kokain esteraze

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Human liver cocaine esterases: ethanol-mediated formation of

0 89 2-6 63 8/9 1/0 00 5-2 73 5/$ O1 .5 0. © F AS EB 2735

ethylcocaine

ROBERT A . DEAN , CHARLES D. CHRISTIAN , R . H . BARRY SAMPLE , AND W ILLIAM F. BOSRON’

Departm ents of Pathology, B iochem istry and Molecular B iology, and Medicine, Indiana University School of Medicine,

Indianapolis, Indiana 46202-5122, U SA

ABSTRACT A new , pharmacologically active metabo-

lite of cocaine, ethylcocaine, has been reported in in-

dividuals after concurrent use of cocaine and ethanol.

Form ation of ethylcocaine may contribute to the common

coabuse of these two drugs and the apparent danger of

th is practice. W e have identified a nonspecific carboxyl-

esterase that catalyzes the ethyl transesterification of co-

caine to ethylcocaine in the presence of ethanol. In the

absence of ethanol, th is human liver esterase catalyzes the

hydrolysis of cocaine to benzoylecgonine, a m etabolite

that is inactive as a psychom otor stim ulant. A second hu-

m an liver esterase is also described. This enzyme cata-

lyzes hydrolysis of cocaine to ecgonine methyl ester, alsoinactive as a stimulant. These two liver esterases m ay play

im portant roles in regulating the metabolic inactivation

of cocaine.-Dean, R . A .; Christian , C . D .; Sample,

R . H . B.; Bosron , W . F. Human liver cocaine enterases:

ethanol-m ediated formation of ethylcocaine. FASEBJ. 5:2735-2739; 1991.

Key Words: cocaine esterase alcohol . human liver

TH E PREVALENCE OF COCAINE ABUSE in the United States has

risen dramatically during the past 2 decades (1). Increased

admissions to drug treatm ent programs, emergency room

visits , and deaths from cocaine use reflect the significance of

th is public health problem (2). Coabuse of ethanol and co-

caine also has become evident in recent years. In one survey,

59% of cocaine abusers reported a diagnosis of lifetime alco-

hol abuse/dependence (3). A second survey revealed that

30% of cocaine users drink ethanol on almost every occasion

of cocaine use (4). Frequent coabuse of these two drugs may

be due to potentiation of the stimulant actions of cocaine by

ethanol (5). For, example, cocaine-induced motor activity in

m ice is significantly greater after coadministration of

ethanol, which itself is a depressant (5). Ingestion of ethanol

increases the cardiotoxicity of cocaine as suggested by a

three- to fivefold increase of ethanol increase in heart rate

over that observed after adm inistration of either drug alone

(6). Moreover, an epidem iological study purports that con-

current use of ethanol increases the risk of cocaine-relatedsudden death 18-fold (7).

A new metabolite of cocaine, ethyleoeaine,2 is present in

plasm a, liver, b rain , and urine of indiv iduals using cocaine

and ethanol concurrently (8-il). E thylcocaine is produced by

incubation of cocaine and ethanol w ith crude human (9) and

rat (12) liver homogenates. In this paper, w e report the first

isolation and characterization of the human hepatie esterase

responsible for the hydrolysis of cocaine to benzoylecgonine

and the ethanol-dependent transesterification of cocaine to

ethyleocaine. W e suggest that the pharm acologic and toxic

effects of cocaine may be potentiated by ethanol through the

enzymatic form ation of the active metabolite, ethylcocaine.

MATERIALS AND M ETHODS

Drugs

Benzoyleegonine tetrahydrate, 1-cocaine HC1, propylbenzoyl-

eegonine HC1, eegonine methyl ester HC1, and eegonine

H C1 w ere from A lteeh Applied Science Laboratories (S tate

College, Pa.). Benzoyleegonine-Ds and cocaine-D3 were

from Radian Corporation (Austin, Tex.). Ethylcocaine (ben-

zoyleegonine ethyl ester) was prepared in our laboratory

from benzoylecgonine using ethyliodide as the alkylating

agent and tetram ethylethylenediam ine in methylene chloride

(Brzezinski et al., unpublished results). The crystalline

product has a melting point of 108-109#{176}C . A nuclear mag-

netic resonance (NMR)3 spectrum of the crystalline product

showed the presence of the expected replacement of the

methoxy group of cocaine by ethoxy (quartet at 5.25 and

triplet at 1.25 ppm). M oreover, the mass spectrum of the

crystalline product (see Fig . 4B ) is consisten t w ith that previ-

ously reported by Smith (11).

