4-Hydroxy-1-(3-pyridyl)-1-butanone-hemoglobin adducts as biomarkers of exposure to tobacco smoke: validation of a method to be used in multicenter studies.

Abstract

Hemoglobin (Hb) adducts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), a metabolite of two tobacco-specific nitrosamines [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine], were measured as biomarkers of exposure to tobacco smoke as part of a study on genetic alterations and susceptibility to lung cancer among nonsmokers. HPB-Hb adducts were measured after collection of RBCs by Ficoll gradient in six collaborating centers, release of HPB by alkaline hydrolysis from Hb, clean-up by solid-phase extraction, and analysis of an electron-capturing derivative by gas chromatography-electron capture mass spectrometry. Prior to analysis of samples from study subjects, the reproducibility of this approach was validated in blood from donors. The coefficient of variation of reproducibility of paired aliquots from five samples ranged from 7 to 25%; the within-sample reproducibilities of four and eight aliquots were 4 and 16%, respectively. The study subjects consisted of 18 smokers and 52 never-smokers. HPB-Hb adduct levels were significantly higher (P = 0.02) in smokers (26 +/- 13 fmol HPB/g Hb) than in never-smokers (20 +/- 8 fmol HPB/g Hb). There was no difference between sexes. These results suggest that the level of HPB-Hb adducts, measured using a method modified to facilitate use in multicenter studies, can be a useful biomarker of exposure to tobacco smoke.

Full text

Page 1

1998;7:817-821. Published online September 1, 1998.Cancer Epidemiol Biomarkers Prev


S E Atawodi, S Lea, F Nyberg, et al.
method to be used in multicenter studies.
biomarkers of exposure to tobacco smoke: validation of a
4-Hydroxy-1-(3-pyridyl)-1-butanone-hemoglobin adducts as



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Vol.7. 81 7--821,September1998
CancerEpidemiology,Biomarkers& Prevention
8/7
ShortCommunication
4-Hydroxy-
of Exposure
1-(3 -pyridyl)-
to Tobacco
1-butanone-Hemoglobin
Smoke:
Multicenter
Adducts
a Method
as B iomarkers
to BeValidationofUsedin
Studies’
Sunday
Anush
Irene
and
E.Atawodi,2 Suzanne
Constantinescu,
Cristina
Lea,3Fredrik
Wolfgang
Fortes,
Nyberg,
Mukeria,
Brueske-Hohlfeld,
Marlin
ValiAhrens,
Boffetta,Paolo
D.Friesen4
Units
Cancer
Cancer.
Epidemiology.
S-17l77
Institute
Federation
Management,
Prevention
LW. Al:
[I. B-H.]:
ofGene-Environment
Epidemiology
F-69372
Interactions
P. B.].
[S.E. A., M.D. F.]andEnvironmental
Research
Environmental
Karolinska
of CancerEpidemiology.
1 15478Moscow.
Health. Health
Brernen Institute
Bremen.
Oberschleissheim.
1-00198 Rome.
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Abstract
Hemoglobin
butanone
nitrosamines
butanone
biomarkers
study
cancer
measured
sixcollaborating
hydrolysis
and analysis
chromatography-electron
Priorto analysis
reproducibility
fromdonors.
reproducibility
ranged from
reproducibilities
16%,respectively.
smokers
weresignificantly
(Hb) adducts
a metabolite
[4-(methylnitrosamino)-1-(3-pyridyl)-1-
N’-nitrosonornicotine],
of exposureto tobacco
geneticalterations
amongnonsmokers.
aftercollection
centers,
from Hb, clean-up
of anelectron-capturing
of 4-hydroxy-1-(3-pyridyl)-1-
of two(HPB),tobacco-specific
andweremeasured
aspart
as
smokeof a
onand
HPB-Hb
of RBCs
release
susceptibility to lung
were
gradient
alkaline
extraction,
adducts
Ficollbyin
of HPB
solid-phase
by
by
derivative
spectrometry.
subjects,
validated
variation
fromfive
within-sample
eightaliquots
subjects
HPB-Hb
0.02)in smokers
by gas
capturemass
study
was
of samples
this
coefficient
of paired
7 to 25%
of
The
52
never-smokers.
higher
fromthe
ofapproachin blood
Theofof
aliquotssamples
; the
fourand
study
were4 and
of
levels
(26
consisted18
and
adduct
(P
=
±
13
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The
page
accordance
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address:
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advertisement
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partially
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(Environment
this paper
S. E. A.
2 Present
Nigeria.
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CA94720.
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for Research
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studybyDG-XlI
Thereported
Awards
in
theSpecial(to
and
NationalVeterinaryResearch Institute.Vom.PlateauState.
address:Schoolof PublicHealth,BerkeleyUniversity. Berkeley.
whomrequests
on
33-472738575:
for reprints
150
shouldbe addressed.
