Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein

Article abstract of DOI:10.1194/jlr.M083279

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more -BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br₂ gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

Full text of DOI:10.1194/jlr.M083279

Bromofatty aldehyde derived from bromine exposure and
myeloperoxidase and eosinophil peroxidase modify GSH
and protein
Mark A. Duerr,1,* Elisa N. D. Palladino,* Celine L. Hartman,* James A. Lambert,†,§,**
Jacob D. Franke,* Carolyn J. Albert,* Sadis Matalon,†,§,** Rakesh P. Patel,§,**,†† Arne Slungaard,§§
and David A. Ford2,*
EdwardꢀA.ꢀDoisyꢀDepartmentꢀofꢀBiochemistryꢀandꢀMolecularꢀBiologyꢀandꢀCenterꢀforꢀCardiovascularꢀ
Research,*ꢀSaintꢀLouisꢀUniversityꢀSchoolꢀofꢀMedicine,ꢀSt.ꢀLouis,ꢀMOꢀ63104;ꢀDepartmentsꢀofꢀAnesthesiology†
andꢀPathology,††ꢀandꢀCentersꢀforꢀFreeꢀRadicalꢀBiology§ꢀandꢀLungꢀInjuryꢀandꢀRepair,**ꢀUniversityꢀofꢀAlabamaꢀ
atꢀBirmingham,ꢀBirmingham,ꢀALꢀ35294;ꢀandꢀDepartmentꢀofꢀMedicine,§§ꢀSectionꢀofꢀHematology,ꢀOncology,ꢀ
andꢀTransplantation,ꢀUniversityꢀofꢀMinnesota,ꢀMinneapolis,ꢀMNꢀ55455
and myeloperoxidase and eosinophil peroxidase modify
Abstract -Chlorofatty aldehydes (-ClFALDs) and -
bromofatty aldehydes (-BrFALDs) are produced in acti-
vated neutrophils and eosinophils. This study investigated
the ability of -BrFALD and -ClFALD to react with the
thiols of GSH and protein cysteinyl residues. Initial stud-
ies showed that 2-bromohexadecanal (2-BrHDA) and 2-
chlorohexadecanal (2-ClHDA) react with GSH producing
the same fatty aldehyde-GSH adduct (FALD-GSH). In both
synthetic and cellular reactions, FALD-GSH production was
more robust with 2-BrHDA compared with 2-ClHDA as
precursor. NaBr-supplemented phorbol myristate acetate
(PMA)-activated neutrophils formed more -BrFALD and
FALD-GSH compared with non-NaBr-supplemented neu-
trophils. Primary human eosinophils, which preferentially
produce hypobromous acid and -BrFALD, accumulated
FALD-GSH following PMA stimulation. Mice exposed to Br2
gas had increased levels of both -BrFALD and FALD-GSH
in the lungs, as well as elevated systemic plasma levels of
FALD-GSH in comparison to mice exposed to air. Similar
relative reactivity of -ClFALD and -BrFALD with protein
thiols was shown using click analogs of these aldehydes.
Collectively, these data demonstrate that GSH and protein
adduct formation are much greater as a result of nucleo-
philic attack of cysteinyl residues on -BrFALD compared
with -ClFALD, which was observed in both primary leuko-
cytes and in mice exposed to bromine gas.—Duerr,ꢀM.ꢀA.,ꢀ
E.ꢀN.ꢀD.ꢀPalladino,ꢀC.ꢀL.ꢀHartman,ꢀJ.ꢀA.ꢀLambert,ꢀJ.ꢀD.ꢀFranke,ꢀ
C.ꢀJ.ꢀAlbert,ꢀS.ꢀMatalon,ꢀR.ꢀP.ꢀPatel,ꢀA.ꢀSlungaard,ꢀandꢀD.ꢀA.ꢀ
Ford.ꢀBromofatty aldehyde derived from bromine exposure
GSH and protein. J. Lipid Res. 2018. 59: 696–705.
Supplementary key wordsꢀ fattyꢀaldehydesꢀ•ꢀglutathioneꢀ•ꢀplasmalogenꢀ•ꢀ
hypochlorousꢀacidꢀ•ꢀhypobromousꢀacidꢀ•ꢀthiols
-Halofattyꢀaldehydesꢀareꢀproducedꢀbyꢀhypohalousꢀacidꢀ
attackꢀofꢀtheꢀvinylꢀetherꢀbondꢀofꢀplasmalogenꢀ(1,ꢀ2).ꢀTheꢀ
mostꢀwell-characterizedꢀ-halofattyꢀaldehydeꢀisꢀ-chlorofattyꢀ
aldehydeꢀ(-ClFALD),ꢀproducedꢀbyꢀreactionsꢀbetweenꢀhy-
pochlorousꢀacidꢀ(HOCl),ꢀderivedꢀfromꢀneutrophils,ꢀmono-
cytes,ꢀandꢀsomeꢀtissueꢀmacrophagesꢀ(3,ꢀ4),ꢀandꢀplasmalogenꢀ
(Fig. 1).ꢀProductionꢀofꢀ-ClFALDꢀhasꢀbeenꢀshownꢀinꢀhu-
manꢀatheroscleroticꢀplaquesꢀ(5)ꢀandꢀinfarctedꢀratꢀmyocar-
diumꢀ(6).ꢀMyeloperoxidaseꢀ(MPO)-derivedꢀ-ClFALD has
alsoꢀbeenꢀimplicatedꢀinꢀendothelialꢀdysfunctionꢀ(7),ꢀneuro-
nalꢀapoptosisꢀ(8),ꢀactivatingꢀtheꢀnuclearꢀfactorꢀBꢀpathway,ꢀ
eNOSꢀinhibitionꢀ(9),ꢀneutrophilꢀchemotaxisꢀ(3),ꢀandꢀmyo-
cardialꢀcontractileꢀdysfunctionꢀ(6).ꢀRecently,ꢀ-ClFALD was
alsoꢀfoundꢀinꢀtheꢀlungsꢀofꢀmiceꢀandꢀratsꢀexposedꢀtoꢀchlo-
rineꢀgasꢀ(Cl2)ꢀalongꢀwithꢀitsꢀGSHꢀadductꢀ[fattyꢀaldehyde-GSHꢀ
adductꢀ(FALD-GSH)],ꢀandꢀitꢀwasꢀfoundꢀtoꢀcontributeꢀtoꢀ
Abbreviations:ꢀ -BrFALD,ꢀ -bromofattyꢀ aldehyde;ꢀ -ClFALD,
-chlorofattyꢀaldehyde;ꢀ2-BrHA,ꢀ2-bromohexadecanoicꢀacid;ꢀ2-BrHDA,ꢀ
2-bromohexadecanal;ꢀ 2-BrHDyA,ꢀ 2-bromohexadec-15-ynal;ꢀ 2-ClHA,ꢀ
2-chlorohexadecanoicꢀacid;ꢀ2-ClHDA,ꢀ2-chlorohexadecanal;ꢀ2-ClHDyA,ꢀ
2-chlorohexadec-15-ynal;ꢀ2-ClHOH,ꢀ2-chlorohexadecanol;ꢀFALD-GSH,ꢀ
fattyꢀaldehyde-GSHꢀadduct;ꢀHDA,ꢀhexadecanal;ꢀHDA-GSH,ꢀ2-halohexa-
decanalꢀ adductꢀ withꢀ GSH;ꢀ HDyA,ꢀ hexadec-15-ynal;ꢀ HOBr,ꢀ hypobro-
mousꢀ acid;ꢀ HOCl,ꢀ hypochlorousꢀ acid;ꢀ MPO,ꢀ myeloperoxidase;ꢀ MTT,ꢀ
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumꢀbromide;ꢀNAC,ꢀ
N-acetylcysteine;ꢀ NEM,ꢀ N-ethylmaleimide;ꢀ ODA-GSH,ꢀ 2-halooctadeca-
nalꢀadductꢀwithꢀGSH;ꢀPMA,ꢀphorbolꢀmyristateꢀacetate.
This work was supported by National Institutes of Health Grant R01GM115552
(D.A.F.) and National Institute of Environmental Health Sciences Grants
U01ES023759 (R.P.P), U01ES026458 (S.M.), and U01ES027697 (S.M.).
Additional support was provided through subcontracts to D.A.F. from National
Institute of Environmental Health Sciences Grants U01ES023759, U01ES026458,
and U01ES027697. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National Institutes of
Health.
1ꢀPresentꢀaddressꢀofꢀM.ꢀA.ꢀDuerr:ꢀDepartmentꢀofꢀNaturalꢀSciences,ꢀ
MissouriꢀBaptistꢀUniversity,ꢀSt.ꢀLouis,ꢀMOꢀ63141.
Manuscript received 4 January 2018 and in revised form 9 February 2018.
Published, JLR Papers in Press, February 14, 2018
DOI https://doi.org/10.1194/jlr.M083279
2ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ
ꢀ e-mail:ꢀdavid.ford@health.slu.edu
Copyright © 2018 Duerr et al. Published under exclusive license by The American
Society for Biochemistry and Molecular Biology, Inc.
696
Journal of Lipid Research Volume 59, 2018
This article is available online at http://www.jlr.org

mentsꢀrequiringꢀLC-MS,ꢀaꢀThermoꢀFisherꢀSurveyorꢀLCꢀsystemꢀwasꢀ
coupledꢀ toꢀ theꢀ Quantumꢀ Ultra.ꢀ LC-MSꢀ dataꢀ analysisꢀ wasꢀ per-
formedꢀusingꢀXCaliburꢀsoftwareꢀ(ThermoꢀFisher).
increasedꢀlungꢀinflammationꢀandꢀpermeabilityꢀasꢀwellꢀasꢀ
systemicꢀlossꢀofꢀeNOS-dependentꢀvasodilationꢀ(10).
Theꢀmetabolismꢀandꢀphysiologicꢀrelevanceꢀofꢀ-bromofattyꢀ
aldehydeꢀ(-BrFALD)ꢀremainsꢀtoꢀbeꢀelucidated.ꢀEosinophilsꢀ
containꢀeosinophilꢀperoxidaseꢀthatꢀselectivelyꢀproducesꢀhy-
pobromousꢀacidꢀ(HOBr),ꢀwhichꢀcanꢀtargetꢀplasmalogensꢀ
yieldingꢀ -BrFALDꢀ (11)ꢀ (Fig.ꢀ 1).ꢀ Althoughꢀ itꢀ hasꢀ beenꢀ
shownꢀ thatꢀ -ClFALDꢀ reactsꢀ withꢀ cellularꢀ GSHꢀ (12),ꢀ anꢀ
analogousꢀpathwayꢀforꢀ-BrFALDꢀhasꢀnotꢀbeenꢀreported.ꢀ
Theꢀreactionꢀbetweenꢀ-ClFALDꢀandꢀGSHꢀisꢀaꢀnucleophilicꢀ
substitutionꢀreactionꢀofꢀtheꢀGSHꢀthiolꢀatꢀtheꢀ-chlorinatedꢀ
carbonꢀcoupledꢀwithꢀejectionꢀofꢀtheꢀchlorineꢀasꢀaꢀleavingꢀ
groupꢀ(12).ꢀBecauseꢀbromineꢀisꢀaꢀbetterꢀleavingꢀgroupꢀinꢀ
nucleophilicꢀ substitutionꢀ reactions,ꢀ itꢀ followsꢀ thatꢀ -
BrFALDꢀwillꢀbeꢀveryꢀreactiveꢀwithꢀGSH.
Theꢀ mechanismsꢀ throughꢀ whichꢀ -ClFALDꢀ andꢀ -
BrFALDꢀ elicitꢀ cellularꢀ dysfunctionꢀ andꢀ signalingꢀ areꢀ un-
known,ꢀbutꢀstudiesꢀhaveꢀshownꢀthatꢀ-ClFALDꢀcanꢀreactꢀwithꢀ
cellularꢀ proteinsꢀ throughꢀ unspecifiedꢀ mechanismsꢀ (13),ꢀ
whichꢀmayꢀaccountꢀforꢀsignalingꢀalterations.ꢀInꢀthisꢀstudy,ꢀ
weꢀshowꢀthatꢀcomparedꢀwithꢀ-ClFALD, -BrFALDꢀisꢀmoreꢀ
potentꢀinꢀreactingꢀwithꢀthiols.ꢀImportantly,ꢀthisꢀselectivityꢀ
ofꢀthiolꢀreactivityꢀisꢀalsoꢀobservedꢀinꢀtheꢀpresenceꢀofꢀendog-
enouslyꢀproducedꢀ-ClFALDꢀandꢀ-BrFALDꢀinꢀeosinophils,ꢀ
neutrophils,ꢀandꢀmiceꢀexposedꢀtoꢀbromineꢀgas.ꢀFurthermore,ꢀ
usingꢀclick-chemistryꢀanalogsꢀofꢀ-ClFALDꢀandꢀ-BrFALD,ꢀ
weꢀdemonstrateꢀtheꢀrobustꢀmodificationsꢀofꢀcellularꢀpro-
teinsꢀbyꢀ-BrFALDꢀinꢀcomparisonꢀtoꢀ-ClFALD.
