l I I \ (le " MINI”! A. METHGD FOR ME VESUAL EST§MAT§ON 0F ERYTHROCYTE CHOLSNESTERASE ACTIVlTY Thesis for the Degree of M. S. MICHIQAN. SYATE UNWERSITY LGUESE K. MUELLER 1970 b“:— THESIS LIBRJP { Michigan bl“ to University IINBING IY - unAs & SONS' Max mm mc. U'MRY IINDE RS ABSTRACT A METHOD FOR THE VISUAL ESTIMATION OF ERYTHROCYTE CHOLINESTERASE ACTIVITY by Louise K. Mueller Reagent and standard impregnated strips were used in a screening procedure for the visual estimation of erythrocyte cholinesterase activity in samples of whole blood. Thiocholine released by enzyme action on acetylthiocholine substrate reacts quantitatively with dithiobisnitro- benzoic acid to yield a yellow anion. The time required for the produc- tion of a standard amount of yellow anion was used as a measure of enzyme concentration. The action of plasma esterase was inhibited by quinidine sulfate. Test reagent strips were sufficiently stable to be used 3.5 months. Standard reagent strips were stable at least one month. The precision of visual estimation of enzyme activity was not affected by varying the intensity of the standard color or the rate of approach to a standard color. Correction factors relating rate of enzyme reaction at tempera- tures ranging from 15-35 C. to a base temperature of 25 C. were calculated. The time of color change was related to temperature and level of erythro- cyte cholinesterase activity at the mean activity of the normal samples and 71% mean activity. A decrease in erythrocyte cholinesterase activity of 45% or more could be detected with a 5.8% index of discrimination. The method had a precision of :_25%. A METHOD FOR THE VISUAL ESTIMATION OF ERYTHROCYTE CHOLINESTERASE ACTIVITY By Louise K. Mueller A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1970 For Bill Jim, Cathy, and Dave 11 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. A. E. Lewis, my major professor, for his suggestions and encouragement in this research project. I thank Dr. T. Brody and Dr. D. Cowan for serving on my committee and for comments and suggestions which helped make this work more meaningful. My thanks to the laboratory staff at the MSU Health Center for their cOOperation in obtaining blood samples. To the faculty, staff, and fellow students of the Department of Pathology, my sincere appreciation for their words of encouragement, assistance, and donation of blood samples. I am grateful for the financial assistance of the Allied Health Professions Advanced Training Grant AHT-69-049. My thanks to Dr. C. C. Morrill, Chairman of the Department of Pathology, and others who made this assistance available. 111 INTRODUCTION . . . . . . REVIEW OF LITERATURE . . TABLE OF CONTENTS Location of the Enzyme. . . . . . Transmission of Nerve Impulses. . . Mechanism of Transmission of Nerve Impulses Effects of Decreased Cholinesterase . Measurement of Cholinesterase Activity. . MATERIALS AND METHODS. . Preparation of Test and Standard Reagent Strips Visual Quantitation of Enzyme Activity. . . . . Reference Method. Modified Visual Method. . . . Temperature-Rate RESULTS. . . . . . . . . DISCUSSION . . . . . . . SUMMARY AND CONCLUSIONS. REFERENCES . . . . . . . APPENDICES . . . . . . v ITA O O O O O O O O O 0 Studies. . . . iv 11' 12 12 13 13 14 15 21 24 26 30 33 Table LIST OF TABLES Page Comparison of reagent absorbances using fresh reagents md lyophilized strips 0 O O O O O O O O O O ‘ O O O Q 0 O O O 17 Comparison of estimates of enzyme activity (in minutes) using 3 and 5 minute color standards and a 3 minute color standard with one-half enzyme concentration . . . . . . . . 18 Comparison of reference method values with modified visual. mthOd values 0 O O O O O O O O O O O O O O O O O O O O O O 19 LIST OF FIGURES Figure Page 1 Time of color change in relation to temperature and level of erythrocyte cholinesterase activity at mean activity of:norma1 sample and 712 mean activity . . . . . . 20 vi Appendix I II LIST OF APPENDICES Reagents O O O I O O O O O O O O 0 Data for Index of Discrimination . vii INTRODUCTION In recent years there has been a sharp increase in the use of the organOphosphate insecticides by agriculturalists. The basis of success for these compounds resides in their ability to combine with and non- competitively inhibit the action of cholinesterase, an enzyme involved in the transmission of nerve impulses. Unfortunately, the compounds are toxic to man as well as to insects. As a result of careless handling techniques and/or faulty equipment, the compounds enter the body through the skin or by inhalation or ingestion. Variable decreases can occur in enzyme activity in the blood and wherever cholinoceptive membranes are located. Since the enzyme is nor— mally present in excess, an initial exposure does not necessarily pro- duce symptoms of poisoning other than transient symptoms at points of entry. Because of the temporary nature of such symptoms as sweating, muscular twitching, headache, and mild nausea, they are often ignored. Repeated small exposures, however, can increase susceptibility to poisoning to a degree where serious illness characterized by rapid onset