that could theoretically react with dichlorvos: tyrosine,
threonine, and two lysines. The interpretation that the OP is
covalently bound to tyrosine relies on comparison with the
MSMS spectrum of pure albumin treated with dichlorvos
where the b2 ion at 256.2 amu carries no label but the b3 ion at
527.5 amu has a mass consistent with dimethoxyphosphor-
ylation of tyrosine (Figure 3B).
The MSMS spectrum for the second dichlorvos-labeled
peptide isolated from a poisoned patient is given in Figure 4.
The triply charged parent ion has a mass of 647.4 m/z,
consistent with the peptide LVRYTKKVPQVSTPTL plus an
added mass of 108 from dichlorvos. The y and b ion series fit
the predicted peptide sequence. The masses of all observed
b ions are consistent with an added mass of 108 amu on
The patient samples were reanalyzed by LCMSMS without
using the MRM feature of the QTRAP 4000. The output
mass spectrometry spectra were manually searched for
the dichlorvos-labeled parent ions 609.4 and 647.4 m/z. Both
parent ions were found. They eluted from the nanocolumn at
about 40 min. The MSMS spectra acquired from LCMSMS
analysis supported an added mass of 108 on peptides
VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. How-
ever, peak intensities were lower than MSMS spectra acquired
MRM Transitions for the Albumin Peptides
added mass þ 108
added mass þ 108
charge þ 3 m/z
Product ion b12,
charge þ 2 m/z
charge þ 3 m/z
Product ion b13,
charge þ 2 m/z
Note. Average masses are listed. The accession number for human albumin
in the NCBI nonredundant database is gi:122920512.
parent ions eluted at the same time from the nanocolumn. The triply charged
parent ion at 609.4 m/z has a product ion at 748.8 m/z. The triply charged parent
ion at 647.4 m/z has a product ion at 805.3 m/z. Q1 is the mass of the parent ion.
Q3 is the mass of the product ion. MRM spectra, such as this, for dichlorvos-
labeled albumin were obtained from the blood of two humans poisoned by
MRM transitions for dichlorvos-labeled albumin peptides. Two
LI ET AL.
by MRM or by infusion. Peak intensities were 4 e4 for
unfractionated digest analyzed by LCMSMS, 1.5 e5 for HPLC-
purified digest analyzed by LCMSMS, 1.3 e6 and 1 e7 for
HPLC-purified digest analyzed by MRM, and 3 e8 for infused
sample. The difference in signal intensity is the result of being
able to collect only one scan per MSMS spectrum in the
LCMSMS protocol, as compared with 10 scans per MSMS
spectrum in the MRM protocol and 500 scans per MSMS
spectrum in the infusion protocol.
The Chlorpyrifos-Poisoned Patient
The pesticide sold for agricultural use is chlorpyrifos.
Patients exposed to chlorpyrifos are analyzed for adducts from
chlorpyrifos oxon because poison symptoms are caused by the
oxon. Chlorpyrifos is a precursor of the active metabolite
chlorpyrifos oxon. Chlorpyrifos undergoes oxidative desulfur-
ation by liver cytochrome P450 enzymes to become the highly
toxic chlorpyrifos oxon (Sams et al., 2004; Tang et al., 2001).
HPLC-purified peptides from a pepsin digest of serum from
the patient poisoned with chlorpyrifos were analyzed on the
QTRAP using LCMSMS and infusion methods. The diethox-
yphosphate adduct on albumin was not observed.
In an effort to understand why no diethoxyphosphate
albumin adducts were detected, the relative reactivity of
albumin with dichlorvos and chlorpyrifos oxon was examined.
