Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol, and 5-hydroxytryptophol

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1 Clinicl Chemist y 42: (1996) Lbortory testing for recent lcohol consumption: comprison of ethnol, methnol, nd 5-hydroxytryptophol ANDRS HLANDR,l* OLOF BCK,2 nd A. WAYN JoNs3 The rtio of 5-hydroxytryptophol to 5-hydroxyindole-3- cetic cid (5HTOIJSHIAA) in urine ws compred with concentrtions of ethnol nd methnol s wy to monitor recent lcohol consumption. During detoxifiction of lcohol-dependent subjects, ethnol persisted longer in urine thn in breth or plsm. Blood nd urinry methnol remined incresed for 2-6 h fter blood ethnol hd returned to bckground concentrtions, wheres 5HTOLI 5HIAA remined incresed fir 6-15 h. In helthy volunteers who hd ingested lcohol (rnge 3-98 g) the previous fternoon or evening, 87% (for men) nd 59% (for women) of ll drinking occsions exceeding 7 g of lcohol were identified by n incresed 5HTOIJ5HIAA in the first morning urine void. This compres with 32% nd 12%, respectively, identified by nlysis of ethnol (>2 xmol/ L). No gender difference in the excretion pttern of SHTOIJSHLAA ws seen. The results demonstrte tht SHTOLJ5IJIAA provides specific nd more sensitive method to detect recent lcohol consumption thn does ethnol or methnol. INDXING mit11s: lcoholism. 5-hydroxyindole-3-cetic cid. lcohol metbolism #{149} serotonin #{149} ldehyde dehydrogense lcohol dehydrogense A mjor problem in the erly detection nd tretment of lcohol dependence is obtining relible informtion bout person s drinking hbits. Self-reports nd dignostic questionnires re widely used for this purpose, but people with lcohol-relted problems my delibertely deny or underreport the ctul Krolinsk Institute, Deprtment of Clinicl Neuroscience, Psychitry Section t St. Gorns Hospitl, Stockholm, Sweden. 2 Deprtment of Clinicl Phrmcology, Krolinsk Hospitl, Stockholm, Sweden. Deprtment of Forensic Toxicology, University Hospitl, Linkoping, Swemounts they consume [1, 2]. For this reson, underdignosis of lcohol misuse is not uncommon by these methods. Wellcontrolled popultion surveys, for exmple, hve ccounted for only bout hlf of the known consumption of lcohol [3]. To help overcome this problem, lrge number of biochemicl nd hemtologicl tests hve been developed to identify lcoholdependent subjects or those engged in hzrdous drinking [4, 5]. However, when biochemicl mrkers re evluted in terms of dignostic sensitivity nd specificity, most studies still rely on self-reports s the gold stndrd for lcohol consumption, which cretes problem. The limited relibility of interview dt underscores the need for more objective tests of recent lcohol consumption, permitting frequent monitoring of individul drinking ptterns. The most obvious wy to prove tht person hs been drinking is by demonstrting the presence of ethnol in blood, urine, sliv, or breth [6]. However, becuse ethnol is rpidly eliminted from the body, this pproch is prcticl only for consumption of lcohol within the pst few hours. Incresed concentrtions of the metbolites of ethnol oxidtion, cetldehyde nd cetic cid, hve been proposed s lterntive indictors of recent drinking. Acetldehyde, however, is rpidly converted to cetic cid by ldehyde dehydrogense (ALDH), nd the concentrtion in peripherl blood is normlly very low f7]4 Its use is further hmpered by problems ssocited with rtifctul formtion fter smpling the blood. The concentrtion of cetic cid, on the other hnd, increses with the development of metbolic tolernce to lcohol, nd might therefore be more suitble s mrker of chronic lcoholism [8]. A metbolic interction between ethnol nd serotonin (5- hydroxytryptmine) cn be utilized for detection of recent drinking [9-11]. 5-Hydroxytryptophol (5HTOL) is normlly minor metbolite of serotonin, but its formtion increses drmticlly fter ingestion of ethnol, wheres 5-hydroxyindole-3-cetic cid (5HIAA), the mjor metbolite under norml Address correspondence to this uthor t: Alcohol & Drug Dependence Unit, St. Gorns Hospitl, Box 125, S Stockholm, Sweden. Fx (+46) Received October 1, 1995; ccepted Jnury 24, Nonstndrd bbrevitions: ALDH, ldehyde dehydrogense; 5HTOL, 5-hydroxytryptophol; 5HIAA, 5-hydroxyindole-3-cetic cid; nd ADH, lcohol dehydrogense. 618