and short course occurs. Results of repeated exposures are reflected by extended periods of decreased erythrocyte cholinesterase activity. By periodically testing the blood of individuals handling organOphos- phates the incidence of serious illness could be reduced. The purpose of this study was to develOp for field use a rapid, simplified procedure for the visual estimation of erythrocyte 2 cholinesterase activity. The procedure is a modification of the spec- trophotometric method of Ellman, Courtney, Ahdres, and Featherstone (1961). With this method erythrocyte cholinesterase activity in a whole blood sample is measured following a coupled reaction sequence. Ellman's at al. (1961) equations for the reactions are as follows: H20 + (CH3)N+CH2CHZSCOCH3 enzmei (CH3)3N+CH20H23- + CH3COO" + 2H+ Acetylthiocholine thiocholine (CH3) 3N+cuzcnzs’ + 02N C) 33 (:3.N02 _.) (CH3)3N+CH2CHZSS (3 N02 thiocholine COO’ 'COO’ ‘ COO- dithiobisnitrobenzoic acid + ‘s. 6:) N02 COO- 5-thio-2-nitrobenzoate In this study, enzyme activity was estimated visually by measuring the time required to produce a standard amount of the yellow anion, 5-thio-2-nitrobenzoate. Reagents and standard were_supplied in a stable, premeasured form on 1yophilized reagent strips. REVIEW OF LITERATURE Cholinesterases are muc0polysaccharide enzymes which catalyze the hydrolysis of the choline esters of fatty acids. Mammalian species con- tain 2 types of the enzyme, acetylcholinesterase-and butyrylcholinesterase. They differ in function, substrate specificity, reaction to inhibitors, and distribution. Acetylcholinesterase (true cholinesterase, acetylcholine acetylhy- drolase) performs the specific function of catalyzing the hydrolysis of_ acetylcholine, a neurohumoral transmittor substance. Following purifica- tion by column chromatography, its molecular weight, as determined from sedimentation and diffusion coefficients, is 230,000. In gel filtration studies, the enzyme moved at the same rate as catalase, suggesting a molecular weight of 250,000. Friction ratio indicates the enzyme is a globular protein. The number of active sites per molecule has been cal- culated as 4 (Kremzer and Wilson, 1964). The enzyme is capable of hydrolyzing a number of choline and non- choline esters. The more closely the alcohol group in noncholine esters simulates the choline configuration, the more rapidly is the ester hydro- lyzed. Expressed as a percentage of acetyl-B-methyl choline hydrolysis, the carbon analog of acetylcholine, 3:3—dimethy1 butyryl acetate, is hydrolyzed most rapidly next to acetylcholine (Adams, 1949). For any given alcoholic group, the optimal acyl group for the enzyme is acetate. Chain branching at the carbon atom of the alcohol.adjacent to the ester link, as in acetyl-B-methyl choline (methacholine),decreases hydrolysis 3 4 by one-third as compared to acetylcholine (Adams and Whittaker, 1949). Mendel and Rudney (1945) found that Optimal acetylcholine concentration for the enzyme's activity varies with ionic strength. In the absence of salts other than 0.025 M NaHCO3, Optimum activity occurs at 0.00025 M acetylcholine. Following addition Of 0.08 M KCl to the NaHC03, optimum. activity occurs at 0.002 M acetylcholine. An absolute increase in the rate of substrate hydrolysis also occurs. The authors suggest that the escape of KT occurring in viva upon stimulation might possibly help to maintain Optimal conditions for enzyme activity. Inhibition of enzyme activity occurs at acetylcholine concentrations in.excess oi:'.10"3 M. Enzyme action is also pH dependent, maximum activity occurring at pH 8 to 8.5 (Wilson and Bergmann, 1950). Butyrylcholinesterase (pseudocholinesterase, acetylcholine acylhy- drolase) differs from acetylcholinesterase in that, for any given alco- holic group, the Optimal acyl grOup is butyrate. The hydrolysis rate of. butyrylcholine is twice that of acetylcholine. Benzoylcholine is also hydrolyzed, but not methacholine (Adams and Whittaker, 1949). Enzyme activity increases-rapidly from pH 6 to pH,8 where it.reaches maximum activity and remains until the enzyme is denatured. NO inhibition of enzyme occurs in the presence of excess acetylcholine (Click, 1937). The normal physiologic-function of the enzyme is not known at present. Butyrylcholinesterase is more sensitive to inhibition by the organo- phosphates, diisoprOpyl phosphorofluoridate (DFP) and tetraethyl pyrophos- phate (TEPP) than acetylcholinesterase (Crab and Harvey, 1949). The sensitivity of both enzymes to antimalarial and related drugs has been studied by Wright and Sabine (1948). With quinidine, quinine, plasmochin, and paludrine, concentrations completely inhibiting butyrylcholinesterase had no effect on acetylcholinesterase. 