Alcohol Determination in the Clinical Laboratory

J)Ubowski, Kllrt M.: Alc:uhol ddermlpalion in the clinic:allaboratory. AmJ Clin Plltbo174: 747-750,1980. Fo"r methodsror blood-akobol analysili-gas thromatography, en,ymatic:D,ud.ation with alcobol ddlydrOCm:lse, chemical oxidation wirll"dd dichromat'!, and usmornt!try -,lire briefly reviewed rromUle point of vie'" 01 the diniarl labor.llK')'. ~dvantas~ artdlilJlilatioRS or these .l1ldhods are discussed. 3I1d theIr keyreaturteS are tabulated. The correlldion of the results of blood-alcohol a'lIl1yses with 'taa.'U or alcoholk influence and theircornsponciwg ._d SYPiPlomsb presePted in tabular f~m.(Key words: AlcoholdeCerminaCion; BlOod-alcoholanalYSIS.)

THE DETERMINATION OF ALCOHOL'" in bloodand other biologic specimens is a frequently requestedservice in clinical laboratories. and is often requestedon an emergency basis in hospitals.

Breath is the preferred specimen whenever the pa-tient is able to participate in the breath-alcohol analysis.The latter has the inherent advantages of simplicity.rapidity, and noninvasive sampling, and reflects thealcohol content of the arterial circulation, which isphysiologically and clinically more significant than thevenous blood-alcohol concentration, especially duringactive alcohol absorption. Current instrumentation in-cludes devices employing aM chromatography, infra-red absorptiometry. fuel cell catalysis, and solid-stategas sensing, as well as the older chemical oxidationdevices.

All quantitative breath-alcohol instruments currentlymarketed employ end-expiratory breath. Features andperformance of'current breath-alcohol analyzers of po-tcntial applicability to clinical laboratory practice havebeen summarizcd.··12.'l Several of the newer devices areportable. battery-powered. and self-contained, andhence are especially practical for bedside or emergency-room use. When necessary or desirable, whole breathOr the alcohol in a measured volume of breath canbe readily stored on simple sorption columns for subse-quent elution and analysis.! Measurement of alcoholin breath involves some special biologic and chemicalconsiderations." and requires a 15-min "deprivation"period preceding breath-sampling to ensure the absence

Rec.:ivcd April )0. 1980; accepted for publicali!)n April 30. 1980Addr~ss rcponl rcquests to Dr. Oubow$ki: Uni".rliIY or Okla-

homa College oC Medicine. P. O. 80r. 269(11. Okl:lhoma City.Oklahomw 7) 190.

• The un modi lied term ..•• <;,,!lol .. in this arti.:le refers 10 ethano].

KURT M. DUaOWSKI. PH.D.

Department of Medicine and TOxicology' Lal:>orarories,The University of Oklahoma H9Blrh SClenCflS Cemer.

Oklahoma City. Oklahoma

of contamination from recently ingested alcohol in theoral cavity.

Blood is the most useful specimen when breath isnot available, with plasma or serum being physiologi-cally more appropriate specimens than whole blood.However. for certain forensic applications (e.g .. in-vesugatlcn of alleged traffic law violations) analysisof whole blood is required because statutory interpre-tations of the results of blood-alcohol analyses an: uni-versally based upon whole blood. Collection of plasmaand whole blood requires the use of anticoagulants-heparin, disodium edetate, and sodium or potassiumcitrate or oxalate are suitable if the specimen is notto be stored. For specimens to be preserved. potassiumoxalate (S mglml of blood) and sodium fluoride (t.5mg/ml of blood) are an appropriate anticoagulant andpreservative combination for short-term storage at 5 Cof initially sterile blood specimens.

The many methods for alcohol analysis in blood fallinto about a dozen different categories, of which thefour listed il\ Table I are the most obvious candidatesfor use in the clinical laboratory. In the author's view.osmolality measurement, while rapid and :iimple, is nota method of choice. Estimation of alcohol in serumrequires the subtraction (rom the actual osmolality de-termination result (by freezing-point depression) of theassumed normal osmolality value.·4 Substantial dis-crepancies between the blood-alcohol concentrations(BACs) thus estimated and the actual alcohol concen-tration!) are possible and have been dccumented.!

