Ach Esterase Estimation

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COLORIMETRIC ESTIMATION AND HISTOCHEMICALDEMONSTRATION OF SERUM CHOLINESTERASE*

BY HERBERT A. RAVIN,t KWAN-CHUNG TSOUJ ANDARNOLD M. SELIGMAN(From the Yamins Laboratory for Surgical Research,eth Israel Hospital, and theDepartment of Surgery, Harvard Medical Scho ol, Boston,

Massachusetts)(Received for publication, April 9, 1951)

The presence of enzymes in mammalian blood which hydrolyze acetyl-choline was first suspected by Dale (1)) and later demonstrated by Plattner(2) and by Engelhart and Loewi (3). A serum cholinesterase isolated byStedman et al. (4) from horse blood was first thought to be the same asthe cholinesterase n the central nervous system. Distinct differences be-tween the cholinesterase of erythrocytes and of serum and species differ-ences n serum cholinesterase and in the cell-serum partition of total cho-linesterase activity have been demonstrated (5, 6).

Two fundamentally different types of cholinesterase are now recognizedand are differentiated primarily with respect to their action on varioussubstrates. Acetylcholinesterase of the central nervous system (and oferythrocytes) hydrolyzes acetylcholine faster than any other ester. It alsoattacks propionylcholine and acetyl+methylcholine, but not benzoylcho-line or butyrylcholine. It exhibits optimal activity with a substrate con-centration of 2 to 3 X 10e3M and is moderately inhibited at higher con-centrations of the substrate.Serum cholinesterase also attacks acetylcholine faster than any otherester. It also attacks propionylcholine, benzoylcholine, and butyrylcho-line, but not acetyl-/%methylcholine. Its activity is not significantly in-fluenced by substrate concentration. Central nervous system cholines-terase is said not to attack non-choline esters,2 such as tributyrin, while* This investigation was supported by a research grant from the National CancerInstitute, National Institutes of Health, United States Public Health Service, andin part by an institutional grant to Harvard University from the American CancerSociety.i Research Fellow of the National Cancer Institute.$ Research Fellow in Chemistry, Harvard University.1 Serum cholinesterase is synonymous with non-specific cholinesterase, pseudo-cholinesterase, or type II cholinesterase. Central nervous system cholinesteraseis synonymous with specific cholinesterase, acetylcholinesterase, true cholines-terase, or type I cholinesterase.a On the other hand, unpublished observations in this laboratory indicate thatfresh rat brain (reported by Mendel and Rudney (8) to contain only acetylcholin-

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844 SERUM CHOLINESTERASEserum choline&erase does attack such simple esters at a significant rate(7, 8).In the course of studies designed to develop a method for the histo-chemical demonstration of acetylcholinesterase, chromogenic substrateswere developed which were highly specific for serum cholinesterase. Thesesubstrates, P-carbonaphthoxycholine iodide (III) and the 6-bromo ana-logue, are homologous to carbonaphthoxy-m-phenylalanine, employed inthe calorimetric estimation of carboxypeptidase activity (10, ll), and tobenzoylcholine, which is susceptible to attack by serum cholinesterase,but not by the acetylcholinesterase of the central nervous system. Theester linkage between choline and a carboxylic acid is split to releaseP-naphthylcarbonic acid, which spontaneously and immediately decarboxy-lates to yield p-naphthol. The latter is coupled with a suitable diazoniumsalt to produce a highly colored, insoluble azo dye, which precipitates ator near the site of enzymatic activity in histochemical preparations, andwhich can be extracted from homogenates or serum into organic solventsfor the quantitative estimation of enzyme activity. Calorimetric methodsbased on the production of p-naphthol have been described for esterase(12)) lipase (12), acid and alkaline phosphatase (13), and carboxypepti-dase (11).

It is the purpose of this paper to describe the synthesis of the substrates,report the evidence for their specificity, present the method for the color-imetric estimation of the enzyme, establish the range of normal values,and give the method for demonstrating the enzyme histochemically.

Synthesis of Substrates3P-Carbonaphthoxy chloride (I) and 6-bromo-2-carbonaphthoxy chloridewere prepared, according to the method of Einhorn and Rothlauf for I(14), by addition of a benzene solution of 2-naphthol and 6-bromo-2-naph-

esterase) is capab le of hydrolyzing the simp le esters, &naphthyl acetate, S-acet-amino-2-naphthyl acetate, and 2-acetoxy-6-naphthoic acid phenyl methyl amide.The se observations wil l form the bas is of a histoc hem ical method for demonstratingacetylcholinesteraseinnervous tissue. 2-Acetoxy-3-dimethylaminonaphthalenemeth-iodide, which was synthesized (9) in the hope that it would be spe cifically hydro-lyzed by acetylcholinesterase , was subsequen tly found to be totally resistant toenzymatic hydrolysis, although spontaneous hydrolysis was rapid. The blue azodye formed from the hydrolysis product with tetrazotized diorthoanisidine wasbas ic and proved to be a powerful nuclear stain. Th is nuc leop hilic property ofpigmen ts with quaternary amino groups makes imp ossib le the interpretation ofhistoc hem ical localization of the enzyme when the chromoge nic moiety of substratesthat are attacked by cholinesteras e contain s choline .