2 Clinicl Chemistiy 42, No. 4, conditions, is concomitntly decresed. This metbolic shift cn be monitored by nlysis of blood or urine, nd the excretion of 5HTOL will not normlize until severl hours fter blood nd urinry ethnol hve reched bckground concentrtions [11]. On the bsis of this observtion, incresed urinry concentrtion of 5HTOL ws introduced s sensitive biochemicl mrker of recent lcohol consumption [11, 12]. An bnormlly high concentrtion of methnol in body fluids or breth hs lso been suggested s n indictor of recent drinking [13]. Methnol ccumultes in the body during the metbolism of ethnol [14, 15], nd, s for 5HTOL, the concentrtion remins incresed for some time fter ethnol is no longer detectble [16, 17]. Thus mesurement of either 5HTOL or methnol will provide higher sensitivity thn nlysis of ethnol lone for the detection of recent lcohol consumption. The present study ws crried out to evlute further the usefulness of 5HTOL s mrker of recent lcohol consumption, nd to compre its sensitivity nd specificity with those of ethnol nd methnol. Mterils nd Methods DTRMINATION #{248}FTHANOL, MTHANOL, AND ACTON The concentrtions of ethnol, methnol, nd cetone in blood nd urine smples were determined by hedspce gs chromtogrphy essentilly s described elsewhere [18]. To increse sensitivity for determintion of methnol, we used slting-out procedure, dding 1.2 g of sodium chloride to.5 ml of specimen [19]. The limits of detection for nlysis of ethnol nd methnol were 2 j.molil nd 15 /.Lmol/L, respectively. The concentrtion of ethnol in breth ws determined with hnd-held nlyzer (Alcolmeter S-D2; Lion Lbs., Brry, UK) nd the result reported s presumed blood lcohol concentrtion. DTRMINATION OF 5HTOL AND 5HIAA The concentrtion of 5HTOL in urine is expressed s rtio to the concentrtion of 5HIAA, becuse ingestion of foods rich in serotonin (such s bnn, pinepple, kiwifruit, nd wlnuts) cn otherwise cuse flse-positive results [2]. 5HTOL nd 5HIAA were determined with sensitive gs chromtogrphic-mss spectrometric nd HPLC methods [1, 12, 2/]. The reference limit used to indicte n incresed urinry 5HTOL/5HIAA rtio ws set t 15 pmol/nmol [12, 22, 23]. CLINICAL PROCDURS Mle lcohol-dependent ptients (ges 4-62 yers) were recruited from the detoxifiction unit t St. G#{246}rns Hospitl. Blood nd urine smples (1 ml ech) for determintion of ethnol, methnol, nd the 5HTOL/51-IIAA rtio were collected from 1 subjects with blood lcohol concentrtion of mmolll (men 43.6) on dmission to the hospitl. Therefter, the breth ethnol concentrtion ws mesured every 4-6 h, nd blood nd urine smples were lso collected. When breth mesurements indicted zero ethnol concentrtion, smpling of blood nd urine ws continued t 3-h intervls for nother h. In previous experimentl protocol [11], blood nd urine smples were collected from helthy volunteers (socil drinkers without ny history of previous excessive consumption of lcohol). thnol (16 g/l in ornge juice) ws dministered t.8 g/kg body weight to five women nd five men (ges yers) fter n overnight fst, nd the drink ws finished within 3 mm. From ll subjects prticipting in tht experiment, the plsm smples collected t the strt of drinking nd then t 2-h intervls for totl of 1 h, nd the urine smples collected t the strt nd then t every 2-3 h for 22 h, were lso used for the present study. All smples hd since (for -3 months) been stored in l-ml portions t -8 #{176}C. The urinry 5HTOL/ 5HIAA rtio nd the concentrtions of plsm nd urinry ethnol nd of urinry methnol (which ws not nlyzed in the previous study) were determined s described bove. In new experiment on helthy volunteers, consecutive urine smples were obtined dily from four women nd four men (ges yers) over period of 4-6 weeks. A lo-inl smple of the first morning void ws collected, nd the subjects kept record of the mount of lcoholic beverges, if ny, consumed the previous dy throughout this period. The concentrtion of ethnol nd the SHTOL/5HIAA rtio were determined. In n erlier study on the use of 5HTOL s mrker of recent drinking [24], the possible interference by metbolic cidosis in subject with dibetes mellitus could not be ruled out. To exmine this further, we ssyed urine