The other methods for blood-alcohol analysis listedin Table I are appropriate for use in clinical labora-tories, and are listed in order of this author's prefer-ence. Alcohol analysis by gas chromatography can beperformed with simple and relatively mexpenslve in-struments and is the current method of choice. Amongits desirable features are rapidity. sensitivity. minimalrequirements for sample manipulation or preparation.and inherently great selectivity for ethanol even withsingle COlumn/single condition analyses. £ssentially

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748 I\.J.C.P .• 1oI\t'I\'8Ib<r 1'11IIIouaOWSKJ

Tab!« J. Summary of Methods fol' Blood-alcohol Analysis

MClhod

Required SampleTreatment!Separation

Specificityfor Ethanol

Requireli ciilibnuion'.11 time of anDlysi~

Gas ehrcma-toCrapby

Selective forethanol; m~ltiplecolumns/coRdi·tions greatlyincreOlseselectivity

Dilution orheadspac:ec:quiUbr:nion

Enzymaticoltidalionwith alc:oholdehydroeenase

Dilution (forpla~1N1orserum) orcJeprolciOlUllion

Selec Iive fore\l1:,"ol; someinterferenee byisopropanol andmethane!

NoneChcmicaloxidation with~iddi.chromate

Distillation.diffusion.dialysis, oracration

Osmometry None for plasmaor serum

None

ApparalU$Rcquiremenl~

FinalMC3,urcmcnt Limit3lion~

G(1S chro'~t~r:lpb."'Olter balll (forhe:ld~pace). ~tripchart ntcordcr orintccr;ltOr

EICClricw vUltil&C orcurrenr. ~iQ ~tripch!\rl re.:o~r orj ntilgratOt

Ullra"iolcl specrro-pholOtneler. "i:llblcphotometer, orsome automaticanalyzers

Distillation ordiffu!>ion ~pp.ralUs.photometer orspectrophotometer,or litralion device.water bath

Phl)tOnletricl~pccttophuto·merrlc readill*

P\)lentiaJ interCerent:~by hicher alcohob,some enZYMeinhibitor.

Till'lllion orphotometricispectrophoto.metrie readina

Time consurn.ins;nonspecitic

Fretzing-poinldep~S5ionosmometer

05molaJity measure-ment

Nonspccific; onlyplasma or serumspecimens uS3blt;poor cone/alionwlIh blood-;alcoholconeentrauon

complete specificity for ethanol is achieved by usingmultiple columns and analysis conditions. Analysis ofthe "headspace" vapor above a blood or other liquidspecimen saturated with sodium chloride, after equili-bration at .sO C or other controlled temperature, is asimple and desirable gas chromatographic technic. Itis usually preferable to direct injection into the chro-matograph of a diluted whole blood specimen becauseit obviates problems of inlet and column contamina-tion and syringe plugging. Direct injection of a dilutedliquid sample." however, may be preferable for erner-gency tests because it eliminates the IS-lO-min head-

Table 2. Principal Variants of Gas ChromatographicMetbods for Blood-alcohol Analysis

DetectersFlame- ionization detectorThermistor detector

Appropriatc packed columnsSolid tlhase (Porapake. Chl'omo~orb*. etc .)Liquid phoul: (CurbUWWl", Halleomid-. etc)

An31ysis technicsHe<,dspacc 53mpline ~fter equilibl'lltionDirect inj.!ction Ilf dilu/ell bloodPrerein pre.:'pitatilln a.nd direct injecuonOptions: inlernal siandard VJ. direct procedures

Quanti~lionElrctronic iritcgDlion'prinlout of pe~k. 3re!l~

Measurement of strlp-chart recordina of detector r~POn$t: pc:\khtight, or peak UU~

space equilibration period. When many blood speci-mens are to be analyzed for alcohol and analyst limeis limited. automated gas chromatographic headspaceanalysis'o. or the. use of automatic liquid sampling at-tachments is indicated and yields excellent results inroutine use. The principal variants of gas chromate-eraphic methods for blood-alcohol analysis are listedin Table 2 in order of the author's preference. Asindicated in Table I, gas chromatography requires theuse of simultaneous reference standards.