5 Location of the Enzyme The entire lengths of neurons giving rise to postganglionic parasyme pathetic, preganglionic autonomic, and somatic motor peripheral cholinergic fibers contain relatively high concentrations of acetylcholinesterase. Postganglionic sympathetic and primary afferent neurons generally have lower concentrations of the enzyme (Koelle, Davis, and Gromadzki, 1967). Levels within the CNS vary considerably. Concentration is high in neurons of the anterior and lateral horn cells and cranial motor nerves (Giacobini, 1956 and 1967). At the skeletal muscle motor end-plates, high concentrations of acetylcholinesterase are found at the surface and invaginations of the postjunctional membrane. According to Koelle (1965), there is also an internal reserve of recently synthesized enzyme which is held within the endOplasmic membrane for replacement in the course of the cell‘s cycle of protein turnover. Inhibition of this enzyme in nerve and muscle tissue produces a functional derangement in the transmission of nerve impulses. The enzyme has also been found in erythrocyte stroma, myotendinous junctions, thrombocytes, and placental tissue (SVensmark, 1965). Func- tion in these areas is unknown, although the enzyme‘s presence in erythro- cytes has been proposed as a_protective mechanism against nerve excita- tion being caused by circulatory transport of acetylcholine (Alles and stes, 1940). Butyrylcholinesterase is found in the glia and white fiber tracts, the liver, pancreas, intestinal mucosa, and blood plasma. Inhibition or lack of this enzyme due to genetic variance or liver disease increases susceptibility to poisoning following administration of muscle relaxants (Koelle, 1965, and Svensmark, 1965). '1 -- - .‘. 1* 6 Transmission of Nerve Impulses The concept Of chemical neurohumoral transmission was first advanced by DuBois-Reymond in 1877. He stated that excitation from motor-nerve terminals to effector cells occurred either electrically as a result of action currents or chemically as a result of excitor substances formed and released at the surface of the nerve endings. Although similarities were noted in response to injections of adrenal gland extracts and stimu- lation of the sympathetic nerves (Elliott, 1905) and injections of acetyl- choline and stimulation of parasympathetic nerves (Dale, 1914), neuro- humoral transmission of nerve impulses was not generally accepted. In 1921, Otto Loewi began his classical experiments with frog hearts and established the first real proof that excitation of a nerve caused the release Of a chemical substance which then transmitted the nerve impulse. The substance, named Vagusstqfif'by Loewi, was subsequently identified by Loewi and Navratil in 1926 as acetylcholine. Final acceptance of neurohumoral transmission came with (1) the demonstration of a concentration of enzyme in nerve and motor end-plate tissue capable of hydrolyzing acetylcholine with a speed consistent with the process of transmission, (2) the localization of the enzyme at the- neuronal surface where bioelectric phenomena occur, (3) the demonstration of choline acetylase, an enzyme involved in the synthesis of acetyl- choline, in peripheral fibers as well as in brain, and (4) the alteration and inhibition of the nerve action potential (NAP) by anticholinesterases (Nachmansohn, 1946). Mechanism of_Transmission of Nerve Impulses The normal resting mammalian axon has an intracellular concentration of potassium approximately 20 times that of the extracellular fluid. The 7 concentration of sodium is in reverse order. The maintenance Of this concentration differential by sodium pump and active transport results in a transmembrane potential of approximately 70 mV, the interior of the axon negative with respect to the exterior. The application of a stimulus above threshold level initiates a nerve action potential. Membrane permeability is altered permitting sodium ions to flow rapidly inward in response to a concentration gradient. Local reversal of membrane polarity results.- Repolarization occurs immediately as permeability is decreased to sodium and increased to potassium. The polarization—repolarization process affects the adjacent resting membrane whose permeability is in turn altered. Conduction of the NAP occurs without decrement along the axon. The arrival of the NAP at the axonal terminal causes the release of several quanta Of the neurohumoral transmittor synthesized in the axon and stored in the synaptic vesicles. The transmittor substance diffuses across the synaptic cleft to receptor sites on the postjunctional membrane and alters the ionic permeability of the membrane. A generalized per- meability to all ions results in an excitatory postsynaptic potential. If this exceeds threshold, an action potential is propagated in the affected nerve or muscle cell. Destruction or dissipation by diffusion of the neurotransmittor then occurs. At cholinergic junctions, the enzyme cholinesterase assists in the destruction of the neurotransmittor by hydrolysis. Effects of Decreased Cholinesterase Excess acetylcholine potentiates responses of glands and muscles innervated by the parasympathetic system. Results are excessive saliva- tion, vomiting, retching, bradycardia, and frequent micturation with incontinence. 