Rate of elimination of gamma-hydroxybutyrate from blood determined by analysis of two consecutive samples from apprehended drivers in Norway

Rate

of

elimination

of

g

-hydroxybutyrate

from

blood

determined

by

analysis

of

two

consecutive

samples

from

apprehended

drivers

in

Norway

Marit

Årnes

*

,

Liliana

Bachs

,

Mohammad

Al

Sammarai

,

Alan

Wayne

Jones

,

Gudrun

Høiseth

a

DepartmentofForensicMedicine,OsloUniversityHospital,Oslo,Norway

b

DivisionofDrugResearch,DepartmentofBiomedicalandClinicalSciences,LinköpingUniversity,SE-58185Linköping,Sweden

ARTICLE INFO

Articlehistory: Received7April2020

Receivedinrevisedform13June2020 Accepted15June2020

Availableonline20June2020

Keywords: Apprehendeddrivers GHB Back-calculation Impairment Pharmacokinetics ABSTRACT

Aim: Gamma-hydroxybutyrate (GHB) is a commondrug of abuse with aneliminationhalf-life of

20 45min.However,thereissomeevidencethatGHBmightexhibitsaturationkineticsafteringesting

highrecreationaldoses.TheaimofthisstudywastoinvestigatetheeliminationkineticsofGHBfrom

bloodinpeopleapprehendedbythepoliceforimpaireddrivingandsecondarytodescribeconcentrations

inallGHB-positivedrivers.

Methods:Twoconsecutivebloodsamplesweretakenabout30 40minapartfromN=16apprehended

driversinNorway.GHBwasdeterminedinbloodbyanUltraHigh-PerformanceLiquid

Chromatography-TandemMassSpectrometry(UHPLC-MS/MS)method.ThechangesinGHBbetweenthetwoconsecutive

blood samples allowedestimatingGHB’seliminationhalf-life, assuming first-orderand zero-order

eliminationkinetics.GHBconcentrationsarealsoreportedforN=1276apprehendeddriverswithGHBin

blood.

Results: The median time interval betweencollecting the two blood samples was 36min (range

20 56min). The median concentration of GHB in the first blood sample was 56.5mg/L (range

14.1 142mg/L)comparedwith47.8mg/Linthesecondsample(range9.75 113mg/L).Themedian

eliminationhalf-lifewas103min(range21 187min),andGHB’smedianzero-ordereliminationrate

constantwas21.0mg/L/h(range6.71–45.4mg/L/h).Back-calculationtothetimeofdrivingresultedin

GHBconcentrationsupto820mg/Lassumingfirst-orderkineticsandupto242mg/Lassuming

zero-orderkinetics.Inalldrivers(N=1276),themedianGHBconcentrationwas73.7mg/Landhighestwas

484mg/L.

Conclusion:Theeliminationhalf-lifeofGHBinbloodsamplesfromapprehendeddriverswaslongerthan

expected compared with results of controlled dosing studies. Zero-order kinetics seems a more

appropriatemodelforGHBwhenconcentrationsareback-calculated,andthemedianeliminationrate

was21mg/L/h.

©2020TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense

(http://creativecommons.org/licenses/by/4.0/).

1.Introduction

Gamma-hydroxybutyrate(GHB)isanendogenoussubstance,a prescriptionmedicineandarecreationaldrugofabuse[1].Itwas originallytestedasapotentialanestheticagent,butthis clinical applicationwasabandoned,duetoalackofananalgesiceffectand instancesofvomiting[1,2].TodayGHBisaregistered pharmaceu-tical usedmainlyfor thetreatment of cataplectic symptomsin

narcolepticpatients[3,4]andinsomecountriestotreatalcohol withdrawalsyndromeandaddiction[5].GHBactsasadepressant of the central nervoussystem and hassimilar dose-dependent effects to that of ethanol, barbiturates and benzodiazepines, includingeuphoria,lossofinhibitions,sedation,unconsciousness anddeath[6,7].Ithasbeenpopularasarecreationaldrugofabuse sincetheearly1990s,andindosesrangingfrom2 5g[8,9]itacts onGABA-A,GABA-BandpostulatedGHB-specificreceptors[10].

