Bull. Org. mond. Sante 1971, 44, 99-107Bull. Wid H/th Org.

Distribution and Determination of Cholinesterasesin MammalsBO HOLMSTEDT I

This paper reviews the distribution of cholinesterases in the central nervous systemn,the ganglia, the striated muscle, and the blood of mammals, and discusses the correlationbetween the histochemical localization and the function of neuronal cholinesterase.

Different methods for the determination of cholinesterase levels are reviewed, withparticular reference to their practical value for field work. The Warburg method and theTintometer and Acholest colorimetric methods are compared on the basis of cholinesteraselevels determined in normal persons and in those suffeering from parathion intoxication.

As with other enzymes, the cholinesterases canbe characterized by the properties of the isolatedprotein, by the substrate specificity (with regardto choline esters and other esters), by the shape ofthe activity-pS curve, and by their reaction withso-called selective inhibitors.

In mammals, two principal groups of cholinester-ases have been distinguished by the use of thesecriteria (Table 1). However, considerable speciesdifferences exist-for example, the central nervoussystem of rats contains a cholinesterase that splitspropionylcholine more rapidly than acetylcholine.The different determinations of cholinesterases thathave been described have mainly been based onknowledge of the substrate specificity.

DISTRIBUTION OF CHOLINESTERASES

The cholinesterases can also be characterized bytheir occurrence and distribution in different organs.Until about 20 years ago, their distribution in tissueswas usually studied manometrically with slices orhomogenates.A serious limitation of this type of study is that

it does not reveal the cells or parts of cells in whichthe activity of the enzyme is localized. Histochemicaltechniques have been used in an attempt to over-come this limitation. These histochemical methodsare primarily of interest to pharmacologists andphysiologists in so far as they can throw light on theproblems of cholinergic function. Thus, the staining

1 Department of Toxicology, Swedish Medical ResearchCouncil, Karolinska Institute, Stockholm, Sweden.

Table 1

Nomenclature of cholinesterases

acetylcholine acetyl-hydrolase acylcholine acyl-hydrolase (3.1.1.8)(3.1.1.7)

acetylcholinesterase cholinesterase

(specific cholinesterase, (butyrylcholinesterase,

true cholinesterase, propionylcholinesterase,

e `-type cholinesterase) nonspecific cholinesterase,

pseudocholinesterase,

" s "-type cholonesterase)

of some cells and the absence of staining in othersmay supplement other sources of information onthe nature of the transmission process at particularsites. For this purpose, detailed localization withincells may be less important than knowledge of whatenzymes or types of enzyme are present. This,of course, does not exclude the need to demon-strate the enzyme at a subcellular level and to mini-mize diffusion artifacts. Histochemical methods havebeen much criticized with respect to the latter factor.

In the ideal histochemical method, the substanceseen on the slide is a product of the naturallyoccurring substrate. None of the methods ofcholinesterase staining now in use complies withthis requirement; the one coming closest to it isthe thiocholine method (in which the enzymesplits thiocholine esters instead of choline esters),especially if the rates of hydrolysis of the thiocholineesters relative to those of their oxygen analogues are

2618 -99-

B. HOLMSTEDT

Fig. 1Histochemical and spectrophotometric methods of determining cholinesterase

0 01i ChE l1

(CH3)3NCH2CH2SCCH3 --(CH3)3NCH2CH2SH + HOCCH3

Histochemical:Thiocholine method(Koelle & Friedenwald, 1949)

Spectrophotometric:Ellman (1 959)

known for the particular material under study(Fig. 1). The method more closely approaches thephysiological situation than do procedures basedon the hydrolysis of naphthyl acetate or indoxylacetate, which up to now have been less convincinglycontrolled biochemically.

Koelle has carried out biochemical control experi-ments with both choline and thiocholine esters assubstrates. The results show that, with few excep-tions (acetyl-p-methyl thiocholine), the thiocholineesters behave like the choline esters towards the twotypes of enzyme.

