AssessmentoftheExhalationKineticsofVolatileCancerBiomarkersBasedontheirPhysicochemical

Properties

AntonAmann,a,b,*PawelMochalski,aVeraRuzsanyi,a,bYoavBroza,candHossamHaickc,*

a BreathResearchInstitute,Leopold-FranzensUniversityofInnsbruck,6850Dornbirn,Austria.

b Department of Anesthesiology and Critical Care Medicine, Innsbruck Medical University, 6020

Innsbruck,Austria.

c TheDepartmentofChemicalEngineeringandRussellBerrieNanotechnologyInstitute,Technion–

IsraelInstituteofTechnology,Haifa3200003,Israel.

*Jointcorrespondingauthors:anton.amann@i-med.ac.at,hhossam@technion.ac.il

Keywords:volatilecancerbiomarkers,bloodsolubility,fatsolubility,exhalationkinetics

Abstract:

Thecurrentreviewprovidesanassessmentoftheexhalationkineticsofvolatileorganiccompounds(VOCs) thathavebeen linkedwithcancer.Towards thisend,weevaluatevariousphysicochemicalproperties, such as ‘breath:air’ and ‘blood:fat’ partition coefficients, of 112 VOCs that have beensuggestedoverthepastdecadeaspotentialmarkersofcancer.Withthesedata,weshowthatthecancer VOC concentrations in the blood and in the fat span over 12 and 8 orders ofmagnitude,respectively, inordertoprovideaspecificcounterpartconcentration intheexhaledbreath(e.g.,1ppb). This finding suggests that these 112 different compounds have different storagecompartmentsinthebodyandthattheirexhalationkineticsdependsononeorcombinationofthefollowingfactors:(i)theVOCconcentrationsindifferentpartsofthebody;(ii)theVOCsynthesisandmetabolism rates; (iii) thepartition coefficients betweendifferent tissueswithblood and air; and(iv) the VOCs' diffusion constants. Based on this analysis, we discuss how this knowledge allowsmodelingandsimulationofthebehaviorofaspecificVOCunderdifferentsamplingprotocols(withandwithout exertion of effort).We end this reviewby a brief discussion on the potential role ofthesescenariosinscreeningandtherapeuticmonitoringofcancer.

1.Introduction

1.1.Background

Volatile organic compounds (VOCs) of cancer havebeen found in breath, blood [1], headspaceofcancer cells [2-10], and in headspace of resected cancer tissues [11]. Exhaled breath, whichmaychange itschemicalsignaturedependingonthephysiologicalorpathophysiologicalstateofcancer[12-24],isconsideredasoneofthemostfascinatingbodyfluids/sources.Samplingofbreathisnon-invasiveandcanbeusedforscreening,atanintensivecareunit(ICU)[25,26],duringsurgery[27-29], ormonitoring pre and post-surgery [30]. Volatile compounds that do not appear normally inexhaledbreathcanbeusedfordetectionofbacterialor fungal infection in the lungs [31-35].Alsohydrogenandmethane [36]areproducedbybacteria in thegut,andshowhighconcentrations inpersons with fructose or lactose malabsorption after ingestion of these carbohydrates [37, 38].Other volatile compounds appear after ingestion of drugs, an example being 3-heptanone duringvalproate therapy [39]. 13C-labeled compounds such as 13C-uracil, 13C-dextromethorphan or 13C-pantoprazol are specifically administered to measure enzyme activity through the respectivepotentialtometabolizetheseprecursorswithproductionof13CO2measuredinexhaledbreath[40-45].Volatilecompoundsdonotonlyappearinexhaledbreath,butalsoinskinemanations[46,47],urine[48],blood[1]andsaliva[49].Inadditiontovolatileorganiccompounds,alsosmallinorganicmoleculeslikehydrogen,nitricoxide[50-61]orcarbonmonoxide[62]aremostinteresting.Anothermost interestingapplicationofvolatiles is theiruse in searchoperations [63,64].Thepotentialofvolatilesandofbreathanalysisforclinicaldiagnosisandtherapeuticmonitoringisenormous,eventhoughatthepresentstagethereareonlyveryfewbreathtestswhichhavegotapprovalbyFDAorby the EuropeanMedicines Agency (EMA). In particular, no breath tests based on endogenouslyproducedvolatilebiomarkersforcancerareFDA-orEMA-approved.

