Distribution and determination of cholinesterases in mammals

Bull. Org. mond. Sante 1971, 44, 99-107Bull. Wid H/th Org. Distribution and Determination of Cholinesterases in Mammals BO 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 correlation between the histochemical localization and the function of neuronal cholinesterase. Different methods for the determination of cholinesterase levels are reviewed, with particular reference to their practical value for field work. The Warburg method and the Tintometer and Acholest colorimetric methods are compared on the basis of cholinesterase levels determined in normal persons and in those suffeering from parathion intoxication. As with other enzymes, the cholinesterases can be characterized by the properties of the isolated protein, by the substrate specificity (with regard to choline esters and other esters), by the shape of the activity-pS curve, and by their reaction with so-called selective inhibitors. In mammals, two principal groups of cholinester- ases have been distinguished by the use of these criteria (Table 1). However, considerable species differences exist-for example, the central nervous system of rats contains a cholinesterase that splits propionylcholine more rapidly than acetylcholine. The different determinations of cholinesterases that have been described have mainly been based on knowledge of the substrate specificity. DISTRIBUTION OF CHOLINESTERASES The cholinesterases can also be characterized by their occurrence and distribution in different organs. Until about 20 years ago, their distribution in tissues was usually studied manometrically with slices or homogenates. A serious limitation of this type of study is that it does not reveal the cells or parts of cells in which the activity of the enzyme is localized. Histochemical techniques have been used in an attempt to over- come this limitation. These histochemical methods are primarily of interest to pharmacologists and physiologists in so far as they can throw light on the problems of cholinergic function. Thus, the staining 1 Department of Toxicology, Swedish Medical Research Council, 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 others may supplement other sources of information on the nature of the transmission process at particular sites. For this purpose, detailed localization within cells may be less important than knowledge of what enzymes 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 have been much criticized with respect to the latter factor. In the ideal histochemical method, the substance seen on the slide is a product of the naturally occurring substrate. None of the methods of cholinesterase staining now in use complies with this requirement; the one coming closest to it is the thiocholine method (in which the enzyme splits thiocholine esters instead of choline esters), especially if the rates of hydrolysis of the thiocholine esters relative to those of their oxygen analogues are 2618 -99- B. HOLMSTEDT Fig. 1 Histochemical and spectrophotometric methods of determining cholinesterase 0 0 1i 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 the physiological situation than do procedures based on the hydrolysis of naphthyl acetate or indoxyl acetate, which up to now have been less convincingly controlled biochemically. Koelle has carried out biochemical control experi- ments with both choline and thiocholine esters as substrates. The results show that, with few excep- tions (acetyl-p-methyl thiocholine), the thiocholine esters behave like the choline esters towards the two types 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 the only one that allows the two types of cholinesterase to be distinguished histochemically with reasonable certainty. In the most recent modifications of the technique, this has been achieved by combining selective substrates with highly selective inhibitors in the same experiment. It is also essential to per- form biochemical control experiments in exactly the same solution as that used for the histochemical studies. Any shift in pH, for example, alters the concentration of inhibitor necessary to block one type of enzyme selectively. Numerous papers have been published on the use of the histochemical technique for the detailed localization of cholinesterases. The results have been summarized by Koelle (1963, 1970). Central nervous system A histochemical survey of the cholinesterases of the brains of rats and cats has disclosed the presence of low to high concentrations of acetyl- cholinesterase in most neurones of a large number of regions. In addition to acetylcholinesterase, the central nervous system contains significant amounts of cholinesterase (3.1.1.8). In contrast to the former, the latter is present in highest concentrations in the white fibre tracts and is localized in the glial cells. Among other places it is found around the supraoptic nucleus and around the third ventricle in the region of the paraventricular nucleus. The capillaries and other blood vessels of the brain and spinal cord, like those of most other organs, also contain the enzyme. A histochemical survey of the spinal cord shows that only the anterior and lateral horns give a positive acetylcholinesterase reaction. The large motor neurones are heavily stained and the product of the histochemical reaction is clearly visible along the neurites. The difference in staining intensity among various cells is not sufficiently marked to permit the quantitative estimation of cholinesterase activity. The spinal cord also contains cholinesterase (3.1.1.8), as noted above. Ganglia The distribution of cholinesterase in ganglia is subject to differences of opinion. In early studies in the cat, high