Preparation of human liver esterases

Five grams of human liver obtained at autopsy was

homogenized in 5 ml 50 mM sodium phosphate, 1 mM

dithiothreitol, 1 mM EDTA , pH 7.5. A homogenate-

supernatant was prepared by centrifugation at 60,000 x g

fo r 40 mm . Nonspecific acetylesterase activ ity was deter-

m ined by incubating 10 fsl of sample w ith 0 .5 mM

4-m ethylumbelliferylacetate, 90 mM potassium phosphate,

40 m M potassium chloride, pH 7.3 , at 37#{176}C .Form ation of

4-m ethy lum beiliferone was detected by UV spectrophotome-

try at 350 nm (E350 12 .2 Am M em’). The units of esterase

activity are expressed as em oles of 4-m ethy lum belliferone

produced per m inute. The hom ogenate-supernatant had an

esterase specific activity of approximately 0 .4 U /mg. Five

m illiliters of the supernatan t was applied to a 1.5 x 100 cm

LKB AeA-44 gel filtration column in 50 mM sodium phos-

phate, 1 mM dith iothreito l, pH 7.5 . E luted protein was

m onitored by UV spectrophotometry at 280 nm (1 .8 absor-

bance units fu ll-scale) and collected as 4-m i fractions.

Nonspecific esterase activity was determ ined in selected frac-

tions. Two separate esterase activity pools (esterase 1 and es-terase 2) were prepared by com bin ing ind ividual fractions

‘To whom correspondence should be addressed , at: D epartment

of B iochem istry and M olecular B iology , M edical Science B ldg . 405 ,

635 Barnhill D r., Indianapolis , IN 46202-5122, USA .

2E thy lcocaine is an abbreviation for benzoylecgonine ethyl ester.

This compound has been called cocaethylene (7-9).

3Abbreviations: GC/MS, gas chromatography w ith mass spec-

trom etry ; HPLC , h igh-pressure liqu id chrom atography; TCA ,

trich loroacetic acid; NaF, sod ium fluoride; ND , not detected;

NM R, nuclear magnetic resonance.



Human Liver Esterase 2 Human Liver Esterase 1

CH ,N

0

OH

Ecgonine Methy l Ester

corresponding to peaks in acety lesterase activity as shown in

Fig. 1. The specific activities of esterase 1 and esterase 2

were about 0.5 and 0.3 U /mg, respectively. Purified horse

liver alcohol dehydrogenase and rabbit muscle aldolase were

applied to the column and eluted in approxim ately the same

fraction numbers as esterase 1 and 2, respectively.

Cocaine m etabolism by esterase 1 and 2

One-m illiliter aliquots of esterase 1, esterase 2, and a buffer

control were incubated with 24.5 iM cocaine in the absence

and presence of 40 mM ethanol. The buffer was 50 mM

sodium phosphate, 1 m M dith iothreito l, pH 7.5. This was

the same buffer used for isolation of the esterase fractions by

gel filtration. The total volume for each reaction m ixture was

1.2 m l. Each esterase and buffer aliquot was combined with

100 el water or ethanol in water, and 100 tl cocaine that was

prepared in water immediately before use. A fter a 3-h incu-

bation at 37#{176}C,he reactions were stopped by addition of 1

m l 5% triehloroacetic acid (TCA) with vortex m ixing. The

tim e of incubation and amount of esterase I and 2 used in

ineubations were chosen so that 40-80% of added cocaine

w as c on ve rt ed t o m et ab ol it es .