Albert
friesen@iarc.fr.
at the
F-69372
International
Lyon
Agency
Cancer,cours
E-mail:
Thomas. CEDEX08.
Fax:
fmol
HPB/g
results
measured
multicenter
exposure
HPB/g
Hb).
suggest
Hb)
There
than in never-smokers
no difference
level
modified
canbe
smoke.
(20
±
8 fmol
sexes.was
the
betweenThese
that
a method
studies,
to tobacco
of HPB-Hb adducts,
usingto facilitate
biomarker
use
of
in
a useful
Introduction
Tobacco
the
oral
uals
smokers
sure
biomarker
tion
can
binds
method
Hb5
nitrosamines,
and
HPB-Hb
mans,
amine
tobacco
Thus,
an indication
which
prior
smoking
as well
pancreas,
develop
(2).A methodological
tobaccosmoke
forthe
to this problem
measure the
to biomolecules
applied
ofHPB, a common
4-(methylnitrosamino)-l-(3-pyridyl)-
N’-nitrosonornicotine.
adducts has
studieshaveestimated
4-aminobiphenyl,
smoke, have
the concentration
of theamount
an individual
to examination.
Thepurposeof this
measuringHPB-Hb
coworkers
(5),
could
plecollection and
methodinvolvedparallel
dient insixcenters
analysis,alkaline hydrolysis
HPB bysolid-phase
capturing derivative
priorto analysis by
spectrometry.
This
ified method to assess
is an
as in cancers
and
lung
important causativefactorin cancer
as larynx.
most
2-10%
study
and
A possible
methods
carcinogen
The analytical
of adducts
tobacco-specific
1-butanone
persistence
determined
of the
as a resultof exposure
in humansof 7-9
releasedfrom
of tobacco-specific
been exposedduring
of
lung,
cavity,
who
of otherorgans,such
bladder
cancer
( 1). Although
smokers.
problem
lackof
effective
application
to whicha tobacco-specific
suchas DNA
studymeasures
metabolite
individ-
are
of expo-
reliable
are non-
in the
tois the a sensitive
dose.
of analytical
biologically
is the
extent
solu-
that
or proteins.
the
of
in this level
two
with
Although
adequately
that
formed
theof
notbeen inhu-
Hbadductsaromatic
alsoto
a half-life
of
weeks
should
nitrosamines
thefew
(3, 4).
give HPBHb
to
hasmonths
study was
first
to test
described
to facilitate
sample
collection
shipment
ofHPB
extraction.
that waspurified
gaschromatography-electron
reportdescribes
exposure
whethera method
Carmella
multicenter
The
ona Ficoll
laboratory
Hb,purification
of
solvent
capture
validation
to tobaccosmoke.
for
and
sam-
adducts,
be modified
to simplify
by
clean-up.
RBCs
a central
from
formation
by
modified
of gra-
for and to
of
andanelectron-
extraction
mass
mod-the of this
MaterialsandMethods
Materials.
mercial
Reagentsand solvents
(+99.9%)
were obtained
or HPLC
fromcom-
(Fluka sourcesin ultrapuregrades
5 The
hutanone:
matography-niass
abbreviationsused are:Hh.hemoglobin:
liquid
(4 CV.
HPB. 4-hydroxy-l-(3-pyridyl)-l-
HPLC.high-perfomiance
spectroscopy:
chromatography:
percentage
GC/MS.
of
gas chro-
coefficientvariation.
American Association for Cancer Research Copyright © 1998
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Page 3

818
ShortCommunication:
HPB-HbAdductsas Markersof TobaccoSmoke
Chemie
Merck,
HPB
(University
The
was
AG,
Darmstadt,
standards
Buchs,Switzerland;
Germany).
a generous
SDS, Peypin,France;
and
Stephen
Minneapolis,
of
procedure
mixing
B. Solution
0.0074g of CaC1,,
17.565 g of
solutionto pH
to 1 literwith distilled
8. 19 g of NaCl in
orE.
[4,4-d2]-HPB
gift
Center,
isotopic
quantification
prepared
of solution
nonlabeled
S. were
Minnesota
standard
for
buffer
9 volumes
1.0 g of glucose,
g of
adjusting
volume
of
fromHecht
MN).
which
ofCancer
an HPB-d,
corrected
Isotonic
A with
had
the
was
purity98.3%,
used.
1 volume
A was
by
by of so-
lution
by dissolving
MgC12,
distilled
increasing
was solution
prepared
0.1992
in 950
with
water.
distilled
g of
0.4026
water,
KC1,andTris
7.6
mlof
theHC1,
Solution
water.
and
theB
1 liter of
Blood
obtained
France).
tion
+4#{176}C until
collection,
Samples.
from
Blood
tubes
Blood
samplesfor method validation
Sanguine
7-mi
Blood
the
were
the
samples
Centerde Transfusion
collected
anticoagulant.
wascarried
outlined
study
framework
susceptibility
were
neoplastic
randomly selected
subjects were
Eighteenwere
hadnever smoked
( or theequivalent
(Lyon,
collec-
stored
following
wereintoblood
was
day
containing
analysis,
using
additional
recruited
alterations
These
diagnosis
subjects
of70
citrate
which
method
set
within
at
out
the below.