GSH in vitro reactions with halogenated lipids and TLC
purification of reaction product
Unlessꢀ otherwiseꢀ indicated,ꢀ allꢀ reactionsꢀ wereꢀ carriedꢀ outꢀ inꢀ
PBSꢀ(pHꢀ7.4)ꢀandꢀethanolꢀatꢀaꢀratioꢀofꢀ30:70.ꢀBriefly,ꢀGSHꢀ(1ꢀmol)ꢀ
wasꢀfirstꢀsuspendedꢀinꢀ60ꢀlꢀofꢀPBSꢀ(pHꢀ7.4)ꢀinꢀaꢀborosilicateꢀre-
actionꢀvessel.ꢀOneꢀmicromoleꢀofꢀlipidꢀ[hexadecanalꢀ(HDA),ꢀ
2-chlorohexadecanalꢀ(2-ClHDA),ꢀ2-chlorohexadecanolꢀ(2-ClHOH),ꢀ
2-chlorohexadecanoicꢀ acidꢀ (2-ClHA),ꢀ 2-bromohexadecanalꢀ
(2-BrHDA),ꢀorꢀ2-bromohexadecanoicꢀacidꢀ(2-BrHA)]ꢀwasꢀaddedꢀ
toꢀtheꢀGSHꢀinꢀ140ꢀlꢀethanolꢀandꢀtheꢀreactionꢀmixtureꢀwasꢀvor-
texed,ꢀcapped,ꢀandꢀincubatedꢀatꢀ37°Cꢀforꢀ4ꢀh.ꢀReactionꢀaliquotsꢀ
(20ꢀl)ꢀwereꢀsubjectedꢀtoꢀTLCꢀusingꢀ40ꢀÅꢀsilicaꢀgelꢀTLCꢀplatesꢀasꢀ
stationaryꢀphaseꢀandꢀaꢀmobileꢀphaseꢀcomprisedꢀofꢀchloroform/
acetone/methanol/water/aceticꢀ acidꢀ (6/8/2/2/1,ꢀ v/v/v/v/v).ꢀ
Reactionꢀproductsꢀwereꢀvisualizedꢀfollowingꢀ2,4-dinitrophenylhy-
drazineꢀstaining.ꢀInꢀsomeꢀcases,ꢀTLCꢀplatesꢀwereꢀnotꢀstainedꢀbe-
causeꢀTLCꢀwasꢀusedꢀforꢀpurificationꢀofꢀreactionꢀproductsꢀ[e.g.,ꢀ
2-halohexadecanalꢀ adductꢀ withꢀ GSHꢀ (HDA-GSH)]ꢀ andꢀ silicaꢀ
associatedꢀwithꢀreactionꢀproductsꢀwasꢀscrapedꢀfromꢀplatesꢀandꢀex-
tractedꢀusingꢀacetonitrile/waterꢀ(7/3,ꢀv/v)ꢀsupplementedꢀwithꢀ
0.25%ꢀformicꢀacid.ꢀSilicaꢀwasꢀsubsequentlyꢀseparatedꢀfromꢀsolventꢀ
byꢀcentrifugationꢀ(2,000ꢀgꢀmaximum)ꢀforꢀ5ꢀmin.ꢀTheꢀsupernatantꢀ
extractꢀwasꢀsubsequentlyꢀdriedꢀunderꢀnitrogenꢀandꢀsuspendedꢀinꢀ
150 lꢀacetonitrile/methanol/waterꢀ(2/1/1,ꢀv/v/v)ꢀsupplementedꢀ
withꢀ0.1%ꢀformicꢀacidꢀforꢀESI-MS/MSꢀanalysis.
2-BrHDA and GSH in vitro product characterization
TLC-purifiedꢀHDA-GSHꢀwasꢀdilutedꢀ2,000ꢀtimesꢀwithꢀacetoni-
trile/methanol/waterꢀ(2/1/1,ꢀv/v/v)ꢀsupplementedꢀwithꢀ0.1%ꢀ
formicꢀacid,ꢀandꢀwasꢀanalyzedꢀbyꢀESI-MS/MSꢀbyꢀdirectꢀinfusionꢀ
atꢀaꢀflowꢀrateꢀofꢀ5ꢀl/min.ꢀForꢀESI-MS/MS,ꢀtheꢀionizationꢀen-
ergyꢀandꢀtemperatureꢀwereꢀsetꢀatꢀ3,900ꢀVꢀandꢀ270°Cꢀinꢀpositiveꢀ
ionꢀ mode.ꢀ Aꢀ collisionꢀ energyꢀ ofꢀ 18ꢀ eVꢀ andꢀ collisionꢀ gasꢀ ofꢀ
1.0ꢀTorrꢀargonꢀwereꢀusedꢀforꢀMS/MSꢀanalysesꢀinꢀpositiveꢀionꢀ
mode.
MATERIALSꢀANDꢀMETHODS
Materials
HPLCꢀgradeꢀmethanol,ꢀacetonitrile,ꢀandꢀisopropanolꢀwereꢀpur-
chasedꢀfromꢀ(FisherꢀScientific).ꢀTLCꢀplatesꢀ(20ꢀ×ꢀ20ꢀcm,ꢀ40ꢀÅꢀsil-
icaꢀgel)ꢀwereꢀobtainedꢀfromꢀEMBꢀMillipore.ꢀAllꢀotherꢀchemicalsꢀ
wereꢀpurchasedꢀfromꢀSigma-AldrichꢀorꢀFisherꢀScientific.
RAW 264.7 cell treatments with 2-BrHDA and 2-ClHDA
Instrumentation
MSꢀwasꢀperformedꢀusingꢀaꢀThermoꢀFisherꢀTSQꢀQuantumꢀUltraꢀ
massꢀspectrometerꢀ(ThermoꢀFisher,ꢀWaltham,ꢀMA).ꢀForꢀexperi-
RAWꢀ264.7ꢀcellsꢀwereꢀgrownꢀtoꢀconfluenceꢀinꢀ6-wellꢀplatesꢀ(9.5ꢀ
cm2ꢀgrowthꢀarea)ꢀandꢀwereꢀtreatedꢀwithꢀeitherꢀ0,ꢀ10,ꢀ25,ꢀorꢀ50ꢀMꢀ
2-BrHDAꢀorꢀ2-ClHDAꢀinꢀDMEMꢀsupplementedꢀwithꢀ2%ꢀFBSꢀforꢀ
4ꢀh.ꢀTheꢀmediumꢀwasꢀthenꢀremovedꢀatꢀ4ꢀhꢀafterꢀtheꢀstartꢀofꢀtheꢀ
treatmentꢀandꢀcellsꢀwashedꢀwithꢀPBS.ꢀTheꢀcellsꢀwereꢀscrapedꢀinꢀ
1.25ꢀmlꢀPBSꢀcontainingꢀ10ꢀmMꢀN-ethylmaleimideꢀ(NEM)ꢀandꢀim-
mediatelyꢀfrozenꢀpriorꢀtoꢀanalysisꢀofꢀFALD-GSH.ꢀInꢀotherꢀexperi-
ments,ꢀ RAWꢀ 264.7ꢀ cellsꢀ wereꢀ treatedꢀ withꢀ eitherꢀ 0,ꢀ 10,ꢀ 25,ꢀ orꢀ
50 Mꢀ2-BrHDA,ꢀ2-ClHDA,ꢀorꢀHDAꢀinꢀDMEMꢀsupplementedꢀwithꢀ
2%ꢀFBSꢀforꢀ4ꢀh.ꢀTheꢀmediumꢀwasꢀthenꢀremovedꢀatꢀ0.5,ꢀ1,ꢀ2,ꢀ3,ꢀandꢀ
4ꢀhꢀafterꢀtheꢀstartꢀofꢀtheꢀtreatmentꢀandꢀcellsꢀwashedꢀwithꢀPBS.ꢀTheꢀ
cellsꢀwereꢀscrapedꢀinꢀ1.25ꢀmlꢀPBSꢀcontainingꢀ10ꢀmMꢀNEMꢀandꢀ
immediatelyꢀfrozenꢀalongꢀwithꢀtheꢀmediumꢀpriorꢀtoꢀanalysisꢀofꢀ
FALD-GSH
Fig. 1.ꢀ MPOꢀselectivelyꢀproducesꢀHOClꢀandꢀEPOꢀselectivelyꢀpro-
ducesꢀHOBr.ꢀMPOꢀfoundꢀinꢀneutrophils,ꢀmonocytes,ꢀandꢀmacro-
phagesꢀ utilizeꢀ Clꢀ ionsꢀ andꢀ H2O2ꢀ toꢀ produceꢀ HOClꢀ followingꢀ aꢀ
respiratoryꢀ burstꢀ andꢀ degranulation.ꢀ However,ꢀ someꢀ Br-ꢀ ionsꢀ areꢀ
usedꢀbyꢀMPOꢀtoꢀproduceꢀHOBrꢀ(notꢀdepicted).ꢀEPO,ꢀfoundꢀinꢀ
eosinophils,ꢀselectivelyꢀutilizesꢀBr-ꢀionsꢀandꢀH2O2ꢀtoꢀproduceꢀHOBrꢀ
evenꢀthoughꢀBr-ꢀionꢀconcentrationsꢀareꢀ1,000ꢀtimesꢀlowerꢀthanꢀtheꢀ
Clꢀconcentrationsꢀphysiologically.ꢀBothꢀHOClꢀandꢀHOBrꢀcanꢀat-
tackꢀtheꢀvinylꢀeitherꢀbondꢀofꢀplasmalogenꢀtoꢀreleaseꢀlysosphospho-
lipidꢀandꢀ-ClFALDꢀorꢀ-BrFALD,ꢀrespectively.
Human neutrophil studies
Wholeꢀbloodꢀ(50ꢀml)ꢀwasꢀtakenꢀfromꢀhealthyꢀvolunteersꢀandꢀ
anticoagulatedꢀwithꢀEDTAꢀ(finalꢀconcentrationꢀ5.4ꢀmM)ꢀpriorꢀtoꢀ
theꢀ isolationꢀ ofꢀ neutrophilsꢀ usingꢀ aꢀ Ficoll-Hypaqueꢀ gradientꢀ asꢀ
previouslyꢀdescribedꢀ(12).ꢀNeutrophilꢀandꢀeosinophilꢀstudiesꢀwereꢀ
approvedꢀandꢀauthorizedꢀbyꢀtheꢀSaintꢀLouisꢀUniversityꢀInstitutionalꢀ
Reviewꢀ Boardꢀ Protocolꢀ 9952.ꢀ Informedꢀ consentꢀ wasꢀ obtainedꢀ
fromꢀtheꢀhumanꢀsubjects.ꢀNeutrophilsꢀwereꢀdilutedꢀtoꢀ1ꢀmillionꢀ
neutrophilsꢀperꢀmilliliterꢀinꢀHBSꢀ±ꢀ1ꢀmMꢀNaBr.ꢀPhorbolꢀmyristateꢀ
Bromofatty aldehyde modifications of glutathione and protein
697

acetateꢀ(PMA)ꢀ(100ꢀnM)ꢀinꢀethanolꢀ(0.1%)ꢀwasꢀthenꢀaddedꢀasꢀ
indicatedꢀandꢀthenꢀtheꢀneutrophilsꢀwereꢀincubatedꢀforꢀ30ꢀminꢀ
atꢀ37°C.ꢀAtꢀtheꢀendꢀofꢀeachꢀreaction,ꢀ10ꢀmMꢀofꢀNEMꢀwereꢀaddedꢀ
andꢀtheꢀneutrophilsꢀwereꢀimmediatelyꢀfrozenꢀpriorꢀtoꢀFALD-GSHꢀ
analysis.
waterꢀcontainingꢀ0.15%ꢀformicꢀacidꢀandꢀsolventꢀBꢀwasꢀacetonitrile/
isopropanolꢀ(3/2,ꢀv/v)ꢀcontainingꢀ0.15%ꢀformicꢀacid.ꢀInitialꢀcol-
umnꢀconditionsꢀwereꢀ65/35ꢀ(A/B)ꢀatꢀaꢀflowꢀrateꢀofꢀ200ꢀl/min,ꢀ
whichꢀwasꢀheldꢀforꢀ2ꢀminꢀfollowingꢀtheꢀinjectionꢀofꢀ25ꢀl of the
Strata-X-purifiedꢀsolutionsꢀontoꢀtheꢀcolumn.ꢀSubsequently,ꢀGSHꢀ
adductsꢀofꢀ-halofattyꢀaldehydesꢀwereꢀelutedꢀbyꢀaꢀ3ꢀminꢀlinearꢀ
gradientꢀtoꢀ100%ꢀsolventꢀB.