Inhib ition of esterases 1 and 2

One-m illiliter aliquots of esterase 1, esterase 2, and thebuffer control were incubated with 24.5 IL M cocaine and 40

mM sodium fluoride (NaF) or 0.5 mM eserine in the absence

and/or presence of 40 mM ethanol. The total volume for

each reaction m ixture was 1.2 m l. Each esterase and buffer

aliquo t was combined w ith 50 el water or ethanol in water,

50 1d inh ibitor (water con trol, sodium fluoride, or eserine),

2.5

Esterase

2

1. 5

I

0

1.0

S

0.5

Benzoylecgonine

Figure 2 . Pathways for cocaine metabolism . In the absence of

ethanol, esterase 1 hydro lyzes the methyl ester of cocaine-producing

benzoylecgonine. In the presence of ethanol, esterase 1 catalyzes the

ethy l transesterification of cocaine to ethy lcocaine, which also can

be hydrolyzed to benzoylecgonine by esterase 1 . Esterase 2 hydro-

lyzes the benzoyl ester of cocaine producing ecgonine methyl ester.

and 100 d cocaine prepared in water immediately before

use. A fter a 3-h incubation at 37#{176}Che reactions were

stopped by addition of 1 m l 5% TCA with vortex m ixing.

Analysis of cocaine and metabolites

Four replicate samples for each incubation condition were

split in to two duplicate sets. One set was analyzed by high-

pressure liquid chromatography (HPLC). The second set

was analyzed by gas chrom atography w ith m as s s pec tro me-

try (GC /MS). For HPLC analysis the internal standard was

100 sl 10 jeg/m l propylbenzoyleegonine. Two internal stan-

dards, 1 benzoylecgonine-Da and 1 eg eocaine-D3, were

added for GC/MS analysis. After thorough mixing, precipi-

tated protein was removed by centrifugation. Cocaine, ben-

zoyleegonine, ecgonine methyl ester, ethylcocaine, and inter-

nal standards were extracted from the incubation

supernatants using Bond Elut Certify solid-phase cartridgesaccording to the manufacturer’s specifications (13). For

HPLC analysis the dried concentrates were reconstituted

with 100 1d mobile phase and 10 tl was injected onto a Beck-

m an Ultrasphere C8 column (15 cm x 4.6 mm x 5 s) main-

tained at 43#{176}C.he mobile phase was 23% acetonitrile in

0.25 M KH2PO4, 100 mM pentane-sulfonic acid at pH 2.7.

The flow rate was 1.4 m l/m in. The column eluant was moni-

tored at 235 nm w ith a full-scale absorbance of 0.01 (14). For

GC/M S analysis, the concentrated extracts were treated with

N -methyl-N -(trimethylsyl)-trifluoroacetam ide and trimethyl’

chlorosilane (1000:1) form ing trim ethylsilane derivatives of

benzoylecgonine and eegonine methyl ester. The derivatized

samples were analyzed on a Hew lett-Packard 5970B MSD

using selective ion monitoring as previously described (15).

RESULTS AND DISCUSSION

The duration of action for cocaine (benzoyleegonine methyl

ester) is lim ited by its rate of m etabolism . This occurs by

hydrolysis of the methyl ester group producing benzoylecgo-

nine or the benzoyl ester group producing ecgonine methyl

ester (F ig. 2). Benzoylecgonine and ecgonine methyl ester

are the major metabolites of cocaine identified in urine after

drug administration (16). Neither of these metabolites pos-

sess cocaine-like stim ulant psychomotor activity when ad-

m inistered peripherally , even at high doses (17, 18). In vitro

0. 0

10 30 400

F ra ctio n N um ber

Figure 1. Separation of hum an liver cocaine esterases 1 and 2 by

gel filtration. The supernatan t from a human liver hom ogenate wasprepared and applied to an LKB AcA-44 gel filtration column as

described under M aterials and M ethods. E luted protein was m oni-

to red by UV spectrophotom etry at 280 nm (1.8 absorbance units

full-scale) as shown by the dashed line. Nonspecific acetylesterase

activ ity was determ ined in elu ted fractions by m easuring hydrolysis

of 4-m ethy lum belliferylacetate to 4-m ethylum belliferone as

described under M aterials and M ethods. Nonspecific acetylesterase

activ ity is indicated by the solid line. Two peaks of esterase activity

elu ted. Ind ividual fractions corresponding to these peaks were

pooled as two separate preparations (esterase 1 and esterase 2). Th e

elution position of esterase I is sim ilar to rabb it muscle aldolase

(158 ,000 daltons) and esterase 2 is sim ilar to horse liver alcohol de-

hydrogenase (80,000 daltons).