Themainreasonsforthepre-maturityofcancerbreathanalysisinrealclinicalsettingsisrelated,ingeneral,tothelackofstandardizationandtothepoorknowledgeofthebiochemicalpathwaysandexhalation kinetics of the cancer-related VOCs. The standardization aspects and the biochemicalpathwaysofthecancer-relatedVOCswerepresentedanddiscussedinearlierpapers[18,65-67].Inthepresentreview,wefocusonthediscussionoftheexhalationkineticsofthecancer-relatedVOCs.The exhalation kinetics can be determined by actual real-time measurements under differentconditions[68-76]orbysimulationoftheflowofsomecompoundwithinthebody[70,77-83].Withthisinmind,wepresentalistof112tentativecancer-relatedVOCspublishedintheliteratureduringthe lastdecade.Then,weclassifythe112cancer-relatedVOCswithrespecttotheir“fat-to-blood”and “blood-to-air” partition coefficients and show how these partition coefficients provideestimationontherelativeconcentrationsinalveolarbreath,bloodandthefatcompartmentofthehumanbody.Basedon thegeneratedensembleof thephysicochemicaldata,wediscusshow thebalancebetweenallofthesefactorsdeterminestheexhalationkineticsofcancerVOCs.

1.2.ExhalationkineticsofcancerVOCs

To illustrate the exhalation kinetics of cancer VOCs, we present isoprene as a representativeexample.Isopreneisthemostprominenthydrocarboninexhaledbreath,appearingat∼100ppbinhealthypersonswhodonotexertanyeffort[1,84].Inhumansitisbelievedtobesynthesizedinthe

mevalonatepathway[85-87].Itcanbemeasuredinreal-timeduringanexperimentperformedatastationarybicycle[69,79,82]orevenduringsleep[68,72].

Recently, it was shown that isoprene appears at lower concentration in exhaled breath of lungcancer [88] and breast cancer patients [89], compared to healthy volunteers. So far, this result iscorroboratedby theobservation that thedecrease inconcentrationof isoprene is correlatedwiththe immune activation as measured by the neopterin concentration in blood [90]. While thesefindings offer isoprene as a potential cancer biomarker, it is important to know that theconcentrationof isoprenedependsverymuchonthespecificsamplingprotocol[69-72,78,81,82,91]. In either case, the clear isoprene’s in-situ signals achieved by on-line mass-spectrometrytechniques make it an ideal candidate for the investigation of cancer-related VOC exhalationkinetics.

Figure1showstheoutputofisoprene(innmol/L)duringanexperimentwithahealthyvolunteeronastationarybicycle.Forthesakeofcomparison,theoutputofacetone(inarbitraryunits)andCO2(inL/min)arepresentedonthesamefigure,too.Thespecificinvestigationsofisopreneandacetonehave the advantage that the respective concentrations in exhaled breath are high (∼100 ppb and∼400 ppb, respectively), which makes measurement and demarcation of exhaled vs. inhaledconcentrations comparatively easy. Theprotocol of this experiment changes between rest phasesand 75 W workload pedaling phases. During the first pedaling phase, the output of isopreneincreasesbyafactorof∼10,andsubsequentlydecreasesexponentially(stillduringpedalingphase),with a further decrease back to the baseline concentration during the rest phase. When thevolunteerstartstopedalagain,theincreaseinisopreneoutputismuchsmallerthanduringthefirstpedalingphase.Afteranotherrestphase,resumptionofthepedalingincreasestheisopreneoutput,butataratherlowlevel.Ittakesapproximately2hourstoresynthesizeisopreneinthebodyandtofilluptheisoprenestoresinsuchawaythatthehugeincreaseinisopreneoutputbyafactorof∼10appears again. Putting these findings in a more specific perspective, it is likely that isoprene isproducedandstoredintheperipheryofthehumanbody,probablyinthemuscles[79,91].Duringexertion of an effort, the blood flow through the muscles increases and leads to transport ofisoprene from the muscles to the lungs, where it is exhaled. The exponential decay of isopreneoutput (still duringpedalingphase) is a consequenceof thedepletionof isoprene in themuscles.During the restphase, theblood flow through themusclesgoesback tobaselineandsodoes theisopreneconcentration.It is importanttonoteinthiscontextthattheincreaseofconcentrationinbreathduringexertionofaneffortindicatesthatthespecificchoiceofthebreathsamplingprotocol(e.g.,quietlysitting,standing,afteraperiodofrest,etc.)isofgreatimportanceforbothhealthyandcancer states [92]. Inparticular,different samplingprotocolsmay result in verydifferent isopreneconcentrations (up to a factor of∼5).We expect that a similar situation is true formany volatilecancermarkers[70].