Enzymatic oxidation with alcohol dehydrogenasetADH) is a sensitive and simple method for alcoholmeasurement. It ili a practical method for occasionalor infrequent use because elaborate preparation is notneeded. and for the same reason, it serves well as aback-up procedure for gas chromatography. The twoprincipal variations are measurement of the change inultraviolet absorbance at 260 or 340 om, and visualphotometry 'of a secondary indicator reaction. Severalcommercial kits arc available for ADH methods; thecharacteristics and performances of four of these werereponed by Redetzki and Dees. I" The principal fca-tures of the ADH methods are given in Table 3. Severalinstrumental variations have been reponed, includingthe use of rapid centrifugal analyzers, II rapid electro-chemical measurement usint a membrane oxygen-sens-ing electrode.! and adaptation to the DuPont AUlO'marie Clinical Analyze •.•. ' All ADH-based enzymatic

~~:~'.

vol. 1'. Nu. )

~VVI

ALCOHOL DETUMJNATION 749Table 3. Principal Features of Enzymatic (ADH)Oxidation Methods for Blood-alcohol Analysis

ReactionsADH

Basic Reliction: C,H.OH +NAD·~CI4)CHO.•. NADH + 14+

Conditions: pH 8.7-9'(): CH3CliO trapped willi semicarbuidc oraminoacetic acid

Oiaphor~cDiapiloraK: IN" + NJI,OH + ~. •. • Red Formaun

.•. IIIAO·

PeroxidueOxyaell ~pletion: NAOH + H+ + \o10t ~

NAO" + H~O Mo'·

CatalystNAD oxidoreductilsc (alcohol dehydrogenase; ac I. \.1.11

Prcpnration or plll$m:\. serum, or bloodDilurion With ..uinc ",lurion or deproteiniUlion with perehlori~

or lricblo~etic acid or Ba(OH), + ZnSO.

final measurementyltr •.•.iolet spectrophotunletry

A Ab~orbancc at 260 nm (NAO· absorbs)II AbsorblUlet :u 340 om '(NADH aiHorbs)

Visible photometry: measurement of ~table rcd rormaian colorat 500 nm

Amperome~ric mcas\lremcnt •••.•ith 0, cltetrode:Fluorometry

oxidation methods are subject to potential interferenceby methanol and, especially. isopropanol to varyingextents.1r..lti Reference standards must be analyzed inparallel with the biologic samples and controls, as theyshould be irrespective of the alcohol analysis methodemployed.

In view of the advantages of other methods. chemicaloxidation with potassium dichromate in sulfuric (ornitric) acid solution is now one of the less preferablemethods. It requires substantial sample treatment andseparation of the alcohol from its matrix before reactionwith the acid-dichromate reagent. tt is tlme-consumingin the usual method variations. in addition to beingessentially nonspecitic for ethanol unless combined withcomplex chemical manipulations. Stability of the re-agent and ~implicilY of the final measurement do notsufficiently counterbalance these disadvantages for usein the general clinical laboratory. The method may beuseful for back-up purposes for the gas chromatographicOf ADH methods. since a negative result conclusivelyeliminates the presence of alcohol in the specimen.and potential chemical iruerferants can be detected bysimple tests 011 the same distillate.'

Table 4 correlates SACs with alcoholic influencestages and the: corresponding ~linical signs and symp-toms. Great caution should be exercised in correlatingBACs with presumed alcohol dosage. and speculative

Table 4. Stages of Acute Alcoholiclnfluen<:e/lntoxication

Blood-alcoholConcen-t[';\tion(% w/v)

Stage ofAlcoholInftuence Clinical Signs/Symptoms

0.01-0.05

Q.03-0.1'2

0.09-0.25

Subriety No apparellt influenceBehallior nearly nutma' by ordin;sry

observatiunSIi3hl chltllJC~ uelectable by 5~ciat

1e:$15

Euphoria Mild euphori a. sociability.talutivcl'en

Incru1ed self-confidence: decreasedinhibition.