An ofsampleswas obtainedfrom
study
among
subjects
on
nonsmokers.
with
healthy
A total
women.
theof a multicenter
to lung
admitted
nonmeoplastic
fromthe general
including
and 52were
than onecigarette
tobaccoproducts).
genetic andcancer
subjects
of
patients
or
to hospital
disease
population.
14 men
never-smokers,
daily
aor
studied, and56
smokers
i.e.
year
, they morefor1
in other
Blood
ous
morphisms
pair
samples
shipment
Collection
analysis
and Isolation ofHb.
adduct
To
permit simultane-
genetic
and DNA
separate
storage
of carcinogen-protein
of carcinogen-metabolizing
activities,
plasma,
to IARC for
Fifteen-mi samples
collectedinto three
containingcitrate
diluted bymixing
sterile50-ml polypropylene
degreeof aggregation
with a minimum
sample wasprocessed
The 30 ml
plasticinsert of
trifugationtube
Germany). These
and9.6%sodium
tubesby a one-way
capped andcentrifuged
aggregatedRBCs
thetubes, whereas
the plasmaand
monlyreferred
After centrifugation,
removed,processed,
crose-sodium metrizoate
thenremoved with
cleantweezers,
movedand discarded.
abovethe RBCs
discardedusing
carefulnot to disturb
TheRBCs were
levels,poly-
enzymesre-
enzymeitwas necessary
and
to blood
into WBCs,
analysis.
of blood,
5-ml
anticoagulant.
with an equal
RBCs beforeand
taken
15-ml
The
volume
by venipuncture,
blood
combined
of isotonic
dilution
so thatthey
entrapment.
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tube
was
in a
the
easily,
or onecollection
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buffer
controlstube.
RBCs
This
of thesediment
Onceof lymphocytediluted,the
immediately.
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a UNI-SEP
(Wak-Chemie
tubes
metrizoate,
plastic
were thenpoured
density
Gmbh,
of 5.6%
is held
barrier.
18-20#{176}C) for
granulocytes
WBC migrated
polysucrose-sodium
to as theFicoll layer.
plasma
and stored
layer
a Pasteurpipette
theporousplastic
Thepolysucrose-sodium
in the bottom of the
a Pasteur pipette,
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transferredto a centrifuge
on
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bottom
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to the bottom
interface
metrizoate
top of the
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Medical
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in the
After
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of the
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andsedimented
to the
of
thebetween
the layers,com-
and
other
WBCs
analysis.
the
and
barrier
werecarefully
polysu-
barrier
Using
carefully
metrizoate
alsoremoved
technicians
bottom of the
tubecontaining
for
above
The
porous
discarded.
was
was
a
re-
layer
and
were
tube.
tube
and
at the
was
the
at
least
suspended
supernatant
cedure
into
and
necessary.
shipment
3 timesthe volume
and
removed
of isotonicbuffer.
1000
TheRBCs
10 mm.
washing
then
were
The
pro-
evenly
was
repeated.
Nunc
amount
The
to IARC
centrifuged at
X g for
and
were
Kamstrup,
buffer
stored
and
washed
discarded, the
was TheRBCstransferred
Denmark),
added,
-80#{176}C until
a 4.5-mi
a small
cryotube
(<0.5
(Nunc/AS,
of isotonic
were
ml)wasif
RBCsolutions
analyses.
at
for
Isolation
addition
Poole,
the
M with
was
by
dialyzed
using
weight
bourg,
content
(Sigma
of Hb.
3 volumes
At IARC,
of
thorough
washed
ice-cold
RBCs
HPLC-grade
mixing
solution
phosphate
cellmembranes
rpm for
20-50volumes
dialysis
12,00015,000
water waschanged
wasdetermined
Co.,St. Quentin
7-mifractions
to 4-5mlof blood,
tubes(26-mm external
caps,forstorage
dialyzedHb
cryotubesand
availablefor analysis
were lysed
water
by
(BDH,
5 mm,
to 0.2
6.5)
removed
Hb
water
a molecular
Block,
12 h (5).
Drabkin’s
France).