2736 Vol. 5 September 1991 The FASEB J ou rn a l DEAN ET AL.

CM ,N

C_OCH,CH3

CM , N Ethanol o__.(: :>0

C-OCH, ,‘ Ethylcocaine

-

CM 3

0ocaine



C OC AIN E E ST ER AS ES 2737

TABLE 1. C ocaine m etabolism by nonspec/ic hunsan liver esterase?

Sam ple 40 m ps ethanol Cocaine, /L M Benzoylecgonine, sM Ethylcocaine, M Ecgonine m ethyl ester, $L M

Esterase I -

+

4. 6

5. 6

18.3

11.4

ND

8. 7

ND

ND

Esterase 2 -

+

14.3

17.0

4. 8

4.5

ND

ND

6. 5

6. 0

Buffer -

+

19.9

20.1

4. 5

4.5

ND

ND

ND

ND

‘1The metabolism of 24.5 M cocaine by nonspecific hum an liver esterases in the absence and presence of 40 m ethanol is shown. Reactionconditions and postreaction sam ple handling and analysis are described under M aterials and M ethods. The data represent m eans for duplicate sam ples

analyzed for cocaine, benzoylecgonine, and ethy lcocaine by GC/M S. Results for these duplicate samples d iffered by no m ore than 10% . Duplicate sam ples

analyzed for ecgonine methyl ester by GCIMS differed by no more than 14% . The resu lts are represen tative of several experim ents. A second set of

duplicate samples was analyzed by HPLC and gave sim ilar results for cocaine, benzoylecgonine, and ethy lcocaine. The HPLC procedure does not detectecgonine methy l ester. Resu lts are expressed in m icromoles. ND represents analyses that were not detected . Recovery of added cocaine as the sum of

unmetabol ized cocaine plus cocaine m etabolites ranged from 98 to 112% .

studies have shown enzymatic hydrolysis of cocaine to ben-

zoylecgonine in human serum (19), canine plasm a (20), and

rat liver m icrosomes (21). D im inished recovery of benzoylec-

gonine from the urine of dogs treated w ith cocaine and the

earboxyesterase inh ibito r, tri-o -to lylphosphate, further sup-

ports enzymatic hydrolysis (20). Despite this, formation of

benzoylecgonine is often described as occurring nonenzy-

m at ic al ly ( 22 ). T hi s v ie w i s ba se d on the chem ical instabilityo f t he m et hy l e st er o f c oc ai ne ( 23 ) a nd i n v it ro s tu di es w i th

liver homogenates and serum that failed to detect the en-

zymatic hydrolysis of cocaine to benzoylecgonine (22). The

present study resolves this issue by identifying a human liver

esterase activity that accelerates the hydrolysis of cocaine to

benzoylecgonine.

In this study, two nonspecific acetyl esterases were isolated

from a human liver extract (F ig. I, esterase I and esterase 2).

These two esterases were separated by size exclusion chro-

matography. The molecular weights of esterases I and 2 were

about 150,000 and 80,000, respectively, by comparison w ith

the elution of rabbit muscle aldolase and horse liver alcohol

dehydrogenase, respectively. In an incubation of 24.5 sM co-

caine with an aliquot of esterase 1 for 3 h at 37#{176}C,5% of

added cocaine was hydrolyzed to benzoylecgonine (Table 1).

T hi s a mo un t o f b e nz oy le cg on in e was fourfold that produced

by spontaneous hydrolysis in buffer alone. The amount of

benzoyleegonine formed on incubation of cocaine w ith ester-

ase 2 was equivalent to that produced by spontaneous

hydro lysis in buffer. Formation of benzoylecgonine by incu-

bation of cocaine w ith esterase 1 was completely inhib ited by

40 mM NaF, a known esterase inhibitor (Table 2) . A second

esterase inhibitor, eserine, decreased benzoylecgonine for-

mation by only 16% (Table 2). These observations establ ish

that cocaine can be hydrolyzed to benzoylecgonine by an

N aF -s en si ti ve h um an l iv er c a rb ox ye st er as e.