3 The substrates and other reagents used in this report may be obtained from theDa jac Labo ratories, Monomer-Polymer, Inc., 511 Lancaster Avenue, Leominster,Massachusetts.

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 845

thol (15), 0.1 mole, respectively, to an equimolar benzene solution of phos-gene (20 per cent) in the presence of an excess of quinoline. The reactionmixture, after standing in a refrigerator overnight, was decanted fromsolids and washed with 1 per cent hydrochloric acid-ice mixture, 2 per

cocL2 ~~CH$&C&I

I\ OCOOCH2CH2(C&

co /II

1Cd

CHOLINESTERASE / \al I

OCOOCH2CH@(CH&\ /

cent sodium hydroxide-ice solution, and water. The benzene solution,dried over anhydrous calcium sulfate, was evaporated to dryness underreduced pressure to give the crude acid chlorides in 75 and 68 per centyield, respectively. Carbonaphthoxy chloride (I) was purified by vacuumdistillation under nitrogen, b.p. 119, 1 mm., m.p. 66-67 (Einhorn andRothlauf (14), m.p. 65-66). The bromo derivative was purified by re-crystallization from ligroin, m.p. 101-102.

Analysis-C11HGBrClOz. Calculated . C 46.27, H 2.11Found. 46.31, 2.40

P-Dimethylaminoethyl-2-naphthoxycarbonate (II) and p-Dimethylamino-ethyl-6-bromo-2-naphthoxycarbonate-Each acid chloride (0.01 mole) in 20cc. of dry benzene was added, with stirring, to a dry benzene solution ofP-dimethylaminoethanol (0.02 mole). The reaction mixture, after stand-ing at room temperature overnight, was removed from the precipitated&dimethylaminoethanol hydrochloride. The benzene filtrate was washedwith water and saturated sodium chloride solution and then dried overanhydrous sodium sulfate. The dried solution was evaporated under re-duced pressure to give II, a pale yellow oil, in 80 per cent yield. In thecase of the 6-bromo derivative, which solidified on long standing (m.p.43-45), an 82 per cent yield was obtained. The latter was purified byrecrystallization from ether and petroleum ether, m.p. 4445.

AnaZysis-ClsH1QN03B r. Calculated. C 53.27, H 4.77, N 4.14Found. 53.44, 4.59, 4.25

Treatment of II and the bromo derivative with HBr yielded the corre-

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846 SERUM CHOLINESTERASEsponding hydrobromides, m.p. 153-153.5 (decomposition) and m.p. 170-171 (decomposition), respectively.

An~Zysis-C~~HlsNOoBr. Calculated. C 52.94, H 5.33Found. 52.70, 5.45GaHdWrBre. Calculated, C 43.03, H 4.09; found, C 42.90, H 4.27,&Carbonaphthoxycholine Iodide (III) and 6-Bromo-%carbonaphthoxycho-line Iodide-A solution of II or the 6-bromo derivative (0.05 mole) in 25cc. of dry benzene was treated with methyl iodide (0.05 mole) at 0 and

allowed to stand at room temperature for 24 hours. The product wasobtained in a yield of 85 per cent for III, m.p. 134-136 (decomposition),and in a yield of 83 per cent for the bromo derivative, m.p. 179-180(decomposition).Analysis-ClsH~oNOs1. Calculated. C 47.89, H 5.02, N 3.73Found. 48.00, I 4.87, 3.73CleHr9NOzBrI. Calculated. C 40.02, H 3.99, N 2.92Found. 39.73, 4.20, 3.27

Calorimetric Estimation of CholinesteraseActivityReagent&--1. Carbonaphthoxycholine iodide (III). A stock solution of carbonaph-thoxycholine iodide was prepared in a concentration of 21.6 mg. per 100cc. (3 X 1O-4M) by solution in 5 cc. of acetone, followed by dilution to100 cc. with distilled water. The pH of this solution was below 7.0; itcould be stored for periods up to 1 week at 4 without any significant

hydrolysis.2. Verona1 buffer (0.1 M) pH 7.4. (a) A stock solution of sodium di-ethyl barbiturate (0.1 M) was prepared by dissolving 20.6 gm. of the so-dium salt in 1 liter of distilled water. (b) A stock solution of hydrochloricacid (0.1 M) was prepared by dilution of concentrated hydrochloric acid160 times. (c) Shortly before use, the desired amount of buffer was pre-pared by mixing the stock solutions (a) and (5) in a ratio of 27.5 cc. ofsodium diethyl barbiturate solution to 20 cc. of the dilute hydrochloricacid solution. The pH of the final buffer solution approximated 7.4 (~1~0.05).