Hbsolution,
aliquoted
100
80#{176}C until HPB
wasaliquoted
at-80#{176}C.These
of additionalbiomarkers.
the
of
England).After byvortex
was
buffer
for
phosphate concentration
0.67
to stand
centrifugation
for
Spectra/Por
cutoff
France).
ofthe
Chemical
Duplicate
corresponding
Pyrexglass
Teflon-sealed
Theremaining
4.5-ml Nunc
tions are
of the adjusted
(pH
were
The
distilled
with
Labo
cold
M potassium
for15 mm.
at
hagainst
4 cellulose
and
leftRed
18,00030mm.
of
was
48
tubing,
(Poly
every
with
Fallavier,
dialyzed
were
diameter
of
Mr
Stras-
The
Reagent
The Hb
dialysate
of theeach
30-ml
with
into
mm),
X
at
-
analysis.
into solution
was
two
storedsolu-
Release
Extraction.
tubes
water
concentration
addition
1 h, and
(NH4)2S04.
at 2600
of
HPB
The
from
Hb
to thaw,
as
of the
1 M NaOH.
proteins
Precipitated
rpmfor
clear supernatant
extraction
containing
preconditioned
anol,
5 mlof water,
6.7).Thesolution
under gravity;
on thecolumn
or 0.001
0.05
if washsolution
wash, the column
gravity with
(Wheaton, Miliville,
was pulled into
methanol eluate
overnightin
this stepwas
Hb
and
stored
and
Clean-up
in the
pg
by Solid-phase
glass
in 20
HPB,
solution Pyrexstorage
pA ofwas
were
allowed
added
100of HPB-d2
To
to 0.1 1 M NaOH
sonically
14-20%
removedby centrifugation
internal
solution
The
precipitated
protein
10 mm
standard.
was
mixture
releasethe
by
for
brought
was
with
was
ofdispersed
(v/v)wereof 20%
at 15#{176}C.
was
column
of C18
washing
and
5 ml
wasallowed
columns were
wasthenwashed
M KH2PO4,which
N NaOHor 0.05
was
was
2 ml of methanol
The applied
(Altech
packing.
3-5
of 0.067
to
not
with
had been
to a 6-mi
Associates,
The
of HPLC-grade
M KH2PO4
drainthrough
allowedto run
5 ml of HPLC-grade
adjusted
(Recoveries
topH
well, and HPB
into a 2-mi
Methanolremaining
undervacuum
todryness
concentrator.
to be-80%.
Extract-Clean
Deerfield,
column
solid-phase
IL) 500
by
mg hadbeen
meth-
(pH
column
The
water
-7.0
inconsistent
Following
eluted
disposable
onthe
(-3mmHg).
at room temperature
Recovery of
withml
buffer
the
dry. HPB
to pH
were
with
N H1P04.
not
drained
buffered -7.)the
wasunder
glass vial
NJ).
tube
taken
column
the
was
The
a SpeedVac
determined
HPB for
Derivatization
ylamine
scribed
tafluorobenzoylchloride
containing
wasallowed
to dryness
To
traces of
of HPB.One
solution
and
in hexane
HPB
at room
a SpeedVac
unreacted
thederivatized
ml
of CH2CI2,
inhexane,
of
were
After
1 ml
prepared
solution
to the
of trimeth-
hydrochloride
previously
asde-
(5),
10 ?la 10%
added
mixing,
for 2 h and
of pen-
vial glass
solution
then
the driedresidue.
temperature
concentrator.
pentafluorobenzoylchloride
HPB solution
the
to stand
with
remove
Hb,
taken
and
was madeacidic
American Association for Cancer Research Copyright © 1998
on July 13, 2011 cebp.aacrjournals.orgDownloaded from

Page 4

SWILl.
from 96_04N6122HPE.EJ
S
0
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0
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amu.
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to contain
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1.
Selected
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ionmonitoring
HPB/g
1.42
standard
mass
peak
B, peak
to the
chromatograms
at 9.887
at 9.883
Hb
for sample
represents
which
before
25208.found
fmol
from
Hb.
A,
mm,which10 pg
100
of
pg
releasedg of Hb.
mm, represents
hydrolysis.internaladdedsample
CancerEpidemiology, Biomarkers& Prevention
8/9
with
for
vacuum
hexane
then
9). HPB-pentafluorobenzoate
ml of hexane
layerswere
macolLtd.,
SpeedVac,
recoveryof
-80%.
100pA 1 N HC1
After
applied),
clean-up
raisedto 7. 1 by
andextracted twicewith 1 mlof hexane
5 mm
and
layer
1.5 mm.centrifugation
the
wasrepeated.
addition
in the
layer
The
of sufficient
wasthen
ofmixing.
to a 1.5-mi
United Kingdom),
storedat-20#{176}Cuntil
HPB-pentafluorobenzoate
SpeedVac
was
pH of the
for(no
the
was
(pH
0.9
hexane discarded,
aqueous
0.5
M Na2PO4
twice
combined
glass
to
GCIMS
wasdetermined
extracted
The
with
hexane
(Chro-
with1.5 mm
transferred
Herts,
and
disposablevial
dryness
analysis.
broughton
The
tobe
GC/MS.
was
analyzed
ization
GC/MS
chromatograph
coupled
analysis
silica
film
trometer
(270#{176}C).Helium
pressure
raphy oven
to280#{176}C at 20#{176}C/mm, where
theseconditions,
zoatederivative
Themass
modewith methane
trometerwas tuned
358.9(HPB-d?-pentafluorobenzoate)
pentafluorobenzoate)
HPB/injection.