TABLE 2. E ffect of es leras e in hib itor s on rela tive activity”

Serum eholinesterase (EC 3.1.1.8) catalyzes the hydrolysis

of the benzoyl group of cocaine producing ecgonine methyl

ester (19, 22, 24, 25). Both NaF and eserine inhibit this reac-

tion with norm al serum in vitro (24, 25). M oreover, in vitro

i nc ub at io n o f c oc ai ne w it h s er um f ro m i nd iv id ua ls w it h an

atypical (low activity) serum cholinesterase phenotype re-

veals low rates for hydrolysis to eegonine methyl ester (22,

25). These observations suggest that indiv iduals w ith loweholinesterase activity (for example, the fetus, the newborn,

pregnant women, patients with liver disease, and especially

individuals w ith atypical or silent serum eholinesterase

phenotypes), may be particularly sensitive to cocaine (1).

Human liver also possesses enzymes capable of hydrolyzing

cocaine to ecgonine methy l ester (22). In th is study, we

identified a human liver esterase (esterase 2) that hydrolyzed

24% of added cocaine to eegonine methyl ester after 3 hours

of incubation (Table 1). Incubation of cocaine w ith buffer or

esterase 1 produced no measurable ecgonine methyl ester.

The esterase inhibitors, fluoride ion and eserine, completely

inhibited eegonine methyl ester form ation catalyzed by ester-

ase 2 (Table 2). The small amount of benzoylecgonine de-

tected after incubation of esterase 2 w ith cocaine is attribut-

able to nonenzymatie hydrolysis (Table I). Thus, esterase 2

specifically hydrolyzed the benzoyl ester of cocaine produc-

ing ecgonine methyl ester. These experim ents show that co-

caine can be metabolized by at least two separate human

liver esterases that specifically hydro lyze the benzoyl or

methyl estersof cocaine, respectively(Fig. 2).Benzoylecgo-

nine has recently been shown to be a potent vasoconstrictor

(26). Therefore, regulation of cocaine metabolism by ester-

ases I and 2 m ay influence cocaine-induced psychom otor ac-

t iv it ya s w el l a s v as oc on st ri et io n pr od uc ed b y c oc ai ne a nd t he

m eta bo lite , b en zo ylec go nin e.

Addition of 40 mM ethanol (200 mg/100 ml, or tw ice th e

Esterase 40 mM ethanol Product m easured

Relati ye a ctiv ity w ith in hib ito r

40 m M NaF 0.5 m M eserine

1 - Benzoylecgonine 0 0.84

1 + Benzoylecgonine 0 0.81

1 + Ethylcocaine 0 1.00

2 - Ecgonine methyl ester 0 0

“This tab le shows the effect of 40 mM NaF and 0 .5 m M eserine on the enzymatic m etabolism of 24.5 LM c oc ain e by esterase 1 and esterase 2 . The

eflects of inhib itors are expressed relative to an incubation of enzyme and cocaine (activ ity = 1.0). The activity of esterase I was exam ined in both the

absence and presence of 40 mM ethanol. The activity of esterase 2 was exam ined in the absence of ethanol. R eaction conditions and postreaction sam ple

handling and analysis are described under M aterials and M ethods. Enzymatic formation of benzoylecgonine was calcu lated as the am ount measured m inus

the amount formed by spontaneous hydro lysis in the butler control. Enzym atic activ ity w ith inh ibito rs is expressed relative to the activity observed with

no inhibitor.



Abundance

240000

160000

120000

00000

40000-

317

M a. . C 6a ,g .

27 2

240

ciAbundance

240000

200000

160000

13.2 13.4 13.6 13.0 14.0 14.2 14.4 14.6 14.0

Tln,e (Inin.)

19 4

Abundance

180000

160000

140000

120000

100000

80000.

40000

(82

lOS

: .ILL io 213 244

80 150 160 200 240

Mess/Charge

317

272

28 0 32 0

(A )

ILlC.)

z4

0(6 )m4

(B )

4

0 10 20 0 10 20

MINUTES

2738 Vol. 5 September 1991 T he F AS EB Jo urn al DEAN ET AL .

legal intoxication lim it in most states) to the incubation of

24.5 eM cocaine w ith esterase 1 resulted in the formation of a

new metabolite, benzoyleegonine ethyl ester or ethylcocaine.

This new metabolite was identified by both HPLC and

GC/MS. W e have also demonstrated ethylcocaine formation

in crude human liver homogenates from 24.5 sM cocaine

and ethanol concentrations ranging from 8 to 67 mM .