Human eosinophil studies
Primaryꢀ humanꢀ eosinophilsꢀ wereꢀ preparedꢀ asꢀ previouslyꢀ de-
scribedꢀ(11).ꢀWholeꢀbloodꢀwasꢀtakenꢀfromꢀhypersensitiveꢀvolun-
teers,ꢀ andꢀ eosinophilsꢀ wereꢀ isolatedꢀ usingꢀ aꢀ Percollꢀ gradientꢀ
followedꢀbyꢀaꢀCD16ꢀimmunomagneticꢀbeadꢀsortingꢀsystemꢀonꢀaꢀ
MiltenyiꢀMACSꢀcolumn.ꢀPurifiedꢀeosinophilsꢀwereꢀresuspendedꢀ
inꢀ Hanks’ꢀ balancedꢀ saltꢀ solutionꢀ (pHꢀ 7.3)ꢀ supplementedꢀ withꢀ
100 MꢀNaBr,ꢀasꢀisꢀfoundꢀphysiologicallyꢀ(14–16).ꢀEosinophilsꢀ(1ꢀ×ꢀ
106ꢀcells/ml)ꢀwereꢀtreatedꢀwithꢀorꢀwithoutꢀ(control)ꢀ200ꢀnMꢀPMAꢀ
forꢀ1ꢀhꢀatꢀ37°C.ꢀReactionsꢀwereꢀterminatedꢀbyꢀsnap-freezingꢀinꢀ
liquidꢀnitrogenꢀandꢀFALD-GSHꢀwasꢀanalyzed.
Extraction and quantification of -BrFALD
-BrFALDꢀwasꢀextractedꢀfromꢀBr2-exposedꢀmice,ꢀasꢀwasꢀprevi-
ouslyꢀdescribedꢀforꢀ-ClFALDꢀextractionꢀfromꢀCl2-exposedꢀmiceꢀ
(10).ꢀ Briefly,ꢀ 10ꢀ mgꢀ ofꢀ frozenꢀ pulverizedꢀ lungꢀ tissueꢀ wereꢀ ex-
tractedꢀbyꢀaꢀBlighꢀandꢀDyerꢀlipidꢀextractionꢀinꢀtheꢀpresenceꢀofꢀ
20ꢀ pmolꢀ [d4]ClHDA.ꢀ Lipidꢀ extractsꢀ wereꢀ thenꢀ derivatizedꢀ withꢀ
pentafluorobenzylꢀhydroxylamineꢀandꢀquantitatedꢀbyꢀGC-MSꢀasꢀ
previouslyꢀdescribedꢀ(11).
Synthesis of HDyA, ClHDyA, and BrHDyA alkyne
Mouse exposure to Br2 gas
Theꢀ alkyneꢀ analogsꢀ ofꢀ HDAꢀ [hexadec-15-ynalꢀ (HDyA)],ꢀ
2-ClHDAꢀ[2-chlorohexadec-15-ynalꢀ(2-ClHDyA)],ꢀandꢀ2-BrHDAꢀ
[2-bromohexadec-15-ynalꢀ(2-BrHDyA)]ꢀwereꢀsynthesizedꢀasꢀprevi-
ouslyꢀdescribed,ꢀwithꢀvariationsꢀ(20).ꢀBriefly,ꢀhexadec-7-ynolꢀwasꢀ
convertedꢀtoꢀhexadec-15-ynolꢀusingꢀsodiumꢀhydrideꢀandꢀdiami-
nopropane.ꢀTheꢀalcoholꢀofꢀhexadec-15-ynolꢀwasꢀoxidizedꢀtoꢀanꢀ
aldehydeꢀinꢀaꢀsolutionꢀofꢀ2-iodoxybenzoicꢀacidꢀinꢀDMSO,ꢀwhichꢀ
resultedꢀinꢀHDyA.ꢀThisꢀaldehydeꢀwasꢀchlorinatedꢀusingꢀN-chloro-
succinimideꢀandꢀprolineꢀforꢀ1ꢀhꢀorꢀbrominatedꢀwithꢀN-bromo-
succinimideꢀ andꢀ prolineꢀ forꢀ 1ꢀ h.ꢀ Aꢀ portionꢀ ofꢀ HDyAꢀ wasꢀ notꢀ
halogenatedꢀ forꢀ useꢀ asꢀ aꢀ control.ꢀ Afterꢀ eachꢀ step,ꢀ theꢀ desiredꢀ
productꢀwasꢀpurifiedꢀbyꢀflashꢀchromatographyꢀusingꢀ30ꢀgꢀsilicaꢀgelꢀ
(highꢀpurityꢀgrade,ꢀporeꢀsizeꢀ60Å;ꢀSigma-Aldrich).ꢀSamplesꢀwereꢀ
loadedꢀandꢀelutedꢀwithꢀthreeꢀcolumnꢀvolumesꢀofꢀ30:10:60:5ꢀ(ethylꢀ
acetate:hexane:toluene:aceticꢀacid).ꢀFractionsꢀ(1ꢀmlꢀeach)ꢀwereꢀ
collected,ꢀanalyzedꢀbyꢀTLCꢀ(seeꢀbelow),ꢀandꢀtheꢀdesiredꢀproductsꢀ
wereꢀcombined.
AllꢀanimalꢀproceduresꢀwereꢀapprovedꢀbyꢀtheꢀUniversityꢀofꢀAla-
bamaꢀatꢀBirminghamꢀInstitutionalꢀAnimalꢀCareꢀandꢀUseꢀCommit-
tee.ꢀWholeꢀbodyꢀexposuresꢀofꢀmaleꢀmiceꢀtoꢀBr2ꢀwereꢀperformedꢀasꢀ
previouslyꢀ describedꢀ (17,ꢀ 18).ꢀ Exposuresꢀ wereꢀ performedꢀ withꢀ
twoꢀmiceꢀinꢀtheꢀsameꢀchamberꢀatꢀanyꢀoneꢀtime,ꢀandꢀallꢀexposuresꢀ
wereꢀperformedꢀbetweenꢀ8:00ꢀAMꢀandꢀ12:00ꢀPM.ꢀExposureꢀcondi-
tionsꢀwereꢀ400ꢀorꢀ600ꢀppmꢀBr2ꢀinꢀairꢀforꢀ30ꢀmin.ꢀFlowꢀmetersꢀwereꢀ
usedꢀtoꢀcontrolꢀflowꢀratesꢀofꢀBr2ꢀandꢀroomꢀairꢀtoꢀachieveꢀtheꢀcham-
berꢀBr2ꢀtargetꢀconcentrations.ꢀAꢀbubbleꢀflowꢀmeterꢀwasꢀusedꢀtoꢀ
validateꢀtheirꢀperformanceꢀonꢀaꢀweeklyꢀbasis.ꢀInꢀeachꢀcase,ꢀimme-
diatelyꢀfollowingꢀexposure,ꢀmiceꢀwereꢀreturnedꢀtoꢀroomꢀair.ꢀFoodꢀ
andꢀwaterꢀwereꢀprovidedꢀadꢀlibitum.
Extraction and quantification of FALD-GSH
FALD-GSHꢀwasꢀextractedꢀfromꢀcellꢀextractsꢀandꢀquantitatedꢀasꢀ
previouslyꢀdescribedꢀ(12).ꢀBriefly,ꢀ90ꢀfmolꢀofꢀ[d4]HDA-GSHꢀwereꢀ
added to 100 lꢀofꢀtheꢀRAWꢀ264.7ꢀcellꢀhomogenateꢀandꢀ45ꢀfmolꢀ
ofꢀ[d4]HDA-GSHꢀwereꢀaddedꢀtoꢀ1ꢀmlꢀofꢀneutrophilꢀorꢀeosinophilꢀ
suspension.ꢀOneꢀvolumeꢀofꢀmethanol/acetonitrileꢀ(1/1,ꢀv/v)ꢀwasꢀ
addedꢀtoꢀeachꢀmixtureꢀandꢀthenꢀeachꢀmixtureꢀwasꢀvortexedꢀforꢀ30ꢀs,ꢀ
sonicatedꢀinꢀaꢀsonicationꢀbathꢀforꢀ15ꢀs,ꢀandꢀvortexedꢀforꢀanotherꢀ
30ꢀs.ꢀTheꢀresultingꢀsolutionꢀwasꢀcentrifugedꢀatꢀ1,000ꢀgꢀforꢀ5ꢀmin,ꢀ
andꢀtheꢀsupernatantꢀwasꢀcarefullyꢀremovedꢀfromꢀtheꢀcellꢀdebrisꢀ
pellet.ꢀTheꢀsupernatantꢀwasꢀloadedꢀonꢀaꢀStrata-Xꢀcolumnꢀ(60ꢀmgꢀ
bedꢀweight)ꢀthatꢀwasꢀpreconditionedꢀwithꢀ1.2ꢀmlꢀmethanolꢀfol-
lowedꢀ byꢀ 1.2ꢀ mlꢀ water/methanolꢀ (4/1,ꢀ v/v).ꢀ Columnsꢀ wereꢀ
washedꢀ twoꢀ timesꢀ withꢀ 0.6ꢀ mlꢀ water/methanolꢀ (4/1,ꢀ v/v),ꢀ andꢀ
thenꢀ 2-ClHDAꢀ conjugatesꢀ ofꢀ GSHꢀ wereꢀ elutedꢀ withꢀ 1.2ꢀ mlꢀ ofꢀ
methanol/acetonitrileꢀ(3/1,ꢀv/v)ꢀcontainingꢀ0.25%ꢀformicꢀacid.ꢀ
Theꢀelutedꢀadductꢀwasꢀdriedꢀunderꢀnitrogenꢀandꢀsuspendedꢀ
inꢀ 100ꢀ lꢀ ofꢀ 6/4/5ꢀ acetonitrile/isopropanol/waterꢀ containingꢀ
0.15%ꢀformicꢀacidꢀforꢀanalysisꢀbyꢀLC-MS/MS.
LungꢀandꢀplasmaꢀofꢀBr2-exposedꢀmiceꢀwereꢀanalyzedꢀforꢀFALD-
GSHꢀasꢀpreviouslyꢀdescribedꢀ(10).ꢀBriefly,ꢀ25ꢀµlꢀofꢀplasmaꢀwereꢀ
spikedꢀwithꢀ110ꢀfmolꢀofꢀ[d4]HDA-GSHꢀand10ꢀmgꢀofꢀpulverizedꢀ
lungꢀtissueꢀwereꢀspikedꢀwithꢀ1,100ꢀfmolꢀ[d4]HDA-GSH.ꢀTheꢀaque-
ousꢀlayerꢀofꢀaꢀBlighꢀandꢀDyerꢀlipidꢀofꢀextraction,ꢀasꢀusedꢀforꢀmostꢀ
halogenatedꢀlipidꢀextractionsꢀ(19),ꢀwasꢀisolated.ꢀTheꢀremainingꢀ
organicꢀlayerꢀwasꢀreextractedꢀwithꢀ1ꢀvolꢀmethanol:waterꢀ(1:1,ꢀv:v)ꢀ
andꢀcombinedꢀwithꢀtheꢀfirstꢀaqueousꢀextraction.ꢀTheꢀcombinedꢀ
aqueousꢀlayersꢀwereꢀextractedꢀonꢀaꢀStrata-XꢀSPEꢀextractionꢀcol-
umnꢀafterꢀdilutionꢀwithꢀ1/3ꢀvolꢀofꢀwaterꢀandꢀthenꢀquantitatedꢀbyꢀ
ESI-LC-MS/MSꢀquantitation.