concentrations of acetylcholinester- ase were found in cholinergic neurones (i.e., neurones giving rise to cholinergic fibres), but the adrenergic and sensory neurones appeared to be practically devoid of the enzyme. A more recent investigation in the cat, the rabbit, and the rhesus monkey demon- strated the presence of acetylcholinesterase in all types of peripheral neurone. In cholinergic neurones, the concentration of the enzyme appeared to be consistently high: in the adrenergic and sensory types, concentrations were lower and varied accor- ding to site and species. The neurones of Auerbach's plexus and the associated interstitial cells were found to contain, in addition, uniformly high concentrations of cholinesterase (3.1.1.8), which in other ganglia occurs only in the glial cells. This enzyme was not found in other neurones of the species studied, although its occasional presence 100 DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS in neurones of the superior cervical ganglion of the rat has been reported. Striated muscle Considerable effort has been devoted to the localization of cholinesterase in muscles. Studies on tissue homogenates have revealed that striated muscle contains approximately 95% of acetyl- cholinesterase and 5% of cholinesterase (3.1.1.8), localized in various places. Small " classical " end-plates are arranged in straight 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 is postsynaptic and belongs to the so-called subneural apparatus; very little is presynaptic. However, it is impossible to make, on the basis of denervation experiments, a statement about the presynaptic and postsynaptic distribution of acetylcholinesterase in nerve-muscle junctions that would be appli- cable to all skeletal muscles. Other " plates " do exist, composed of parallel gutters arranged as a " cake frill " or " palisade ", the gutters being oriented in the same direction as the long axis of the muscle fibre. Staining also occurs in large cap-shaped areas situated over the ends of muscle fibres, with gutters arranged in a parallel or in a somewhat reticular pattern. These endings are, in fact, situated at the muscle-tendon junctions. They are also found where muscle fibres end in the middle of the bundle. It is possible that these may not be motor end-plates but stretch receptors; if this is so, then the presence in them of acetylcholinesterase is of some interest. Cholinesterase is also present in muscle spindles, both in the end-plates of the intrafusal fibres and in the fibres themselves. On the basis of histochemical studies, some authors have suggested that cholinesterase is present in the ordinary fibres of striated muscles. This has recently been confirmed by other methods, but the nature of the enzyme is unknown. In some studies it may have been an artifact produced by inadequate histochemical technique. In any event. there is overwhelming evidence that the enzyme content of the motor end-plates is far greater than that of any other structure in striated muscles. Other tissues Acetylcholinesterase is found in a number of other tissues, such as the smooth muscles of the bronchioles and the urinary bladder and the effector cells of the salivary glands. Cholinesterase (3.1.1.8) is found in the carotid body, the hepatic cells, the muscularis mucosa of the intestinal canal, and the cortical cells of the zona glomerulosa of the adrenals. Correlation between histochemical localization and function of neuronal cholinesterase Pharmacological and enzymological investigations have repeatedly led to the suggestion that acetyl- cholinesterase is located both inside and outside the cell membrane. Such suggestions have been based upon comparisons of the effects of tertiary and quaternary ammonium bases with anticholin- esterase activity. It is known that the quaternary compounds 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 " portion external to a relatively impermeable cellular mem- brane and an internal portion has been elaborated upon by Koelle and his co-workers. It has been suggested that the internal or "reserve" portion represents the source for the replacement of the functional enzyme of the cell surface and could possibly be identical with the endoplasmic reticu- lum. Comparison of the distributions of acetyl- cholinesterase in autonomic ganglia indicated that practically all the external acetylcholinesterase of the sympathetic ganglia is associated with the presynaptic axonal terminations; most of the cyto- plasmic acetylcholinesterase of the occasional cholinergic neuronal perikarya is internal to a relatively impermeable membrane. It has been pro- posed that an important function of ganglionic acetylcholinesterase may be protection of the preganglionic terminations from depolarization and antidromic firing in response to acetylcholine libe- rated by these and adjacent terminations. The phar- macological actions of anticholinesterase agents probably result from inactivation of the external or functional acetylcholinesterase. If this represents only a small portion of the total acetylcholinesterase, its inactivation (and reactivation) might not be detectable by the usual techniques. The role of acetylcholine as a neurohumoral trans- mitter at several synaptic and neuroeffector sites is now firmly established. Furthermore, in the region of any junction, acetylcholine may also act at the presynaptic terminal to effect the liberation of additional 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 initiation or propagation of induced action potentials; how- ever, this does not exclude the likelihood that they are involved in the maintenance of the excitable state in those fibres in which they are present in significant