Figure 3 and Fig. 4 show the high-pressure liquid and gas

chromatographie separations of cocaine, benzoylecgonine,

an d t he n ew m et ab ol it e, e th yl co ca in e, p ro du ce d b y i nc ub a-

tion of cocaine and ethanol with esterase 1. H igh-pressure li-quid chrom atography and GC /MS analysis of this new

metabolite revealed abso lute retention times and reten tion

tim es relative to in ternal standards consisten t w ith that of

ethyleocaine standard . Furthermore, mass spectral analysis

of this reaction product revealed ions (Fig. 4) and ion ratios

identical w ith ethyleocaine standards. In the presence of

ethanol, 36% of the cocaine added to esterase 1 was con-

verted to ethyleocaine (Table 1). A concurrent, 38% decrease

in benzoylecgonine form ation suggests that a single enzyme

catalyzes both reactions (Table 1). M oreover, the esterase in-

hibitor NaF completely inhibited form ation of both ethylco-

caine and benzoyleegonine. The esterase inhibitor eserine

(physostigm ine) had no effect on ethylcocaine form ation (Ta-

ble 2), which is consistent with the observation of Hearn et

a l. ( 9). Based on sim ilar observations, Hearn et al. concluded

that the ethyl transesterification of cocaine to ethyleocaine is

no t catalyzed by an esterase (9). However, our studies show

that the lack of inhib ition by eserine is not sufficient to ex-

cluded esterase catalyzed ethy l transesterification of cocaine.

W e suggest that ethylcocaine is synthesized by ethyl trans-

esterification of cocaine in human liver and that this reaction

is indeed catalyzed by an esterase.

In an incubation of ethy leocaine w ith esterase I for 3 h at

37#{176}C,5% of added ethylcocaine was hydrolyzed, producing

benzoyleegonine. This rate of ethylcocaine hydrolysis was

7.6-fold that produced by spontaneous hydrolysis in buffer

alone. Addition of ethanol to the incubation of esterase 2

with cocaine did not substantially alter ecgonine methyl ester

form ation or produce ethylcocaine (Table 1). Incubation ofc oc ai ne a nd e th an ol w it h b uf fe r o r e st er as e 2 a nd i nc ub at io n

of esterase I with benzoylecgonine and ethanol for 3 h at

37#{176}Croduced no measurable ethylcocaine (Table I). Thus,

ethylcocaine is not form ed by a spontaneous chem ical reac-

Figure 3. Identification of cocaine

metabolites by HPLC. The HPLC

chromatogram (A ) shows the sepa-

ration of the in ternal standard,

propylbenzoyleegonine (4), from

benzoylecgonine (1) and cocaine (2)

recovered after incubation of 24.5

IL M cocaine w ith esterase 1. The

4 H PL C chrom atogram (B ) shows

the separation of propylbenzoylec-

gonine (4 ) fr om b en zo yle cg on in e

(1), cocaine (2), and ethylcocaine

(3) recovered after incubation of

24.5 LM cocaine w ith esterase 1 in

the presence of 40 mM ethanol.

The HPLC assay does not detect

ecgonine m ethy l ester. The abso-

lute retention tim es and retention

times relative to the in ternal stan-

__________ dard for the com pounds detected

were consisten t w ith those of ben-

zoylecgonine, cocaine, and ethylco-

c ain e s ta nd ar ds .

F igure 4 . Iden tification of cocaine metabolites by GC /MS . The

GC/M S total ion chromatogram (A ) shows separation of cocaine

(1), ethylcocaine (2), and benzoylecgonine (3) standards prepared in

p lasmanate pretreated w ith trichloroacetic acid and extracted and

analyzed as described under Methods. The m ass spectrum of the

ethylcocaine standard (peak 2, A) is shown in panel B. The G C/M S

to tal ion chromatogram in panel C show s cocaine (1), ethy lcocaine

(2), and benzoylecgonine (3) recovered after incubation of esterase

I with 24 .5 tM cocaine and 40 mM ethanol. The mass spectrum of

ethylcocaine produced in this incubation (peak 2 , panel C) is shown

in panel D . The absolu te retention tim es and retention times rela-

tive to the in ternal standard for the com pounds recovered from the

incubation were consistent w ith those of benzoylecgonine, cocaine,

and ethy lcocaine standards. In addition , the ions and ion ratios ob-

tained by mass spectral analysis of the reaction products recovered

after incubation of esterase 1 w ith 24.5 tM cocaine and 40 m M

ethanol were consisten t w ith those of benzoylecgonine, cocaine, and

ethy lcocaine standards. The reaction catalyzed by esterase 1 does

not produce ecgonine m ethy l ester. Ecgonine m ethyl ester was

form ed in incubations of esterase 2 with cocaine and resolved com-

pletely from cocaine, ethy lcocaine, and benzoylecgonine in th is

assay.

tion, esterase 2, or by direct enzymatic esterification of ben-

zoylecgonine.