THP-1 cell experiments
THP-1ꢀhumanꢀleukemicꢀmonocyteꢀcellsꢀwereꢀgrownꢀinꢀRPMIꢀ+ꢀ
10%ꢀ FBS.ꢀ Fiveꢀ millionꢀ THP-1ꢀ cellsꢀ wereꢀ centrifugedꢀ andꢀ sus-
pendedꢀinꢀ4ꢀmlꢀofꢀRPMIꢀwithꢀnoꢀFBS.ꢀCellsꢀwereꢀpretreatedꢀwithꢀ
orꢀ withoutꢀ 1ꢀ mMꢀ NEM,ꢀ orꢀ 5ꢀ mMꢀ N-acetylcysteineꢀ (NAC)ꢀ forꢀ
30ꢀminꢀatꢀ37°C.ꢀThenꢀTHP-1ꢀcellsꢀwereꢀtreatedꢀwithꢀeitherꢀ5ꢀorꢀ
10 MꢀHDyA,ꢀ2-ClHDyA,ꢀorꢀ2-BrHDyAꢀforꢀ30ꢀmin.ꢀThenꢀcellsꢀ
wereꢀpelletedꢀbyꢀcentrifugationꢀatꢀ1,500ꢀgꢀforꢀ5ꢀmin.ꢀTheꢀsuper-
natantꢀwasꢀremovedꢀandꢀtheꢀpelletꢀwasꢀsuspendedꢀinꢀ4ꢀmlꢀice-
coldꢀPBSꢀfollowedꢀbyꢀcentrifugationꢀforꢀ5ꢀminꢀatꢀ1,500ꢀg.ꢀTheꢀ
supernatantꢀ wasꢀ removedꢀ andꢀ 300ꢀ µlꢀ ofꢀ ice-coldꢀ RIPAꢀ bufferꢀ
withꢀHALTꢀproteaseꢀinhibitorꢀwereꢀadded,ꢀmixedꢀbyꢀpipetting,ꢀ
andꢀthenꢀleftꢀtoꢀsitꢀonꢀiceꢀforꢀ20ꢀmin.ꢀTheꢀextractedꢀTHP-1ꢀcellsꢀ
wereꢀthenꢀcentrifugedꢀatꢀ14,000ꢀgꢀforꢀ15ꢀmin.ꢀTheꢀprotein-con-
tainingꢀsupernatantꢀwasꢀextractedꢀandꢀtheꢀproteinꢀcontentꢀwasꢀ
quantitatedꢀusingꢀaꢀBradfordꢀassay,ꢀandꢀthenꢀfrozenꢀatꢀ20°Cꢀ
untilꢀclickꢀchemistryꢀvisualization.
Click chemistry visualization of THP-1 cell protein
AliquotsꢀofꢀextractedꢀTHP-1ꢀcellꢀproteinꢀ(100ꢀg)ꢀwereꢀtrans-
ferredꢀintoꢀ1.5ꢀmlꢀEppendorfꢀtubes.ꢀOneꢀhundredꢀmicrolitersꢀofꢀ
250ꢀ mMꢀ PBSꢀ wereꢀ addedꢀ toꢀ eachꢀ sampleꢀ andꢀ theꢀ volumeꢀ wasꢀ
broughtꢀtoꢀ170ꢀlꢀwithꢀdiH2O.ꢀTwentyꢀmicrolitersꢀofꢀaꢀmixtureꢀofꢀ
75ꢀmMꢀTHPTAꢀandꢀ15ꢀmMꢀCu(II)SO4ꢀinꢀwaterꢀwereꢀthenꢀaddedꢀ
andꢀtheꢀsampleꢀwasꢀvortexedꢀforꢀ5ꢀs.ꢀFourꢀmicrolitersꢀofꢀ2.5ꢀmMꢀ
TAMRA-azideꢀinꢀDMSOꢀwereꢀaddedꢀtoꢀeachꢀsampleꢀandꢀthenꢀ
vortexedꢀforꢀ5ꢀs.ꢀTenꢀmicrolitersꢀofꢀ400ꢀmMꢀsodiumꢀascorbateꢀ
wereꢀthenꢀaddedꢀtoꢀeachꢀreactionꢀfollowedꢀbyꢀ5ꢀsꢀofꢀvortexing.ꢀ
LCꢀ wasꢀ performedꢀ usingꢀ aꢀ Phenomenexꢀ Onyxꢀ C-18ꢀ columnꢀ
(50ꢀ×ꢀ2.0ꢀmm)ꢀwithꢀESI-MS/MSꢀdetectionꢀusingꢀselectedꢀreactionꢀ
monitoring.ꢀTwoꢀmobileꢀphasesꢀwereꢀusedꢀforꢀLC.ꢀSolventꢀAꢀwasꢀ
698
Journal of Lipid Research Volume 59, 2018

Theꢀreactionꢀmixtureꢀwasꢀprotectedꢀfromꢀtheꢀlightꢀandꢀincubatedꢀ
atꢀroomꢀtemperatureꢀforꢀ30ꢀminꢀwithꢀgentleꢀrocking.ꢀToꢀpurifyꢀ
theꢀclickedꢀprotein,ꢀ600ꢀlꢀofꢀmethanol,ꢀ150ꢀl of CHCl3,ꢀandꢀ
400ꢀlꢀofꢀwaterꢀwereꢀaddedꢀfollowedꢀbyꢀ5ꢀsꢀofꢀvortexing.ꢀSamplesꢀ
wereꢀthenꢀcentrifugedꢀforꢀ5ꢀminꢀatꢀ13,000–18,000ꢀg.ꢀTheꢀupperꢀ
layerꢀwasꢀcarefullyꢀremovedꢀtoꢀmakeꢀsureꢀnotꢀtoꢀdisturbꢀtheꢀinter-
face.ꢀFourꢀhundredꢀandꢀfiftyꢀmicrolitersꢀofꢀmethanolꢀwereꢀthenꢀ
addedꢀtoꢀtheꢀremainingꢀsolutionꢀandꢀvortexedꢀforꢀ5ꢀs.ꢀTheꢀho-
mogenousꢀsolutionꢀwasꢀcentrifugedꢀforꢀ5ꢀminꢀatꢀ13,000–18,000ꢀg,
andꢀ thenꢀ theꢀ supernatantꢀ wasꢀ removedꢀ whileꢀ notꢀ disturbingꢀ
theꢀpellet.ꢀAnꢀadditionalꢀ450ꢀµlꢀofꢀmethanolꢀwereꢀaddedꢀtoꢀtheꢀ
pellet,ꢀvortexedꢀforꢀ5ꢀs,ꢀandꢀcentrifugedꢀforꢀ5ꢀminꢀatꢀ13,000–
18,000 gꢀ followedꢀbyꢀcarefulꢀremovalꢀofꢀtheꢀsupernatant.ꢀTheꢀ
pelletꢀwasꢀthenꢀallowedꢀtoꢀairꢀdryꢀforꢀ30–60ꢀmin.ꢀSampleꢀload-
ingꢀbufferꢀwasꢀthenꢀaddedꢀtoꢀtheꢀdryꢀpelletꢀatꢀaꢀconcentrationꢀofꢀ
1–2ꢀmg/ml.ꢀTheꢀsamplesꢀwereꢀthenꢀvortexedꢀforꢀ10ꢀminꢀatꢀroomꢀ
temperatureꢀfollowedꢀbyꢀheatingꢀatꢀ70°Cꢀforꢀ10ꢀmin.ꢀAfterꢀtheꢀ
samplesꢀhadꢀcooledꢀtoꢀroomꢀtemperature,ꢀtheyꢀwereꢀcentrifugedꢀ
forꢀ1ꢀminꢀatꢀ1,500ꢀg.ꢀSamplesꢀwereꢀthenꢀloadedꢀontoꢀatꢀ12%ꢀBis-
Trisꢀ gelꢀ inꢀ theꢀ statedꢀ quantities.ꢀ Gelꢀ electrophoresisꢀ wasꢀ per-
formedꢀatꢀ100ꢀVꢀinꢀMOPSꢀrunningꢀbuffer.ꢀOnceꢀcompleted,ꢀtheꢀ
gelꢀwasꢀremoved,ꢀbrieflyꢀwashedꢀinꢀwater,ꢀandꢀthenꢀimmediatelyꢀ
visualizedꢀatꢀ532/580ꢀnmꢀ(excitation/emission).ꢀAfterꢀvisualiza-
tion,ꢀtheꢀgelꢀwasꢀfixedꢀandꢀstainedꢀbyꢀCoomassieꢀforꢀtotalꢀproteinꢀ
visualization.
Dunnett’sꢀpostꢀhocꢀtest.ꢀWhenꢀstatisticallyꢀappropriate,ꢀTukey’sꢀ
postꢀhocꢀtestꢀwasꢀused.ꢀAllꢀdataꢀareꢀpresentedꢀasꢀmeanꢀ±ꢀSEMꢀun-
lessꢀotherwiseꢀindicated.
RESULTS
Previousꢀstudiesꢀhaveꢀshownꢀthatꢀ-BrFALDꢀisꢀproducedꢀ
inꢀ humanꢀ neutrophilsꢀ andꢀ eosinophilsꢀ (2,ꢀ 11).ꢀ Becauseꢀ
bromineꢀisꢀaꢀbetterꢀleavingꢀgroupꢀthanꢀchlorine,ꢀitꢀwasꢀan-
ticipatedꢀthatꢀ-BrFALDꢀwouldꢀreactꢀwithꢀGSHꢀthroughꢀaꢀ
thiol-dependentꢀ mechanismꢀ similarꢀ toꢀ thatꢀ ofꢀ -ClFALD
withꢀGSHꢀ(12).ꢀInitialꢀstudiesꢀshownꢀinꢀFig. 2ꢀcomparedꢀtheꢀ
reactionꢀofꢀGSHꢀwithꢀeitherꢀtheꢀ16-carbonꢀ-BrFALDꢀorꢀ
-ClFALD,ꢀ2-BrHDAꢀorꢀ2-ClHDA,ꢀrespectively.ꢀTheꢀbandꢀ
visualizedꢀfromꢀtheꢀreactionꢀproductꢀofꢀ2-BrHDAꢀandꢀGSHꢀ
migratedꢀtoꢀtheꢀsameꢀpositionꢀonꢀtheꢀTLCꢀasꢀthatꢀofꢀ2-Cl-
HDAꢀandꢀGSH.ꢀHowever,ꢀtheꢀreactionꢀproductꢀofꢀ2-BrHDAꢀ
andꢀGSHꢀwasꢀmuchꢀdarkerꢀasꢀvisualizedꢀbyꢀTLCꢀstainingꢀ
withꢀphosphomolybdicꢀacid,ꢀandꢀGSHꢀwasꢀcompletelyꢀcon-
sumedꢀ inꢀ reactionsꢀ withꢀ 2-BrHDAꢀ comparedꢀ withꢀ 2-Cl-
HDA.ꢀInꢀcomparisonꢀtoꢀtheꢀreactivityꢀofꢀ2-ClHDAꢀandꢀ
2-BrHDAꢀwithꢀGSHꢀafterꢀ4ꢀh,ꢀTLCꢀanalysesꢀdidꢀnotꢀrevealꢀ
anyꢀreactionsꢀbetweenꢀGSHꢀandꢀeitherꢀ2-ClHOH,ꢀ2-ClHA,ꢀ
2-BrHA,ꢀ orꢀ HDAꢀ (Fig.ꢀ 2A).ꢀ Importantly,ꢀ theꢀ nonhaloge-
natedꢀfattyꢀaldehyde,ꢀHDA,ꢀdidꢀnotꢀreactꢀwithꢀGSH,ꢀwhichꢀ
confirmsꢀtheꢀreactionꢀtoꢀbeꢀdependentꢀonꢀtheꢀpresenceꢀofꢀ
the -halogenatedꢀcarbon.
Previously,ꢀtheꢀreactionꢀproductꢀofꢀ2-ClHDAꢀwithꢀGSHꢀ
wasꢀfoundꢀtoꢀbeꢀaꢀpeptidofattyꢀaldehydeꢀ(HDA-GSH)ꢀ(12).ꢀ
Toꢀconfirmꢀtheꢀsameꢀproductꢀofꢀtheꢀreactionꢀofꢀ2-BrHDAꢀ
andꢀGSH,ꢀtheꢀTLC-purifiedꢀproductꢀwasꢀanalyzedꢀbyꢀESI-
MS/MS.ꢀTheꢀparentꢀ[M+H+]ꢀionꢀofꢀm/zꢀ546ꢀ(Fig.ꢀ2Bꢀinsert)ꢀ
andꢀitsꢀfragmentꢀionsꢀm/zꢀ399ꢀandꢀ296ꢀ(Fig.ꢀ2B)ꢀareꢀidenti-
calꢀtoꢀthoseꢀmolecularꢀionsꢀpreviouslyꢀreportedꢀforꢀHDA-
GSH,ꢀwhich,ꢀinꢀconjunctionꢀwithꢀcomigrationꢀofꢀreactionꢀ
productsꢀbyꢀTLC,ꢀindicateꢀthatꢀHDA-GSHꢀisꢀtheꢀproduct.