3. Tetrazotized diorthoanisidine4 (2 mg. per cc.) was dissolved in coolwater immediately before use.4. Trichloroacetic acid solution (80 per cent).5. Anhydrous ethyl acetate.4 Available in powder form containing 20 per cent tetrazotieed diorthoanisidine,5 per cent zinc chloride, and 20 per cent aluminum sulfate; trade name duPont naphthanil diazo blue B.

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 847Procedure

Serum (0.1 cc.) was diluted with 16 cc. of distilled water (final concen-tration 0.00625 cc. of serum per cc. of solution).

Just prior to use, the stock substrate solution was mixed with an equalvolume of freshly prepared 0.1 M Verona1 buffer. 5 cc. of the bufferedsubstrate solution were then added to 1 cc. of the diluted serum in a 20 CC.test-tube and mixed. A test-tube containing only 5 cc. of the buffer-substrate solution was prepared as a control for non-enzymatic hydrolysis.All determinations were performed in duplicate. To avoid errors in timeof incubation, serum and substrate were not allowed to stand at roomtemperature for longer than 5 minutes. The tubes were then incubatedat 37 for 1 hour. After incubation, 1 cc. of a solution of tetrazotizeddiorthoanisidine* was added to each tube, and the tubes were gently shakento permit complete mixing. 3 minutes were then allowed for the comple-tion of the coupling reaction, and 1 cc. of 80 per cent trichloroacetic acidwas added to each tube to stop all hydrolysis, spontaneous and enzymatic,and to facili tate extraction of the dye.

The azo dye was extracted from the aqueous trichloroacetic acid-proteinphase by shaking vigorously with 10 cc. of ethyl acetate. The mixtureof ethyl acetate and water was allowed to separate, and any emulsionformed was readily broken by centrifugation at 2500 r.p.m. for 10 minutes.The ethyl acetate layer, containing the extracted azo dye, was transferredwith a pipette to a Klett tube, and the color density measured in a photo-electric calorimeter with a green filter (540 mr.L).

The readings were converted to mg. of /I-naphthol with a calibrationcurve prepared from ,&naphthol in the presence of serum, according to thesteps of the procedure outlined above. The visible absorption spectrumof this dye and the linear calibration curve obtained with 8-naphthol havebeen published (12). A calibration curve falling within the range of ac-curacy of the calorimeter may be obtained with 0.01 to 0.08 mg. of p-naphthol.

Definition of Unit-l unit of serum cholinesterase activity is defmedas that amount of enzyme which liberates 10 mg. of ,f3-naphthol per hourfrom carbonaphthoxycholine iodide under the standard conditions of thetest (pH 7.4, temperature 37, substrate concentration 1.2 X lo-* M).

1 unit of total esterase activity is defined as that amount of enzymewhich liberates the color equivalent of 10 mg. of P-naphthol per hour from/3-naphthyl acetate under the standard conditions of the test.

Culcu&ztction---The number of units of total esterase or of serum cholin-esterase activity per 100 cc. of serum was obtained by multiplying by1600 the number of mg. of ,&naphthol released in 1 hour from p-naphthyl

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848 SERUM CHOLINESTERASEacetate or from carbonaphthoxycholine iodide, respectively, when 0.00625cc. (1 cc. of a 1: 160 dilution) of serum was used.