smples collected from 2 dibetic ptients (12 women nd 8 men, ges yers) dmitted to St. G#{246}rns Hospitl for tretment of ketocidosis or com. Therefter, we ssyed dditionl smples collected t 1-week intervls for totl of 3-5 weeks, hving sked the ptients not to consume ny lcohol during this period. The concentrtions of ethnol nd cetone nd the 5HTOL/5HIAA rtio were determined. All blood smples were collected from n ntecubitl vein into Vcutiner Tubes contining DTA (Becton Dickinson, Meyln Cedex, Frnce) for preprtion of plsm by centrifugtion. The urine smples were collected into plstic tubes without preservtive. Before nlysis, the plsm nd urine smples were stored t -8 #{176}C. Smples were thwed overnight t 4 #{176}C. The study ws pproved by the locl thicl Committee (VSO/SSO) in Stockholm. Results Figure 1 shows the time course of concentrtions of ethnol in breth, plsm, nd urine, nd of methnol in plsm nd urine, s well s the 5HTOL/5HIAA rtio in urine, for ech of the 1 lcohol-dependent subjects during detoxifiction. thnol ws usully detectble longer in urine thn in breth or plsm. The concentrtion of methnol ws mrkedly incresed in the first smple [men.57 ±.15 (SD) mmol/l in plsm nd.62 ±.17 mmol/l in urine] nd did not strt to fll until the blood ethnol hd returned to bckground concentrtion. Therefter, methnol remined incresed for further 2-6 h (men 3.5 h). The urinry SHTOL/51-HAA rtio in the first smple rnged between 331 nd 181 pmol/nmol (men 684 ± 259 pmol/ nmol), nd remined bove the cutoff for 6-15 h (men 1 h) fter ethnol ws no longer detectble in blood smples. Figure 2 shows the time course of concentrtions of ethnol

3 62 Helnder et l.: Lb testing for lcohol consumption Breth thnol D Urinry thnol U Urinry Methnol Plsm thnol A Plsm Methnol * Urinry 5HTOL/5HIAA 8 1. :; 6.8 o 6 4 #{149}6 A C #{149} #{149} #{149} #{182} U, o _ #{149}. 2 o.z -..,..T1TTT #{149} #{182}6 #{149}I so u, C l A - #{182} -1. U) -j I- U) o 6.4Q -.4 C #{149}.c 2 uj UJ 2 C #{182} Tim. (hi to Tim. (hi - 1 too U, #{149}I ii, #{149} iooo ;:I 4 _ v, 3 U, 2 to Ag. 1. Time course of concentrtions of ethnol in breth, plsm, nd urine, nd of methnol in plsm nd urine, s well s the rtio of 5HTOL/5HIAA in urine, in smples collected from 1 mle lcohol-dependent subjects (A-fl during detoxifiction. The dshed line represents the cutoff limit used to indicte n bnorml 5HTOL/51-IIM rtio (>15 pmol/nmol). in plsm nd urine, nd of methnol in urine, s well s the 5HTOL/5HIAA rtio in urine, for the five femle nd five mle socil drinkers fter dministrtion of ethnol t.8 g/kg body weight. The concentrtion of methnol ws highest in the 6-h smple (-5 times bseline) nd decresed gin to the bseline concentrtion (<.4 mmolll) 1-12 h fter the strt of drinking. The 5HTOL/5HIAA rtio ws incresed >1-fold in the 6-h smple nd did not return to bseline until fter h. No significnt gender differences in ethnol elimintion rte or in the urinry excretion ptterns of methnol nd 5HTOL/5HIAA were seen. In the experiment involving nlysis of consecutive morning

4 Clinicl Chemistry 42, No. 4, P-ethnol U-ethnol U-methnol U-5HTOL/5HIAA U P-ethnol 9 #{149} U-ethnol 9 A U-methnol 9 #{149} U-5HTOL/5HIAA g io.5 C Time (h) #{149} Ag. 2. Concentrtion-time profiles of ethnol in plsm (P) nd urine (U), methnol in urine, nd the rtio of 3 5HTOL/5HIAA in Urine, in smples collected from helthy 2 socil-drinking volunteers (five women nd five men, ges I yers) fter orl dministrtion of ethnol t.8 1 g/kg body weight. The drink ws finished within 3 mm in the morning fter n overnight fst. Dt represent men vlues ± SD for the 5HTOL/ 5HIAA rtio, but, for clrity, SOs hve been omitted for the other vribles. The dshed line represents the cutoff limit used to indicte n bnorml 5HTOL/5HIAA rtio (>15 pmol/nmol(. urine smples, n verge of 29 g of lcohol (rnge 3-98 g/dy) hd been consumed the previous dy on 155 of totl of 261 smpling dys, ccording to the self-reported drinking protocols. Usully, drinking occurred in the evening or lte fternoon. Anlysis of the urinry 5HTOL/5HIAA rtio in the first morning void identified on the verge 73% (sensitivity) of ll drinking occsions exceeding 7 g of lcohol, s compred with 22% by nlysis of ethnol (>2 mol/l) (Tble 1). On some occsions when