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide assay
TheꢀmetabolicꢀactivityꢀofꢀTHP-1ꢀcellsꢀwasꢀexaminedꢀusingꢀtheꢀ
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumꢀ bromideꢀ
(MTT)ꢀassay.ꢀCellsꢀwereꢀplatedꢀinꢀaꢀclearꢀ96-wellꢀplateꢀandꢀgrownꢀ
toꢀconfluence.ꢀCellsꢀwereꢀtreatedꢀwithꢀtheꢀindicatedꢀconcen-
trationsꢀ ofꢀ lipidsꢀ inꢀ phenolꢀ red-freeꢀ growthꢀ mediumꢀ withꢀ 10%ꢀ
FBSꢀforꢀ2ꢀh.ꢀMTTꢀ(Sigma-Aldrich,ꢀM2128;ꢀ1.2ꢀmMꢀinꢀ100ꢀl se-
rum-freeꢀ phenolꢀ red-freeꢀ medium)ꢀ wasꢀ addedꢀ toꢀ cellsꢀ forꢀ 4ꢀ h.ꢀ
Seventy-fiveꢀmicrolitersꢀofꢀmediumꢀwereꢀremovedꢀandꢀ50ꢀl of
DMSOꢀwereꢀaddedꢀforꢀ10ꢀminꢀtoꢀlyseꢀtheꢀcells.ꢀAbsorbanceꢀwasꢀ
readꢀatꢀ540ꢀnmꢀonꢀanꢀEnspireꢀmultimodeꢀplateꢀreaderꢀandꢀcor-
rectedꢀ forꢀ backgroundꢀ absorbance.ꢀ Tritonꢀ (0.1%)ꢀ inꢀ PBSꢀ wasꢀ
usedꢀasꢀaꢀpositiveꢀcontrol.ꢀMTTꢀreductionꢀisꢀexpressedꢀasꢀpercentꢀ
MTTꢀreductionꢀ(blankꢀdesignatedꢀasꢀ100%).
HDA-GSH formation in RAW 264.7 cells treated with
exogenous 2-BrHDA and 2-ClHDA
Statistical analyses
Furtherꢀstudiesꢀwereꢀperformedꢀtoꢀdetermineꢀwhetherꢀ
theꢀ sameꢀ preferredꢀ reactivityꢀ ofꢀ GSHꢀ withꢀ 2-BrHDAꢀ
StatisticalꢀanalysisꢀforꢀtheꢀpresentedꢀdataꢀutilizedꢀANOVAꢀtoꢀ
compareꢀthreeꢀorꢀmoreꢀgroupsꢀtoꢀaꢀcontrolꢀconditionꢀfollowedꢀbyꢀ
Fig. 2.ꢀ Theꢀ2-BrHDAꢀreactsꢀwithꢀGSHꢀtoꢀformꢀHDA-
GSH.ꢀReactionꢀproductsꢀofꢀGSHꢀ(1ꢀµmol)ꢀwithꢀ1ꢀµmolꢀ
ofꢀeitherꢀHDA,ꢀ2-ClHDA,ꢀ2-ClHOH,ꢀ2-ClHA,ꢀ2-BrHDA,ꢀ
orꢀ2-BrHAꢀwereꢀvisualizedꢀbyꢀphosphomolybdicꢀacidꢀ
stainingꢀfollowingꢀTLCꢀchromatographyꢀasꢀdescribedꢀ
inꢀtheꢀMaterialsꢀandꢀMethodsꢀ(A).ꢀTLCꢀpurifiedꢀreac-
tionꢀ productsꢀ fromꢀ 2-BrHDAꢀ andꢀ GSHꢀ wereꢀ subse-
quentlyꢀanalyzedꢀbyꢀESI-MS/MS.ꢀBothꢀtheꢀQ1+ꢀparentꢀ
molecularꢀionꢀatꢀm/zꢀofꢀ546.3ꢀ[(B)ꢀinsert]ꢀandꢀCADꢀ
analysesꢀofꢀm/zꢀ546.3ꢀ(B)ꢀwereꢀtheꢀsameꢀasꢀthatꢀprevi-
ouslyꢀreportedꢀforꢀtheꢀreactionꢀproductꢀofꢀ2-ClHDAꢀ
(12)ꢀandꢀassignmentsꢀforꢀtheꢀdepictedꢀmolecularꢀionsꢀ
inꢀ(B)ꢀareꢀprovidedꢀinꢀ(C).
Bromofatty aldehyde modifications of glutathione and protein
699

comparedꢀwithꢀ2-ClHDAꢀoccurredꢀinꢀRAWꢀ264.7ꢀcells.ꢀAtꢀ
allꢀconcentrationsꢀtestedꢀ(10,ꢀ25,ꢀandꢀ50ꢀM),ꢀ2-BrHDAꢀ
conversionꢀ toꢀ HDA-GSHꢀ wasꢀ approximatelyꢀ 25ꢀ timesꢀ
greaterꢀthanꢀthatꢀinꢀcellsꢀtreatedꢀwithꢀ2-ClHDAꢀ(Fig. 3A).ꢀ
RAWꢀ 264.7ꢀ productionꢀ ofꢀ HDA-GSHꢀ followingꢀ treat-
mentsꢀwithꢀeitherꢀ2-ClHDAꢀorꢀ2-BrHDAꢀ(20ꢀM)ꢀtreatmentꢀ
wasꢀrapid,ꢀoccurringꢀwithinꢀtheꢀfirstꢀ30ꢀminꢀofꢀtreatmentꢀ
withꢀnoꢀfurtherꢀsignificantꢀincreasesꢀatꢀlaterꢀtimeꢀpointsꢀex-
aminedꢀ(Fig.ꢀ3B,ꢀC).ꢀAtꢀallꢀtimeꢀpointsꢀanalyzed,ꢀtheꢀconver-
sionꢀofꢀ2-BrHDAꢀtoꢀHDA-GSHꢀwasꢀatꢀleastꢀ25ꢀtimesꢀgreaterꢀ
usingꢀ 2-BrHDAꢀ comparedꢀ withꢀ 2-ClHDA,ꢀ suggestingꢀ thatꢀ
theꢀ kineticsꢀ ofꢀ thisꢀ reactionꢀ isꢀ overꢀ 25-foldꢀ greaterꢀ usingꢀ
2-BrHDAꢀcomparedꢀwithꢀ2-ClHDA.ꢀInꢀbothꢀ2-ClHDAꢀandꢀ
2-BrHDAꢀmedia,ꢀexportꢀofꢀHDA-GSHꢀwasꢀobservedꢀatꢀ30ꢀ
minꢀ(Fig.ꢀ3C).ꢀHowever,ꢀwhileꢀmediaꢀlevelsꢀinꢀtheꢀ2-ClHDAꢀ
treatmentꢀremainedꢀunchangedꢀafterꢀ30ꢀmin,ꢀtheꢀmediaꢀofꢀ
cellsꢀtreatedꢀwithꢀ2-BrHDAꢀatꢀ1,ꢀ2,ꢀ3,ꢀandꢀ4ꢀhꢀhadꢀelevatedꢀ
HDA-GSHꢀlevelsꢀwhenꢀcomparedꢀwithꢀ30ꢀmin.ꢀTheꢀrequire-
mentꢀ forꢀ halogenꢀ atꢀ theꢀ -positionꢀ forꢀ theꢀ reactionꢀ withꢀ
GSHꢀwasꢀfurtherꢀconfirmedꢀinꢀtheꢀcellꢀcultureꢀstudiesꢀbe-
causeꢀHDA-GSHꢀwasꢀnotꢀdetectedꢀinꢀeitherꢀtheꢀmediaꢀorꢀ
cellsꢀofꢀHDA-treatedꢀRAWꢀ264.7ꢀcellsꢀ(Fig.ꢀ3B,ꢀC).
FALD-GSH production in activated human neutrophils
and eosinophils
Primaryꢀhumanꢀneutrophilsꢀwereꢀutilizedꢀtoꢀanalyzeꢀtheꢀ
productionꢀofꢀFALD-GSHꢀasꢀaꢀresultꢀofꢀHOBrꢀorꢀHOClꢀat-
tackꢀonꢀplasmalogenꢀandꢀsubsequentꢀreleaseꢀofꢀ-BrFALDꢀ
orꢀ-ClFALD.ꢀActivatedꢀhumanꢀneutrophilsꢀcontainꢀMPO,ꢀ
which,ꢀunderꢀphysiologicꢀconditions,ꢀprimarilyꢀproducesꢀ
HOClꢀandꢀ-ClFALDꢀ(1).ꢀHowever,ꢀinꢀtheꢀpresenceꢀofꢀ1ꢀ
mMꢀNaBr,ꢀneutrophilsꢀcanꢀproduceꢀHOBrꢀandꢀ-BrFALDꢀ
(2,ꢀ11).ꢀNeutrophilsꢀstimulatedꢀwithꢀPMAꢀinꢀtheꢀpresenceꢀ
ofꢀ1ꢀmMꢀNaBrꢀproducedꢀsignificantꢀamountsꢀ-BrFALDꢀ
comparedꢀwithꢀneutrophilsꢀthatꢀwereꢀnotꢀsupplementedꢀ
withꢀ1ꢀmMꢀNaBrꢀ(Fig. 4A).ꢀFurthermore,ꢀtheꢀproductionꢀ
of -BrFALDꢀinꢀneutrophilsꢀincubatedꢀwithꢀNaBrꢀledꢀtoꢀ
overꢀanꢀorderꢀofꢀmagnitudeꢀgreaterꢀaccumulationꢀofꢀtheꢀ
FALD-GSHꢀmolecularꢀspecies,ꢀHDA-GSHꢀandꢀ2-haloocta-
decanalꢀ adductꢀ withꢀ GSHꢀ (ODA-GSH),ꢀ comparedꢀ withꢀ
conditionsꢀwithꢀnoꢀNaBrꢀsupplementation.
UnlikeꢀhumanꢀneutrophilꢀMPO,ꢀhumanꢀeosinophilꢀper-
oxidaseꢀselectivelyꢀproducesꢀHOBrꢀandꢀ-BrFALDꢀinꢀcom-
parisonꢀtoꢀHOClꢀandꢀ-ClFALD,ꢀrespectively,ꢀevenꢀwithꢀCl
levelsꢀexceedingꢀBrꢀlevelsꢀbyꢀoverꢀthreeꢀordersꢀofꢀmagni-
tude.ꢀAccordingly,ꢀstudiesꢀwereꢀdesignedꢀtoꢀassessꢀFALD-GSHꢀ
productionꢀinꢀPMA-stimulatedꢀprimaryꢀhumanꢀeosinophils.ꢀ
TheꢀdataꢀinꢀFig. 5ꢀshowꢀthatꢀPMA-stimulatedꢀeosinophilsꢀ
producedꢀ theꢀ FALD-GSHꢀ molecularꢀ species,ꢀ HDA-GSHꢀ
andꢀODA-GSH.ꢀAdditionallyꢀtheꢀproductionꢀofꢀFALD-GSHꢀ
producedꢀbyꢀstimulatedꢀeosinophilsꢀcanꢀalsoꢀbeꢀinhibitedꢀ
byꢀpretreatmentꢀwithꢀtheꢀhemeꢀenzymeꢀinhibitor,ꢀsodiumꢀ
azideꢀ(NaN3)ꢀ(21).
Fig. 3.ꢀ HDA-GSHꢀproductionꢀinꢀRAWꢀ264.7ꢀcellsꢀtreatedꢀwithꢀex-
ogenousꢀ2-BrHDAꢀandꢀ2-ClHDA.ꢀRAWꢀ264.7ꢀcellsꢀwereꢀtreatedꢀwithꢀ
0,ꢀ1,ꢀ10,ꢀ25,ꢀorꢀ50ꢀMꢀ2-ClHDAꢀorꢀ2-BrHDAꢀforꢀ4ꢀhꢀ(A)ꢀandꢀHDA-
GSHꢀwasꢀquantifiedꢀasꢀdescribedꢀinꢀtheꢀMaterialsꢀandꢀMethods.ꢀDataꢀ
inꢀpanelsꢀBꢀandꢀCꢀareꢀRAWꢀ264.7ꢀcellularꢀ(C)ꢀandꢀmediaꢀ(B)ꢀlevelsꢀ
ofꢀ HDA-GSHꢀ followingꢀ treatmentsꢀ withꢀ eitherꢀ 20ꢀ Mꢀ HDA,ꢀ 2-Cl-
HDA,ꢀorꢀ2-BrHDAꢀforꢀtheꢀindicatedꢀtimeꢀintervals.ꢀ***Pꢀ<ꢀ0.001ꢀforꢀ
HDA-GSHꢀproductionꢀinꢀcellsꢀtreatedꢀwithꢀ2-BrHDAꢀcomparedꢀwithꢀ
thoseꢀtreatedꢀwithꢀ2-ClHDA;ꢀnꢀ=ꢀ3ꢀforꢀeachꢀtreatment.ꢀ#Pꢀ<ꢀ0.05ꢀandꢀ
###Pꢀ<ꢀ0.001ꢀforꢀcomparisonsꢀofꢀHDA-GSHꢀinꢀtheꢀmediaꢀofꢀ2-BrHDAꢀ
treatmentsꢀ atꢀ 30ꢀ minꢀ comparedꢀ withꢀ HDA-GSHꢀ inꢀ theꢀ mediaꢀ ofꢀ
2-BrHDAꢀtreatmentsꢀatꢀotherꢀtimeꢀpoints;ꢀnꢀ=ꢀ3ꢀforꢀeachꢀtreatment.