SpeciJicity of SubstrateThe proof of the specificity of this substrate for the demonstration of

serum cholinesterase activity was based on simultaneous observations ofthe effect of homogenates of rat brain, rat liver, human serum, humanwashed red cell membranes, and a highly purified preparation of serumcholinesterase (human plasma, Fraction IV-6-35) on the rates of hydroly-sis of ,B-naphthyl acetate6 (16) and carbonaphthoxycholine iodide (III),and on the observed inhibitory effect of low concentrations of physostig-mine on the enzymatic activity of these preparations toward both sub-strates. Physostigmine salicylate was added to the serum, and the mixturewas incubated at room temperature for 1 hour before adding the bufferedsubstrate solution. This period of preincubation with physostigmine wasnecessary to allow the reaction of enzyme and inhibitor to come to equi-librium, thereby providing a more accurate estimate of residual enzymaticactivity (17).All five of the above preparations attacked ,&naphthyl acetate in thefollowing order of decreasing activity: liver, serum cholinesterase, humanserum, rat brain, human red cell membranes. The activity of brain ho-mogenate, serum cholinesterase, and red cell membranes toward /3-naph-thy1 acetate was inhibited 100 per cent, human serum was inhibited only40 to 60 per cent, and the activity of rat liver homogenate was entirelyunaffected. It is claimed that only cholinesterase, of the entire familyof esterases, is inhibited by such low concentrations of physostigmine (18).This observation was confirmed by comparison of the inhibition of theenzymatic activity of liver homogenate and purified human plasma cho-linesterase5 toward P-naphthyl acetate by increasing concentrations ofphysostigmine salicylate (Table I). A homogenate of rat liver did notcontain a physostigmine-sensitive esterase (cholinesterase), whereas ho-mogenates of mouse liver and human liver did demonstrate such an en-zyme.

Only serum cholinesterase and human serum (of the series noted above)6 Serum c holinesteras e was provided through the courtesy of Dr. Edw in J. Cohn,

University Laboratory of Phy sical Chemistry Related to Medicine and Pu blicHea lth, Harvard University.

6 A stock solution of fi-naphthyl acetate was prepared in a concentration of 10mg. per 100 cc. (3 X 1OW M) by solution in 5 cc. of acetone, followed by dilution to100 cc. with distille d water. The pH of this solution was below 7.0; it couldbe stored for 1 month at room temperature (22) withou t any sig nific an t hydrolys is.The substrate in this concentration precipitated at 4, but could be redissolved with-out signific ant hydrolysis on warming. The determination of esterase activity inserum was performed with this stock solution exactly as described for cholinesteras e.

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 849attacked carbonaphthoxycholine iodide, the former at a much greater ratethan the latter, per mg. of protein N concentration, thus demonstratingthat carbonaphthoxycholine is not attacked by other aliesterases (7) orby acetylcholinesterase of the central nervous system. Homogenates ofmouse and human liver, in contrast to rat liver, attacked carbonaphthoxy-choline iodide.

The activity of human serum toward carbonaphthoxycholine was almostcompletely abolished by physostigmine salicylate in a concentration of 2 yper cc. When the concentration of purified plasma cholinesterase wasdecreased so that the activity of the aqueous solution was roughly equiva-lent to that of human serum in the amounts used in the standard methodoutlined above (0.00625 cc.), there was a corresponding degree of inhibi-

TABLE IComparison of Inhibition of Physos tigmine on Serum Choline&erase and

Esterases of Liver

Enzyme source

Human serum*cholinesterase

Rat liverhomogenate

I hysostignksalicylate

y per CC.0.01.02.04.00.04.0

:E

_-

-

P-Naphthyl acetate:sterase activitymg. naphtholmg. enzymeper hr.

Inhibition

10.440.380.060.00

15.315.6

per cent0.0

96.399.3

100.00.00.0

I Carbonaphthoxycholine iodideE terase activitymg. naphtholmg. enzymeper hr.

20.077.055.243.850.00.0

-

Inhibition

per cmt0.0

65.074.081.0

0.00.0

* See foot-note 5.tion of cholinesterase activity for carbonaphthoxycholine by the same con-centration of physostigmine (Table II). The degree of inhibit ion of theenzymatic activity of serum toward fl-naphthyl acetate by physostigminesuggested that 60 per cent of the esterase activity of human serum is dueto cholinesterase (Table II).

When a solution of purifled serum cholinesterase, having 40 times theactivity of the preparation of diluted serum used in the routine measure-ments, was tested against equimolar concentrations of /3-naphthyl acetateand carbonaphthoxycholine iodide (1.2 X lo-* M), the ,9-naphthyl acetatewas hydrolyzed at only one-half the rate of the latter substrate (Table I).At this level of enzyme concentration, the activity of the enzyme towardP-naphthyl acetate was almost completely abolished by physostigmine ina concentration of 2 y per cc., while activity toward carbonaphthoxycho-line was reduced only 74 per cent. The reduction in cholinesterase activitywas only 81 per cent with 4 y per cc. of physostigmine (Table I). Since

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850 SERUM CHOLINESTERASEthe inhibition of cholinesterase by physostigmine is known to be competi-tive (16), the increased resistance of the enzyme toward physostigmine inthe presence of carbonaphthoxycholine, as contrasted to ,&naphthyl ace-tate, demonstrates the greater affinity of carbonaphthoxycholine for theactive enzyme centers.