concentrations. Apart from its functions in the nervous system, acetylcholine probably serves as a local hormone in many autonomic effector organs-i.e., it appears both to be synthesized by and to act on cardiac and smooth muscle in the maintenance of excitability and rhythm. At several sites where acetylcholinesterase acti- vity has been demonstrated (e.g., the musculotendi- nous junctions of skeletal muscle) it is not possible to assign even a tentative function to the enzyme or its substrate. The same may be said for nearly all the localizations of the nonspecific cholinesterases. Further studies may uncover roles at such sites that hitherto have been unsuspected. Blood cholinesterases Even the function of the cholinesterases in blood remains unknown, but both the erythrocyte acetylcholinesterase and the plasma cholinesterase (3.1 .1 .8) serve as very useful indices of the degree of absorption of anticholinesterase insecticides. The great variation in acetylcholinesterase activity from one person to another makes it necessary that the level in each person who might be occupationally exposed to cholinesterase inhibitors be determined before such exposure can occur. It also seems ad- visable that workers who from the beginning show only a feeble enzyme activity should not be per- mitted to work in situations where exposure is likely to occur. In practical work and field work, the cholinesterase activity in the plasma of man can usually be moni- tored. The enzyme is almost always inhibited before (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 forms of cholinesterase, and evidence for the electro- phoretic heterogeneity of the enzyme in human serum has been demonstrated by several workers. As many as six or seven forms are present in certain sera. These isoenzymes have been the subject of intense study during recent years, not least from the pharmacogenetic point of view. The poly- morphism of serum cholinesterase is discussed by Goedde et al. (1967) and by Latner & Skillen (1968). These workers carried out their studies in Europe and in North America. So far, similar studies have not been undertaken in developing countries in the tropics. When large-scale cholin- esterase screening programmes are performed in connexion with malaria eradication, anomalous plasma values-depending upon the substrate and the methods used-may indicate the presence of an atypical plasma cholinesterase. However, erro- neous results may also be caused by impaired liver function. CHOLINESTERASE DETERMINATION Many methods of cholinesterase determination have been suggested and compared, but only a few of them are appropriate for work in the field. Such methods should be simple and should not require electricity or complicated equipment. In developing countries one of the most important requirements for a field method is that it be simple enough to be used by nonspecialists. Different methods for determining cholinesterase are reviewed below with particular reference to their practicality in field work. The review by Augustins- son (1971) of different methods of determining cholinesterase levels should be consulted for details and references. "Remaining substrate " methods The earliest method for determining cholinesterase consisted of assaying the acetylcholine remaining after incubation with the enzyme-containing material (Fig. 2). At first, this was usually carried out by bioassay. Today, other methods are available- the Hestrin method, which is based on colorimetric determination of the iron(III) hydroxamate; the Schonemann 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)3NCH2CH20CCH3 I~~~~~~~~~~~,_ " Remaining substrate ": Bioassay Hestrin method Schonemann method Gas chromatography method, which has become available only in recent years, has not yet been used for enzyme assays. The "remaining substrate" methods suffer from the disadvantage of not measuring the rate of disappearance of the substrate, and in this respect they are inferior to other means of determining cholinesterase 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 used techniques are discussed below. Warburg manometric technique. This method, introduced many years ago, is still one of the most accurate and reliable methods available for assaying cholinesterase activity; according to Augustinsson (1971) the accuracy can be within ± 2%. The method 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 the medium cannot be varied and that the rate of hydro- lysis cannot be studied at substrate concentrations below 0.4 mm since insufficient gas is liberated at such concentrations. A further limitation is the difficulty of assaying reversible esterase inhibitors. The results obtained with the Warburg technique agree fairly satisfactorily with those obtained with the Hestrin, Michel, titrimetric, and Acholest methods. 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 which whole 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 investigated and successfully used for 10 years in the temperate zone (Augustinsson, 1955). The technique is based on the use of two selective substrates, butyrylcholine ChE 0 (CH3)3NCH2CH20H + HOCCH3 Acid formation: Warburg Michel pH-stat Acholest for plasma cholinesterase and acetyl-p-methylcholine for erythrocyte acetylcholinesterase. As it had not been shown whether the filter- paper technique gave reliable results in the conditions prevailing in the tropics (Witter, 1963), it was decided to check it. The stability of the enzyme activity of blood on paper was first studied under various laboratory and tropical conditions. It was found that the blood samples can be dried in the sun, in the shade, or in a desiccator with no loss of enzyme activity. From