Plasma was incubated with various concentrations of chlorpyr-
ifos oxon and dichlorvos for 16 h at 37?C. Cluster areas of
labeled and unlabeled peptides observed in MALDI-TOF mass
spectra were used to calculate percent labeling of tyrosine 411
(see the ‘‘Materials and Methods’’ section on ‘‘Reaction of
human plasma with chlorpyrifos oxon and dichlorvos’’ for
quantitation details). Figure 5 shows that 60% of the tyrosine
411 residues in albumin were labeled when human plasma was
treated with 0.2mM dichlorvos. In contrast, less than 1%
labeling occurred with 0.2mM chlorpyrifos oxon. It was not
until 1.5mM chlorpyrifos oxon was used in the reaction that
a significant amount of albumin adduct was observed (about
30%). Thus, it appears that chlorpyrifos oxon is substantially
less reactive than dichlorvos toward albumin. The decreased
reactivity of chlorpyrifos oxon indicates that a lower fraction of
labeled albumin would be expected from sera of chlorpyrifos
oxon–poisoned individuals. This, in turn, argues that a volume
of plasma larger than the 0.010 ml used in the present study
TKKVPQVSTPTL where dimethoxyphosphate is on tyrosine 411. (A) MSMS
spectrum of the dichlorvos-labeled albumin peptide from the serum of a
patient poisoned with dichlorvos. The spectrum was acquired in conjunction
with the MRM method and is the sum of 10 MSMS spectra. The triply
charged parent ion shows an m/z of 609.5 in this spectrum. (B) MSMS
spectrum of the dichlorvos-labeled peptide prepared by in vitro treatment of
pure human albumin with dichlorvos. The spectrum is the sum of 500 MSMS
spectra acquired during infusion of purified peptides. The mass at 226.2 m/z
is the dimethoxyphosphotyrosine immonium ion minus water. Masses are
MSMS spectra of dichlorvos-labeled albumin peptide VRY411
peptide, LVRY411TKKVPQVSTPTL. Serum from a patient poisoned with
dichlorvos yielded the peptide modified on tyrosine 411 by dimethoxyphos-
phate. The triply charged parent ion has m/z 647.3.
MRM triggered MSMS spectrum of the dichlorvos-labeled albumin
DICHLORVOS ADDUCT ON ALBUMIN IN HUMANS
would need to be analyzed to detect the low level of albumin
expected to have been modified by chlorpyrifos oxon.
Comparison of Peptide Analyses for BChE and Albumin from
Dichlorvos- and Chlorpyrifos-Poisoned Patients
Sera from the group of patients described in this report were
previously analyzed for BChE adducts (Li et al., 2010). In
those experiments, partially purified BChE was digested with
trypsin and analyzed by mass spectrometry. Adducts on serine
198 were found. Table 2 summarizes the results for adducts on
both BChE and albumin from the sera of four poisoned
patients. The three patients who attempted suicide by drinking
pesticides have low serum BChE activity, with inhibition levels
from 62 to 84%. BChE in the accidental exposure case was
only slightly inhibited (8%). OP adducts on BChE were found
for all three samples where BChE inhibition was high but not
for the sample where BChE inhibition was low.
who ingested a relatively large dose of dichlorvos, based on
on albumin for the patient whose BChE was inhibited 8%. No
chlorpyrifos oxon adduct was found on albumin for the patient
whose BChE was inhibited 62%. The absence of a detectable
(1) Chlorpyrifos oxon added to plasma is less reactive with
hydrolyzed by paraoxonase in plasma and is sequestered by
albumin in noncovalent binding sites from which it can be
extracted with pentane (Eyer et al., 2009; Heilmair et al., 2008).
This diminishes theconcentration of chlorpyrifos oxonavailable
for covalent reaction with tyrosine 411 of albumin. (3) The
chlorpyrifos poison ingested by patients is converted to the toxic
oxon by hepatic cytochrome P450 enzymes. There is a 10-fold
variability in the efficiency of this step, as shown in studies that
measured plasma levels of both the chlorpyrifos and the oxon
(Eyer et al., 2009). Patients with high CYP2B6 and CYP3A4
levelswouldproduce more of theoxon and therefore mighthave
detectable levels of OP-albumin adducts in 0.01 ml plasma.
However, detection of albumin adducts in other patients is
expected to require plasma volumes larger than 0.01 ml.
OP-Albumin Adduct in Poisoned Humans
This is the first report to identify OP-albumin adducts in
humans poisoned by OP. The amino acid modified by covalent
attachment of dichlorvos is tyrosine 411. In previous work, we
have found that many proteins can be modified by OP on
tyrosine and that the OP-tyrosine adduct is stable and does not
undergo aging (Li et al., 2008; Schopfer et al., 2010).