GHBisadrugthatisoccasionallydetected inbloodsamples from apprehended drivers in many countries [11,12], and is a problem for traffic safety, owing to impairment effects on performance and behavior [6,13,14]. When GHBis detected in druggeddrivingorotherforensiccases,thereissometimesaneed

* Correspondingauthorat:DepartmentofForensicSciences,OsloUniversity Hospital,POBox4950Nydalen,N-0424Oslo,Norway.

E-mailaddress:b32652@ous-hf.no(M.Årnes).

http://dx.doi.org/10.1016/j.forsciint.2020.110374

0379-0738/©2020TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

ContentslistsavailableatScienceDirect

Forensic

Science

International

toback-calculatethebloodconcentrationfromtimeofsamplingto thetimeofdriving,whichisoftenseveralhoursearlier[15].

GHB is oxidized by GHB dehydrogenase to succinic semi-aldehydeandfurtherbysuccinicsemialdehydedehydrogenaseto succinicacid, which enters theKrebs’ cycle toproduce carbon dioxideandwater[16,17].Theterminalhalf-lifeforGHBaccording to controlled human dosing studies mainly is reported to be 20 45min [18–26], however, there are studies presenting a slightlylongerhalf-lifeofGHBespeciallywithhighconcentrations [27–29].PreviouspublicationshavealsoquestionedwhetherGHB eliminationisbetterdescribedusinga zero-orderkineticmodel [26,30].Inacasereport[30]inwhichapersonwasintoxicated with alcohol and GHB, back-calculation to determine ingested amountofGHBusingfirst-orderkineticsgaveanimprobablehigh dose,thatifingestedprobablywouldhaveprovenfatal.

Many papers havereported concentrationsof GHB in blood samples from drugged drivers [11,12,31,32], but there are no publisheddataonitseliminationhalf-lifeinthispopulation.The primaryaimofthisstudywastocalculatebothafirst-order half-life and a zero-order elimination rate constant for GHB in apprehendeddriversbasedontheconcentrationsdeterminedin twoconsecutivebloodsamples.Wealsousedtheseresultsto back-calculate GHB concentrations in the actual forensic cases. A secondaryaimofthisstudywastodescribeGHBconcentrationsin allGHBpositive driversapprehendedinNorwayoverthesame studyperiod.Thisallowedevaluatingwhetherthecasesusedto estimate elimination kinetics were representable of the larger populationofGHB users.To furtherdescribe thepopulationof driversinfluencedbyGHB,weinvestigatedtheprevalenceofother psychoactivesubstancespresentinthesamebloodsamples. 2.Methods

2.1.Studypopulation

Informationwasextractedfromatoxicologydatabase belong-ing to the Department of Forensic Medicine, Oslo University Hospital.Thisdatabaseincludesresultsoftoxicologicalanalysesof bloodand other biologicalspecimens fromapprehended drunk anddruggeddrivers.Thestudyincluded16casesfromtheperiod between01.01.2008–05.11.2018inwhichtwoconsecutiveblood samplesweresubmittedfortoxicologicalanalysis.Thesesamples were taken 20 56min apart and both specimens contained measurableamountsofGHB.

Inall16cases,GHBconcentrationsinblooddecreasedbetween thetwosamplingtimes,whichwasanecessaryrequirementfor makinga kinetic evaluation.In thefollowing analysis, thefirst bloodsamplingtimepointis referredtoasT1whilethesecond

timepointisreferredtoasT2,andthetimeintervalbetweenthem

is

D

For the secondary aim of our study, we compared GHB concentrations in blood from these 16 special cases with the concentrations found in all GHB positive apprehended drivers (N=1276) from the same study period. During this time approximately60,000impaireddrivingcasesunderwent toxico-logicalanalysisandGHBwasdetectedinabout2%ofthese. 2.2.BloodsamplesandanalysisofGHB

Allbloodsamplesweretakenfromacubitalveinandtheblood was collected in 5mL Vacutainer tubes, containing 20mg of sodiumfluoride(afinalconcentrationof0.4%),asapreservative,in additionto 143IU of heparin (BDVacutainer Systems, Belliver Industrial Estate, Plymouth, UK). The samples were routinely screenedforpresenceofethanolandalargenumberoflicitand illicitdrugsincludingGHB.