Because of the interest in the two types of cholin-esterase-acetylcholinesterase (3.1.1.7.) and cholin-esterase (3.1.1.8)-it is clearly important to have a his-tochemical method capable of distinguishing be-tween them. At present, the thiocholine method is theonly one that allows the two types of cholinesteraseto be distinguished histochemically with reasonablecertainty. In the most recent modifications of thetechnique, this has been achieved by combiningselective substrates with highly selective inhibitorsin the same experiment. It is also essential to per-form biochemical control experiments in exactly thesame solution as that used for the histochemicalstudies. Any shift in pH, for example, alters theconcentration of inhibitor necessary to block onetype of enzyme selectively.Numerous papers have been published on the

use of the histochemical technique for the detailedlocalization of cholinesterases. The results havebeen summarized by Koelle (1963, 1970).

Central nervous systemA histochemical survey of the cholinesterases

of the brains of rats and cats has disclosed thepresence of low to high concentrations of acetyl-cholinesterase in most neurones of a large number ofregions.

In addition to acetylcholinesterase, the centralnervous system contains significant amounts of

cholinesterase (3.1.1.8). In contrast to the former,the latter is present in highest concentrations in thewhite fibre tracts and is localized in the glial cells.Among other places it is found around the supraopticnucleus and around the third ventricle in the regionof the paraventricular nucleus. The capillariesand other blood vessels of the brain and spinalcord, like those of most other organs, also containthe enzyme.A histochemical survey of the spinal cord shows

that only the anterior and lateral horns give apositive acetylcholinesterase reaction. The largemotor neurones are heavily stained and the productof the histochemical reaction is clearly visible alongthe neurites. The difference in staining intensityamong various cells is not sufficiently marked topermit the quantitative estimation of cholinesteraseactivity. The spinal cord also contains cholinesterase(3.1.1.8), as noted above.

GangliaThe distribution of cholinesterase in ganglia

is subject to differences of opinion. In early studiesin the cat, high concentrations of acetylcholinester-ase were found in cholinergic neurones (i.e., neuronesgiving rise to cholinergic fibres), but the adrenergicand sensory neurones appeared to be practicallydevoid of the enzyme. A more recent investigationin the cat, the rabbit, and the rhesus monkey demon-strated the presence of acetylcholinesterase in alltypes of peripheral neurone. In cholinergic neurones,the concentration of the enzyme appeared to beconsistently high: in the adrenergic and sensorytypes, concentrations were lower and varied accor-ding to site and species. The neurones of Auerbach'splexus and the associated interstitial cells werefound to contain, in addition, uniformly highconcentrations of cholinesterase (3.1.1.8), whichin other ganglia occurs only in the glial cells.This enzyme was not found in other neurones ofthe species studied, although its occasional presence

100

DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS

in neurones of the superior cervical ganglion of therat has been reported.

Striated muscleConsiderable effort has been devoted to the

localization of cholinesterase in muscles. Studieson tissue homogenates have revealed that striatedmuscle contains approximately 95% of acetyl-cholinesterase and 5% of cholinesterase (3.1.1.8),localized in various places.

Small " classical " end-plates are arranged instraight rows across muscle fibres or in groups.The cholinesterase in the end-plates consists pre-dominantly of acetylcholinesterase, but cholinesterase(3.1.1.8) is also present. Most of the former ispostsynaptic and belongs to the so-called subneuralapparatus; very little is presynaptic. However,it is impossible to make, on the basis of denervationexperiments, a statement about the presynapticand postsynaptic distribution of acetylcholinesterasein nerve-muscle junctions that would be appli-cable to all skeletal muscles.

Other " plates " do exist, composed of parallelgutters arranged as a " cake frill " or " palisade ",the gutters being oriented in the same direction asthe long axis of the muscle fibre.

Staining also occurs in large cap-shaped areassituated over the ends of muscle fibres, with guttersarranged in a parallel or in a somewhat reticularpattern. These endings are, in fact, situated at themuscle-tendon junctions. They are also found wheremuscle fibres end in the middle of the bundle. Itis possible that these may not be motor end-platesbut stretch receptors; if this is so, then the presencein them of acetylcholinesterase is of some interest.Cholinesterase is also present in muscle spindles,both in the end-plates of the intrafusal fibres andin the fibres themselves.On the basis of histochemical studies, some authors

have suggested that cholinesterase is present in theordinary fibres of striated muscles. This has recentlybeen confirmed by other methods, but the natureof the enzyme is unknown. In some studies it mayhave been an artifact produced by inadequatehistochemical technique. In any event. there isoverwhelming evidence that the enzyme contentof the motor end-plates is far greater than that ofany other structure in striated muscles.