TABLE IIComparison of Inhibition of Physostigmine on Serum Choline&erase and

Esterases of SerumSub strate 1.2 X 10-r Y

@-Naphthyl acetateCarbonaphthoxycholineiodide

Serum* cholin-&erase activitymg. najV$h ol fieg

0.0150.023

Inhibition byphysostigmine,2 y per cc.per cent

9893

S&n .&eraseactivitym g. na~3tho2 fe,

0.0200.008

Inhibition byphysostigmine,2 y per cc.per cent

5896

* See foot-note 5. Serum cholinesterase was diluted until its activity was com-parable to 0.00625 cc. of serum.SERUM ESTERASE

I

25 : _15 65 75 152 45gg 35; 30x 25b 40 I20l-z 150B IO51 SERUM CHOLINESTERASEI 1 I55 65UNITS PER 100 CC. UNITS PER 100 CC.

FIG. 1. Distribution of total esterase and cholinesterase activity in 50 normal seraas determined by calorimetric methods.Choline&eraseActivity of Normal Serum

The sera of 50 normal adults contained an average of 45 units of totalesterase activity per 100 cc., with a range of 28 to 62 units per 100 cc.The sera of these same normal adults contained an average of 29 units ofcholinesterase activity per 100 cc., with a range of 18 to 46 units per 100 cc.(Fig. 1). Repeated observations of cholinesterase activity in three normal

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 851subjects for 1 to 2 weeks showed only slight fluctuation in enzymaticactivity in each.The concentration of cholinesterase in units per 100 cc. of serum com-prised 40 per cent to 60 per cent of the total esterase activity, measuredsimultaneously and expressed in comparable units. However, since twodifferent substrates, with different rates of hydrolysis by cholinesterase,were used, this fractionation should be considered only approximate. Inal l cases physostigmine salicylate in a concentration of 2 y per cc. com-pletely abolished cholinesterase activity as measured by the enzymatic

TIME IN HOURSFIQ. 2

600-

CC. OF SERUMFIG 3

FIG. 2. Hydrolysis of fi-naphthyl acetate by serum ester&se and hydrolysis of@-carbonaphthoxycholine iodide by serum cholinesterase plotted as a function oftime.FIG. 3. Total esterase and cholinesterase activ ity of normal human serum plottedas a function of serum concentration.

hydrolysis of carbonaphthoxycholine iodide. Under the same conditionsof testing, physostigmine salicylate produced a 60 to 80 per cent fall intotal esterase activity, as measured by the enzymatic hydrolysis of p-naphthyl acetate.The course of hydrolysis of carbonaphthoxycholine iodide by serum fol-lowed zero order kinetics over a period of 4 hours. The hydrolysis of p-naphthyl acetate by serum followed first order kinetics over the sameperiod of time (Fig. 2).Sera were tested at several dilutions over a range of an 8-fold increasein concentration (from 0.00625 to 0.05 cc.) with respect to both totalesterase and cholinesterase activity. Cholinesterase activity per 100 cc.of serum was unaltered over this entire range of concentration. In marked

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852 SERUM CHOLINESTERASEcontrast, total esterase per 100 cc. of serum decreased rapidly as the amountof serum employed in the measurement was increased above 0.025 cc.In the range of concentration of serum from 0.00625 to 0.0125 cc. per tube,there was no significant change in the activity of serum with respect tototal esterase (Fig. 3).

Sera with abnormally low cholinesterase activity could be incubatedfor a longer period of time (up to 4 hours), or a larger amount of serum(up to 0.1 cc.) could be used, without introducing any special correctionfactors into the calculation of unit activity. In such cases it was advis-able to select the larger specimen of serum, because of the larger blankcorrection due to spontaneous hydrolysis when prolonged incubation wasused. When more than 0.1 cc. of serum was used, a slight error wasintroduced, owing to the binding of the azo dye to serum protein. If,however, standard calibration curves were prepared in the presence ofsimilar amounts of serum, this error could be largely obviated (13).

Sera with abnormally low total esterase activity could be incubated upto 2 hours, or a larger sample of serum could be used, in order to obtaina reasonable amount of ,&naphthol for accurate measurement. Becauseof the first order nature of the enzymatic hydrolysis of ,&naphthyl acetateby serum it was generally simpler, in this circumstance, to use a largerspecimen of serum, in order to avoid the factor of retardation of enzymaticactivity as the reaction proceeded. In actual practice it was seldom neces-sary to alter the standard technique for the determination of these en-zymes. The dilution factor for serum employed in the standard methodwas selected in order to provide a color density falling within the rangeof most accurate measurement with a standard photoelectric calorimeter(Klett). In the few cases with too great a color density, the ethyl acetateextract was diluted with 1 to 3 volumes of ethyl acetate.