Theanalytical methodusedfor quantitative analysisof GHB bothscreeningandconfirmationanalysiswaspreviouslydescribed elsewhere [33]. Inbrief,this involvedusingultra highpressure liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS).Themethodisfullyvalidatedforroutineuseinforensic toxicology.Until2011,thelaboratoryusedacut-offlevelforGHBof 10.4mg/L,andafterthata cut-offlevelof8.3mg/L.Onlyvalues abovethiswerereportedaspositive.

2.3.Pharmacokineticcalculations

Thebloodsamplesfromapprehendeddriverswereanalysedat leastonce at each time point for GHBcontent. Whenmultiple determinationsweremade,ameanconcentrationfromeachtime pointwascalculated.TheconcentrationofGHBatT1isreferredto

asC1,whiletheconcentrationatT2isreferredtoasC2.

For a drug eliminated from blood according to first-order kinetics, the following equation can be used to calculate eliminationrateconstant:

First-orderslope=-K1=-[Ln(C1)–Ln(C2)]/

D

Half-life(T1/2)=Ln2/K1=0.693/K1

Foradrugthatobeyszero-orderkinetics,theeliminationrate frombloodiscalculatedasfollows:

Zero-orderslope=-K0=-[C1–C2]/

D

To back-calculateGHBconcentrationstothetimeof driving, referredtoas“C0(first-order)”and“C0(zero-order)”,respectively,

weusedthetimeelapsedfromtimeofdrivingaccordingtothe laboratorypolicereportformandtimewhenthe_firstbloodsample wastaken,referredtoas

D

Forthepost-absorptiveeliminationphaseitisgiventhatCt=C0

–k0xt

To back-calculate the concentrations to the time of driving assumingzero-ordereliminationwethereforeuse

C0(zero-order)=C1+

D

Assumingfirst-ordereliminationkinetics,eliminationisgiven byLnCt=LnC0–k0t

Forourback-calculationswethereforeuse LnC0=LnC1+K1

D

WhichcanbeexpressedasC0=C1eK1DT0

ThisfurthersimplifiestoC0=C12(DT0/T1/2)

2.4.Statisticalanalysis

MicrosoftExcelsoftwareversion2010andIBMSPSS1_Software

version 22.0 were used for the statistical analyses. The mean, medianandhighestand lowestvalues wereusedasdescriptive statistics.Thetoxicologicalresultswereassessedretrospectively and no new determinations of GHB or other drugs were undertaken.

3.Results

Duringthestudyperiod,sixteencasesmatchedourinclusion criteriaandwereusedinthepharmacokineticanalysis(Table1).A summary of GHBconcentrations and calculated first-order and zero-ordereliminationrateconstantsareshowninTable2.Inone subject (No.16),thecalculated half-lifewas exceptionally long, whichsuggeststhatinthiscaseabsorptionofGHBwasincomplete whenthefirstbloodsamplewastaken.Wethereforepresentthe

datawithandwithoutthisaberrantcaseincluded.Excludingthis case,themediancalculatedeliminationhalf-lifewas103minand themedianzero-ordereliminationrateconstantwas21.0mg/L/h. There was a significant correlation between the measured C1

concentrationandtheeliminationhalf-life(Spearmansrho=0.56, p=0.025).

The resultsof theback-calculationsof GHBconcentrationto timeofdrivingarepresentedinTable3.Informationonthetimeof consumption ofGHBin relationtothetimeof drivingwas not availableforthiscaseseries.Thismayleadtooverestimatingthe true concentration, since somedrivers might have beenin the absorptionphasewhendrivingandwouldreachtheirmaximum

Table2

GHBconcentrations,calculatedeliminationhalf-livesandzero-orderrateconstants.