Production of -BrFALD and FALD-GSH in Br2-exposed
mice
wereꢀ producedꢀ inꢀ theꢀ lungsꢀ ofꢀ chlorineꢀ gasꢀ (Cl2)-exposedꢀ
miceꢀandꢀratsꢀ(10).ꢀSimilarꢀtoꢀthoseꢀpreviousꢀstudiesꢀinꢀchlo-
rineꢀgas-exposedꢀrodents,ꢀtheꢀdataꢀinꢀFig. 6A show that
-BrFALDꢀwasꢀelevatedꢀinꢀtheꢀlungsꢀofꢀmiceꢀexposedꢀtoꢀ30ꢀminꢀ
Whileꢀ mostꢀ physiologicꢀ -halofattyꢀ aldehydeꢀ productionꢀ
isꢀlikelyꢀdueꢀtoꢀhypohalousꢀacidꢀproducedꢀbyꢀperoxidases,ꢀaꢀ
recentꢀstudyꢀdemonstratedꢀthatꢀ-ClFALDꢀandꢀFALD-GSHꢀ
700
Journal of Lipid Research Volume 59, 2018

Fig. 5.ꢀ Eosinophilsꢀ produceꢀ FALD-GSH.ꢀ Primaryꢀ humanꢀ eosino-
philsꢀwereꢀincubatedꢀinꢀHank’sꢀbalancedꢀsaltꢀsolutionꢀsupplementedꢀ
withꢀ100ꢀMꢀNaBr,ꢀandꢀwereꢀsubsequentlyꢀactivatedꢀwithꢀ200ꢀnMꢀPMA,ꢀ
asꢀindicatedꢀinꢀtheꢀpresenceꢀorꢀabsenceꢀofꢀ100ꢀMꢀNaN3ꢀinꢀduplicate.ꢀ
PMA-stimulatedꢀ eosinophilsꢀ readilyꢀ produceꢀ FALD-GSHꢀ andꢀ NaN3
blocksꢀtheꢀproductionꢀofꢀtheꢀFALD-GSH.ꢀFALD-GSHꢀproductꢀwasꢀde-
terminedꢀasꢀdescribedꢀinꢀtheꢀMaterialsꢀandꢀMethods.
notꢀ examined,ꢀ withꢀ theꢀ mostꢀ likelyꢀ mechanismsꢀ beingꢀ
throughꢀthiolꢀmodificationꢀorꢀSchiffꢀbaseꢀadductꢀformationꢀ
withꢀamines.ꢀWeꢀinvestigatedꢀtheꢀroleꢀofꢀthiolꢀmodificationꢀ
of -BrFALDꢀandꢀ-ClFALDꢀreactivityꢀwithꢀproteinꢀusingꢀ
theꢀ clickableꢀ alkyneꢀ analogs,ꢀ 2-BrHDyA,ꢀ 2-ClHDyA,ꢀ andꢀ
HDyA.ꢀ Figure 7Bꢀ demonstratesꢀ thatꢀ bothꢀ 2-ClHDyAꢀ andꢀ
2-BrHDyAꢀformꢀproteinꢀadducts,ꢀwhileꢀHDyAꢀadductsꢀareꢀ
barelyꢀpresent.ꢀBecauseꢀtheꢀ2-BrHDyAꢀadductedꢀproteinꢀtoꢀ
aꢀ greaterꢀ extentꢀ thanꢀ 2-ClHDyA,ꢀ lessꢀ totalꢀ proteinꢀ fromꢀ
2-BrHDyA-treatedꢀcellsꢀwasꢀloadedꢀtoꢀallowꢀforꢀsimultane-
ousꢀvisualizationꢀofꢀallꢀconditions.ꢀTheꢀdifferentialꢀproteinꢀ
loadingꢀforꢀtheseꢀconditionsꢀcanꢀbeꢀvisualizedꢀbyꢀtheꢀCoo-
massieꢀstainingꢀinꢀFig.ꢀ7A.
Theꢀ enhancedꢀ proteinꢀ adductionꢀ ofꢀ 2-BrHDyAꢀ com-
paredꢀwithꢀthatꢀofꢀ2-ClHDyAꢀsuggestedꢀthatꢀthiolꢀreactivityꢀ
wasꢀtheꢀmechanismꢀofꢀproteinꢀbinding,ꢀasꢀstudiesꢀwithꢀGSHꢀ
showedꢀthatꢀbromineꢀwasꢀaꢀbetterꢀleavingꢀgroupꢀthanꢀchlo-
rineꢀinꢀreactionsꢀwithꢀthiols.ꢀToꢀconfirmꢀtheꢀimportanceꢀofꢀ
thiolsꢀtoꢀ2-ClHDyAꢀandꢀ2-BrHDyAꢀproteinꢀbinding,ꢀTHP-1ꢀ
cellsꢀ wereꢀ pretreatedꢀ withꢀ theꢀ thiol-reactiveꢀ compound,ꢀ
NEM.ꢀ NEMꢀ pretreatmentꢀ greatlyꢀ decreasedꢀ bothꢀ 2-BrH-
DyAꢀandꢀ2-ClHDyAꢀproteinꢀbinding.ꢀInꢀcomparison,ꢀtheꢀ
thiolꢀcompetitorꢀbait,ꢀNAC,ꢀdecreasedꢀ2-BrHDyAꢀproteinꢀ
modificationꢀtoꢀaꢀgreaterꢀextentꢀcomparedꢀwithꢀ2-ClHDyA,ꢀ
whichꢀisꢀconsistentꢀwithꢀdataꢀshownꢀinꢀFig.ꢀ2ꢀindicatingꢀthatꢀ
2-BrHDAꢀreactsꢀwithꢀGSHꢀtoꢀaꢀgreaterꢀextentꢀcomparedꢀ
withꢀreactionsꢀwithꢀ2-ClHDA.
Fig. 4. -HaloFALDꢀ andꢀ FALD-GSHꢀ producedꢀ inꢀ NaBr-supple-
mentedꢀneutrophils.ꢀPrimaryꢀhumanꢀneutrophilsꢀwereꢀisolatedꢀandꢀ
treatedꢀwithꢀeitherꢀPMAꢀorꢀvehicleꢀinꢀtheꢀpresenceꢀorꢀabsenceꢀofꢀ
1ꢀmMꢀNaBr.ꢀ-Halo-FALDꢀandꢀFALD-GSHꢀwereꢀquantitatedꢀusingꢀ
GC-MSꢀandꢀESI-MS/MS,ꢀrespectively,ꢀasꢀdescribedꢀinꢀtheꢀMaterialsꢀ
andꢀMethods.ꢀ***ꢀandꢀ****ꢀindicateꢀPꢀ<ꢀ0.001ꢀandꢀPꢀ<ꢀ0.0001,ꢀre-
spectively,ꢀforꢀcomparisonsꢀofꢀeachꢀspeciesꢀwithꢀandꢀwithoutꢀPMAꢀ
present.ꢀ###ꢀandꢀ####ꢀindicateꢀPꢀ<ꢀ0.001ꢀandꢀPꢀ<ꢀ0.0001,ꢀrespec-
tively,ꢀforꢀcomparisonsꢀofꢀeachꢀspeciesꢀwithꢀandꢀwithoutꢀNaBrꢀsup-
plementation;ꢀnꢀ=ꢀ3ꢀforꢀeachꢀtreatment.
ofꢀ400ꢀorꢀ600ꢀppmꢀBr2ꢀandꢀthenꢀreturnedꢀtoꢀroomꢀairꢀforꢀ1ꢀh.ꢀ
Lungꢀ(Fig.ꢀ6B)ꢀandꢀplasmaꢀ(Fig.ꢀ6C)ꢀlevelsꢀofꢀFALD-GSHꢀlev-
elsꢀwereꢀalsoꢀelevatedꢀfollowingꢀBr2ꢀexposure,ꢀindicatingꢀthatꢀ
-BrFALDꢀderivedꢀfromꢀeitherꢀBr2ꢀorꢀHOBr-targetingꢀplas-
malogensꢀisꢀreadilyꢀconvertedꢀtoꢀGSHꢀadductsꢀinꢀtheꢀlungꢀ
andꢀpotentiallyꢀtheꢀpulmonaryꢀcirculation.ꢀAlternatively,ꢀsys-
temicꢀbloodꢀincreasesꢀinꢀFALD-GSHꢀpostꢀBr2ꢀexposureꢀmayꢀ
beꢀtheꢀresultꢀofꢀwashoutꢀofꢀFALD-GSHꢀfromꢀitsꢀproductionꢀ
site,ꢀtheꢀlung.
Metabolic activity of THP-1 cells following treatments
with halogenated aldehydes
Basedꢀonꢀtheꢀenhancedꢀreactivityꢀofꢀ2-BrHDAꢀwithꢀthiolsꢀ
comparedꢀwithꢀ2-ClHDA,ꢀfurtherꢀstudiesꢀexaminedꢀaltera-
tionsꢀinꢀcellꢀmetabolicꢀactivityꢀelicitedꢀbyꢀtheseꢀhalogenatedꢀ
aldehydes.ꢀ Dataꢀ shownꢀ inꢀ Fig. 8ꢀ demonstrateꢀ thatꢀ treat-
mentsꢀwithꢀ10ꢀandꢀ25ꢀMꢀ2-BrHDAꢀsignificantlyꢀdecreaseꢀ
THP-1ꢀcellꢀmetabolicꢀactivity,ꢀasꢀascertainedꢀbyꢀMTTꢀassay,ꢀinꢀ
comparisonꢀtoꢀchangesꢀelicitedꢀbyꢀthoseꢀconcentrationsꢀofꢀ
2-ClHDAꢀandꢀHDA,ꢀwhichꢀdidꢀnotꢀsignificantlyꢀalterꢀmeta-
bolicꢀactivity.ꢀAdditionally,ꢀmetabolicꢀactivityꢀdecreasedꢀasꢀtheꢀ
2-BrHDAꢀconcentrationꢀwasꢀincreasedꢀfromꢀ10ꢀtoꢀ25ꢀM.