Serum could be stored at 4 for 1 month without any significant changein either total esterase or cholinesterase activity. Slight to moderate he-molysis did not introduce error into the determination of cholinesterase,since the acetylcholinesterase of erythrocytes did not hydrolyze carbo-naphthoxycholine iodide. Hemolysis did increase total esterase activityowing to the presence in erythrocytes of aliesterases (in the red cell con-tents) and acetylcholinesterase (in the red cel l membranes) of the centralnervous system type, which is able to hydrolyze fi-naphthyl acetate.2

The sera of three patients with cirrhosis of the liver with various degreesof severity of liver impairment were found to have serum cholinesteraseactivity well below the lower limit of the normal (1.4, 7.5, and 8.2 units).

The sera of four patients with myocardial infarction and shock werefound to have low serum cholinesterase activity and low prothrombic ac-tivity. Normal levels of serum cholinesterase activity were reached 4 to7 days later.

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 853Histochemical Demonstration of Cholinesterase

Fresh tissues were cut at 10 p thickness by a rotary microtome in aLinderstrom-Lang freezing cabinet and mounted on glass slides coated witha film of dried 1.0 per cent gelatin solution. Sections prepared in thismanner could be stored in the cold (0) for at least 1 month without anyloss of enzymatic activity. Fixation of tissue (mouse liver) in cold ace-tone for 2 hours, or in 10 per cent neutral formalin for 30 minutes, followedby cold acetone for 2 hours, resulted in a loss of 75 to 80 per cent of serumcholinesterase activity, although non-specific esterase activity remainedunaffected by this short fixation treatment. Formalin fixation was moredestructive of cholinesterase than other hydrolytic enzymes for which his-tochemical techniques have been devised (19).

Either carbonaphthoxycholine iodide or 6-bromocarbonaphthoxycholineiodide could be used for the histochemical demonstration and localizationof cholinesterase. The 6-bromo derivative offered an advantage over car-bonaphthoxycholine in that the bromo azo dye formed in the reactionprovided a more bril liant blue color than the azo dye employed in thecalorimetric estimation of the enzyme. The stabilized salt of diazotizedac-naphthylamine described in the histochemical methods for alkaline phos-phatase (20) and esterase (21) gave a red azo dye. Since the zinc saltwhich is used to stabilize tetrazotized diorthoanisidine in diazo blue B4did not inhibit the activity of this enzyme, the blue azo dye was foundpreferable.Procedure-6-Bromocarbonaphthoxycholine iodide (20 mg.) was dis-solved in 50 cc. of normal saline. Calcium chloride (2 cc. of 1.5 per centstock solution) and 20 mg. of diazo blue B were added and stirred untilthe diazonium salt was completely dissolved, to give a pale yellow solution.Then 50 cc. of 0.1 M Verona1 buffer, pH 7.4, were added and the solutionwas filtered rapidly through a medium pore filter. Tissue sections wereincubated in this buffered substrate solution for 20 to 30 minutes at 37.In order to intensify areas of low enzymatic activity, the slides were trans-ferred to a freshly prepared solution for an additional 20 to 30 minuteperiod of incubation. Permanent sections were washed in tap water andmounted in glycerogel. Slides that were not intended to be permanentwere mounted in clear glycerol saturated with cadmium chloride to pro-vide a high index of refraction.

Serum cholinesterase activity was demonstrated in the muscularis mu-cosae, Meissners plexus, and Auerbachs plexus in the ileum of man, dog,rat, and mouse. There was slight staining of the superficial layers of themucous membrane of the intestine in the dog, but in none of the otherspecies noted above. Sections of the brain of rat, mouse, dog, and manwere uniformly negative for serum cholinesterase activity. Rat liver was

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854 SERUM CHOLINESTERASEnegative for cholinesterase activity, but the liver of the mouse, dog, andman was positive, with moderate accentuation of the stain in the periportalareas. Sections of rat heart were also negative, while the interventricularseptum of dog heart was positive for serum cholinesterase activity.

Staining was entirely cytoplasmic in liver, muscularis mucosae, and doginterventricular septum. Ganglia of Meissners and Auerbachs plexusesoccasionally showed a pun&ate distribution of dye around the cel l in ad-dition to cytoplasmic staining, suggestive of a synaptic network. Nuclearstaining was not observed in any sections. Detailed studies of tissue sec-tions stained by this method will be published elsewhere.