Withoutlier Withoutoutlier

Parameter Mean Median Highestandlowestvalues Mean Median Highestandlowestvalues C1(mg/L)a 67.7 56.5 14.1-142 65.9 46.9 141–142

C2(mg/L)b 54.2 47.8 9.75–113 51.6 39.1 975–113

T1/2(min.)c 168 106 21–1149 103 103 21–187

Zero-orderrateconstant(mg/L/h)d

23.6 20.4 3.42–45.4 24.9 21.0 6.71–45.4

a

ConcentrationofGHBinfirstbloodsample.

b

ConcentrationofGHBinsecondbloodsample.

c

First-orderkineticseliminationhalf-life.

d

Zero-orderkineticseliminationrateconstant. Table1

Individualconcentrationsandcalculatedhalf-livesandzero-ordereliminationrateconstantsforGHBinbloodofapprehendeddrivers.

Subject C1(mg/L)a C2(mg/L)b DT1(min)c T1/2(min)d Zero-orderrateconstant(mg/L/h)e

1 26.7 12.3 24 21 36.0 2 38.1 25.0 32 53 24.6 3 46.3 34.5 23 54 30.9 4 14.1 9.75 39 73 6.71 5 72.9 60.5 21 78f 35.5 6 30.6 20.0 49 80f 13.0 7 36.9 29.6 29 91 15.2 8 46.9 39.1 27 103 17.3 9 129 99.9 41 109 43.7 10 142 111 42 115 45.4 11 142 113 40 122 43.3 12 76.0 56.4 56 130 21.0 13 28.9 26.4 20 154 7.50 14 90.8 77.2 41 176 19.9 15 66.1 59.1 30 187 13.9 16 95.7 93.2 42 1149f 3.42 a

ConcentrationofGHBinfirstbloodsample.

b

ConcentrationofGHBinsecondbloodsample.

c

Timeintervalbetweenfirstandsecondbloodsample.

d

Calculatedterminalhalf-life.

e

Zero-orderkineticseliminationrateconstant.

f _{Ethanolalsodetectedinthesample.}

Table3

Back-calculatedconcentrationsofGHBinbloodtothetimeofdrivingusingbothfirst-andzero-ordereliminationkinetics.

Subject C0(mg/L)a(firstorder) C0(mg/L)b(zero-order) DT0(min)c

1 820 90.4 106 2 812 133 232 4 37.5 25.6 103 5 151 121 82 6 113 63.1 150 7 146 82.5 180 8 78.1 68.8 76 9 151 147 24 10 202 186 58 11 312 242 139 12 127 110 97 13 42.3 39.4 84 14 102 101 30 16 104 104 144 Mean Median

Highestandlowestvalues

229 137 37.5–820 108 102 25.6–242 108 100 24-232 a

ConcentrationofGHBatthetimeofdrivingcalculatedassumingfirst-orderkinetics.

b

ConcentrationofGHBatthetimeofdrivingcalculatedassumingzero-orderkinetics.

c

concentration of GHB in blood at a later time point. For two subjects(No.3 and 15), time of drivingwas not available. The median

D

was 100min. By back-calculating using first-order kinetics, the resultsforthetwosubjectswithshortestestimatedhalf-livesare 820and812mg/L,respectively,comparedto90.4and133mg/Lfor thesamesubjectswhenapplyingzero-ordereliminationkinetics. TheconcentrationsofGHBinbloodfromN=1276apprehended driversare summarized in Table 4. Of these onlyN=110 were mono-drugcases.ThedrugsmostoftencombinedwithGHBare showninTable5withamphetaminetoppingthelist.Morethan oneadditionaldrugwasdetectedinN=985(77%)ofthecases. 4.Discussion

Thepresentstudyindicatesthattheeliminationhalf-lifeofGHB when calculated from two consecutive blood samples from impaireddriversislongerthanpreviouslyreported.Additionally, it supportsthe ideathat zero-orderelimination kineticsseems moreappropriateasapharmacokineticmodelformakinga back-calculationinforensiccasework.