reltively lrge mounts of lcohol hd been consumed, the second nd, in one cse, even the third void of urine the next dy confirmed tht the 5HTOL/5HIAA rtio remined incresed for considerbly longer thn ethnol could be detected (Fig. 3). The men sensitivity for nlysis of ethnol nd the 5HTOL mrker ws somewht less for women thn for men (Tble 1). However, two women reported tht they occsionlly voided during the night. The specificity ws 1% for both ethnol nd 5HTOL/5HIAA in ll subjects except one. This mle subject showed one slightly incresed metbolite rtio (21 pmol/nmol), lthough no lcohol consumption ws reported the previous evening. We observed significnt correltion between the mount of lcohol consumed nd the urinry 5HTOL/5HIAA rtio in the first morning void the next dy (Fig. 4). In the urine smples collected from 2 dibetic ptients with metbolic ketocidosis or dmitted to hospitl in dibetic com, the highest concentrtion of cetone ws 4.4 mmol/l. The 5HTOL/5HIAA rtios were 6.5 ± 3.4 pmol/nmol (men ± SD, n 85), nd ll smples except one showed rtios below the cutoff limit of 15 pmol/nmol. This single vlue ws only slightly incresed (17 pmol/nmol) nd ws present in the urine smple collected immeditely upon dmission to the hospitl. Tble 1. Sensitivity nd specificity of urinry (U) ethnol nd 5HTOL/5HIAA rtio to detect recent lcohol consumption in helthy socil drinkers. Incresed U-ethnol Norml U-ethnol No. of dys 5lITOL/5HIAA >2 moi/l 5HIOL/5HIAAb <2 gemol/l No. of Age, Totl With >7 g Alcohol intke No. No. lcohol- No. No. yers study ethnol rnge, g (men) dys Sens., % dys Sen.., % free dys dys Spec., % dys Spec., % Men (29.8) (24.5) (27.8) (45.) Men Women Men >15 pmol/nmol. <i pmol/nmol. 8-5 (27.1) 8-71 (27.1) 8-6 (21.4) 9-64 (24.)

5 622 Flelnder et l.: Lb testing for lcohol consumption 4 A- 3 2 to 4-4 to H 11!I too 1 o lot void #{149} 2nd void e 3rd void Fig. 3. Urinry ethnol concentrtions nd 5HTOL/5HIAA rtios in the first, second, nd, in one cse, third morning voids collected on 16 dys from three of the socil drinkers. The clshed line represents the cutoff limit used to indicte n bnorml 5HTL/5HlA rtio (>15 pmol/nmol). Discussion Hitherto, the determintion of ethnol in body fluids or breth hs represented the only generlly vilble objective method to prove recent consumption of lcohol. However, becuse ethnol is rpidly eliminted from the body, this pproch will only detect very recent drinking, even though ethnol cn be detected for some hours longer in urine thn in blood or breth, owing to retention of urine in the bldder. The use of even more sensitive nlyticl ssys my increse dignostic sensitivity, but lso the risk of flse-positive results, becuse severl microorgnisms occsionlly found in humns re cpble of producing ethnol by metbolic conversion of glucose or other endogenous substrtes /25]. An incresed concentrtion of methnol in body fluids or breth hs been suggested s n lterntive indictor of recent drinking [13]. The concentrtion of methnol increses during drinking spree owing to competitive inhibition of lcohol dehydrogense (ADH). The methnol might originte from endogenous sources, but it is lso present s congener in lcoholic beverges nd cn be derived from vrious foodstuffs (e.g., fruits nd fruit juices) [26]. In the present experiments, methnol remined incresed even fter the blood ethnol concentrtion hd returned to bckground concentrtions in both lcoholdependent subjects nd helthy volunteers. This time lg between ethnol nd methnol clernce grees with previous reports [14-17] nd implies tht methnol is more sensitive thn ethnol s indictor of recent drinking. However, n bnormlly high concentrtion of methnol in blood or urine could reflect recent s well s long-term continuous drinking. After cute intke of lcohol by helthy volunteers, the concentrtion of methnol incresed to -.14 mmol/l on verge before returning to bseline vlues fter 1-12 h. During chronic drinking, methnol ccumultes grdully to rech much higher concentrtions, nd complete clernce fter termintion of drinking might require severl dys [14]. This ws further confirmed in the lcohol-dependent subjects during detoxifiction, where methnol leveled out within the concentrtion rnge reched in 4 4 U) I- U, Amount of