Identification of the dichlorvos-albumin adduct in human
serum required only 0.010 ml of serum because the exposure
levels were high, as indicated by plasma BChE inhibition
levels of 80%. It is anticipated that detection of OP-albumin
adducts in people exposed to low doses will also be possible
but that larger volumes of plasma will need to be processed
in preparation for analysis by mass spectrometry. OP-albumin
adducts have been found in the plasma of guinea pigs treated
in vivo with the nerve agents tabun, soman, sarin, and
cyclosarin (Read et al., 2010; Williams et al., 2007).
OP-Albumin as a Biomarker of Exposure
Butyrylcholinesterase in human plasma reacts rapidly with
a wide range of OP. Upon reaction with OP, the enzymatic
chlorpyrifos oxon. Percent labeling was calculated from cluster areas observed
in the MALDI-TOF mass spectrometer of pepsin-digested plasma that had
been treated with OP. The unlabeled peptides VRYTKKVPQVSTPTL and
LVRYTKKVPQVSTPTL have masses of 1717 and 1830 amu. The dichlorvos-
labeled peptides have masses of 1825 and 1938 amu. The chlorpyrifos oxon–
labeled peptides have masses of 1853 and 1966 amu.
Reactivity of tyrosine 411 in human albumin with dichlorvos and
Detection of OP Adducts on BChE and Albumin in
and blood draw
of BChE %
None2.50 0No No
aChlorpyrifos oxon was identified by mass spectrometry of the BChE adduct
in a sample for which the poison was originally unknown. Results for BChE
have been previously reported (Li et al., 2010). Mass spectrometry analysis
used 2 ml serum for detection of BChE adducts and 0.010 ml serum for
detection of albumin adducts. The concentration of BChE in human plasma is
4 mg/l, whereas that of albumin is 40,000 mg/l. This difference in protein
abundance explains the necessity of using a larger plasma sample for detection
of BChE adducts.
LI ET AL.
activity of BChE is inhibited. Loss of activity has long served
as an indicator of OP exposure (Eddleston et al., 2008; Namba
et al., 1971). However, use of BChE activity as a biomarker for
OP exposure has major drawbacks. (1) The normal activity of
BChE can vary widely so that only severe exposure can be
confidently diagnosed (Eddleston et al., 2008; Kalow and
Staron, 1957). (2) Hepatocarcinoma and malnutrition can cause
depression of BChE activity (Whittaker, 1980). (3) Other
compounds, such as carbamates, can inhibit BChE activity (Li
et al., 2009) so that the true identity of the inhibitor is always in
question. Consequently, efforts have shifted to the application
of mass spectrometry for identification of the actual adducts
formed upon reaction of BChE with inhibitors (Fidder et al.,
2002; Li et al., 2009). Mass spectrometry provides a direct
measure of the nature of the inhibitor attached to BChE.
However, analysis of OP adducts on BChE as biomarkers for
OP exposure also has drawbacks. The principal problem is that
OP adducts on the active site serine of BChE are unstable.
Instability arises from two sources. (1) Treatment with oximes
releases the OP from serine, and oxime treatment is part of the
normal medical response to OP poisoning. (2) Spontaneous
dealkylation of the OP on BChE occurs via the aging process
(Masson and Lockridge, 2010). Dealkylation reduces the
information content of the resulting adduct. For example,
aging converts adducts with soman, sarin, and cyclosarin
to identical methylphosphonate structures, as indicated in
Figure 6. The products of the aging of dichlorvos and
chlorpyrifos oxon adducts on BChE are illustrated in Figure 1.
To circumvent these drawbacks, new OP targets have been
sought. Serum albumin is a promising candidate (Li et al.,
2007, 2008; Means and Wu, 1979; Noort et al., 2009;
Ortigoza-Ferado et al., 1984; Read et al., 2010; Sogorb
et al., 2008; Tarhoni et al., 2008; Williams et al., 2007; Yeung
et al., 2008).