8 Voluntary muscles react to excessive acetylcholine with fibrilla- tion, fasciculations, weakness, and paralysis. In the autonomic ganglia and in the CNS, acetylcholine has first an excitatory action and then, at higher concentrations, an inhibitory reac- tion. Transient hyperpnea, followed by gasping and respiratory failure, changes in blood pressure, failure of circulation, convulsions, hyper- excitability to stimuli, hyperglycemia, and acidosis occur. Hemodynamic effects are dependent on drug, dose, route of administration, and species (Gleason, Gosselin, and Hodge, 1963; Grob, 1956; Heath, 1961; Holmstedt, 1959; and Koelle, 1965). Measurement of Cholinesterase Activity The reaction of acetylcholinesterase with its natural substrate, acetylcholine, forms the basis for the in vitro measurement of the enzyme's activity. Through the use Of DFP, an irreversible inhibitor of the enzyme, the sequence and mechanism of action of the enzyme's active sites have been elucidated. The enzyme has 2 active sites: one, an anionic site bearing a unit charge and the second, an esteratic site containing a serine and a histidine residue. The quaternary N of acetyl— choline forms an ionic bond with a dissociated carboxyl group at the anionic site while the carbonyl carbon of the substrate forms a covalent bond with the enzyme's serine residue at the esteratic site. The pro- ton released in the reaction is accepted by the imidazoyl group of the histidine. Two hydrolysis reactions follow: the first releasing choline and acylated enzyme and the second releasing acetic acid and regenerated enzyme (Krupka, 1966; and Wilson, 1967). Equations for the reactions are as follows:‘ acetylcholine + enzyme #3 enzyme-substrate complex enzyme-substrate complex + HOH -——-§ choline + acylated enzyme acylated enzyme + HOH . > acetic acid + enzyme Measurement of enzymatic activity based on decrease in amount of added substrate can be made by reacting the remaining choline ester with alka- line hydroxylamine to form hydroxamic acid. The latter reacts with acid ferric chloride to form a brown-colored complex, whose absorbsnce is prOportional to the amount of reacting substrate (Bonting and Feather- stone, 1956; Hestrin, 1949; and de la Huerga, Yesinick, and Pepper,-1952). By using thiocholine ester as substrate, Tabachnick (1956) measured decrease in substrate concentration of an enzyme-substrate mixture at 250 nm. Measurements Of enzymatic activity based on product formation can be divided into 2 categories: those measuring acetic acid or C02 released by acetic acid and those measuring thiocholine. Manometrically, in a procedure devised by Ammon, the acetic acid produced by the action of enzyme on substrate can be reacted with NaHCO3 in a Warburg flask and the amount of C02 released measured.' Activity is vexpressed in ul of C02 evolved or uM of substrate hydrolyzed per 0.1 m1. of erythrocytes per 30 min. (Augustinsson, 1948). Change in pH (ApH) of the reaction mixture caused by acetic acid production can be measured electrometrically with a glass electrode. The method, as published by Michel (1949), includes correction factors for ApH resulting from nonenzymatic hydrolysis of substrate and for change in enzyme reaction rate with decrease in pH. Enzyme activity is eXpressed as the rate of ApH/hr. Witter, Grubbs, and Farrior (1966) and Cestaric (1964) have introduced simplified modifications of this method. 10 By using indicators, [ipH can be measured spectrOphOtometrically. Croxatto, Croxatto, and Huidabro (Augustinsson,,l957); Reinhold, Tourigney, and Yonan (1953); and Caraway (1956) used phenol.red indicator. Rappaport, Fischl, and Pinto (1959) used menitrOphenol. Limperos and Rants (1953), using bromothymol blue, rated whole blood enzyme activity according to the color that developed in 20 minutes. Fleisher, Woodson, and Simet (1956), after modifying Limperos' and Ranta's test to measure the time required tO reach a specific color, were able to detect a decrease of 50% or more of mean pOpulation activity in plasma (butyrylcholine substrate) or erythrocyte (acetyl-B-methyl choline substrate) enzyme activity. Ellman et a2. (1961) measured enzyme activity using acetylthio— choline as substrate. The thiocholine formed from substrate hydrolysis reacts with 5:5-dithiobis-Z-nitrobenzoate (DTNB) to yield a yellow anion whose concentration is prOportional to substrate consumed. By adding quinidine sulfate, an inhibitor of plasma esterase, to the reaction mix- ture, erythrocyte activity in whole blood was measured. Activity was expressed in moles of substrate hydrolyzed/min./RBC. MATERIALS AND METHODS The procedure of Ellman et a2. (1961) was modified for use in the visual estimation of erythrocyte cholinesterase activity. The mean normal value of enzyme activity in moles of substrate hydrolyzed/min./m1. of blood and moles of substrate hydrolyzed/min./gm. of hemoglobin were calculated. Blood from 38 male students was collected in heparin tubes.