the data given in Table 2 it is obvious that cholinesterase activities are highly stable, even under seemingly unfavourable conditions. Only when the filter-papers are not allowed to dry com- pletely or are contaminated with moisture after drying is there a decrease of enzyme activity. The normal variation of human blood cholinester- ase activity obtained in this way was determined and the dispersion was calculated statistically. The plasma cholinesterase activity of males was found to be significantly higher than that of females, but no such difference was found in erythrocyte acetyl- cholinesterase activity. Table 2 Cholinesterase activity in blood samples collected on filter-paper from various places and mailed to Stockholm for analysis Place Stockholm Geneva Dakar Lagos Calabar Manaus Time interval between 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 samples dried on filter-paper and mailed to a distant labora- tory for determination of cholinesterase activities. The simplicity and practicality of the filter-paper method make it ideally suitable for monitoring levels in spray-men and exposed populations when insecticides with anticholinesterase properties are used in malaria eradication and other public health programmes. It also forms an ideal standard with which values determined by other methods can be compared. Michel method. This method, first described in 1949, has always been one of the most popular methods for routine determinations of blood cholinesterase. The procedures described by Michel have usually been followed, but many more or less significant modifications have been described. The method has often been used in field studies, owing to its relative simplicity. In this method the enzyme is allowed to act on acetylcholine in a standard buffer solution for a fixed period of time, and the pH of the mixture is measured at the beginning and at the end of this interval. The rate of change of pH (usuallyz pH/h) is a measure of enzymatic activity. Augustinsson (1971) has pointed out that the absolute accuracy and the sensitivity of the Michel method are not as great as those of the titrimetric and Warburg methods, primarily because in the Michel method the pH, which is a logarithmic function of the acid concentration, is measured rather than the acid production itself. The change of pH with respect to time is not usually recorded for each assay unless a recording pH meter is used. This improved equipment, introduced by Tammelin and his co-workers, simultaneously registers six enzyme reactions and is calibrated to relate the pH of the solution to the rate of addition of acetic acid. The change in pH resulting from the production of acid in a solution of a choline ester incubated with cholinesterase can also be estimated by the change in colour of an indicator rather than with a pH meter. A number of techniques have been described 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 procedures employing an acid-base indicator have been used for serum or plasma, but some are also available for whole blood or erythrocytes. The majority are " wet" methods, in that pre-made solutions are used and the colour change is estimated either visually or with a spectrophotometer. Commercial kits are available and some colorimetric methods have been adapted for use with the Auto Analyzer. Fig. 3 Comparison of Acholest, Warburg, and Tintometer methods for the determination of cholinesterase * 4 -J z 50- z w0 w a. 0 4 -J 4 z z w 0 w a. Tintometer whole blood DAYS Acholest DAYS * The arrows beneath the x-axes indicate the times at which the antidote (pralidoxime methanesulfonate) was administered. 104 DISTRIBUTION AND DETERMINATION OF CHOLINESTERASES IN MAMMALS Acholest method. A " non-wet " method, the Acholest test is particularly suitable for use in the field. It is a screening test that is easy to perform and requires a minimum of equipment. A slip of filter paper is impregnated with the choline ester and bromothymol blue and is subsequently dried. The serum or plasma is added directly to this paper and the colour change is observed, comparison being made with a similar paper on which no choline ester is present. The Acholest technique has been used 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 slowly changes the colour of the indicator in the paper from deep blue to yellow-green. The activity is measured by the time needed for the colour to match that of the control paper: the higher the cholin- esterase activity, the shorter the time. The Acholest test paper can be used only if the plasma is separated from the red blood cells, and there must be a complete absence of haemolysis. The difficulty of centrifugation in the field can be circumvented by the use of an easily made hand-powered micro- centrifuge developed for this purpose (Holmstedt, 1965). The mean error of the Acholest method has been calculated, using the Warburg method as a standard, as ±6.5 % for colour-development times of 11-56 minutes; when the time is less than 11 minutes the mean error is ±16%. In other words, the error is lower in the region that is of interest in the diagnosis of cholinesterase inhibition by insecticides. Comparison of Acholest, Warburg, and Tintometer methods. Oudart & Holmstedt (1970) have compared the results given by the Warburg method (using filter paper), the Tintometer colorimetric method, and the Acholest method in determinations of cholinesterase levels in normal people and in those with parathion intoxication. The enzyme activi- ties obtained are shown in Fig. 3 as percentages of the initial cholinesterase activity, making it possible to compare all methods. The curve ob- tained by using the Acholest test paper corresponds to