Other tissues

Acetylcholinesterase is found in a number ofother tissues, such as the smooth muscles of the

bronchioles and the urinary bladder and the effectorcells of the salivary glands. Cholinesterase (3.1.1.8)is found in the carotid body, the hepatic cells, themuscularis mucosa of the intestinal canal, andthe cortical cells of the zona glomerulosa of theadrenals.

Correlation between histochemical localization andfunction of neuronal cholinesterasePharmacological and enzymological investigations

have repeatedly led to the suggestion that acetyl-cholinesterase is located both inside and outsidethe cell membrane. Such suggestions have beenbased upon comparisons of the effects of tertiaryand quaternary ammonium bases with anticholin-esterase activity. It is known that the quaternarycompounds have a relatively small ability to pene-trate, owing to their insolubility in lipids.

This indication that the total neuronal acetyl-cholinesterase consists of a " functional " portionexternal to a relatively impermeable cellular mem-brane and an internal portion has been elaboratedupon by Koelle and his co-workers. It has beensuggested that the internal or "reserve" portionrepresents the source for the replacement of thefunctional enzyme of the cell surface and couldpossibly be identical with the endoplasmic reticu-lum. Comparison of the distributions of acetyl-cholinesterase in autonomic ganglia indicated thatpractically all the external acetylcholinesterase ofthe sympathetic ganglia is associated with thepresynaptic axonal terminations; most of the cyto-plasmic acetylcholinesterase of the occasionalcholinergic neuronal perikarya is internal to arelatively impermeable membrane. It has been pro-posed that an important function of ganglionicacetylcholinesterase may be protection of thepreganglionic terminations from depolarization andantidromic firing in response to acetylcholine libe-rated by these and adjacent terminations. The phar-macological actions of anticholinesterase agentsprobably result from inactivation of the externalor functional acetylcholinesterase. If this representsonly a small portion of the total acetylcholinesterase,its inactivation (and reactivation) might not bedetectable by the usual techniques.The role of acetylcholine as a neurohumoral trans-

mitter at several synaptic and neuroeffector sites isnow firmly established. Furthermore, in the regionof any junction, acetylcholine may also act at thepresynaptic terminal to effect the liberation ofadditional quanta of acetylcholine (if the neurone is

101

B. HOLMSTEDT

cholinergic) or of other neurohumoral transmitters(if the neurone is non-cholinergic).

It is unlikely that acetylcholine and acetyl-cholinesterase participate directly in the initiationor propagation of induced action potentials; how-ever, this does not exclude the likelihood thatthey are involved in the maintenance of the excitablestate in those fibres in which they are present insignificant concentrations.Apart from its functions in the nervous system,

acetylcholine probably serves as a local hormonein many autonomic effector organs-i.e., it appearsboth to be synthesized by and to act on cardiac andsmooth muscle in the maintenance of excitabilityand rhythm.At several sites where acetylcholinesterase acti-

vity has been demonstrated (e.g., the musculotendi-nous junctions of skeletal muscle) it is not possibleto assign even a tentative function to the enzymeor its substrate. The same may be said for nearlyall the localizations of the nonspecific cholinesterases.Further studies may uncover roles at such sitesthat hitherto have been unsuspected.

Blood cholinesterasesEven the function of the cholinesterases in blood

remains unknown, but both the erythrocyteacetylcholinesterase and the plasma cholinesterase(3.1 .1 .8) serve as very useful indices of the degreeof absorption of anticholinesterase insecticides.The great variation in acetylcholinesterase activity

from one person to another makes it necessary thatthe level in each person who might be occupationallyexposed to cholinesterase inhibitors be determinedbefore such exposure can occur. It also seems ad-visable that workers who from the beginning showonly a feeble enzyme activity should not be per-mitted to work in situations where exposure islikely to occur.

In practical work and field work, the cholinesteraseactivity in the plasma of man can usually be moni-tored. The enzyme is almost always inhibitedbefore (or at the same time as) the red cell cholin-esterase, and only rarely afterwards.The determination of plasma cholinesterase acti-

vity is of value for:(1) the diagnosis of intoxication, including

chronic intoxication;(2) guidance in treatment with antidotes; and(3) determination of the time when an individual

can resume his activities.