To investigate thepharmacokineticpropertiesof adrug,the ideal method entails giving a bolus dose under controlled conditionsandanalysisofbloodorplasmasamplestodetermine aconcentration-time-profile.Thepost-peakbloodsampleswould thenbeusedtodeterminetheeliminationrateconstantwithand withoutmakinga logarithmictransformationoftheblood-drug concentrations. In forensicscience it is obviously ethically and practicallychallengingtostudythepharmacokineticsofdrugsby controlledhumandosingstudies.Instead,usefulinformationcan beobtainedfromforensicpracticecaseworkwhentwoormore bloodsamplesattimedintervalsapartareavailable,suchasfrom apprehendeddrivers[34].Bymakingcertainassumptions,suchas that both specimens are taken on the post-absorptive phase, informationcanbegleanedaboutrateofdrugeliminationfrom bloodinthispopulation.Thisispreviouslyperformedforethanol inanumberofstudies[35–38].

Ourdata(excludingtheoutlier)showedamedianhalf-lifeof GHBof103min,whichisconsiderablylongerthanthehalf-lives frompreviousstudiesoflessthan60min[18–26].Wecalculateda zero-ordereliminationrateconstantof21.0mg/L/h,whichisvery

well in accordance with a previously published zero-order elimination rate constant for GHB of 18mg/L/h [30]. Also, in twopreviouslypublishedstudies[14,20];wehavebeenabletoget detailedinformationabouttheGHB-concentrations,and permis-sion touse the data tocalculate a zero-order elimination rate constant. In an experimental study byLiakoni et al.[14] of 16 subjectswhoreceived50mg/kgGHB,themedianGHB concentra-tion in plasma after one and three hours was 83.1mg/L and 24.4mg/Lrespectively(bloodplasmaratioforGHBis0.8–1.2[39]). The calculatedmedian zero-order eliminationrate constant for thispopulationwas29mg/L/h.

InthestudyofBrailsfordetal.[20],25mg/kgGHBwasgivento 12healthyvolunteers.Theconcentration-timeprofileofGHBwas usedtodetermineazero-ordereliminationrateconstantof23mg/ L/hwhenvaluesfromCmaxtotwohourspostdosingwereused.

When a shorter time period was used, the elimination rate constantwassomewhathigher.Themedianzero-order elimina-tionrateconstantfromthepresentstudywasthereforeingood agreementwithvaluesderivedfromthesetwocontrolleddosing studies.

TheconcentrationsofGHBinbloodfromthe16apprehended drivers agreedwell with theconcentrations determined for all driversarrestedinNorwaywithGHBinblood(Table4).Themean and median concentrations in all drivers were 85.0mg/L and 73.7mg/L,respectively(N=1276),beingingoodagreementalso with impaired drivers from Sweden (mean 90mg/L, median 84mg/L, N=473) [11] and Australia (mean 89mg/kg, median 87mg/kg,N=160)[40].Thepresentstudyadditionallyshowsthat GHBimpaireddrivers,likeotherdruggeddrivers[41],aremostly poly-drugusers,withapreferenceforamphetamines,butwhoalso take other psychoactive substances (Table 5). It is especially interesting to observe the frequent combination of GHB with centralstimulants,acombinationthatisalsodemonstratedfore.g. benzodiazepinesandopioids[41].TheprevalenceofGHBinthe present population of apprehended drivers is comparable to a studydoneinAustralia[40].

Fordrugswithfirst-orderkinetics, theeliminationrate from blood or plasma is directly proportional to the concentration present, whereas for drugs withzero-order kineticsa constant amountiseliminatedper unittime (independent of concentra-tion).Thepresentback-calculations,especiallyinthosecaseswith a calculated short elimination half-life, yield improbable high concentrationsat thetimeof drivingwhenapplyingfirst-order kinetics, higher than concentrationsobserved in our1276GHB positivecasesover10years.Thissupportsthenotionthat zero-order elimination kinetics is a more appropriate model when makingaback-calculationinthispopulationofdrugusers.