Identification
dependenton it retention
thatof the HPB-d2
samplewerecalculated
peakareas of m/z
amountof HPB
obtainedover
above a cutoff
This corresponds
backgroundpeak
HPB-d?Jl00pg HPB-d2,
internalstandard
from a particular
HPB
(infmol)
g).Fig.1 shows
HPB-d?(m/z 358.9)
thatwas found
The
up
by
mass
was
purified
in 9 p?l of
gaschromatography-negative
spectrometry
carriedout
equipped
to a Hewlett
wascarried
capillarycolumn
thickness, 0.33
ionsource
was
of 150kPa.
wasraised
HPB-pentafluorobenzoate
isopropanol,
derivative
taken and3-?tl
ion
aliquots
chemical
monitoring.
5980A
injector
spectrometer.
polarDB17O1
mm inner
to the
aheated
gasata column
of thegas
temperature
held for2 mm.
of the pentafluoroben-
were
ion-
by
a Hewlett
a
5988A
a slightly
mX
coupled
( 150#{176}C)through
usedas
The temperature
fromam initial
selected ion
onPackard gas
with splitless(270#{176}C)
The
fused
diameter;
mass spec-
interface
Packard
out
mass
on
(300.25
directly p.m),
carrier head
chromatog-
of100#{176}C
Under it was
time
-9
was
gas
negative
the retention
HPBof was mm.
spectrometeroperated in the negative
mass
ions
ion
as reagent
to monitor
(-
1 torr).
molecular
andm/z
limit
The spec-
at m/z
360.9
HPB
(HPB-d2-
was
. Thedetection for1 pg
ofa chromatographic
time
standard.
froma plot
and m/z
X axis.
of2-100
of 2 pg/sample
to an approximate
resulting
which
was injected.
sample of Hb
divided by
theselected
and HPB-d2
to release
peakas HPBwas
thanbeing-0.007
Amounts
of the
360.9
A linear
pg
were
doubling
the isotopic
was observed
Theamount
wasexpressed
amount
ion
(m/z
fmolHPB/g
mm
of HPB
of the
Y axis
greater
internal in each
ratio
the
calibration
sample.
reported
integrated
358.9
on
range
point
on
versus
curve
the
wasthe
theperOnly
as detected.
area
of
theHPB-d2
HPB
as the amount
in thesample
chromatogram
fora Hb
Hb.
results
of the
impurity
when
of
of the
1.7 pgfrom
released
of
(in
for
theof Hb
mass
360.9)sample
44
Statistical
pies
the
seven
determined,
included
values
according
ofHPB-Hb
nonparametric
peated
Analysis.
analyzed,
HPB
For 52 subjects
mean
for purposes
HPB-Hb
of the
analysis.
for
smoking
levelvalues
Kruskal-Waliis
analysisafter
forwhom
values
of grouped
levels
limit
mean
study
Because
notnormal,
rank
duplicate
was
analysis.
were too
for that
and
subjects
the
sam-
were
subject’s
subjects
the
level
of the tworecordedas
For
whose
value
statistical
calculated
to sex
adduct
adductlow to be
was
HPB
thedetectionsample
SDin The of
were groupsof defined
andstatus.
was
test
transformation.
distribution
weused
and
the
re-of sum,we
the log
Results
A
m!z 358.9
HPB-d0
9.6 9.8
Time
1?’.B
(ml,,.)
102
B
m/z 360.9
HPB-d2
The
ously
increase
existing
HPB
solid-phase
derivatized
extraction
An
published
proaches
analytical
published
method
method
of HPB,
(a)
from
extraction
HPBwas
derived
method
(7). Our
in thatFicoll
used in this
To
two
studyis based
analysis
on
time
made
extraction
method
clean-up
two-step
aromatic
modification
differsfrom
tubeswereused
a previ-
and
to the
(5).
shortento
recovery
method:
released
modifications
organic
replaced
a lengthy
with
with
first
also
were
a multiple,
Hb
and
replaced
from
using
method
gradient
solvent
with
HPLC
of
was
(b)
ausing
of the
organica simple
otherwork amines
was
previously
to collect
(6).
analyticaltherecently
ap-
RBCs
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Page 5

.0
Dl
a-
I
0
.
300
200
100
0
Fig.
aliquots
2.