TogetamorethoroughlookontheexhalationkineticsofVOCs,itisimportanttoknowwhichVOCsbehaveaccordingtotheFarhiequation[93]:

valveolar

b:a A Q

CCV Vλ

=+ & & (1)

TheFarhiequationisafirstveryimportantstepinmodelingoftheexhalationkinetics[69-72,78,80-82,91].Thisequation relates thealveolarconcentration (Calveolar)ofaVOCto theconcentration inmixedvenousblood( vC ).Asseeninthisequation,adecreaseintheconcentrationduringexertion

ofanefforthappensbecausethealveolarventilation( AV& )usuallyincreasesmorestronglythanthe

cardiacoutput ( QV& ).Anexampleofacompound thatdecreases inconcentrationduringeffort (in

linewithFarhi’sequation)isbutane.[70]

AnimportantphysicochemicalconstantintheFarhiequationisthe‘blood:air’partitioncoefficient(

b:aλ ). For isoprene, this partition coefficient is approximately 0.95 (mol/L)/(mol/L) [94, 95]. This

means that the concentration of isoprene in blood is ∼95% of the concentration in alveolar air.Incidentally,the‘blood:air’partitioncoefficientcanbequitedifferentfromthe‘water:air’partitioncoefficient ( w:aλ )at37°C. Incaseof isoprene, the w:aλ isapproximately0.28.The reason for this

differenceisthatbloodcontainslipids,whichtakeupalargeramountofisoprenethanwater.

Another important physicochemical constant is the ‘fat:blood’ partition coefficient ( f:bλ ). For

isoprene, this λf:b is approximately 82.0 (mol/L)/(mol/L) [96]. This means that the equilibriumconcentration of isoprene in fat is about 82 times the concentration of isoprene in blood. For anisoprene concentration of 150 ppb (∼5.8×10-9 mol/L) in alveolar air, the estimated equilibriumconcentrationinbloodis∼5.5×10-9mol/L(=0.95*5.8×10-9mol/L)andtheestimatedconcentrationinfatis4.5×10-7mol/L(=82*5.5×10-9mol/L).

For a 70 kgpersonof 1.80mheight, the volumeofblood is∼6 L and theamountof fat tissue isestimatedtobe12.78L[97].In6Lofbloodand12.78Loffat,weestimatetheamountofisoprenetobe∼3.3×10-8mol (=6×5.5×10-9mol)and∼5.8×10-6mol (=12.78×4.5×10-7L),respectively.Hencetheamountofisopreneinfatisestimatedtobe∼175timestheamountinblood.Withthisinmind,it is reasonable to claim that the exhalation kinetics of cancer-related VOCs depends on theconcentration of the respective VOC in different compartments of the body, on the partitioncoefficients between the different compartments (e.g.,λb:a and on the fat:air partition coefficientλf:a),andonthediffusionkineticsbetweendifferentcompartments.Inthefollowingsection,wewillpresentwaystoobtaineachoftheseparametersanddiscusstheinter-relationshipbetweentheseparametersaswellaswiththeexhaledVOC-relatedcancer.