The OP-albumin adduct has several advantages as a bio-
marker of OP exposure. (1) OP binds to tyrosine 411 on
albumin, and tyrosine adducts are not reversed by oximes
(Read et al., 2010). (2) OP-tyrosine adducts do not age (Li
et al., 2007; Williams et al., 2007). This means that exposure to
soman can be distinguished from exposure to sarin and to
cyclosarin. (3) The OP-albumin adduct persists longer in the
blood than the OP-BChE adduct. Studies in guinea pigs treated
with nerve agents found OP-tyrosine adducts 24 days after
exposure to OP, at a time when OP-BChE adducts were
undetectable (Read et al., 2010)
The major drawback for use of albumin-OP adducts as
a biomarker for exposure to OP is the slow reaction of OP with
albumin (Li et al., 2008). However, despite the low reactivity,
spectral techniques, as has been demonstrated in this report.
The principal advantage of working with albumin is the
stability of its OP-tyrosine adduct. Another significant factor is
that the OP adduct is located on the surface of albumin, as
opposed to the adduct on BChE that is located at the bottom of
a deep pocket in the protein (Nachon et al., 2005). A novel
outgrowth of the stability and accessibility of the albumin
adduct is the potential to develop antibodies to OP-tyrosine.
Such antibodies could be used for detection of OP exposure.
Hypothesis to Explain Neurotoxicity because of Chronic Low-
Dose Exposure to OP
It is generally agreed that acute toxicity because of exposure
to OP comes from inhibition of acetylcholinesterase in the
synapses and nerve muscle junctions (Mileson et al., 1998).
However, inhibition of acetylcholinesterase does not explain all
of the clinical sequelae that arise from exposure to OP,
especially low-dose exposure. It has been proposed that excess
acetylcholine or inhibition of serine hydrolases with greater OP
reactivity than acetylcholinesterase may explain low-dose
neurotoxicity (Pernot et al., 2009; Richards et al., 2000).
Symptoms of low-dose toxicity are neurological in nature (e.g.,
headache, memory loss, anxiety, fatigue) (Ray and Richards,
2001; Salvi et al., 2003). Investigation into the causes of this
neurotoxicity is ongoing.
OP binding to albumin would not be expected to explain the
neurotoxicity of OP but can serve as a model for what could be
happening to other proteins. The special reactivity of tyrosine
411 in human albumin suggests that other proteins may have
similarly reactive tyrosine residues. In fact, we have demon-
strated that OP react with tyrosine on tubulin and that this
reaction can disrupt the structure of microtubules in vitro and
in vivo (Grigoryan and Lockridge, 2009; Jiang et al., 2010). If
the function of key proteins important for axonal transport
(such as tubulin) is disrupted by OP, the neuron could lose
synaptic connectivity and nerve function (Gearhart et al., 2007;
Terry et al., 2007).
The reactions of soman, sarin, and cyclosarin with serine 198 of BChE yield
distinct initial covalent adducts with added masses of þ 162 for soman, þ 120
for sarin, and þ 160 for cyclosarin. These adducts age to the identical
methylphosphonate structure with an added mass of þ 78.
Aging yields the identical methylphosphonate adduct on BChE.
DICHLORVOS ADDUCT ON ALBUMIN IN HUMANS
Epidemiologists have linked chronic low-dose OP exposure
to Parkinson’s disease, neurologic dysfunction, Gulf War
illness, and depression (Beseler et al., 2008; Hancock et al.,
2008; Kamel et al., 2007; Toomey et al., 2009). Disruption of
axonal transport has been suggested as the mechanism to
explain neurodegenerative diseases, including Parkinson’s,
Alzheimer disease, and amyotrophic lateral sclerosis (Morfini
et al., 2009). Our finding that tyrosine in albumin is covalently
labeled by OP in clinically relevant human cases suggests that
OP labeling of tyrosine in other proteins may also be occurring
under these conditions. This concept provides a new direction
in the search for a mechanism of OP-induced chronic
The U.S. Army Medical Research and Materiel Command
(W81XWH-07-2-0034 to O.L.); the National Institutes of
Health (U01 NS058056 to O.L.) and CA36727; De ´le ´gation
Ge ´ne ´rale pour l’Armement (DGA/PEA 03CO10-05/01 08 7 to
P.M.) (DGA/PEA 08CO501 to F.N.); Agence Nationale de la
Recherche (ANR-06-BLAN-0163 and ANR-09-BLAN-0192
Mass spectra were obtained with the support of the Mass
Spectrometry and Proteomics core facility at the University of
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