DISCUSSIONCarbonaphthoxycholine iodide (III) and its 6-bromo derivatives are

hydrolyzed only by a cholinesterase of the type found in human plasma orserum. No supplementary tests need be performed to determine which ofthe two major types of cholinesterase is being measured, as is necessarywhen acetylcholine is used as a substrate. In this respect the presentmethod is analogous to methods in which benzoylcholine (22) is used inthe measurement of serum cholinesterase activity. The calorimetricmethod presents advantages inherent in calorimetry over the titrimetric(23), electrometric (24), and manometric methods (25) for estimating cho-linesterase activity.

The substrate employed in this method is readily adapted to the histo-chemical demonstration of the plasma type cholinesterase in tissues, andthe method for the quantitative estimation of the enzyme in serum maybe applied directly to other biologic fluids or tissue homogenates. It is,therefore, possible simultaneously to determine quantitatively and to dem-onstrate histochemically the serum cholinesterase act,ivity of tissues. Be-cause both techniques uti lize the same substrate, there is an advantageover combinations of microtitrimetric methods (22-24) based on the useof acetylcholine or benzoylcholine, with histochemical techniques util izingmyristoylcholine (26) or butyrylthiocholine (27).

The distribution of cholinesterase in tissues demonstrated by the tech-nique reported here is in good agreement with the results obtained by pre-vious methods (26, 27). There is, however, a significant difference in thedetails of cytologic distribution of staining between the azo dye method,the method of Gomori (26), and the copper thiocholine method of Koelle(27) and Koelle and Friedenwald (28). The latter methods show ex-tensive nuclear staining, presumably indicating a nuclear localization ofthe enzyme, whereas the method reported here shows no nuclear stainingat all. Enzymatic activity was demonstrated in the cytoplasm and innervous tissue elements (myenteric plexus). Staining was obtained at the

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 855

periphery of the cell as well, suggesting localization of the enzyme at ornear the cell membrane. A membranous localization of the enzyme, atleast of the axon, is supported by the observations of Boell and Nachman-sohn (29), who demonstrated high concentrations of acetylcholinesterasein the axon sheath of the giant nerve of the squid, and a low concentrationof this enzyme in the axoplasm expressed from the sheath.

The different localization of staining in the copper thiocholine method(27, 28) is due to the fact that copper thiocholine bears a strong positivecharge on the quaternary amine group of thiocholine, in contrast to theabsence of a strong charge on the naphthyl azo dye obtained by enzymaticactivity and coupling. Copper thiocholine is, in addition, more solublethan the azo dye, and therefore, in order to obtain precipitation, the re-action must be conducted in a saturated solution of copper thiocholine.The supersaturated solution produced by enzymatic action, therefore, canresult in precipitation on basophilic structures in nuclei rather than at thesite of enzymatic activity. This does not happen with the azo dye, whichpresumably demonstrates the localization of enzymatic activity more ac-curately.

Similar nuclear affinity for the calcium soaps of long chain fatty acidsmay explain the results with Gomoris methods. Nuclear staining in themethod for alkaline phosphatase has recently been shown to be largely dueto nuclear affinity for calcium phosphate (30,31).

Numerous studies have been carried out on the variations in the con-centration of serum cholinesterase in disease states (see Augustinsson (5)for complete bibliography). Conflicting results have been reported. Ofthe mass of observations made to date, it appears that only liver diseaseis consistently associated with a fall in serum cholinesterase activity andthat this fal l may be correlated with the concentration of serum albumin.The occurrence of low serum cholinesterase activity in liver disease wasconfirmed in a few cases by the calorimetric method. It is claimed in arecent report that serum cholinesterase activity reflects hepatic functionmore accurately than a number of tests currently in use (32). The deter-mination of serum cholinesterase activity has allegedly been abandonedbecause of the marked variability in activity present in normal sera (5).While the entire range of cholinesterase activity in normal sera is broad,80 per cent of normal sera fall within a fairly narrow range of activity.The range of cholinesterase activity of normal sera by the present methodis comparable to that obtained by a titrimetric method (32). Althoughisolated observations of serum cholinesterase activity may not be diag-nostic, serial determinations have been useful in following the course ofliver disease (32).

7 Personal communication from Dr. E. H. Leduc and Dr. E. W. Dempsey.

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856 SERUM CHOLINESTERASEThe simultaneous determination of total esterase activity revealed a

general parallelism between serum esterase and serum cholinesterase ac-tivity. It, was also shown, by the use of selective inhibition of serumcholinesterase with low concentrations of physostigmine, that an appre-ciable fraction of the esterolytic activity of human serum is due to a vari-able amount of a specific enzyme, serum cholinesterase, within the generalclass of carboxylic acid esterases. The identification of a specific andmajor component of serum esterase may be expected to provide a morerational approach to the study of variations in esterase activity associatedwith disease states.