Ourdatathereforeindicateasaturationofeliminationcapacity, asweseeasignificantcorrelationbetweenconcentrationofGHBat thefirsttime pointandthefirst-ordereliminationhalf-life.The concentrationatwhichsaturationoccurscannotbedetermined from our study. In addition to saturation of the elimination pathway,theremightbeadegreeofcapacity-limitedabsorptionas well,aspreviouslydocumentedforGHB[23].Sincewearedealing withrecreationalusersofGHBwhotakehighdoses,longerTmax Table4

Mean,medianandhighest/lowestGHBconcentrationsinbloodofdriversapprehendedinNorway. Typeofcase Na

Meanconc.(mg/L) Medianconc.(mg/L) Highestandlowestvalues

GHBmono-drugb _{110 104 98.7 9.26–251} GHBpoly-drugc 1166 83.2 70.2 8.85–484 GHBallcasesd 1276 85.0 73.7 8.85–484 a N=numberofcases. b

GHBastheonlydrugidentifiedinblood.

c

GHBidentifiedwithoneormoreotherpsychoactivesubstances.

d

AllcaseswithGHBidentifiedinblood. Table5

MostcommondrugsdetectedincombinationwithGHB.

DrugidentifiedtogetherwithGHB Na _%ofallcases

Amphetamine 773 60.6 Clonazepam 631 49.5 Methamphetamine 542 42.5 N-desmethyldiazepam 414 32.4 Diazepam 368 28.8 THC 289 22.6 Alprazolam 145 11.4 Ethanol 112 8.78 a

N: number of occurrences, although in manycases multiple drugswere identified,sothepercentageofallcasesexceeds100%.

(timeintervalfromconsumptiontoamaximumblood concentra-tion is reached) and thereby increased possibility of ongoing absorptionatC1cannotbeexcluded.IfaprecursorofGHB,

gamma-butyrolactoneor1,4butanediolwasingestedinsomecases,this couldtheoreticallyleadtoprolongedhalf-lifeofGHB.

Onestrengthofthepresentstudyistheabilitytoinvestigate pharmacokineticsinreallifedruggeddrivingcases.Thereisonly scant information of drugpharmacokinetics in drugabuse and overdosecasesincomparisonwiththerapeuticdrugmonitoring cases.Wealsomadeuseofalargebackgroundmaterialofblood concentrationsof GHBinthepopulationof impaireddriversin Norway. The blood concentrations in cases with double blood samplesagreedwellwithvaluesinallapprehendeddrivers.

Onelimitationofthestudyisthatwelackdetailedinformation aboutthedoseofGHBtakenandthetimeoflastuseinrelationto time of driving and time of sampling blood for toxicological analysis.Theavailabilityofonlytwobloodsamplesseparatedin timebyabout30minisalsoopentocritiqueasawaytocalculate eliminationrateconstants.Forlonghalf-lifedrugs,suchshorttime intervalwillnotgiveagoodestimateoftheelimination,butfora shorthalf-lifedruglikeGHBthistimespanismoreappropriate. Oneimportantquestionisalsowhetherstillongoingabsorptionat thefirsttimepointmightaccountforthelongerestimatedhalf-life inourstudythaninpreviouspublications.Itshouldalsobenoted thatthreeofthe16doublebloodsamplecasesincludedethanol(as seeninTable1)andinterestinglyenough,oneofthemwastheone showing an extremely long half-life. The other two, however, showedrelativelyshorthalf-lives.Apharmacokineticinteraction betweenethanolandGHBhasnotbeendocumented[27]andwe arenotabletothrowanylightonthisquestion.Inconclusion,this article reports GHB concentrations in blood from over 1000 apprehended drivers in Norway. In a sub-population of traffic offendersdoublebloodsamplesweretakenandusedtodetermine rates of elimination from blood assuming both first-order and zero-orderkinetics.Forthispopulationofdrugusers,theresults supportzero-orderratherthanfirst-ordereliminationkinetics. Creditauthorstatement

MÅparticipatedinplanningthestudy,performedthestatistical analysesanddraftedthemanuscript.LB,MASandAWJ participat-ed in planning the study and gave useful feedback on the manuscript.GHparticipatedinplanningthestudyandthedesign ofthemanuscriptandsupervisedtheproject.Allauthorsaccepted thelastversionofthemanuscript.

Funding

Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors. Declaration

The study was conducted according to the data processing agreementwiththeHigherProsecutingAuthority,whichstandsas theownerofforensicmaterialsinNorway.Inaccordancewiththis agreement,onlyanonymousdatawereusedinthepresentstudy. DeclarationofCompetingInterest

Noneoftheauthorshaveanyconflictsofinterests. References

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