-ClFALD and -BrFALD modification of cellular protein
thiols
Previousꢀ studiesꢀ haveꢀ shownꢀ thatꢀ -ClFALDꢀ modifiesꢀ
proteinsꢀ(13),ꢀbutꢀtheꢀmechanismꢀforꢀthisꢀmodificationꢀwasꢀ
Bromofatty aldehyde modifications of glutathione and protein
701

usingꢀ40ꢀÅꢀsilicaꢀgelꢀTLCꢀplatesꢀandꢀaꢀmobileꢀphaseꢀcom-
prisedꢀ ofꢀ chloroform-acetone-methanol-water-aceticꢀ acidꢀ
(6:8:2:2:1,ꢀ v/v/v/v/v)];ꢀ andꢀ 2)ꢀ ESI-MS/MSꢀ analysesꢀ ofꢀ
TLC-purifiedꢀ productsꢀ havingꢀ theꢀ sameꢀ parentꢀ ionꢀ andꢀ
productꢀions.ꢀTheseꢀreactionsꢀalsoꢀindicatedꢀtheꢀreactionꢀ
ofꢀ 2-BrHDAꢀ withꢀ GSHꢀ wasꢀ muchꢀ moreꢀ robustꢀ thanꢀ thatꢀ
withꢀ2-ClHDA.ꢀFurthermore,ꢀwhileꢀbothꢀ2-BrHDAꢀandꢀ2-Cl-
HDAꢀ reactedꢀ withꢀ GSH,ꢀ noꢀ reactionꢀ productsꢀ wereꢀ ob-
servedꢀbyꢀTLCꢀusingꢀnonhalogenatedꢀfattyꢀaldehyde,ꢀHDA,ꢀ
orꢀ-halogenatedꢀfattyꢀacidsꢀandꢀ-chlorofattyꢀalcohol.ꢀTheꢀ
selectiveꢀsynthesisꢀofꢀFALD-GSHꢀusingꢀ2-BrHDAꢀ(com-
paredꢀwithꢀthatꢀusingꢀ2-ClHDA)ꢀwasꢀsupportedꢀby:ꢀ1)ꢀsyn-
theticꢀ reactionsꢀ ofꢀ 2-BrHDAꢀ withꢀ GSHꢀ resultingꢀ inꢀ aꢀ
moreꢀintenseꢀstainedꢀreactionꢀproductꢀbyꢀTLCꢀanalyses,ꢀ
asꢀwellꢀasꢀnearꢀtotalꢀconsumptionꢀofꢀGSHꢀinꢀcomparisonsꢀ
toꢀreactionsꢀofꢀ2-ClHDAꢀwithꢀGSH;ꢀ2)ꢀcellularꢀproduc-
tionꢀofꢀFALD-GSHꢀwasꢀ25ꢀtimesꢀgreaterꢀinꢀcellsꢀtreatedꢀ
withꢀ2-BrHDAꢀcomparedꢀwithꢀ2-ClHDA;ꢀandꢀ3)ꢀenhanc-
ingꢀ-BrFALDꢀinꢀcomparisonꢀtoꢀ-ClFALDꢀproductionꢀinꢀ
neutrophilsꢀbyꢀNaBrꢀsupplementationꢀinꢀtheꢀmediaꢀledꢀ
toꢀanꢀorderꢀofꢀmagnitudeꢀincreaseꢀinꢀFALD-GSHꢀcom-
paredꢀwithꢀneutrophilsꢀsynthesizingꢀonlyꢀ-ClFALDꢀ(noꢀ
NaBrꢀ supplementation).ꢀ Theꢀ findingꢀ thatꢀ endogenousꢀ
-BrFALDꢀinꢀneutrophilsꢀisꢀreadilyꢀconvertedꢀtoꢀFALD-
GSHꢀindicatesꢀthatꢀsomeꢀ-BrFALDꢀmayꢀbeꢀmadeꢀunderꢀ
normalꢀconditionsꢀ(noꢀNaBrꢀsupplementation),ꢀbutꢀmayꢀ
notꢀbeꢀdetectedꢀdueꢀtoꢀtheꢀrapidꢀreactionꢀwithꢀGSH.ꢀIn-
terestingly,ꢀapplyingꢀexogenousꢀ2-BrHDAꢀandꢀ2-ClHDAꢀ
toꢀRAWꢀ264.7ꢀcellsꢀledꢀtoꢀHDA-GSHꢀproductionꢀinꢀcellsꢀ
asꢀwellꢀasꢀtheꢀappearanceꢀofꢀthisꢀpeptidoaldehydeꢀinꢀtheꢀ
cellꢀcultureꢀmedia.ꢀTheꢀexportꢀofꢀHDA-GSHꢀfromꢀcellsꢀ
treatedꢀ withꢀ eitherꢀ 2-ClHDAꢀ orꢀ 2-BrHDAꢀ suggestsꢀ thatꢀ
FALD-GSHꢀmayꢀbeꢀaꢀstableꢀmetaboliteꢀofꢀ-halofatty al-
dehydesꢀthatꢀmayꢀbeꢀfoundꢀinꢀplasmaꢀduringꢀinflamma-
tionꢀinvolvingꢀleukocyteꢀactivation,ꢀandꢀFALD-GSHꢀmayꢀ
beꢀtargetedꢀforꢀfurtherꢀmetabolismꢀbyꢀotherꢀorgansꢀorꢀ
uptakeꢀforꢀexcretion.
-BrFALDꢀandꢀ-ClFALDꢀareꢀproducedꢀbyꢀeitherꢀHOBrꢀ
orꢀBr2,ꢀandꢀeitherꢀHOClꢀorꢀCl2,ꢀrespectively,ꢀmediatedꢀoxi-
dationꢀofꢀtheꢀvinylꢀetherꢀbondꢀatꢀtheꢀsn-1ꢀpositionꢀofꢀplas-
malogens.ꢀHOBr-mediatedꢀoxidationꢀofꢀplasmalogensꢀhasꢀ
notꢀbeenꢀinvestigatedꢀinꢀdetail.ꢀHOBrꢀisꢀproducedꢀby:ꢀ1)ꢀ
MPOꢀinꢀtheꢀpresenceꢀofꢀNaBr;ꢀ2)ꢀeosinophilꢀperoxidase;ꢀ
andꢀ3)ꢀtheꢀhydrationꢀofꢀBr2ꢀunderꢀconditionsꢀofꢀBr2ꢀexpo-
sure.ꢀ Regardingꢀ MPOꢀ productionꢀ ofꢀ HOBr,ꢀ activatedꢀ
neutrophilsꢀ produceꢀ HOBrꢀ andꢀ 2-BrHDAꢀ whenꢀ supple-
mentedꢀwithꢀbuffersꢀcontainingꢀ100ꢀMꢀNaBrꢀ(2).ꢀItꢀshouldꢀ
beꢀappreciatedꢀthatꢀnormalꢀphysiologicalꢀconcentrationsꢀofꢀ
bromideꢀinꢀhumanꢀplasmaꢀrangeꢀfromꢀ20ꢀtoꢀ150ꢀMꢀ(16),ꢀ
andꢀ thusꢀ itꢀ isꢀ likelyꢀ thatꢀ neutrophilsꢀ doꢀ indeedꢀ produceꢀ
HOBrꢀ underꢀ physiologicalꢀ concentrations.ꢀ Additionally,ꢀ
brominationꢀofꢀhostꢀmoleculesꢀbyꢀactivatedꢀneutrophilsꢀhasꢀ
previouslyꢀbeenꢀreportedꢀ(2,ꢀ22).ꢀInꢀthisꢀstudy,ꢀweꢀexam-
inedꢀwhetherꢀFALD-GSHꢀisꢀproducedꢀunderꢀconditionsꢀ
leadingꢀ toꢀ endogenousꢀ -BrFALDꢀ production.ꢀ Accord-
ingly,ꢀinꢀadditionꢀtoꢀ-BrFALDꢀreactingꢀwithꢀGSHꢀtoꢀyieldꢀ
FALD-GSHꢀunderꢀsyntheticꢀconditionsꢀandꢀcellꢀcultureꢀ
conditionsꢀwithꢀexogenouslyꢀsuppliedꢀ-BrFALD,ꢀtheꢀ
dataꢀhereinꢀshowꢀFALD-GSHꢀproductionꢀunderꢀconditionsꢀ
Fig. 6.ꢀ Theꢀ 2-BrFALDꢀ andꢀ FALD-GSHꢀ inꢀ miceꢀ exposedꢀ toꢀ bro-
mineꢀgas.ꢀC57bl/6ꢀmiceꢀwereꢀexposedꢀtoꢀBr2ꢀgasꢀ(400ꢀorꢀ600ꢀppm,ꢀ
30ꢀmin)ꢀandꢀthenꢀbroughtꢀbackꢀtoꢀroomꢀair.ꢀAtꢀ1ꢀhꢀfollowingꢀexpo-
sure,ꢀ lungsꢀ andꢀ plasmaꢀ wereꢀ collected.ꢀ Theꢀ 2-BrFALDꢀ (A)ꢀ andꢀ
FALD-GSHꢀ(B)ꢀwereꢀthenꢀmeasuredꢀinꢀtheꢀlungꢀandꢀFALD-GSHꢀ(C)ꢀ
wasꢀ measuredꢀ inꢀ theꢀ plasma.ꢀ FALD-GSHꢀ wasꢀ extractedꢀ fromꢀ theꢀ
lungꢀtissueꢀandꢀplasmaꢀandꢀquantitatedꢀusingꢀESI-MS/MSꢀselectiveꢀ
reactionꢀmonitoringꢀasꢀdescribedꢀinꢀtheꢀMaterialsꢀandꢀMethods.ꢀ
*Pꢀ<ꢀ0.05,ꢀ**Pꢀ<ꢀ0.005,ꢀandꢀ***Pꢀ<ꢀ0.001ꢀforꢀproductionꢀofꢀFALD-
GSHꢀcomparedꢀwithꢀairꢀcontrols;ꢀnꢀ=ꢀ3ꢀforꢀeachꢀtreatment.
DISCUSSION
-ClFALDꢀreactsꢀwithꢀGSHꢀtoꢀproduceꢀFALD-GSHꢀ(12).ꢀ
Becauseꢀ MPOꢀ andꢀ eosinophilꢀ peroxidaseꢀ canꢀ produceꢀ
HOBrꢀand,ꢀsubsequently,ꢀ-BrFALD,ꢀoneꢀofꢀtheꢀgoalsꢀofꢀ
thisꢀstudyꢀwasꢀtoꢀexamineꢀtheꢀreactivityꢀofꢀ-BrFALDꢀcom-
paredꢀwithꢀ-ClFALDꢀwithꢀGSHꢀandꢀotherꢀthiol-containingꢀ
targetꢀmolecules.ꢀInitialꢀexperimentsꢀshowedꢀtheꢀreactionꢀ
productꢀofꢀGSHꢀwithꢀeitherꢀ2-ClHDAꢀandꢀ2-BrHDAꢀyieldedꢀ
theꢀsameꢀreactionꢀproductꢀ(FALD-GSH)ꢀasꢀascertainedꢀby:ꢀ1)ꢀ
TLCꢀanalysesꢀ[eachꢀreactionꢀproductꢀhadꢀanꢀrfꢀ=ꢀ0.27ꢀ
702
Journal of Lipid Research Volume 59, 2018

Fig. 7.ꢀ NEMꢀblocksꢀ2-BrHDyAꢀandꢀ2-ClHDyAꢀreac-
tionꢀ withꢀ THP-1ꢀ cellꢀ protein.ꢀ THP-1ꢀ cellsꢀ wereꢀ pre-
treatedꢀwithꢀorꢀwithoutꢀ1ꢀmMꢀNEMꢀorꢀ5ꢀmMꢀNACꢀforꢀ
30ꢀ minꢀ atꢀ 37°Cꢀ followedꢀ byꢀ treatmentꢀ withꢀ 5ꢀ Mꢀ
HDyA,ꢀ2-ClHDyA,ꢀorꢀ2-BrHDyAꢀforꢀ30ꢀminꢀatꢀ37°C.ꢀ
TAMRA-clickedꢀTHP-1ꢀcellꢀproteinꢀwasꢀvisualizedꢀafterꢀ
gelꢀelectrophoresis.ꢀPanelꢀAꢀshowsꢀCoomassieꢀblueꢀto-
talꢀproteinꢀstain.ꢀNEMꢀpretreatmentꢀblockedꢀtheꢀfluo-
rescenceꢀsignalꢀofꢀ2-BrHDyAꢀandꢀ2-ClHDyA,ꢀbutꢀNACꢀ
pretreatmentꢀonlyꢀsignificantlyꢀdecreasedꢀtheꢀ2-BrH-
DyAꢀsignalꢀ(B).
ofꢀ neutrophilꢀ activationꢀ leadingꢀ toꢀ endogenousꢀ -
BrFALDꢀ production,ꢀ conditionsꢀ leadingꢀ toꢀ eosinophilꢀ
activationꢀandꢀ-BrFALDꢀproduction;ꢀandꢀinꢀinꢀvivoꢀcon-
ditionsꢀwithꢀmiceꢀexposedꢀtoꢀbromineꢀwithꢀsubsequentꢀ
-BrFALDꢀproduction.