It should be borne in mind, however, that verte-brate blood sera generally contain several formsof cholinesterase, and evidence for the electro-phoretic heterogeneity of the enzyme in humanserum has been demonstrated by several workers.As many as six or seven forms are present in certainsera. These isoenzymes have been the subject ofintense study during recent years, not least fromthe pharmacogenetic point of view. The poly-morphism of serum cholinesterase is discussed byGoedde et al. (1967) and by Latner & Skillen(1968). These workers carried out their studiesin Europe and in North America. So far, similarstudies have not been undertaken in developingcountries in the tropics. When large-scale cholin-esterase screening programmes are performed inconnexion with malaria eradication, anomalousplasma values-depending upon the substrate andthe methods used-may indicate the presence ofan atypical plasma cholinesterase. However, erro-neous results may also be caused by impaired liverfunction.

CHOLINESTERASE DETERMINATION

Many methods of cholinesterase determinationhave been suggested and compared, but onlya few of them are appropriate for work in the field.Such methods should be simple and should notrequire electricity or complicated equipment. Indeveloping countries one of the most importantrequirements for a field method is that it be simpleenough to be used by nonspecialists.

Different methods for determining cholinesteraseare reviewed below with particular reference to theirpracticality in field work. The review by Augustins-son (1971) of different methods of determiningcholinesterase levels should be consulted for detailsand references.

"Remaining substrate " methods

The earliest method for determining cholinesteraseconsisted of assaying the acetylcholine remainingafter incubation with the enzyme-containing material(Fig. 2). At first, this was usually carried out bybioassay. Today, other methods are available-the Hestrin method, which is based on colorimetricdetermination of the iron(III) hydroxamate; theSchonemann method, another colorimetric tech-nique, which has been used only on rare occasions;and gas chromatographic determination of de-methylated choline esters. However, the latter

102

DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS

Fig. 2' Remaining substrate" methods and methods based on formation of acid

0

(CH3)3NCH2CH20CCH3I~~~~~~~~~~~,_

" Remaining substrate ":BioassayHestrin methodSchonemann methodGas chromatography

method, which has become available only in recentyears, has not yet been used for enzyme assays.The "remaining substrate" methods suffer from

the disadvantage of not measuring the rate ofdisappearance of the substrate, and in this respectthey are inferior to other means of determiningcholinesterase activity.

Methods based on formation of acid

Methods of this type are also based on the equa-tion shown in Fig. 2. The most commonly usedtechniques are discussed below.

Warburg manometric technique. This method,introduced many years ago, is still one of the mostaccurate and reliable methods available for assayingcholinesterase activity; according to Augustinsson(1971) the accuracy can be within ± 2%. Themethod is useful in a variety of experimental con-ditions, including those with inhibitors and reacti-vators. Rate curves can be obtained. The disad-vantages of the technique are that the pH of themedium cannot be varied and that the rate of hydro-lysis cannot be studied at substrate concentrationsbelow 0.4 mm since insufficient gas is liberated atsuch concentrations. A further limitation is thedifficulty of assaying reversible esterase inhibitors.The results obtained with the Warburg technique

agree fairly satisfactorily with those obtained withthe Hestrin, Michel, titrimetric, and Acholestmethods. For references see Augustinsson (1971).A variation of the Warburg technique that is

suitable for field studies has been used by Augustins-son & Holmstedt (1965). This method, in whichwhole blood is absorbed on filter-paper, dried,and shipped to a laboratory where Warburg deter-minations can be performed, presents great advan-tages. It had previously been carefully investigatedand successfully used for 10 years in the temperatezone (Augustinsson, 1955). The technique is basedon the use of two selective substrates, butyrylcholine

ChE0

(CH3)3NCH2CH20H + HOCCH3

Acid formation:WarburgMichelpH-statAcholest

for plasma cholinesterase and acetyl-p-methylcholinefor erythrocyte acetylcholinesterase.As it had not been shown whether the filter-

paper technique gave reliable results in the conditionsprevailing in the tropics (Witter, 1963), it was decidedto check it. The stability of the enzyme activity ofblood on paper was first studied under variouslaboratory and tropical conditions. It was foundthat the blood samples can be dried in the sun, inthe shade, or in a desiccator with no loss of enzymeactivity. From the data given in Table 2 it is obviousthat cholinesterase activities are highly stable, evenunder seemingly unfavourable conditions. Onlywhen the filter-papers are not allowed to dry com-pletely or are contaminated with moisture afterdrying is there a decrease of enzyme activity.The normal variation of human blood cholinester-

ase activity obtained in this way was determined andthe dispersion was calculated statistically. Theplasma cholinesterase activity of males was foundto be significantly higher than that of females, butno such difference was found in erythrocyte acetyl-cholinesterase activity.