I Dimillulion of 1I1h!1I1ion.juda~nl, andCOlltrol

Lnss of efficiency in finer pcrt'orm;U\cetC$I~

Excitement Emotion~1 instability: decreasedi"hibilion~

Lon DC. eritielli judgmentImpainnent of memory and

comprehensionDecreased $!:n~itory reSponse:

IRcreaseQ reaction timeSome mu~cular incoordina(ion

Disorientation. mental eonw5ion:dizzilH:u

6uxgerated emotion;;\! 1tateS l fear .anger, srief. f'/I.'.)

Disturbance of SCn);llion (diplopia.f'1C'.) and of perception or color.form: motion, dimell,ion~

Decreased pain senseImpaired balance: l'['IU~CUIM

incoordinillion; stllggering gait.slurred speech

0.18-0.30 ConFusion

0.27-0.40 Stupor ~pilthy; gencral inertia. appf()achiog.paralySis

Markedly ckCreiLSCd response to~limuli

Mark~ n1u!cular incoordinOltion:inabililY to sl"od or ",alII

Vomiting: incontinence of urine :1I1dIeces

Impilited eun5ciou~M$~; .Iecp orstupor

C()mpletc unconsciouseess: com".anc$thesiu

Dcpre~~d or aboliShed rellexesSubnormlll tempcralOrcIncontinence of urine and fecesEnlharrlSSment of circulation and

reSpirationPo$sible death

Dc.'\th from resriratory paraly$i~

0_35-0.50 Comll

Q.45'" Death

retrograde extrapolation of an experimentally deter-mined BAC 10 an earlier time should be avoided becauseof its many pitfalls."

750References

I. A(;A Test MelhodQlOJ:YALe ~Ihyl Akohol. Wilmington. ~lu·w3rt'. DuPont Co .• In51,ument Products. /97&

2. Ch:\mpion HR. tt :\1: Alcohol intoxication n,le! \oe,'um osmolalilY.Lancet I: 1402- 1404. 197)

l Cheng FS. Chrislilln GO: enzymatic delermin<lliun or bluodeth3nol. wilh 3mpcromerric rne:l5111'ement of rare of olly;end~pl~linn. CI;rl ('Irern ~4:621-626. 1978

4. Dubowski KM: Mdhoo ror the dcterminuricn ur I!lhyl ilh::uhulin biological specimens. Alcohol :tnd thl! impaired driver.Chi~.1t:O. N~rioMI S:tfcry Cuuncil. 1970. pp 78-81: 91-94

~. Ol,bllWski KM: An"ly_i:. uf ethane]: Type: C procedure. Merh·Qdulugy (ur loInillytical loxicOIOSY' €diled Q)' I Sun"hinc:.Cleveland. eRe Prc~~. Inc .. 1975. "" 145_ t51

b. n"bllw~ki KM: Rd~c:"t developments in br"ath·al~ohl)llIn.:Ilysis.Alcohol. dn'gs. and If.uti.: SafO!IY· Pro.:lleding:> of the SixlhInt"malion",' Conference on Alcohol. Drug~. lInd Tr;.aflkS~ft1y. Toronto. Septrtmbel' 8-13. 1974. Ediled by S IsI~el·sr::"n and S Lambert, Toronto. I\ddicilun Research Foull(l;l.lion of Ontllriu. t91.s. pp 481-494

1. Dubow.~kl KM: Silldi~~ in brnlh·:alc:ohol an:lly~i~: biolugic;,1((\~to,·s. Z Rtchrsme,lizi'l 7t.:93- I 17. J975

j.·Dubow$ki KM: Humiln pharmilCQkim,lic;s uf clhunul. I. PCilkblood concentrauons atld elimination in mille and female sub-jeers. Alcohol Technical R.ep5:55-fo~, 1976

DUBOWSKI A.l.C.r .• N••.•••",bc, I"",

9 Dub¢ •.••sk. KM: C"Ueetion 'lnd laler Dn~y~is oT bre;lIh·;,knhulafiercaiCiunl sulfult sorptjQn. Clio Chc:m 23: 137~-1313. I'.m

