Biochemical Verification of Tobacco Use and Cessation: An ...€¦ · NICOTINE COTININE...
Transcript of Biochemical Verification of Tobacco Use and Cessation: An ...€¦ · NICOTINE COTININE...
BiochemicalVerificationofTobaccoUseandCessation:AnUpdate
NealLBenowitzMDUniversityofCalifornia,SanFrancisco
SRNTWebinarOctober13,2017
Disclosures
• Consultanttopharmaceuticalcompaniesthatmarketsmokingcessationproducts,includingPfizer,GlaxoSmithKlineandMcNeil.
• Paidexpertinlitigationagainsttobaccocompanies.
Overview
• Majorbiomarkers:cotinine,CO,NNAL,minortobaccoalkaloids
• Optimalcutpoints• Windowsofdetection• Individualfactorsinfluencingbiomarkerlevels• Overthecountertests• DistinguishingVapingfromSmoking
ConstituentsandBiomarkersofTobacco
Use
ChemicalConstituentsofTobaccoSmoke
Nicotine Formaldehyde*CarbonMonoxide 1,3-butadiene*Acrolein Benzo(a)pyrene*Ammonia Variousnitrosamines*NitrogenOxides Hydrazine*Benzene* Nickel*2-naphylamine* Polonium-210*4-aminobiphenyl* Cadmium
* Known or suspected human carcinogen*ns
Benzene
Carbon Monoxide
1,3-Butadiene
Formaldehyde
AcroleinNitrogen Dioxide
Nicotine
NicotinePolycyclic
AromaticHydrocarbons
PAH
Solanesol
Benzo(a)pyreneMetals
Courtesy of Brett Singer, LBNL
TobaccoSmokeisaMixtureofChemicalsIncludingGasesandParticles
Challenges in Assessing Exposure:Extent of Sorption and Stability Varies
Fraction in air at 2 h (furnished room)
0.0
0.2
0.4
0.6
0.8
1.0
Courtesy of Brett Singer, LBNLMultiple tracers/biomarkers are desirable
TobaccoSpecificBiomarkers
• Nicotineandmetabolites– Cotinine– Urinetotalnicotineequivalents
• Minortobaccoalkaloids– Anabasine,anatabine– Nicotelline
• Tobaccospecificnitrosamines– NNAL
OtherTobaccoSmokeBiomarkers
• Carbonmonoxide• Thiocyanate(cyanide)• Polycyclicaromatichydrocarbons
– 1hydroxypyreneandothers
• Volatileorganiccompounds– Acrylonitrile,acrolein,butadieneandothers
OptimalCotinineLevelstoDistinguishSmokingand
Nonsmoking
PlasmaCotinineLevelsThroughouttheDay
SRNTSubcommitteeonBiochemicalVerification(NTR2002)
Optimalcut-offpointstodistinguishtobaccousevs.notobaccouse.
• Plasmaorserumcotinine15ng/mL• Urinarycotinine 50ng/mL• ExpiredCO 8-10ppm• PlasmaSCN 78-84mmol/L
NHANESParticipants• 1999-2000,2001-2002,2003-2004• NationallyrepresentativesampleofU.S.civilianpopulation
• Adolescents(12-19y.o.)andadults(mean45y.o.)
• 13,078nonsmokersand3,078smokers• NHW(6,896);NHB(3,671);MA(4,472)
All Adults
Fixed: 14.0 ng/mL Specificity = 98.5%Sensitivity = 92.4%
0
2
4
6
8
10
12
14
16
0.035 0.1 1 10 100 1000
Actual % = 57%(cropped at 16%)
ROC: 3.08 ng/mL Specificity = 97.4%Sensitivity = 96.3%
%Po
pula
tion
Overall
Serum Cotinine, ng/mL
RecommendedCotinineCutpointsbyRace/Ethnicity
Adults Adolescents*
NHW- 5ng/ml 3ng/ml
NWB- 6ng/ml 3ng/ml
MA- 1ng/ml 1ng/ml
* sensitivity of cut-point lower in adolescents –likely due to higher prevalence of occasional smoking and more under-reporting of smoking
Caveatsregardingcutpoints
• Theoptimalcutpointisinfluencedbytheprevalenceandintensityofactivetobaccouseandsecondhandsmokeexposure.
• Higherprevalenceandintensityoftobaccoexposurewillresultsinahigheroptimalcutpoint
• Race- andphysiology-relatedmetabolicdifferencescaninfluencecutpoints
Comparisonofbiofluidsforcotininemeasurement
• Blood(serum,plasma)isgoldstandard• Saliva/plasmaratio=1.04(0.95– 1.14)
– Ratioinfluencedbysalivaflowrate• Urine/plasmaratio=4.6(4.0– 5.3)
– RatioinfluencedbyurineflowrateandpH
()– 95%C.I.