that obtained by means of the manometric method. Both methods show the decrease in cholin- esterase activity at the same time. The effects of the injections of the antidote are also clearly indicated by both methods. The curve obtained with the Tinto- meter is much less satisfactory and the decrease in enzyme activity is smaller than could be recorded with the other two methods. Fig. 4 The Eliman method +8(1) (CH3)3NCH2CH2S-CCH3 + H20 acetylthiocholine (2) (CH3)3NCH2CH2S thiocholine -(CH3)3NCH2CH2S-S NO2 coo acetylcholinesterase + + ON (CH3)3NCH2CH2S + CH3COO + 2H thiocholine if + 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 during the hydrolysis of the ester can be determined by titration with standard alkali at constant pH using either an indicator or a potentiometer. The auto- mated pH-stat method is one of the most convenient and precise of the methods available for assaying cholinesterase. Its great advantage is that the acid liberation can be continuously recorded. A recent comparison of the pH-stat and the Michel methods led to the conclusion that the former offers better control 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 of sulfhydryl groups. It was later modified for the determination of cholinesterase activity in whole blood, erythrocytes, and tissue homogenates (Fig. 1 and 4). The thiocholine (II) formed during the hydrolysis of acetylthiocholine (I) reacts rapidly and irreversibly with 5-dithiobis-2-nitrobenzoic acid, releasing a coloured product, 5-thio-2-nitrobenzoic acid (III). Cholinesterase activity may be determined spectrophotometrically by following the production of 5-thio-2-nitrobenzoic acid. In contrast to other colorimetric methods, the ElIman method is based on the measurement of a reaction product instead of the remaining intact substrate, thus making it more sensitive. Its other advantages include simplicity, high precision, pH constancy, flexibility, short incubation periods, and continuous increase in colour density as a function of incubation time. Augustinsson shares the opinion of Voss and others that it is one of the most straight- forward methods available. It can also be used with the Auto Analyzer. Advantage has recently been taken of the Ellman method for studies of anticholinesterase agents during malaria eradication campaigns. Radiometric methods Radiometric methods have also been used recently for cholinesterase determinations (Fig. 5). The me- thod of Winteringham & Disney (1962) is based on the incubation of blood or tissue with acetate- labelled acetylcholine. Enzyme action is halted by the addition of acid and excess of inhibitor (physo- stigmine). A direct assay of acetic acid-14C formed by cholinesterase 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 unsatisfactory for use in the field. Fig. 5 Radiometric determination of cholinesterase i ChE (CH3)3NCH2CH2014CCH3 -(CH3)3NCH2CH20H +: H014CCH3 0 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 of biogenic amines and their related enzymes, New York, Wiley, pp. 217-269 Augustinsson, K.-B. (1955) Acta physiol. scand., 35, 40 Augustinsson, K.-B. & Holmstedt, B. (1965) Scand. J. clin. lab. Invest., 17, 573 Ellman, G. L. (1959) Arch. Biochem. Biopliys., 82, 70 Eliman, G. L., Courtney, K. D., Andres, V., Jr & Feather- stone R. M. (1961) Biochem. Pharmacol., 7, 88 Goedde, H. W., Doenicke, A. & Altiand, K. (1967) Pseudocholinesterasen. Pharmakogenetik, Biochemie, Klinik, Berlin, Springer Holmstedt, B. (1965) Science, 149, 977 Holmstedt, B. & Oudart, J.-L. (1966) Bull. Soc. Path. exot., 59, 411 Koelle, 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 in biology and medicine, London and New York, Academic Press 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, 500 Reed, D. J., Goto, K. & Wang C. H. (1966) Anal. Biochem., 16, 59 Winteringham, F. P. W. & Disney, R. W. (1962) Nature (Lond.), 195, 1303 Witter, R. (1963) Arch. environ. Hlth, 6, 537 DISCUSSION VANDEKAR: Routine cholinesterase determination for monitoring exposure to anticholinesterase insecticides is much less important when the latter are carbamates than when they are organophosphorus compounds. However, such determinations are still of great value in confirming the diagnosis in a case of accidental poisoning. In this connexion, it should be pointed out that some widely used carbamates, in contrast to organophosphorus compounds, show much greater activity against erythro- cyte cholinesterase than against plasma cholinesterase- e.g., the inhibitory power of propoxur and carbaryl (in terms of their '50 values) against erythrocyte cholinesterase is about 50 and 20 times greater, respectively, than their inhibitory power against plasma cholinesterase. The former enzyme thus provides a much more sensitive index of absorption than does the latter. GAGE: The measurement of blood cholinesterase for the surveillance of individuals exposed to cholinesterase inhibitors involves the possibility that there may be a change in activity in the blood between the times of sampling and measurement. If inhibitor is present in the sample, inhibition may increase; if the enzyme is in the non-aged state of inhibition, there may be a recovery in activity. Does the drying of the blood sample on filter paper prevent any such changes? AUGUSTINSSON: As long as the samples are stored dry and at a temperature below 4°C, no changes in cholin- esterase activity are observed. Samples have been stored for more than 10 years without appreciable decrease in cholinesterase activity, and without any increase in the activity of samples from persons intoxicated with organo- phosphorus compounds. Samples treated in vitro with organophosphorus compounds have likewise shown no increase in activity.