* Imme- diately after collection the blood samples were refrigerated (2-10 C.) and analyzed within 24 hours. Hemoglobin concentration was measured on each sample by the cyan- methemoglobin method of Drabkin. ' Erythrocyte cholinesterase activity was measured on each sample by the method of Ellman et a1. (1961). The procedure was modified to include sterox** as a cell lysing agent in the phosphate buffer (pH 8.0, 0.1 M). (See Appendix I for reagent preparation.) Enzyme activity was calculated using the formulas: moles of substrate hydrolyzed/min./m1. of blood -. (4 .50) (10'5) AA moles of.substrate hydrolyzed/min./gm. of hgb. - (4.5o)(10'3) AA Hgb. *B—D Vacutainers 3200 RA **Sterox SE, Item #64049. Harleco. Hartman—Leddon Co., Philadelphia, Pa. ’ 11 12 where (4.50)(10’5)and (4.50)(10‘3) are conversion factors reflecting dilution, extinction coefficient of yellow anion, and changes in units; AA - change in absorbance/min.; Hgb. - concentration of hemoglobin in gm./100 m1. of blood. Preparation of Test and Standard Reagent Strips Strips, 6 x 54 mm., were cut from electrophoresis paper wicks.* Test reagent strips were prepared by adding DTNB (0.025 m1.), quinidine sulfate (0.01 ml.), and acetylthiocholine iodide (0.02 ml.) to strips at points 2, l6, and 31 mm. from one end. Standard reagent strips were pre- pared by adding glutathione (0.02 ml.) and DTNB (0.025 ml.) to stripe at points 2 and 31 mm. from one end. Each strip was placed in a 10 x 75 mm. test tube, flash frozen in a mixture of dry ice and alcohol, and 1yOphilized for 3 hours. Following 1yOphilization, the test tubes_con- taining the strips were sealed and stored at -5 C. until used. Visual Quantitation of Enzyme Activity Enzyme activity was estimated from time in minutes required for the enzymatic reaction to produce sufficient yellow anion to match a standard color. The extinction coefficient of the yellow-colored anion is 13,600 and therefore: moles/L. Of standard/min. - AA X 122 X 1.25 13,600 where 122 - 3.05 - correction factor for dilution of DTNB; 0.025 1.25 - 0.025 -. correction factor for dilution of standard. 0.02 Standards representing colors produced by the average normal enzymatic reaction in 3 and 5 minutes were prepared. *Beckman #319329. 13 The average normal enzymatic reaction proceeded at the rate of 0.122 A./min. There are presently no available methods useful for stOp- ping the reaction. Correction for color produced as a result of thiol material released from the cells required the addition of blood to both standard and test reagent mixtures. Therefore, the time required for the production of yellow anion sufficient to match a single standard was measured. Repeated assays with visual matching of.enzyme reaction colors to 3 and 5 minute standard colors and to a 3 minute standard color with one- half enzyme concentration were performed. Reference Method Whole blood, 0.005 ml., was added to 3.0 m1. of phosphate—sterox buffer in a 1 cm. cuvette. The photometer* slit was adjusted so that the absorbance (at 412 nm.) of the solution in the cuvette was zero. Quinidine sulfate (0.01 m1.), DTNB (0.025 ml. ). and acetylthiocholine iodide (0.02 ml.) were added to the cuvette. Changes.in absorbance (AA total) were recorded for 5 minutes. The reaction of blood thiol groups with DTNB in a mixture of blood, buffer, and DTNB was similarly recorded (AASH). The time required for AAtOtal' AASH to equal the absorbance of a standard was calculated. Modified Visual Method Whole blood, 0.005 ml., was added to each of.2 10 x 75 mm. test tubes. Each tube contained 3.0 m1. of phosphate-sterox buffer. At zero time a standard reagent strip was added to one tube; a test reagent strip was added to the other tube. Both tubes were vigorously shaken for 3 *Beckman DU, Beckman Instruments, Inc., Fullerton, Calif. 92634. 14 minutes, the strips removed, and clasped time recorded when the color of the enzymatic reaction mixture matched that of the standard. Temperature-Rate Studies Five samples of blood were analyzed by the reference mthod at 15, 20, 25, 30, and 35 C. Factors relating rates to‘a 25 C. base temperature were calculated. RESULTS The mean nOrmal value of erythrocyte cholinesterase activity in moles of substrate hydrolyzed/min./m1. of blood at 25 C. was 5.48 x 10"6 :p0.57 (1 s.d.). The mean normal value in moles of.substrate hydrolyzed] min./gm. of hemoglobin at 25 C. was 3.49 x 10"5 1:0.41 (l s.d.). The stability of the strips impregnated with reagents was.investi- gated following 1yOphilization and storage. The data in Table 1 indicate some deterioration in test reagent strips probably resulting from sub- strate instability. If adjustment is made for the initial decrease in absorbance and deterioration incurred during the 1yOphilization process, the absorbance contributed by reagent breakdown following 3.5 months of storage at -5 C. as compared to the total absorbance of the enzymatic reaction at 5 minutes is 7.83%. No significant deterioration Of standard reagent strips occurred during 1 month of storage following 1yOphilization. Results of repeated assays in which enzyme activity was estimated in minutes required to match a standard color are shown in Table 2. The data show that precision of visual matching, as indicated by the per- centage ts/x, was only slightly affected by increasing the intensity of the standard color. Increasing the time interval required to match the 3 minute standard color (enzyme concentration reduced to one-half) had only a slight effect on precision. In practice, it was found that at least 3 minutes were required to remove all of the standard from the reagent strip. 