Windowofcotininedetection
• Detection~5half-lives,dependingoninitiallevelandbiofluidtested(longerdetectioninurine)
• Cotininehalf-liferange10to30hr• Detectionrange50to150hours
LimitationsofCotinineasBiomarkerofNicotineandTobacco
SmokeExposure
NICOTINE
COTININE TRANS-3’-HYDROXYCOTININE
TRANS-3’-HYDROXYCOTININE
NICOTINE-1’-N-OXIDE NORNICOTINENICOTINE
NICOTINEGLUCURONIDE
COTININE
COTININEGLUCURONIDE COTININE-N-OXIDE NORCOTININE
TRANS-3’HYDROXYCOTININE
GLUCURONIDE
-80%
4.4%9.8%
4.2%
13.0%
12.6% 2.4% 2% 7.4%
0.4%
33.6%
NicotineMetabolicProfile
Cotinine as a Quantitative Measure of Nicotine Intake from Tobacco Use
Daily Generation of Cotinine =
Daily Nicotine Dose (D) × % Conversion of Nicotine to Cotinine (f)
Elimination rate for Cotinine =
Clearance of Cotinine (ClCOT) × Blood Cotinine Concentration (COT)
Cotinine as a Quantitative Measure of Nicotine Intake from Tobacco Use
• The conversion factor K = ClCOT / f
• Cl cot is determined by activity of CYP2A6, UGT2B10 and the renal clearance of cotinine
• f is determined by activity of CYP2A6, UGT2B10, FM03 and the renal clearance of nicotine
CotinineasaQuantitativeMeasureofNicotineIntakefromTobaccoUse
• TypicalK=0.08mg/24h/ng/ml
• A20cigaretteperdaysmokershasabloodcotininelevelof300ng/ml
D=(0.08)(300)=24mg/day=1.2mg/cigarette
FactorsPotentiallyInfluencingCotinineasaBiomarkerofNicotine
Intake
Genetics KidneyfailureSex Oralcontraceptive/HormoneRace replacementCigarette Anticonvulsantdrugs/rifampinsmoking Menthol
SexDifferencesinRateofNicotineMetabolism
15.617.6
22.5
27.0
0
10
20
30
Nico
tine
Clea
ranc
e (m
l/min
/kg)
Men Women(no OCP)
Women(+ OCP)
PregnantWomen
All Groups Significantly Different from One Another
JPET 2002; 301:594CPT 2006; 79:480
Kvaluesindifferentpopulationsofsmokers
Units:mgnicotine/24hr/ng/ml cotinineAverageK=0.08LowerinmenHigherinwomenVeryhighinpregnancyLowerinAfricanAmericansandAsians
OvertheCounterCotinineTests
OTCCotinineTests
• Fastest,easiestandleastexpensivetest• Immunoanalysisusingcotinineantibodies• Urineandsalivatests• Semi-quantitativewithrelativelyhighcotininethreshold– highspecificity,lowsensitivity
• InadequatesensitivityforSHSandoccasionaltobacco/nicotineuse
• PossiblebetterclassificationwhencombinedwithexpiredCO
COT iScreen OFD NicCheck ISalimetricsELISA Kit
SafeCare
Saliva Saliva Urine UrineSaliva
99% 99% 98% 99%99%
4 ng/ml 30 ng/ml 200 ng/ml 200 ng/ml20 ng/ml50 ng/ml
Active Use Cutoff
Sensitivity
Specimen
NicAlertStrips
SalivaUrine
99%
10 ng/ml (S)100 ng/ml (U)
Marrone, Addiction, 2011
Marrone, Addiction 2011; 106:1325-1334
UrineandSalivaNicAlert andSmokingStatus
Urine
6 (>1000)
5 (500-1000)
4 (200-500)
3 (100-200)
2 (30-100)
1 (10-30)
0 (0-10) Heavy
SmokersLight
SmokersNot ExposedNonsmokers
ExposedNonsmokers
Nic
Ale
rt Z
ones
, Cot
inin
e C
once
ntra
tions
(ng/
ml)
Saliva
Heavy Smokers
LightSmokers
Not ExposedNonsmokers
ExposedNonsmokers
OptimalCOLevelstoDistinguishSmokingandNonsmoking
Marrone, Addiction, 2011
Marrone, Addiction 2011; 106:1325-1334
BreathCOandSmokingStatus
Heavy Smokers
LightSmokers
Not ExposedNonsmokers
Exposed Nonsmokers
Bre
ath
CO
(ppm
)70
60
50
40
30
20
10
0
ExpiredCOandNicAlertPerformance
Measures Cut Point Sensitivity (%) Specificity (%)Expired CO ≥ 5 ppm 94.4 97.6
Urine NicAlert ≥ 3 92.2 97.6
Saliva NicAlert ≥ 1 76.7 85.4
CO / Urine NicAlert ≥ 5 / ≥ 3 97.6 95.1
WindowofCOdetection• Detection~5half-lives• COhalf-life:
– Average4hr– Exercise2hr– Sleep8hrDetectionrange10to20hr (COlevelscanstillbeelevatedafterovernightabstinence)ConsiderothersourcesofCO(vehicleexhaust;marijuanasmoking)
OptimalNNALLevelstoDistinguishSmokersfromNonsmokers
TobaccoSpecificNitrosamines:NNK,NNNandNNAL
• NNKandNNNarepotentlung,esophagealandoralcavitycarcinogensthatarederivedbynicotineinthetobaccocuringandcombustionprocess.