Acknowledgment is due Mr. David V. Berry for technical assistance,to Mrs. Shirley Golden for microanalyses, and to Miss Miriam Schwartzof the Blood Bank, Beth Israel Hospital.

SUMMARY1. The preparation and properties of P-carbonaphthoxycholine iodide, a

chromogenic substrate for serum cholinesterase, and 6-bromo+carbo-naphthoxycholine iodide for the histochemical demonstration of the enzymeare described.

2. The specificity of these substrates for the serum type of cholines-terase is demonstrated.

3. A standardized calorimetric method is outlined for the estimation ofserum cholinesterase acbivity, and the range of normal values establishedfor this method.4. A procedure is described for the histochemical demonstration andlocalization of serum type cholinesterase in tissues.

BIBLIOGRAPHY1. Dale, H. H., J. Pharma col. and Ezp. Therap., 6, 147 (1914).2. Plattner, F., Arch. ges. Physiol., 214, 112 (1926).3. Eng elhart, E., and Loew i, O., Arch. ezp. Path. u. Phurm ukol., 160, 1 (1930).4. Stedman, E., Stedman, E., and Eass on, L. H., Bioche m. J., 26, 2056 (1930).5. Aug ustinsson , K.-B., Acta physiol. Scan &, 16, supp l. 52 (1948).6. Alle s, G. A., and Hawes, R. C., J. Bio l. Chem., 133, 375 (1942).7. Rich ter, D., and Croft, P. G., Bioc hem . J., 36, 746 (1942).8. Mendel, B., and Rudney, H., Bioche m. J., 37, 59 (1943).9. Wo lf, G., Friedman, 0. M., Dickinson, S. J., and Seligm an, A. M., J. Am. Chem.

Sot., 72, 390 (1950).10. Wo lf, G., and Seligma n, A. M., J. Am. Chem. Sot., 73, 2080 (1951).11. Ravin, H. A., and Seligm an, A. M., J. BioZ. Chem., 191, 391 (1951).12. Selig ma n, A. M., and Na chla s, M. M., J . CZin. Invest., 29, 31 (1950).13. Selig ma n, A. M., Chaun cey, H. H., Na chla s, M. M., Manheimer, L. H., and

Ravin, H. A., J. BioZ . Chem ., 190, 7 (1951).14. Einhorn, A., and Rothlauf, L., Ann. Chem., 382, 256 (1911).

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H. A. RAVIN, K.-C. TSO U, AND A. M. SELIGMAN 85715. Koe lsch, F., Org. S yntheses, 20, 18 (1940).16. Nac hlas, M. M., and Seligman, A. M., J. Bio l. Chem., 181, 343 (1949).17. Krayer, O., Goldstein, A., and Plachter, F. L., J. Pharma col. and Exp. T herap.,

80, 8 (1944).18. Tau ber, H., Chem istry and techno logy of enzymes, New York (1949).19. Seligm an, A. M., Chauncey, H. H., and Nach las, M. M., Stain Tech noE., 26, 19

(1951).20. Manheimer, L. H., and Se ligm an, A. M., J. Nat. Cance r Inst., 9, 181 (1949).21. Na chla s, M. M., and Se ligm an, A. M., J. Nat. Cance r Inst., 9, 415 (1949).22. Sawyer, C. H., J. Ezp. Zool., 94, 1 (1943).23. Glick, D., Biochem. J., 31, 521 (1937).24. Glick, D., J. Gen. Ph ysiol., 21, 289 (1938).25. Friend, D. G., and Krayer, O., J. Pha rmac ol. and Exp. Thera p., 71,246 (1941).26. Gomo ri, G., Proc. Sot. Exp. BioZ . and Med., 68, 354 (1948).27. Koe lle, G. B., J. Pharmaco l. and Exp. Therap., 100, 158 (1950).28. Koe lle, G. B., and Friedenwald, J. S., Proc. Sot. Exp. BioZ. and Med., 70, 617

(1949).29. Boe ll, E. J., and Nachm ansohn, D., Scie nce, 92, 513 (1940).30. Novikoff, A. B., Scien ce, 113, 320 (1951).31. Gomori, G., J. Lab. a nd CZin. Med., 37, 526 (1951).32. Vorha us, L. J., II, Scud amo re, H. H., and Kark, R. M., Am . J. Med. SC., 221,

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Ach Esterase Estimation

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