15 16 Seventeen samples Of blood were assayed by the reference.and modi- fied methods. Results are shown in Table 3. The mean and standard devi- ation of the reference method was 5.19 i_0.66 as compared to 5.08 i 0.72 for the modified method. The index or error of discrimination (see Appendix II) at 552 of normal activity was.5.82. The modified visual methodhad a precision of i 25%. Using the reference method, the effect of temperature on enzyme rate was investigated at 15, 20, 25, 30, and 35 C. Correction factors relating rates to 25 C. were calculated.' The time of color change was related to temperature and level of cholinesterase activity at mean activity of the normal sample and 712 of mean activity (Figure 1). 17 TABLE 1. Comparison of reagent absorbances using fresh reagents and 1yOphilized strips m Number of Average initial * Sample determinations absorbance A./0.609 Test Reagents Fresh 2 0.0340 5.57% Strips at 1 day 2 0.0438 7.20% Strips at 1 month 2 0.0368 6.042 Strips at 3.5 months 2 0.0917 15.052 Standard Reagents Fresh 11 0.659 t. 0.004“ 100.00% Strips at 1 week 10 0.658 1 0.020 99.80% Strips at 1 month 5 0.656 1 0.012 99.50% *5 min. A. of average normal enzymatic reaction **s.d. 18 TABLE 2. Comparison of estimates of enzyme activity (in minutes) using 3 and 5 minute color standards and a.3 minute color standard with one-half enzyme concentration m Standard Mean estimate* Standard Deviation Z precision 3 minute 3.06 0.30 22.2 5 minute 4.97 0.39' 17.5 3 minute- 1/2 enzyme 6.06 0.51 18.8 *N - 10 **ts/§ 19 TABLE 3. Comparison of reference method values with modified visual method values Sample Reference Modified 1 4.38 4.60 2 5.64 5.02 3 5.53 5.50 4 6.10 5.17 5 4.40 5.18 6 5.14 5.27 7 5.33 5.22 8 4.56 3.43 9 5.63 5.50 10 6.25 5.87 11 4.28 5.67 12 5.57 6.15 13 5.51 4.83 14 4.65 4.97 15 4.92 5.05 16 6.01 5.47 17 4.38 3.47 E 5.19 5.03 s 0.66 0.72 32 0.43 0.52 20 9 8 Time (min.) 7 4 6 B 5 A 20 25 30 35 Temperature C. A - mean activity of normal sample B - 712 mean activity Figure 1. Time of color change in relation to temperature and level of erythrocyte cholinesterase activity at mean activity of normal sample and 712 mean activity. DISCUSSION Procedures that have been developed to date for the routine screen- ing of Cholinesterase activity either measure serum activity or use aqueous reagents lacking long term stability. Although toxic exposure to the organOphosphate insecticides has a greater effect on plasma activity, regeneration of the plasma enzyme is a rapid process. The cumulative effects of repeated small exposures can be.more reliably detected when erythrocyte activity is measured since the return of this enzyme.activity to pre-exposure levels is a relatively slow process. dependent on hematOpoiesis. Decrease of 702 to 752 of original erythro— cyte activity can be tolerated before symptoms Of serious illness occur (Crab and Harvey, 1949). Therefore, a test permitting the measurement of erythrocyte activity was sought. LyOphilized reagent strips provided premeasured reagents in a form convenient for use. Stability of aqueous substrate stored at refrigera- tor temperature does not exceed 15 days. Substrate deterioration on the strips at 3.5 months contributed only a small portion Of the total absorb- ance-Of the enzymatic reaction. NO corrections were made-for substrate deterioration, nonenzymatic hydrolysis of substrate (0.0011 A./min.) or absorbance of materials other than sulfhydryl groups in the modified visual procedure. Adjustment of the standard would be required to cor- rect for these reactions and should be considered in further work. 21 22 The original procedure published by Ellman at al. (1961) measured enzyme activity of whole erythrocytes.‘ A mean value of 1.08 x 10‘15 moles of substrate hydrolyzed/min./erythrocyte was.found by this group. The addition of sterox to the phosphate buffer (pH 8.0, 0.1 M) as a cell lysing agent simplified visual and spectrOphotometric reading of enzyme activity. Assuming a mean value of 29YY of hemoglobin/erythrocyte and converting the moles of substrate hydrolyzed/min./erythrocyte from above to moles of substrate hydrolyzed/min./gm. of hemoglobin a value of 3.72 x 10'”5 moles is obtained. This compares with a value of 3.49 x 10"5 moles obtained using the modified buffer. No effect was noted on linearity of reaction. The sample of blood required for the test, 0.01 ml., can be Obtained by ear or finger puncture. NO specialized equipment is required. Enzyme activity can be estimated in less than 15 minutes with a precision of i 25%. There is a wide variation in erythrocyte cholinesterase activity from individual to individual. Levels within an individual, however, remain relatively constant, showing no variation in diurnal or seasonal studies (Callaway, Davies, and Rutland, 1951). Ideally individual base line values should be obtained prior to contact with anticholinesterase agents for comparison with later values. Practically the time calculated for 29% decrease in mean population normal activity can be used with a 5.82 index.of discrimination. Observation of the test and standard tubes can be made at 7.16 minutes and activity considered as greater than, less than, or equal to 55% of normal value. Erythrocyte-cholinesterase concentration is dependent on total ery- throcyte count. Decreased enzyme levels are usually seen only in pernicious anemia in relapse and paroxysmal nocturnal hemoglobinuria (Auditors and 23 Hartmann, 1959). Therefore, a decrease in activity cannot be attributed solely to exposure to anticholinesterase chemicals. SUMMARY AND CONCLUSIONS Repeated small exposures to the organOphosphate insecticides, result- ing in an increased susceptibility to poisoning, cause variable decreases in the activity of erythrocyte cholinesterase.