Table 2

Cholinesterase activity in blood samples collected onfilter-paper from various places and mailed to Stockholm

for analysis

Place

Stockholm

Geneva

Dakar

Lagos

Calabar

Manaus

Time intervalbetween sampling

and analysis(days)

13

3

6

9

17

ChE levels (units/ml) in:

plasma R. B.C._I___L _

3.42

3.48

3.45

3.48

3.55

3.60

0.85

0.90

0.87

0.85

0.82

0.75

103

B. HOLMSTEDT

The results conclusively proved the usefulness,even under tropical conditions, of blood samplesdried on filter-paper and mailed to a distant labora-tory for determination of cholinesterase activities.The simplicity and practicality of the filter-papermethod make it ideally suitable for monitoringlevels in spray-men and exposed populations wheninsecticides with anticholinesterase properties areused in malaria eradication and other public healthprogrammes. It also forms an ideal standardwith which values determined by other methods canbe compared.

Michel method. This method, first described in1949, has always been one of the most popularmethods for routine determinations of bloodcholinesterase. The procedures described by Michelhave usually been followed, but many more or lesssignificant modifications have been described. Themethod has often been used in field studies, owingto its relative simplicity.

In this method the enzyme is allowed to acton acetylcholine in a standard buffer solution fora fixed period of time, and the pH of the mixtureis measured at the beginning and at the end of thisinterval. The rate of change of pH (usuallyz pH/h)is a measure of enzymatic activity.

Augustinsson (1971) has pointed out that theabsolute accuracy and the sensitivity of the Michelmethod are not as great as those of the titrimetricand Warburg methods, primarily because in theMichel method the pH, which is a logarithmicfunction of the acid concentration, is measuredrather than the acid production itself. The change ofpH with respect to time is not usually recorded foreach assay unless a recording pH meter is used.This improved equipment, introduced by Tammelinand his co-workers, simultaneously registers sixenzyme reactions and is calibrated to relate the pHof the solution to the rate of addition of aceticacid.The change in pH resulting from the production

of acid in a solution of a choline ester incubatedwith cholinesterase can also be estimated by thechange in colour of an indicator rather than witha pH meter. A number of techniques have beendescribed and a number of indicators have been used,including phenol red, m-nitrophenol, cresol red,bromocresol purple, litmus, and (most commonly)bromothymol blue. Most of these proceduresemploying an acid-base indicator have been usedfor serum or plasma, but some are also available

for whole blood or erythrocytes. The majorityare " wet" methods, in that pre-made solutions areused and the colour change is estimated eithervisually or with a spectrophotometer. Commercialkits are available and some colorimetric methodshave been adapted for use with the Auto Analyzer.

Fig. 3

Comparison of Acholest, Warburg, and Tintometermethods for the determination of cholinesterase *

4-J

z 50-

zw0

wa.

04-J4

z

zw0

wa.

Tintometerwhole blood

DAYS

Acholest

DAYS

* The arrows beneath the x-axes indicate the times at which theantidote (pralidoxime methanesulfonate) was administered.

104

DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS

Acholest method. A " non-wet " method, theAcholest test is particularly suitable for use in thefield. It is a screening test that is easy to perform andrequires a minimum of equipment. A slip of filterpaper is impregnated with the choline ester andbromothymol blue and is subsequently dried. Theserum or plasma is added directly to this paperand the colour change is observed, comparison beingmade with a similar paper on which no cholineester is present. The Acholest technique has beenused in the field by Oudart and Holmstedt (Holm-stedt & Oudart, 1966; Oudart & Holmstedt,1970).The plasma cholinesterase hydrolyses the sub-

strate in the paper, liberating acid, which slowlychanges the colour of the indicator in the paper

from deep blue to yellow-green. The activity ismeasured by the time needed for the colour to matchthat of the control paper: the higher the cholin-esterase activity, the shorter the time. The Acholesttest paper can be used only if the plasma is separatedfrom the red blood cells, and there must be a

complete absence of haemolysis. The difficulty ofcentrifugation in the field can be circumventedby the use of an easily made hand-powered micro-centrifuge developed for this purpose (Holmstedt,1965).