Enzym atic ethyl transesterification of cocaine is consistent

w ith the reported detection of ethy lcocaine in plasm a, liver,

brain, an d urine of indiv iduals using cocaine and ethanol

concurrently (8-Il). D etection of other ethylated metabo-

lites , ecgonine ethyl ester, 3’-hydroxybenzoylecgonine ethy l

ester and 4-hydroxybenzoylecgonine ethyl ester, in urine of

individuals exposed to both cocaine and ethanol (ii) lend fur-

ther support to the presence of human enzymes catalyzing

the ethyl transesterifleation reaction.


http://slidepdf.com/reader/full/kokain-esteraze 5/5C OC AIN E E ST ER AS ES 2739

The identification of ethylcocaine requires careful atten-

tion to experimental procedures. Ethylcocaine cannot be

adequately resolved from cocaine by thin-layer chromatogra-

phy (22). In our study , ethyleocaine was iden tified by both

HPLC (Fig . 3) and GC/MS (Fig. 4). E thylation procedures

used to convert benzoyleegonine and eegonine methyl ester

to vo latile , nonpolar derivatives for GC analysis (27) cannot

be used because they would confound the identification of

ethylcocaine. M oreover, cocaine and ethy leocaine must not

be exposed to high pH or o ther conditions causing spontane-

ous hydro lysis . The solid-phase extraction m ethods usedhere do not contribute to artifactual hydro lysis of cocaine or

ethyleocaine. Precautions are also required to identify en-

zymatic hydrolysis of cocaine. For eam ple, the use of concen-

trated ethanol to d isso lve radiolabeled cocaine probably in-

hibited the enzymatic hydrolysis of cocaine to

benzoyleegonine in one study (22).

Recent reports show that ethy leocaine is equipoten t to co-

caine in stimulating motor activ ity in m ice (12) and inh ibit-

ing binding of dopam ine uptake b lockers (8). A s w ith co-

caine, system ic adm inistration of ethylcocaine increases the

ex tracellular concentrations of dopam ine in the nucleus ac-

eumbens of the rat (8). Thus, the central stim ulant activity

of ethylcocaine appears to be mediated through the same

mechanisms as that of cocaine. Our study shows that

ethanol-induced form ation of the active metabolite, ethylco-

caine, is accompanied by dim inished inactivation of cocaine

to benzoylecgonine. If this occurs in vivo, coadministration

of cocaine and ethanol m ight prolong or enhance the central

stim ulant activity of cocaine, and thus reinforce the coabuse

of these two drugs.

Sudden death after cocaine adm inistration has been at-

tribu ted to cocaine eard iotoxicity (28). A recen t report sug-

gests that at h igh concen trations cocaine binds to m usearinic

eholinergie receptors, functioning like the competitive an-

tagonist, atropine (29). This atropine-like effect could

produce unopposed adrenergie stim ulation of the heart, dra-

matical ly elevating heart rate and thus increasing the risk of

cardiac arrhythm ia and death (29). Coadm inistration of co-

caine and ethanol increases heart rate three- to fivefold thatobserved w ith either drug alone (6). The mechanism for this

effect is not known, but a prelim inary report shows that

ethylcocaine has a relatively h igh affin ity for m usearinie

recep tors (30). Additional stud ies are needed to assess

whether coabuse of ethanol and cocaine represents a greater

cardiotoxic risk than exposure to cocaine alone.

Supported by gran t P50-AA07611 . The authors thank D rs. T.-K .

L i, David Henry , and Edw in Harper for suggestions and Kwabena

Owusu-Dekyi, M onica Brzezinski, and Nati Dum aual for as-

sistance with experim ents .

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