• NNKisonlyfoundintobaccoproducts;itslevelsdifferfromproducttoproduct,dependingontypeoftobaccoandnitratecontent.
• NNKismetabolizedinthebodytoNNAL,whichcanbemeasuredintheurineoftobaccousers.
UrinaryNNALinCigaretteSmokersandNon-Smokers
NHANES 2011-2012 • 6705 Participants • Ages ≥ 6 years old• 62% Overall Detection
Rate• 95% percentile urine
NNAL in nonsmokers=11.4 pg/mg creat Urinary NNAL, pg/mg
creatinine
Perc
ent
OptimalurinecotinineandNNALcutpointstodistinguishactivevs
passivesmoking
Goniewiczetal.2011:Cot31.1ng/ml;NNAL47.3pg/ml[337smokers,228heavySHS]
Benowitzetal2017:Cot30ng/ml;NNAL14.4pg/ml [54smokers,303lightSHSornon-exposed]
UrineNNALwithUseofDifferentTobaccoProducts
0
100
200
300
400
Non-Tobacco use
Pipe or Cigar Cigarette Snuff or Chewing Tobacco
NN
AL
(pg/
mg
crea
tinin
e)
AmongNon-SmokersUrineNNALGreatestinChildren
WindowofNNALdetection
• Detection~5half-lives• NNALhalf-liferange10to16days• Detectionrange6to12weeks
MinorTobaccoAlkaloidsasBiomarkers
ofTobaccoUse
MinorNicotineAlkaloids
Anabasine andAnatabine areBiomarkersofTobacco(asOpposedtoNicotine)
Consumption• NotpresentinNRT.NoneorminimalinE-cigaretteliquid
• Urineconcentrationsof2ng/mlhighlyspecificfortobaccousewhileusingNRT
• CanestimatenicotineintakecomingfromtobaccoevenwithconcomitantNRTuse
• Potentialbiomarkertoassesstobaccovse-cigarettevsdualuse
Nicotelline
• Becauseofstabilityandlowvolatility,nicotellinehasbeenproposedasanenvironmentaltracerandbiomarkerforparticulatematterderivedfromtobaccosmoke
Nicotelline HighlyCorrelatedwithPMinCigaretteEmissions
ElectronicCigarettes
ECLiquidandAerosol• Nicotine• Propyleneglycol• Glycerin• Particulates(includingmetals)• Volatileorganicchemicals(acrolein,formaldehyde)
• Flavorants• Miscellaneouscontaminants
ReducedNNKexposureafterSwitching
**
E-CigStudy- Nicotelline inUrine
Changes in Biomarkers after Switching from Cigarettes to E-Cigarettes
TNE
NNAL
NICOTELLINE
Days0 1 2 2 Months
SummaryofBiomarkerstoDistinguishE-cigarettefromTobaccoUse
• Nicotineintakesimilartocigarettesmoking,sonicotinebiomarkersarenotuseful
• Nicotellineisusefulbiomarkertodistinguishsmokingvsnon-combustibletobaccouse.
• Anabasine/abatabinepotentiallyuseful,butsomeE-liquidscontainsubstantialamountsofthesealkaloids
• ForstudiesofE-cigderivedtoxicantbiomarkers,toexcludedualtobaccouseIrecommendurineNNALmeasurement(<1pg/mgcreat)
Summary
Mainpoints
• Cotinine,COandNNALalluseful• OptimalcutpointsdependonprevalenceandheavinessofsmokingandSHSexposure
• Differencesinwindowofdetection:– CO– hours– Cotinine– days– NNAL- weeks
Mainpoints• Cotininelevelsinfluencedbyindividualmetabolicdifferences
• COinfluencedbyenvironmentalsourcesandsmokingmarijuana
• OTCcotininetestseasyandcheap,specificbutlacksensitivityforlight/occasionaluse
• MinortobaccoalkaloidsmostusefulfordistinguishingtobaccofromnicotineorE-cigaretteuse
OptimalBiochemicalCutPointsSeparatingSmokersand
Nonsmokers
Biomarker Biospecimen Concentration
Cotinine Blood/saliva 3-5 ng/ml
Cotinine Urine 30 ng/ml
Carbon Monoxide Breath 5-6 ppm
NNAL Urine 10 pg/mg creat
AssayCost/SensitivityBiomarker Specimen Method LLOQ Cost Per Sample Exhaled CO Breath Portable
Instrument0.5 ppm $200 - 3000
per instrument
Cotinine Urine OTC Kit NA $0.50-5.00 Nicotine and Cotinine
Urine Saliva Blood
GC-NPD 1 &10 ng/ml, respectively
$50-100
Nicotine Urine Saliva Blood
GC-MS/MS 0.2 ng/ml $75-200 nic+cot
Cotinine Urine SalivaBlood
GC-MS/MS 2 ng/ml $75-200nic+cot
Cotinine Urine LC-MS/MS 0.05 ng/ml $100-200cot +3-OH cot
Total Nicotine Equivalents
Urine LC-MS/MS NA $180-300
NNAL Urine LC-MS/MS 0.25 pg/ml $180-300