* A rapid, simplified pro- cedure applicable for large scale survey testing in the detection of these small exposures was sought. The procedure developed is a modification Of the procedure of Ellman at al. and makes use of 1yophilized reagent strips. Enzyme activity is visually estimated by measuring the time required for a reaction mix- ture of whole blood, buffer, and test reagent strip to match the color of a mixture of whole blood, buffer, and standard reagent strip. Erythrocyte cholinesterase activity Of 38 male college students in moles of substrate hydrolyzed/min./gm. of hemoglobin at 25 C. was 3.49 x 10"5 i;0.41 (1 s.d.) or in moles of substrate hydrolyzed/min./ml. of blood, 5.48 x 10"6 i 0.57 (1 s.d.). Seventeen samples of blood assayed by the reference and modified visual methods had a mean and standard deviation of5.19‘i_0.66 and 5.08 i 0.72, respectively. LyOphilized test reagent strips could be used for at least 3.5 months if stored at -5 C. Standard reagent strips showed no loss of activity after 1 month of storage under similar conditions. The precision of visual estimation of enzyme activity was not affected by varying the intensity of the standard color or the rate of approach to a standard color. 24 25 Correction factors relating rate of enzyme reaction at temperatures ranging from 15—35 C. to a base temperature of 25 C. were calculated. The time of color change was related to temperature and level of erythro- cyte cholinesterase activity at the mean activity of the normal sample and 71% mean activity. A decrease in erythrocyte cholinesterase activity of 45% or more could be detected with a 5.8% index of discrimination. The method had a precision of i_25%. REFERENCES REFERENCES Adams, D. H.: The specificity of human erythrocyte cholinesterase. Biochim. BiOphys. Acta, 3, (1949): 1-14. Adams, D. H., and Whittaker, V. P.: The cholinesterases of human blood. I. The specificity of the plasma enzyme and its relation to the erythrocyte cholinesterase. Biochim. BiOphys. Acta, 3, (1949): 358-366. Alles, G. A., and Hawes, R. C.: Cholinesterases in the blood Of man. J. Biol. Chem., 133, (1940): 375-390. Auditore, J. V., and Hartmann, R. C.: Paroxysmal nocturnal hemoglobin- uria. II. Erythrocyte acetylcholinesterase defect. Am. J. Med., 27, (1959): 401-410. ’ Augustinsson, K-B.: Cholinesterases: A study in comparative enzymology. Acta Physiol. Scand., 15, Supp. 52, (1948): 1-181. Bonting, S. L., and Featherstone, R. M.: Ultramicro assay Of the cholin- esterases. Arch. Biochem. BiOphys., 61, (1956): 89-98. Callaway, 8., Davies, D. R., and Rutland, J. P.: Blood cholinesterase. levels and range of persona1.variation in a healthy adult pOpue lation. Brit. Med. J., 2, (1951): 812~816. Caraway, W. T.: Determination of serum cholinesterase activity. Am. J. Cestaric, E. S.: Cholinesterase. In Workshop on Clinical Enzymology. Technical Manual. Ed. by Henry, J. B., (1964), American Society of Clinical Pathologists. Dale, H. H.: The action of certain esters and ethers of choline and their relation to muscarine. J. Pharm. Exp. Ther., 6, (1914): Durham, W. F., and Hayes, W. J.: Organic phosphorus poisoning and its therapy. Arch. Environ. Health, 5, (1962): 21-47. Elliott, T. R.: The action of adrenalin. J. Physiol., 32, (1905): 401- 467. 26 27 Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone,.Re M.: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, (1961): 88-95. Fleisher, J. H., Woodson, G. S., and Simet, L.: A visual method for estimating blood cholinesterase activity. A.M.A. Arch. Ind. Health, 14, (1956): 510-520. Fristedt, B., and Ovrum, P.: Rapid semi-quantitative determination of cholinesterase activity in serum. Acta Med. Scand., 184, (1968): Giacobini, E.: Histochemical demonstration of AcChE activity in isolated nerve cells. Acta Physiol. Scand., 36, (1956): 276-290. Giacobini, E.: Cholinergic and adrenergic cells in sympathetic ganglia. Ann. N. Y. Acad. Sci., 144, (1967): 646-656. Gleason, M. N., Gosselin, R. E., Hodge, H. C., and Smith, R. P.: Clinical Toxicology of Commercial Products. The Williams and Wilkins Co., Baltimore, Md., 1969. Click, D.: PrOperties of choline esterase in human serum. Biochem. J., 31, (1937): 521-525. Grob, D.: The manifestations and treatment of poisoning due to nerve gas and other organic phosphate anticholinesterase compounds. A.M.A. Arch. Int. Med., 98, (1956): 221-239. Grob, D., and Harvey, A. M.: Observations on the effects of tetraethyl perphosphate (TEPP) in man, and on its use in the treatment of myastenia gravis. Bull. Johns HOpkins Hosp., 84, (1949): 5322567. Hestrin, S.: The reaction of acetylcholine and ether carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem., 180, (1949): 249-261. Holmstedt, 3.: Pharmacology of organophosphorus cholinesterase inhibi- tors. Pharmacol. Rev., 11, (1959): 567-688. de‘la Huerga, J., Yesinick, C., and POpper, H.: Colorimetric method for the determination of serum cholinestearse.