cysteine,ꢀtheꢀabilityꢀofꢀ-ClFALDꢀandꢀ-BrFALDꢀtoꢀreactꢀwithꢀ
THP-1ꢀhumanꢀmonocyteꢀcellularꢀproteinꢀwasꢀexaminedꢀ
usingꢀalkyneꢀanalogsꢀofꢀHDAꢀ(HDyA),ꢀ2-ClHDAꢀ(2-ClH-
DyA),ꢀandꢀ2-BrHDAꢀ(2-BrHDyA).ꢀTheseꢀexperimentsꢀandꢀ
analysesꢀ wereꢀ performedꢀ withoutꢀ Schiffꢀ baseꢀ reductionꢀ
and,ꢀtherefore,ꢀHDyAꢀdidꢀnotꢀmodifyꢀproteinsꢀ(Fig.ꢀ7).ꢀ
Theꢀreactivity,ꢀhowever,ꢀofꢀ2-ClHyDAꢀandꢀ2-BrHyDAꢀwithꢀ
proteinsꢀinꢀtheꢀabsenceꢀofꢀSchiffꢀbaseꢀreductionꢀsuggestedꢀ
Inꢀtheseꢀstudies,ꢀweꢀexaminedꢀtheꢀinꢀvivoꢀproductionꢀofꢀ
lungꢀ -BrFALDꢀ andꢀ FALD-GSHꢀ asꢀ wellꢀ asꢀ plasmaꢀ FALD-
GSHꢀinꢀmiceꢀexposedꢀtoꢀsublethalꢀlevelsꢀofꢀbromineꢀgas.ꢀ
Inhalationꢀofꢀchlorineꢀandꢀbromineꢀgasꢀhasꢀbeenꢀshownꢀtoꢀ
elicitꢀsevereꢀsystemicꢀphysiologicalꢀeffects,ꢀincludingꢀcardiacꢀ
dysfunctionꢀ andꢀ vasoregulatoryꢀ dysfunctionꢀ (10,ꢀ 23,ꢀ 24).ꢀ
Bromineꢀgasꢀhasꢀmanyꢀindustrialꢀapplications,ꢀandꢀisꢀmanu-
facturedꢀandꢀtransportedꢀworld-wide.ꢀAccordingly,ꢀbromineꢀ
asꢀwellꢀasꢀchlorineꢀpresentꢀaꢀpersistentꢀhazardousꢀmaterialꢀ
threatꢀbecauseꢀtheirꢀreleaseꢀcanꢀcauseꢀmassiveꢀhumanꢀcasu-
alties.ꢀInꢀhumans,ꢀinhalationꢀofꢀhighꢀconcentrationsꢀofꢀBr2
causesꢀrespiratoryꢀandꢀmyocardialꢀinjury,ꢀcardiacꢀarrest,ꢀandꢀ
circulatoryꢀcollapseꢀ(25,ꢀ26).ꢀFollowingꢀbromineꢀexposure,ꢀ
bromideꢀionsꢀandꢀbrominatedꢀcompoundsꢀformedꢀonꢀtheꢀ
pulmonaryꢀepithelialꢀsurfaceꢀareꢀstableꢀandꢀmayꢀpersistꢀinꢀ
theꢀcirculationꢀforꢀdaysꢀfollowingꢀexposureꢀ(27,ꢀ28).ꢀTheꢀ
discoveryꢀ ofꢀ biomarkersꢀ ofꢀ bromineꢀ exposureꢀ thusꢀ isꢀ ofꢀ
greatꢀimportance,ꢀandꢀtheꢀdataꢀhereinꢀindicateꢀthatꢀFALD-
GSHꢀisꢀanꢀexcellentꢀblood-borneꢀbiomarkerꢀofꢀbromineꢀex-
posure.ꢀBasedꢀonꢀourꢀfindingsꢀofꢀ-BrFALDꢀandꢀFALD-GSH,ꢀ
itꢀisꢀlikelyꢀthatꢀtheꢀprimaryꢀsourceꢀofꢀcirculatingꢀFALD-GSHꢀ
isꢀfromꢀtheꢀlung.ꢀFurtherꢀfutureꢀstudiesꢀwillꢀneedꢀtoꢀbeꢀper-
formedꢀtoꢀdetermineꢀtheꢀtemporalꢀcourseꢀthatꢀFALD-GSHꢀ
remainsꢀelevatedꢀpostꢀbromineꢀexposure.
Fig. 8.ꢀ Disparateꢀeffectꢀofꢀ2-BrHDAꢀandꢀ2-ClHDAꢀonꢀTHP-1ꢀcellꢀ
metabolicꢀactivity.ꢀTHP-1ꢀcellsꢀwereꢀtreatedꢀwithꢀtheꢀindicatedꢀcon-
centrationsꢀofꢀeitherꢀHDA,ꢀ2-ClHDA,ꢀorꢀ2-BrHDAꢀforꢀ2ꢀhꢀatꢀ37°C.ꢀ
MetabolicꢀactivityꢀofꢀHCAECꢀwasꢀmeasuredꢀusingꢀanꢀMTTꢀassayꢀfol-
lowingꢀtheꢀmanufacturer’sꢀprotocol.ꢀMTTꢀreductionꢀisꢀexpressedꢀasꢀ
percentꢀofꢀcontrolꢀMTTꢀreductionꢀ(vehicleꢀonly,ꢀdesignedꢀasꢀ100%).ꢀ
Dataꢀareꢀexpressedꢀasꢀmeanꢀ±ꢀSEM;ꢀnꢀ=ꢀ4ꢀforꢀeachꢀtreatment.ꢀTꢀin-
dicatesꢀtreatmentꢀofꢀTHP-1ꢀcellsꢀwithꢀTritonꢀX-100.ꢀ#Pꢀ<ꢀ0.05ꢀforꢀ
comparisonsꢀ betweenꢀ 2-BrHDAꢀ atꢀ theꢀ twoꢀ concentrationsꢀ exam-
ined.ꢀ****Pꢀ<ꢀ0.0001ꢀforꢀcomparisonsꢀbetweenꢀ2-BrHDAꢀandꢀeitherꢀ
2-ClHDAꢀorꢀHDAꢀatꢀtheꢀindicatedꢀconcentrations.
Afterꢀdeterminingꢀthatꢀtheꢀpresenceꢀofꢀaꢀthiolꢀwasꢀnec-
essaryꢀ forꢀ bothꢀ 2-ClHDAꢀ andꢀ 2-BrHDAꢀ reactionsꢀ withꢀ
Bromofatty aldehyde modifications of glutathione and protein
703

targetꢀtheꢀvinylꢀetherꢀbondꢀofꢀplasmalogens:ꢀdisparateꢀutilizationꢀofꢀ
sodiumꢀhalidesꢀinꢀtheꢀproductionꢀofꢀalpha-haloꢀfattyꢀaldehydes.ꢀJ.
Biol. Chem. 277:ꢀ4694–4703.
theꢀrequirementꢀofꢀtheꢀ-halogenꢀforꢀreactivityꢀwithꢀthiolsꢀ
forꢀthisꢀcovalentꢀmodificationꢀofꢀproteins.ꢀFurthermore,ꢀ
2-BrHDyAꢀmodificationꢀofꢀproteinꢀwasꢀgreaterꢀthanꢀthatꢀ
byꢀ2-ClHDyA,ꢀsimilarꢀtoꢀthatꢀwithꢀreactionsꢀofꢀ2-BrHDAꢀ
andꢀ2-ClHDAꢀwithꢀGSHꢀthatꢀreflectsꢀbromineꢀasꢀaꢀbetterꢀ
leavingꢀgroupꢀforꢀthiolꢀnucleophilicꢀattack.ꢀNEMꢀgreatlyꢀ
diminishedꢀproteinꢀbindingꢀinꢀbothꢀtheꢀ2-ClHDyA-ꢀandꢀ
theꢀ 2-BrHDyA-treatedꢀ samples,ꢀ supportingꢀ theꢀ mecha-
nismꢀofꢀcovalentꢀmodificationꢀbeingꢀthroughꢀproteinꢀthi-
ols.ꢀ NACꢀ pretreatmentꢀ wasꢀ shownꢀ toꢀ blockꢀ 2-BrHDyAꢀ
proteinꢀbinding,ꢀbutꢀnotꢀ2-ClHDyA.ꢀTheꢀdiscrepancyꢀwasꢀ
likelyꢀdueꢀtoꢀtheꢀreducedꢀreactivityꢀofꢀtheꢀthiolꢀofꢀNACꢀ
withꢀ2-ClHDyAꢀcomparedꢀwithꢀ2-BrHDyA,ꢀwhichꢀallowedꢀ
2-ClHDyAꢀtoꢀenterꢀtheꢀcellꢀandꢀbindꢀprotein.ꢀCollectivelyꢀ
theseꢀstudiesꢀdemonstrateꢀthatꢀ-BrFALDꢀandꢀ-ClFALD
covalentlyꢀmodifyꢀcellularꢀproteinꢀthroughꢀthiol-dependentꢀ
mechanisms.ꢀInterestingly,ꢀNussholdꢀetꢀal.ꢀ(13)ꢀdemon-
stratedꢀaꢀnumberꢀofꢀendothelialꢀcellꢀproteinsꢀthatꢀreactꢀ
withꢀ2-ClHDA.ꢀProteomicꢀanalysesꢀrevealedꢀthatꢀproteinsꢀ
associatedꢀwithꢀcytoskeletalꢀstructure,ꢀmetabolicꢀactivity,ꢀ
andꢀoxidativeꢀstressꢀwereꢀmodifiedꢀbyꢀ2-ClHDA.ꢀAlso,ꢀtheyꢀ
showedꢀdecreasedꢀmetabolicꢀactivity,ꢀdeterminedꢀbyꢀMTTꢀ
assays,ꢀinꢀendothelialꢀcellsꢀtreatedꢀwithꢀ25ꢀandꢀ50ꢀMꢀ2-
ClHDA.ꢀMTTꢀactivityꢀwasꢀnotꢀstatisticallyꢀloweredꢀinꢀendo-
thelialꢀcellsꢀtreatedꢀwithꢀ10ꢀMꢀ2-ClHDA.ꢀTheꢀresultsꢀhereinꢀ
areꢀsimilarꢀtoꢀthoseꢀobservedꢀbyꢀNussholdꢀetꢀal.ꢀ(13),ꢀwithꢀ
noꢀdecreasesꢀinꢀMTTꢀactivityꢀobservedꢀatꢀ10ꢀMꢀ2-ClHDAꢀinꢀ
THP-1ꢀ cellsꢀ andꢀ aꢀ slight,ꢀ yetꢀ statisticallyꢀ insignificant,ꢀ de-
creaseꢀinꢀMTTꢀactivityꢀobservedꢀatꢀ25ꢀMꢀ2-ClHDA.ꢀTheꢀ
presentꢀ studiesꢀ revealedꢀ thatꢀ 2-BrHDAꢀ significantlyꢀ de-
creasedꢀ metabolicꢀ activity.ꢀ Itꢀ isꢀ possibleꢀ thatꢀ futureꢀ pro-
teomicꢀstudiesꢀmayꢀalsoꢀrevealꢀthatꢀproteinsꢀassociatedꢀwithꢀ
metabolicꢀactivityꢀwillꢀbeꢀmodifiedꢀbyꢀ2-BrHDA.
ꢀ 3.ꢀ Thukkani,ꢀA.ꢀK.,ꢀF-F.ꢀF.ꢀHsu,ꢀJ.ꢀR.ꢀCrowley,ꢀR.ꢀB.ꢀWysolmerski,ꢀC.ꢀ
J.ꢀAlbert,ꢀandꢀD.ꢀa.ꢀFord.ꢀ2002.ꢀReactiveꢀchlorinatingꢀspeciesꢀpro-
ducedꢀduringꢀneutrophilꢀactivationꢀtargetꢀtissueꢀplasmalogens:ꢀpro-
ductionꢀofꢀtheꢀchemoattractant,ꢀ2-chlorohexadecanal.ꢀJ. Biol. Chem.
277:ꢀ3842–3849.
ꢀ 4.ꢀ Thukkani,ꢀA.ꢀK.,ꢀC.ꢀJ.ꢀAlbert,ꢀK.ꢀR.ꢀWildsmith,ꢀM.ꢀC.ꢀMessner,ꢀB.ꢀ
D.ꢀMartinson,ꢀF.ꢀF.ꢀHsu,ꢀandꢀD.ꢀA.ꢀFord.ꢀ2003.ꢀMyeloperoxidase-
derivedꢀ reactiveꢀ chlorinatingꢀ speciesꢀ fromꢀ humanꢀ monocytesꢀ
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36365–36372.
ꢀ 5.ꢀ Thukkani,ꢀA.ꢀK.,ꢀJ.ꢀMcHowat,ꢀF.ꢀF.ꢀHsu,ꢀM.ꢀL.ꢀBrennan,ꢀS.ꢀL.ꢀHazen,ꢀ
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manꢀatheroscleroticꢀlesions.ꢀCirculation. 108:ꢀ3128–3133.
ꢀ 6.ꢀ Thukkani,ꢀA.ꢀK.,ꢀB.ꢀD.ꢀMartinson,ꢀC.ꢀJ.ꢀAlbert,ꢀG.ꢀA.ꢀVogler,ꢀandꢀD.ꢀ
A.ꢀFord.ꢀ2005.ꢀNeutrophil-mediatedꢀaccumulationꢀofꢀ2-ClHDAꢀdur-
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mineꢀtheꢀmetabolicꢀclearanceꢀofꢀFALD-GSH,ꢀincludingꢀ
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