2.Estimatingtheλb:aandλ f:b

For the majority of VOCs of interest, the λb:a and λf:b have not been measured. Nevertheless, anumberofpaperspresentrelevantdataonλb:a,λf:aandλf:bofVOCs[94,95,98-106].Inaddition,theλb:aofmanyhydrocarbonscanbeestimatedbasedontherespectiveλb:aofsimilar(butnotidentical)alkanesandisoalkaneswhichhavebeeninvestigatedinRef.[95].Finally,onemayestimateλb:ausingtheformulagivenbyPoulin&Krishnan[107]

λb:a = λo:w· λw:a· (a+0.3b) + λw:a· (c+0.7b) (2)

Here,a≈0.0033isthefractionofneutrallipidsintheblood,b≈0.0024isthefractionofphospholipidsin the blood, and c≈0.82 is the fraction of water in the blood. The octanol:water partitioncoefficients (λo:w) canbe compiled fromScifinder (https://scifinder.cas.org),whereas thewater:airpartitioncoefficients(λw:a;Henry’sconstants)at25°CcanbetakenfromthecompilationofSander[108]orestimatedbytheEPISuiteTMsoftwaredevelopedattheUSenvironmentalprotectionagency(EPA, http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm). Otherwise, λw:a can beestimatedbyuseofsurrogatecompounds,forwhichλw:aisknown,withcorrectionbythequotientof the respective vapor pressures (of the compound in question and its surrogate compound).Furthermore, in order to estimate the Henry constants at 37°C, the derivative dln(λw:a)/d(1/T) asgiveninthecompilationbySander[108]canbeused,orthecorrespondingenthalpyofvaporization(ΔHvap)dividedbythegasconstant,R.

Similartotheλb:a,onemayestimateλf:ausingthemethodofPoulin&Krishnan,[107]givenbytheequation:

λf:a = λo:w· λw:a·(A+0.3B) + λw:a· (C+0.7B) (3)

Here,A≈0.798 is thefractionofneutral lipids intheadiposetissue(fat),B≈0.002 is thefractionofphospholipidsintheadiposetissue,andC≈0.15isthefractionofwaterintheadiposetissue.

3.Partitionscoefficientsofcancer-relatedVOCs

Duringthepastdecade,some112tentativecancerVOCsinexhaledbreathhavebeenreported[7,30, 67, 109-121]. There were 36 hydrocarbons, 7 alcohols, 8 aldehydes, 2 acids, 12 ketones, 12aromatic compounds, 2 heterocycles, 2 nitriles, 5 terpenes, 7 esters, 2 ethers, 1 sulfide, 2halogenated compounds, and 15 compounds from other chemical classes. Examples ofhydrocarbonsamongthislistof112VOCsare2-methyl-propane(CAS75-28-5)or5-methyl-tridecane(CAS25117-31-1).Exampleofanalcoholis1-octen-3-ol(CAS3391-86-4).Examplesofaldehydesarepentanal,hexanal,octanalandnonanal.Anexampleforaketone is6-methyl-5-hepten-2-one(CAS110-93-0).Anexampleofanaromaticcompoundisbenzophenone(CAS119-61-9).Anexampleofaterpene is "trans-caryophyllene" (CAS 87-44-5). In addition, also biomarkers in exhaled breathcondensatehavebeenpublished[122].

The112cancerVOCsinexhaledbreathcouldbeconsideredasbeingonanequalfootinginexhaledbreath. Nevertheless, the very different blood:air partition coefficients (λb:a) imply that therespectiveconcentrationsinbloodareratherdifferent,eveniftheconcentrationinbreathwouldbethe same. Each of the mentioned 112 compounds can be looked upon from many differentviewpoints.Hereareafewexamples:

• 2-Methyl-propane(CAS75-28-5)commonlyappearsinhumanbreath, insmokersandnon-smokers[123].ItisalsoreleasedbyStreptococcuspneumonia[31].Ithasbeensuggestedasvolatilebiomarkerforbreastcancerinexhaledbreath[111].

• 5-Methyl-tridecane (CAS 25117-31-1) has been suggested to be a volatile biomarker ofbreastcancer[111]andbelongstotheclassofmonomethylatedhydrocarbons,whichwere

proposed to be used in the "breathmethylated alkane contour" (BMAC) for detection ofcancerandotherdiseases[124-130].

• Benzophenone (CAS 119-61-9) was observed in the context of feces,[131] axillary sweat[132]andsaliva[133]andwassuggestedasavolatilebiomarkerforlungcancer[134].

• Trans-Caryophyllene (CAS 87-44-5) has been observed on the surface of juicy grapefruits[135],infeces[131],skinemanations[133],humanbreastmilkandsaliva[133,136].