Day-to-day
ofan
reproducibility
control
over
sample.
a 4-month period of HPB-Hblevels in
analytical Hb
0
50
Day of analysis
I 00
Table3
Mean HPB-Hbadduct levelsbysexandsmokingstatus
C, Kruskal-Wallis
I, Seven
HPB/g
rank
had
sumtest.
subjects
Hb).
HPB-Hbadductlevels belowthelimit ofdetection (9fmol
820
Short
Communication:HPB.HbAdducEsas Markers ofTobaccoSmoke
T,l’le
aliquots
I
W
of
thin-sample
blood
reproducibility
twodonors:
formultiple.
released
same-day
hydrolysis
analysis
from
of
from
HPB byHb
Sample
NfMean
-?---
fmol
±SD
.
?Hh(g/sample)
-
?c?CV
aliquotsHPB/g Hh
DonorI8 I .8065±10
IS
l6’?
4?i
Donor
24
1.14 434±
Taith’2
Wit hin-samplerepr
h
oducibility
uman
forthe blindduplicatean
bloodsamples
alysis offive
Sample
no.
Hh
5/tO ml)
HPB-Hhadductlevel
fmol/g HhMean ± SD
,?,
CV
I
4
2
0
3
7
5
9
8
6
.23
1.19
1.50
.34
.37
.18
1.04
1.24
1.21
.42
75
6168± 10
51
37 44± tO
47
52 49±3
45
35 40±7
32
2327±7
l5Ck
22%
7%
17%
25%
in six
lect
after
sucrose-sodium
with
participating
WBCs
separation.
centers. Thesetubes were designed
been
that
does
HPB.
to col-
andplasma:
Thus,
metrizoate
derivatization,
RBCs
it was
have generallydiscarded
the
interfere
important
Ficoll
or analysis
to show poly-
mediumnot
isolation, of
Within-Sample
els. The
reproducibility
blood
four,
shown
of these
tively.
Reproducibility
method
in two
donors.
other was
1, within-sample
expressed
forHPB-Hb
tested
samples
sample
eight
reproducibilities
were
Adduct
within-sample
prepared
wasdivided
equalaliquots.
Lev-
modified
was first for
pooled blood
pooled
fromthe
into
As
from
and
in Table
samples.
two One
dividedtheinto
foranalysis
respec-as % CV.4 andI 6%,
Reproducibility
Adducts
yses using
were
from
isolated
analyzed
codes
dividual
25e/efor the
of Blind
To test
blood
two
a colleague
the 10
themethod
broken
reproducibility.
fivesamples
Duplicate
thereproducibility
samples
aliquots
fromoutside
samples,
outlined
whenall results
expressed
witha mean
Analysis ofHPB-Hb
in Blood.
the method,
divided
I to I0 by
from
using
were
of duplicate
other
numbered
thelaboratory.
hydrolyzed
Sample
were available.
CV, ranged
17%(Table
anal-
fromfive blooddonors
into equaland at random
Hb
was
andand HPB
above.
was
identification
only Intrain-
as?from
2).
7 to
of
Day-to-Day
Sample.
analysis
sample
that
analytical
about
levels
period.
from
16 fmol
aliquots
ibility
CV,
Reproducibility
control
a large
mixed
frozen
control
samples
determined
Thelevel
150 to 198
HPB/g
were analyzed
foranalysis
rangedfrom
ofan
of
Analytical
results
sample
into
ormore
included
2 shows
over
control
theaverage
17%).On
sameday.
controlHb
10%.
Control
the
prepared.
identical
aliquots
witheach
thevariation
4-month
Hbsample
level
4 separate
Same-day
sample,
Hb
To
period.
was
were
the
quality
pooled
and
-80#{176}C. One
sample
analyzed.
these
in theanalytical
fmolHPB/g
Hb (cF CV
on
ofthe
4 to
over
was
4-month
HbThe
well
at
Hb
divided40aliquots
of
batch
in the
analysis
ranged
178
days,
reproduc-
this
were
Fig.
aliquots
of
eight
forthe
Hb:was
±
=
three
the
expressed as%
.
.
. #{149}#{149}
.
a
HPB-Hb(fmol/gHb)
PC
RangeMean±SD
Sex
Male 14(12-38)
22±9 0.50
Female56
(9-67) 21± 10
Smoking status
Smokers18
(9-67)26±120.02
Never-smokers”
52(9-38) 19 ± 8
HPB-Hb
HPB-Hb
10 fmol
adduct
never-smokers
sexes.
subjects,
ex-smokers
HPB/g
HPB-Hb
Adducts
adduct
HPB/g
levels
in StudySubjects.
subjects
fmol
higher
difference
between
24
adduct
conducted
virtually
The
overall
study
Hb).
smokers
found
current
7 fmol
level
on
identical
mean
21 level
Hb
in the
(range,
significantly
3).
was
level
70
9-67
in the
HPB/g
in
was
was±
HPB-Hb
than
between
were
(Table
in
No
seen Nodifference
mean
(8
Hb).