The mean error of the Acholest method has beencalculated, using the Warburg method as a standard,as ±6.5 % for colour-development times of 11-56minutes; when the time is less than 11 minutes themean error is ±16%. In other words, the error islower in the region that is of interest in the diagnosisof cholinesterase inhibition by insecticides.

Comparison of Acholest, Warburg, and Tintometermethods. Oudart & Holmstedt (1970) have comparedthe results given by the Warburg method (usingfilter paper), the Tintometer colorimetric method,and the Acholest method in determinations ofcholinesterase levels in normal people and in thosewith parathion intoxication. The enzyme activi-ties obtained are shown in Fig. 3 as percentagesof the initial cholinesterase activity, making itpossible to compare all methods. The curve ob-tained by using the Acholest test paper correspondsto that obtained by means of the manometricmethod. Both methods show the decrease in cholin-esterase activity at the same time. The effects of theinjections of the antidote are also clearly indicated byboth methods. The curve obtained with the Tinto-meter is much less satisfactory and the decreasein enzyme activity is smaller than could be recordedwith the other two methods.

Fig. 4The Eliman method

+8(1) (CH3)3NCH2CH2S-CCH3 + H20

acetylthiocholine

(2) (CH3)3NCH2CH2S

thiocholine

-(CH3)3NCH2CH2S-S NO2

coo

acetylcholinesterase + +

ON (CH3)3NCH2CH2S + CH3COO + 2H

thiocholineif

+ 02N S-S NO2

coo coo

5-dithiobis-2-nitrobenzoic acid( DTNB ) III

+ 02N S

coo

5 - thio - 2 - nitrobenzoic acid( Xmax = 412 nm)

WIO 10231

105

106 B. HOLMSTEDT

Titrimetric determination. The acid liberated duringthe hydrolysis of the ester can be determined bytitration with standard alkali at constant pH usingeither an indicator or a potentiometer. The auto-mated pH-stat method is one of the most convenientand precise of the methods available for assayingcholinesterase. Its great advantage is that the acidliberation can be continuously recorded. A recentcomparison of the pH-stat and the Michel methodsled to the conclusion that the former offers bettercontrol of the assay conditions, higher sensitivity,and better reproducibility. Its complexity, however,makes it less suitable for field work.

Ellmani method. The Ellman method was origi-nally proposed in 1959 for the determination ofsulfhydryl groups. It was later modified for thedetermination of cholinesterase activity in wholeblood, erythrocytes, and tissue homogenates (Fig.1 and 4). The thiocholine (II) formed during thehydrolysis of acetylthiocholine (I) reacts rapidlyand irreversibly with 5-dithiobis-2-nitrobenzoic acid,releasing a coloured product, 5-thio-2-nitrobenzoicacid (III). Cholinesterase activity may be determinedspectrophotometrically by following the productionof 5-thio-2-nitrobenzoic acid.

In contrast to other colorimetric methods, theElIman method is based on the measurement of a

reaction product instead of the remaining intactsubstrate, thus making it more sensitive. Its otheradvantages include simplicity, high precision, pHconstancy, flexibility, short incubation periods, andcontinuous increase in colour density as a functionof incubation time. Augustinsson shares the opinionof Voss and others that it is one of the most straight-forward methods available. It can also be usedwith the Auto Analyzer. Advantage has recentlybeen taken of the Ellman method for studies ofanticholinesterase agents during malaria eradicationcampaigns.

Radiometric methods

Radiometric methods have also been used recentlyfor cholinesterase determinations (Fig. 5). The me-thod of Winteringham & Disney (1962) is based onthe incubation of blood or tissue with acetate-labelled acetylcholine. Enzyme action is halted bythe addition of acid and excess of inhibitor (physo-stigmine). A direct assay of acetic acid-14C formed bycholinesterase activity (in rat blood) with acetyl-1-14C-choline as a substrate has also been described(Reed et al., 1966; McCaman et al., 1968).Although radiometric methods are extremely

sensitive, they have so far proved to be unsatisfactoryfor use in the field.