10. Dub.>wski KM. ~hnu ••1 rur :m4lysis or ethanol on bluIUI:II.::o1fluids. Report No. DOT·TSC·NHTSA·76-4. Washingllln.D. C .. U. S Dcp:lnmcnl uf Trolnsp0l'tt\\ion. NlIliuoal HiSh.W3y Traffic SafelY AdminiSlr:llion. Jotn\laty 1977

11. Hadjiioannou TP. Hadjii~nnou lA. to1ll1mstadtHV: AUlomatedenzymic delerminalion of o!thanol in blood. serum. andurine wilh ~ mini:Jlurc centrifulal analyzer. Clin Chem U:802-805. 1976

12. HarSt'r RN: Recenlly pul)lish •.'d !lnalylie.1 mt'lhods fordC:lermil).in~ :alcohol in bOOy miueriaJ~. Rcport No, OOT.HS·8U1·242.W:\shjn~ton. D. C.. tJ. S. Department of Tralls~n:!ti,tn.NOlt;l)n:!1 Hiahl>l:l)l Tr:lffic $2fel), Administralion. October 1974

13. M:a~n MF. Dubowski KM: Brc:\th·alc:ohol ~l\llIysi~:uses. mellt.ods and ~ome forensic problcmi-l'eViCw ;)lid opinion. JForenstc Sci 21:9,;,41, 197b

14. Redel2lci HM, KocmcrTA, Hughes JR. et 0\1:Osmometry in th~'e ••"luation or alcohol inlo11ication. CliD Toxil:ol ~:343-36311m

IS. R~delzkiHM. Dees WL: Comparison offour lIits for enZymatifdetermination orcrh!lnol in blood. Clin Chem 22;113~~6. 1971

16. V:\Sili3des J. Pollock .I. R(lbinson CA: Pirfall~ of the .h:C\h",do:hydro~asc pfI)CellUfe fo)l' the erner,enc)I :\ssay of alcoho]a case j;ludy of iseprepancl ovcrdoulc. CliD Chern 24:383-l8S, 1918

1140 SECTION IV. Chemicals.

Table 55-20Stages of Acute Alcchollc Influence/Intoxicationin Nontolerant lndlviduals"

Blood Alcohol Stage ofConcentration Alcohol

(% w/v) Influence Clinical Sign/Symptom

0.01-0.05 Sobriety No apparent influenceBehavior nearly normal by ordl-

nary observationSlight changes detectable by

special tests0.03-0.12 . Euphoria Mild euphoria, sociability, talk-

atlvenessIncreased self-confidence; de-

creased inhibitionsDiminution of attention, Judg-

ment, and controlLoss of efficiency in finer perter-

mance tests0.09-0.25 Excitement Emotional instability; decreased

inhibitionsLoss of critical judgmentImpairment of memory and com-

prehensionDecreased sensory response; in-

creased reaction timeSome muscular Incoordination

.0.18-0.30 Confusion Dlsorlentatlcn, mental confuelcn;dlzzlnoss

Exaggerated emotional states(fear, anger, grief, etc.)

Disturbance of sensation (dlplo-pia, etc.) and of perception ofcolor, form, motion, olmen-slcne '

Decreased pain senseImpaired balance; muscular

incoordination; staggering gait,slurred speecn

0.27-0.40 Stupor .Apathy; general inertia, ap-proaching paralysis

Markedly decreased response tostimuli

MarKed muscular Incoordination;inability to stand or walk

Vomiting; incontinence of urineand feces

Impaired consciousness; sleep

0.35-0:50Dr stupor .

Coma Ccrnplete .unconsciousness;coma; anesthesia

Depressed or abolished reflexesSubnormal temperatureIncontinence of urine and fecesEmbarrassment of circulation

and respirationPossible death

0.45+ Death Death from respiratory paralysis

-Adapted from Dubowski KM, Am J Clin PathoI19BOj74:747-750,

.:: Ellenhorn'sedical

To icology:Diagnosis andTreatment of Human PoisoninqSecond Edition