adduct
smokers( I0
and
fmol
adduct
subjects,
=
±HPB/g
29
log-transformed
results,
Hb)
17mean
=
±
Resultsof analyses
values yielded
Discussion
The
adducts
tobacco
ular,
integrate
months
permit
sites
ysis
purposeofthis
blood
in
study
could
never-smokers
wasto determine
useful
and
this approach
fromtobacco
method
of plasma,
of analysis
per week.
methods
from
whether HPB-Hb
in
smoke
thebeto
ever-smokers.
measureexposure
In partic-
be useful
from
modified
and RBCs
to allow
to
it was hypothesized
low-level
inthe
simultaneous
distant from
of at least
In previously
adducts(5,
analysis.However,
idemiology
thatwould
smoke
(5)
WBCs,
and
to
exposure
A published
collection
the laboratory
eight samples
published
Hb was
in multinational,
studies,a
several
past.was to
at
anal-
for
fresh
multicenter
reliable
analysis
RBCs
of
just
HPB-Hb
prior8),isolated to
molecular ep-
simpleandmethodisneeded
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Page 6

CancerEpidemiology,Biomarkers & Prevention
821
whereby
and
Ficoll
WBCs,
results
time
crose
interfere
of adducts.
and
same
standing
changes
types
permits
types
bloodcan
stored
have
be collected;
frozen
primarily
plasma
indicate
WBCs
metrizoate
GCIMS
Ficoll
collection
techniques
relationship
it will
sameindividual.
standardization
geographically
eta!.
(5)
tobacco-specific
4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone,
exposure to tobacco
HPB-Hbadducts
range,4.8-1160)
range, 8.5-95)
smoke.However,
heterogeneity was
Thisstudywas later
Hb) and37 nonsmokers
study(10) on smokers
(34±16 fmol/g
(8)in pregnant
nonsmoking(27±35 fmol/g
Smokingstatusin these
“nonsmoker,”andit was
was assessedat thetime
Themeanlevels
both smokers(26±
(19± 8 fmolHPB/gHb)
and nonsmokers in previous
because mostparticipants
and mayhave smoked
smokein theperiod
pantsin previousstudies.
long-time formersmokers.
smokersweregenerally
mayhave includedformer
resultsmay not befully
levels were measured
thegeneralpopulation
were comparableto those
separated
a period
been
andRBCs
thatHb can
andthat
inthe
method
tubesfor
into plasma,
several
the
are discarded.
WBCs,
months.RBC; andover of
tubes
in which
of our
as plasma
and
with
usedforisolation of
theoften
be collected
Ficoll
collection
or quantification
isolation
ofthese
becomeavailable
between
important
Moreover,
in collection
distant study
used Hb
nitrosamines
The
studyat the
of polysu-
does
of the
WBCs,
from
our
intracellular
different
of a Ficoll
isolation
sites.
ofHPB,
N’ -nitrosonorni-
same
andthelayer
sodiumtube not
ourlevel
Use
facilitates
As
the
increases,
the
some
between
Carmeila
ofthe
and
markersof
levelsof
HPB/gHb;
fmol/g Hb;
tobacco
large
levels.
HPB/g
similar
smokers
group
of of RBCs,
cellplasma
subjects.
types
and
the
under-
of inter-
to
and
behave
use
and
cell
tubefrom
of cell
first adductsa me-
tabolite
cotineas
smoke
smokers
21
clearly
smokers
based
in humans.
(80
Elevated
190
(29
in 40
and
±fmol
±nonsmokers
related
26
to were
for
observed,
extended
to exposure
nonsmokers,
range
both and
the
smokers
a
onofHPB
fmol
9).
non-
same
and
results.
to 100
fmol
±44
a recent
(55
women
was
that
(163
Ref.
and
the
Hb)
(68
(69
and
HPB/g
fmoi/g
Hb;
Hb)
A
Hb) study
fmol/g
gave
as “smoker”
“current”
by
smoking±46
Hb) similar
articles
assumed
of interview.
of HPB
12 fmoi
were
defined and
smokingstatus
adducts
HPB/g
lower
studies
in the
or have
before
In addition,
In previous
current
smokers
comparable.
in blood
to validate
reported
reported
Hb)
than
(5,
8, 9,
study
been
blood
many
in this
never-smokers
among
10). This
hospital
to less
than
ever-smokers
studies,it appears
whereas
recentquitters,
When HPB-Hb
takenat random
method (Table
in previous
studyin
and
those smokers
may
patients
tobacco
partici-
be
were
exposed
collection
less
just
were
that
smokers,nonsmokers
or so
adduct
from
results
that
samples
our 2),
studies.
Within-sample,
ducibilities
methodology
significantly
never-smokers.