lcohol (g) Fig. 4. Correltion between the urinry 5HTOL/5HIAA rtio in the first morning void nd the mount of lcohol consumed (rnge 3-98 g) on the dy before smpling in eight socil-drinking femle nd mle subjects. Accordingto the self-report protocols, drinking usully occurred in the evening or lte fternoon. The dshed line represents the cutoff limit used to indicte n bnorml 5HTOL/5HIAA rtio (>15 pmol/nmol). socil drinkers fter the ingestion of ethnol t.8 g/kg body weight. Accordingly, n incresed methnol concentrtion is not specific for recent lcohol consumption, but my be useful for detection of chronic drinking [27, 28]. The results of the present study demonstrte tht the urinry 5HTOL/5HJAA rtio provides more sensitive method to detect recent lcohol consumption compred with nlysis of ethnol or methnol in blood, breth, or urine. The specificity for detecting lcohol ws lso very high with the 5HTOL/ 5HIAA rtio [29]. A dose-dependent increse in the urinry excretion of 5HTOL is seen fter ech drink, owing to competitive inhibition of ALDH by ethnol-derived cetldehyde nd (or) the chnge in heptic redox stte [3]. In contrst to methnol, the bseline vlue for 5HTOL/5HIAA is not incresed fter prolonged misuse nd cn therefore be used to identify recent drinking in both socil drinkers nd chronic consumers. The mjor dvntge of 5HTOL/5HIAA compred with ethnol nd methnol is tht the serotonin metbolite rtio stys incresed for severl hours longer thn ethnol nd methnol, thereby incresing dignostic sensitivity. However, the exct time vilble for detection of cute lcohol consumption by nlysis of urine depends not only on the dose nd time since the lst drink, but lso on voiding between the time of drinking nd smpling. To enhnce the specificity of the mrker, 5HTOL is expressed s rtio with 5HIAA rther thn cretinine, becuse dietry serotonin might otherwise cuse flse-positive results owing to generl increse in the urinry output of serotonin metbolites [2]. To discriminte between norml nd incresed vlues of the urinry 5HTOL/5HIAA rtio, the cutoff limit ws originlly set t 2 pmollnmol [12]. However, fter creful study of lrge number of bstinent subjects of both Cucsin nd Orientl origin [22/, lower limit of 15 pmollnmol ws introduced into clinicl prctice. Actully, ll those subjects hd rtios

6 Clinicl Chemistry 42, No. 4, <1 pmol/nmol, but we used higher cutoff to reduce the risk of reporting flse positives. The rtio is stble both within dy nd between dys during periods of bstinence [11, 12, 31], nd both metbolites re reltively stble in urine smples during trnsport, hndling, nd storge [21, 31]. Vrition in ADH nd ALDH isoenzyme pttern does not influence the 5HTOL/ 5HIAA bsl vlue [22], despite the well-known genetic effects on the metbolism of ethnol [32]. The present results further indicte tht gender nd dibetic ketocidosis do not mrkedly influence the urinry rtio of 5HTOL/5HIAA. Tretment with the lcohol-sensitizing drugs disulfirm (Antbuse#{174};Dumex, Copenhgen, Denmrk) nd cynmide (Dipsn#{174};Lederle, Wyne, NJ), both potent inhibitors of ALDH, represent the only known cuse for flse positives [33]. However, the chnge in rtio resulting from tking ALDH inhibitors ws less pronounced thn fter drinking lcohol. Moreover, lrge interindividul differences in response were evident, nd, in some subjects, there ws no effect t ll on the 5HTOL/5HIAA rtio. The effect of disulfirm lsted longer thn for cynmide, which is in good greement with their respective clinicl effects [34]. In conclusion, the possibility of detecting recent intke of lcohol by lbortory tests decreses in the order: urinry 5HTOL/5HIAA > urinry or plsm methnol > urinry ethnol > blood or breth ethnol. Mesuring 5HTOL/5HIAA cn detect consumption of even moderte mounts of lcohol within the preceding -24 h nd, by frequent urine smpling, detiled picture of person s drinking pttern cn be obtined. This new biochemicl mrker cn be useful for investigting single relpses in lcoholic outptients, to prove person ctully hs bstined from lcohol [35, 36], nd possibly in connection with workplce lcohol testing, e.g., s postccident test of recent hevy drinking (possible impirment cused by hngover). 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