‘ Amer. J. Clin. Path., 22, (1952): 1126-1133. Koelle, G. B.: Neurohumoral transmission of the autonomic nervous sys- tem. In The Pharmacological Basis of Therapeutics. The Macmillan Co., New York, 1965. Koelle, G. B., Davis, R., and Gromadzki, C. 0.: Electron microscopic localization of cholinesterases by means of gold salts. Ann. N.Y. Acad. Sci., 144, (1967): 613-622. Kremzer, L. T., and Wilson, D. B.: A partial characterization Of acetyl- cholinesterase. Biochemistry, 3, (1964): 1902-1905. 28 Krupka, Rn M.: Chemical structure and function of the active center of acetylcholinesterase. Biochemistry, 5, (1966): 1988-1998. Lewis, A. E.: Biostatistics. Reinhold Publishing Corp., New York, 1966. Limperos, G., and Ranta, K. E.: A rapid screening test for the determi- nation of the approximate cholinesterase activity of human blood. Science, 117, (1953): 453-455. Mendel, B., and Rudney, H.: Some effects of salts on true cholinesterase. Science, 102, (1945): 616-617. Michel, H. 0.: An electrometric method for the determination of red blood cell and plasma cholinesterase activity. J. Lab. Clin. Med., 34, (1949): 1564-1568. Nachmansohn, D.: Chemical mechanism of nerve activity. Ann. N. Y. Acad. Sci., 47, (1946): 395-425. Rappaport, F., Fischl, J., and Pinto, N.: An improved method for the estimation of cholinesterase activity in serum. Clin. Chim. Acta, 4, (1959): 227-230. Reinhold, J. G., Tourigny, L. G., and Yonan, V. L.: Measurement Of serum cholinesterase activity. Amer. J. Clin. Path., 23, (1953): 645- 653. Svensmark, 0.: Molecular prOperties of cholinesterases. Acta Physiol. Scand., Supp. 245, (1965): 1-74. Tabachnick, I. A.: A rapid spectrOphOtometric assay of purified cholin- esterase. Biochim. BiOphys. Acta, 21, (1956): 580-581. Wilson, I. B.: Conformational changes in acetylcholinesterase. Ann. N. Y. Acad. Sci., 144, (1967): 664-673. Wilson, I. B., and Bergmann, F.: Acetylcholinesterase. VIII. Dissocia- tion constants of the active groups. J. Biol. Chem., 186, (1950): ’ 683-692. Witter, R. F., Grubbs, L. M., and Farrior, W. L.: A simplified version Of the Michel method for plasma or red cell cholinesterase. Clin. Chim. Acts, 13, (1966): 76-78. Wright, C. I., and Sabine, J. C.: Cholinesterases of human erythrocytes and plasma and their inhibition by antimalarial drugs. J. Pharm. 29 Generalgeferences Heath, D. F.: OrganOphosphorus Poisons. Pergamon Press, London, 1961. Nachmansohn, D.: Chemical and Molecular Basis of Nerve Activity. Academic Press, New York, 1959. APPENDICES APPENDIX I Reagents Potassium dihydrogen phosphate, 0.1 M: Dissolve 13.609 gm. potassium dihydrogen phosphate in distilled water and dilute to l L. Sodium hydroxide, 0.1 M: Dissolve 4.0 gm. sodium hydroxide in dis- tilled watar and dilute to 1 L. Phosphate-sterox buffer, pH 8.0, 0.1 M: To 50 m1. of 0.1 M potassium dihydrogen phosphate add 46.1 ml. of 0.1 M sodium hydroxide. To each 100 m1. of buffer add 0.1 m1. of sterox.* Phosphate buffer, pH 7.0, 0.1 M: To 50 m1. of 0.1 M potassium dihydrogen phosphate add 29.1 ml. of 0.1 M sodium hydroxide. Acetylthiocholine iodide, 0.075 M: Dissolve 21.67 mg. acetylthio- choline iodide in 1 ml. distilled water. Dithiobisnitrobenzoic acid.(5:S-dithiobis-Z-nitrobenzoic acid, DTNB), 0.01 M: Dissolve 39.6 mg. DTNB in 10 m1. pH 7.0, 0.1 M phosphate buffer. Add 15 mg. sodium bicarbonate. Quinidine sulfate, 0.1%: Dissolve 100 mg. quinidine sulfate in dis- tilled water and dilute to 100 m1. Pa. *Sterox SE, Item #64049. Harleco. Hartman-Leddon Co., Philadelphia, 30 31 Glutathione standard, 0.00683 M: Dissolve 20.97 mg. glutathione in distilled water and dilute to 20 ml. A mixture of 3.0 m1. phosphate- sterox buffer, 0.025 ml. DTNB, and 0.02 ml. glutathione standard should have an A. of 0.609 when read at 412 nm. in a 1 cm. cuvette against a water blank. APPENDIX II Data for Index of Discrimination Index or errOr of discrimination - intersection of upper confidence limit of normal pOpulation and lower confidence limit of abnormal pOpu- lation (Lewis, 1966). Normal population Abnormal pOpulation i - 5.08 5? - 9.24“ s - 0.72 3 ' 0'72 n - 17 n ' 17 x - i + £§_+ Zs n-l VF sz *By calculation 55% of average normal activity. 32 VITA Louise Kegel Mueller was born in LaCrosse, Wisconsin, on November 11, 1933. She-graduated from St. Mary‘s Academy in June, 1949. She received her 3.8. degree in chemistry and biology from Mt. Mary College, Wauwatosa, Wisconsin, in January, 1954. She completed her professional training in medical technology at St. Mary's Hospital, Milwaukee, Wisconsin, in May, 1954. 33