• 6-Methyl-5-hepten-2-one (CAS 110-93-0), is produced on skin by degradation of squalene(and, in particular so during the influence of ozone). This compound is, for example,observedwhenhealthyvolunteerssitinachambersothattheskinemanationsarecollectedand observed in the chamber indoor air. There are three volatiles, acetone, 6-methyl-5-hepten-2-one,andacetaldehyde,whichexhibitespeciallyhighemission rates throughskinexceeding 100 fmol×cm-2×min-1 without ozone influence [137]. Interestingly, 6-methyl-5-hepten-2-onehasrecentlybeobservedinexhaledbreathofgastriccancerpatients.[121]

Figure 2 presents the estimated values for the negative logarithm of the blood:air partitioncoefficients, viz. –log(λb:a). These λb:a are distributed over 12 orders of magnitude. Hence, if theconcentrations of the cancer VOCs would be equal in exhaled breath − e.g., at 1 ppb − theirconcentrations in blood would be very different and also be distributed over 12 orders ofmagnitude. An analogous situation holds for the λf:b (see Figure 3). Similarly as above, if theconcentrations of the cancer VOCs would be equal in exhaled breath − e.g., at 1 ppb − theirconcentrationsinfatwouldbeverydifferentandbedistributedover8ordersofmagnitude.Forthisreason when studying VOCs’ concentrations in cancer and relating them to potential metabolicpathwaysoneshouldestimate theblood/tissueconcentrationandby thesevalidate theunderlinemetabolicassumption.Thatis,elevatedconcentrationsinthebreathofacertainmoleculewillnotnecessaryreflectthesamemagnitudeofelevationinthetissue.

That volatile cancer biomarkers vary in concentration over many orders of magnitude is mostsurprising,andshouldchangethewaywelookatthesecompounds.Typically,ahighλf:b(as,e.g.,for3-methyl-hexane) will lead to high concentration of the respective volatile compound in lipidmembranes(e.g.,inendothelialcellsdemarcatingthebloodvessels).Highconcentrationswithinthelipid membranes may change their permeability properties. It can be expected, that lipophilicvolatilesbearconsiderableinfluenceonpathophysiologyofdisease.

Thefactthatthesecancer-relatedVOCshaveverydifferentphysicochemicalpropertiesisexpectedto be an advantage. The different λb:a and λf:b imply that the concentrations in differentcompartments(blood,fat,muscle,etc.)areverydifferent.Asaconsequence,theexhalationkineticscan be expected to be different. The release of these compounds from their “main storage”compartment depends on the blood flow through this compartment during sampling. Differentsamplingprotocols (withorwithoutexertionof aneffort, orduring increasedblood flow throughintestinal part of the body due to digestion processes after a meal, etc.) lead to differentconcentrationsofvolatiles.Measurementswithdifferent samplingprotocols couldbedone foranarbitraryVOCand,inparticular,forthe112cancer-relatedVOCsmentionedabove.Inthiscontext,lungandupperairways cancer cellsholdadistinguished statusasdue to their anatomic location,

which allows them to release cancer-related VOCs directly into the exhaled air. Thus, the breathlevels of these species can be enriched as compared to the levels expected from their bloodconcentrations.Consequently,thebreathandbloodconcentrationsofthesevolatilecancermarkerscannoteasilyberelatedusinge.g.theFarhiequation.Nevertheless,fromthebreathanalysispointofviewthelocationofthesecancersisbeneficialastheirVOCs’fingerprintscanbedirectlydetected.

Detailedinvestigationsandevenreal-timeanalysisshouldbepossibleformanyofthese112cancerVOCsintheforeseeablefuture.Whileallofthese112compoundsaretheresultofempiricalstudies,additionalimportantaspectsthatwouldbeneededtobeaddressedconcerntheconnectionofsuchcompounds to real logical metabolic processes related to the disease. By doing so, one couldbiologically relate the concentrations emitted in breath, to concentrations in the different bodycompartments, and thus suggest indication for the disease development status. From atechnological point of view it would be wanted to specify certain threshold concentrations ofvolatiles in breath to guide changes in the clinical treatment. This would result in a therapeuticmonitoringguidedbytheexhaledconcentrationsofvolatiles.Byadaptingtheanalyticalandnano-technologyinthisdirectiononecanachieveanon-lineresponsebyphysicians.