Fig. 5

Radiometric determination of cholinesterasei ChE

(CH3)3NCH2CH2014CCH3 -(CH3)3NCH2CH20H +: H014CCH30 0

Winteringham & Disney (1962) Reed et al. (1966)Potter (1 967)McCaman et al. (1 968)

REFERENCES

Augustinsson, K.-B. (1971) In: Glick, D., ed., Analysis ofbiogenic amines and their related enzymes, New York,Wiley, pp. 217-269

Augustinsson, K.-B. (1955) Acta physiol. scand., 35, 40Augustinsson, K.-B. & Holmstedt, B. (1965) Scand.

J. clin. lab. Invest., 17, 573Ellman, G. L. (1959) Arch. Biochem. Biopliys., 82, 70Eliman, G. L., Courtney, K. D., Andres, V., Jr & Feather-

stone R. M. (1961) Biochem. Pharmacol., 7, 88Goedde, H. W., Doenicke, A. & Altiand, K. (1967)

Pseudocholinesterasen. Pharmakogenetik, Biochemie,Klinik, Berlin, Springer

Holmstedt, B. (1965) Science, 149, 977Holmstedt, B. & Oudart, J.-L. (1966) Bull. Soc. Path.

exot., 59, 411Koelle, G. B. (1963) In: Handbuch der experimentellen

Pharmakologie, Suppi. XV, Cholinesterases and anti-cholinesterase agents, pp. 187-298

Koelle, G. B. (1970) In: Heilbronn, E. & Winter, A.,ed., Drugs and choliniergic mechanisms in the CNS,Stockholm, Research Institute of National Defence,pp. 431-440

Koelle, G. B. & Friedenwald, J. S. (1949) Proc. Soc. exp.Biol. (N.Y.), 70, 617

DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS 107

Latner, A. L. & Skillen, A. W. (1968) Isoenzymes inbiology and medicine, London and New York, AcademicPress

McCaman, M. W., Tomey, L. R. & McCaman, R. E.(1968) Life Sci., 7, 233

Oudart, J.-L. & Holmstedt, B. (1970) Arch. Toxikol., 27, 1

Potter, L. T. (1967) J. Pharmacol. exp. Ther., 156, 500Reed, D. J., Goto, K. & Wang C. H. (1966) Anal.

Biochem., 16, 59Winteringham, F. P. W. & Disney, R. W. (1962) Nature

(Lond.), 195, 1303Witter, R. (1963) Arch. environ. Hlth, 6, 537

DISCUSSION

VANDEKAR: Routine cholinesterase determination formonitoring exposure to anticholinesterase insecticides ismuch less important when the latter are carbamates thanwhen they are organophosphorus compounds. However,such determinations are still of great value in confirmingthe diagnosis in a case of accidental poisoning. In thisconnexion, it should be pointed out that some widelyused carbamates, in contrast to organophosphoruscompounds, show much greater activity against erythro-cyte cholinesterase than against plasma cholinesterase-e.g., the inhibitory power of propoxur and carbaryl (interms of their '50 values) against erythrocyte cholinesteraseis about 50 and 20 times greater, respectively, than theirinhibitory power against plasma cholinesterase. Theformer enzyme thus provides a much more sensitiveindex of absorption than does the latter.

GAGE: The measurement of blood cholinesterase for the

surveillance of individuals exposed to cholinesteraseinhibitors involves the possibility that there may be achange in activity in the blood between the times ofsampling and measurement. If inhibitor is present in thesample, inhibition may increase; if the enzyme is in thenon-aged state of inhibition, there may be a recovery inactivity. Does the drying of the blood sample on filterpaper prevent any such changes?

AUGUSTINSSON: As long as the samples are stored dryand at a temperature below 4°C, no changes in cholin-esterase activity are observed. Samples have been storedfor more than 10 years without appreciable decrease incholinesterase activity, and without any increase in theactivity of samples from persons intoxicated with organo-phosphorus compounds. Samples treated in vitro withorganophosphorus compounds have likewise shown noincrease in activity.