HPB-Hb
tofacilitate
biomarker
interindividual,
shown
HPB-Hb
(P
These
measured
in multicenter
exposure
and day-to-day
for
were
repro-
wereto be
adduct
0.01
acceptablethe
shown
modified
and
higher
levelsto be
in
of
=
) in
suggest
our
studies,
smoke.
ever-smokers
that
method
than
level
modified
a
resultsthe
and
be
adducts,using
usecan useful
oftotobacco
Acknowledgments
We
ohs,
Arno
blood
Nadejda
thank
MN)
Helbringer
samples
Stephen
for
S. Hecht(University
and
Transfusion
ofMinnesotaCancer
standards
Lyon.
of the
management.
Center.
used
Minneap-
the[4.4-D,]-HPB
(Center
usedin development
Jourenkova
nonlabeledHPBin this
for
method:
study:
deSanguine.
validation
with
France)
analytical
providing
and and
forherassistancesample
References
I .
Risk
IARC.TobaccoSmoking.
toHumans.
Monographs
Vol.
on the
Lyon.
Evaluation
France:
ofthe
986.
Carcinogenic
ofChemicals38. IARC.
2.
Schottenfeld
637-665.
Blot. W.J..
and
and
J. F. Fraumeni
York:
Fraumeni, J.F. Cancers
Cancer
ofthelungand
and
pleura.
Prevention.
In:
D.
(eds.),
University
Epidemiology
1996.
pp.
NewOxford Press.
3.
the
Cancer
Maclure,
hemoglobin
M..Bryant.
adduct
I 8 I - I 84,
M.
of
S..
4-aminobiphenyl
1990.
Skipper. P. L..and
during
Tannenhaum.
withdrawal
S. R. Decline
from
of
smoking.
Res.,
50:
4.
Randall,
A.
biomarkers
Biomark.
Mooney,L.
C..
A..
Cooper.
T-L.,
Santella,R.M.. Covey.
R..
P.
L..Jeffrey.
W.
of
cessation.
A. M..Bigbee.
L..
W..
M. T.B..
Perera,
Ottman.Tsai.
Decline
smoking
Y..Wazneh,
DNA
Glassman.
and
Epidemiol.
H..Young, andF.damage
Cancer
other
in peripheral
Prey..
blood following
1995. 4:627-634.
5.
A. M.,
nitrosamine
Cancer
Carmella,S. G..
E., and
hemoglobin
50:
Kagan.
Hecht,
S. S..
S. S. Mass
adducts
1990.
Kagan.M.. Foiles.P. G..
analysis
smokers.
Palladino,
of tobacco-specific
and
G.. Quart,
Quart,spectrometric
snuff indippers. nonsmokers.
Res., 5438-5445.
6.
Kadlubar,
dazo[4.5-h]pyridine
chromatography/electron
with32P-postlabeling.
Friesen,M. D..
F.
Kaderlik,
Analysis
in
K., Lin.D..
adducts
Garren. L.,
2-amino-t-methyl-6-phenylimi-
by alkaline
spectrometry:
Toxicol..7: 733-739.
Bartsch.H.. Lang.N. P.. and
F. ofDNAof
ratand human
mass
Res.
tissueshydrolysisand gas
capturevalidation by comparison
Chern.1994.
7.
solid-phase
tion
amines.
Kutzer,C..
extraction
hemoglobin
J. Chromatogr.
Branner. B.. Zwickenpflug.
gas chromatographic-mass
fromtobacco-specific
35:
1-6.
W..and Richter.
spectrometric
nitrosamines
E. Simultaneous
determina-
and
and
of adductsaromatic
Sci.. 1997.
8.
A.
tobacco
tobacco-specific
M. Maroni
University
Richter,
R.. and
E., Branner,B.. Kutzer, C..
of biomarkers
Donharl,A.M. E.,Scherer. G..Tricker.
Heller.
smoke:
F. A. Comparison
cotinine
nitrosamines
Proceedings
Milano.
for exposure
from
and
‘95.
to environmental
aromatic
nonsmoking
pp.
599-605.
and haemoglobin
in pregnant
ofHealthy
adducts
smoking
Buildings
arnines
women.
and
In:
(ed).
of
Milan:
1995.
9.
DNA
specific
Foiles. P. G..
hemoglobin
carcinogens.
Murphy. S. E..
adducts
Cancer
Peterson.
as
Res..
L. A..Carmella.
metabolic
2698s-270ls,
S. G..
activation
and Hecht.
of
S. S.
andmarkers
52
oftobacco-
(Suppl.).1992.
10.
aromatic
I 994.
Falter.B., Kutzer, C.. andRichter, E. Biomonitoring
nitrosamines.
of hemoglobin
Invest..
adducts:
364-37 aminesandtobacco-specific Clin. 72:I.
American Association for Cancer Research Copyright © 1998
on July 13, 2011cebp.aacrjournals.org Downloaded from