4.Conclusions

The 112 volatile cancer biomarkers published during the last decade have very differentphysicochemicalproperties.Uptonow,noadvantagehasbeendrawnfromthisfact.Inthisreview,weshowedthattheblood:breathandblood:fatpartitioncoefficientsareverydifferentforthe112cancer-related VOCs. Even if the concentration of these 112 compounds would be identical inexhaled breath (e.g., 1 ppb), the respective concentrations in blood and in the fat compartmentwouldvary,respectively,over12and8ordersofmagnitude.Thismeansthatdifferentcompoundsmay be stored (or exist in equilibrium) in different compartments of the body. To gain a morecomprehensiveunderstandingonthecancer-relatedVOCs,acombinedinformationonappearanceand concentration of these VOCs in breath and blood [1], or in breath and urine or saliva, iscritical.[138]. Further information can be achieved by investigating exhaled breath and resectedtumortissuefromthesamepatient[11,30].Forasimplepointofcarescreeningtool,knowingtheexhalation kinetics and optimization of the sampling procedure is important. For research andeventualpharmaceuticaltreatmentalsotheunderstandingofbloodandtissueconcentrationwouldbecritical.

Acknowledgment

Anton Amann gratefully appreciates funding from the Oncotyrol-project 2.1.1. The CompetenceCentre Oncotyrol is funded within the scope of the COMET - Competence Centers for ExcellentTechnologies through BMVIT, BMWFJ, through the province of Salzburg and the TirolerZukunftsstiftung/StandortagenturTirol.TheCOMETProgramisconductedbytheAustrianResearchPromotion Agency (FFG). PawelMochalski acknowledges support from the Austrian Science Fund(FWF) under Grant No. P24736-B23. Veronika Ruzsanyi gratefully acknowledges a Lise-Meitnerfellowship from the Austrian Science Fund (FWF) under Grant No. M1213-B18. A.A. and V.R.appreciate funding from the Austrian Agency for International Cooperation in Education andResearch(OeAD-GmbH,projectSPA04/158-FEM_PERS).A.A.,P.M.andV.R.thankthegovernmentofVorarlberg(Austria)foritsgeneroussupport.HossamHaickgratefullyacknowledgesthefundingfrom the FP7-Health Program under the LCAOS (grant agreement no. 258868) and the FP7’s ERCgrantunderDIAG-CANCER(grantagreementno.256639).

Figure 1:Output of isoprene (nmol/min), acetone (arbitrary units) and CO2 (L/min) for a healthyvolunteerduringrestphasesandexertionofaneffortof75Wonastationarybicycle.IncaseofCO2,15exhalationsperminutewith3Litersofalveolarairwith4%ofCO2correspondto1.8Liters/minofCO2(=15*3*0.04L/min). Isopreneoutputthroughexhaledbreathmayincreaseuptoafactor∼10duringexertionofaneffort,whereastheconcentrationinbreathincreasesuptoafactor∼5.Inthisexperiment,twosteady-statesofisopreneexhalationappear,(A)at∼25nmol/min(correspondingtoproductionofisopreneintheliver)and(B)at∼100nmol/L(correspondingtoproductionofisopreneinthemuscles).Inapersonwithhighcholesterolbloodlevel,theproductionofisopreneintheliverwould be decreased under the influence of statins [139]. Reproduced from Ref. [69], DOI10.1088/1752-7155/3/2/027006,©IOPPublishing.ReproducedbypermissionofIOPPublishing.Allrightsreserved.

Figure2:Partitioncoefficient,-log(λb:a),for112volatilecancerbiomarkerspublishedduringthelastdecade[7,30,67,109-121],aswellasacetoneand2-pentanoneforcomparison.Thecolorforthedifferent compound names is chosen according to the chemical class. Compounds with a higherlog(λb:a)willtendtobeinthebloodandviceversa.

Figure3:Partitioncoefficient,-log(λf:b),for112volatilecancerbiomarkerspublishedduringthelastdecade[7,30,67,109-121],aswellasacetoneand2-pentanoneforcomparison.Thecolorforthedifferent compound names is chosen according to the chemical class. Compounds with a higherlog(λf:b)willtendtobeinthefatandviceversa.

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