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141 ~U.S. ARMY MEDICAL RESEARCH /

INSTITUTE OF CHEMICAL DEFENSE

USAMRICD-TR-90-05

N SPECIFIC RADIOISOTOPIC ASSAY FOR CHOLINESTERASE¢N

DTICI DELTIC Brian G. Talbot

SELECTE F%, Dana R. AndersonI JUL 2 !990 Larrel W. Harris

U C4'i U Willard J. Lennox

May 1990

Approved for public release; disrribunon unlimited

U. S. ARMY MEDICAL RESEARCH INSTITUTE OF CHEMICAL DEFENSEAberdeen Proving Ground, MD 21010-5425

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jPROC RAM I RJECT I TASK IWORK UNITIELEMENT No. 140N. 1ACINNo.

-_____________________________ 62302 3M26300O)I 995BA___11. TITLE (iNclud Securoty Ciamfiction)

Specific Radioisotopic Assay for Chiolinesterase

12. PERSONAL AUTHOR(S)

Talbot, B.G., Anderson, D.R., Harris, L.W., and Lennox, W.J.13s. TYPE OF REPORT 3~b TIME COVERED Bl14. DATE OF REPORT (Year. MPJ'ithDay) IlS. PAGE COUNT

Tehia FROM Dec 87 TO Mar8 May 1990 19

16. SUPPLEMENTARY NOTATION

17. COSATI CODES 16. SUBJECT TERMS,(...... ..... r a nr q...

FIELD GROUP SUB-~GROUP lAcetylcholi.nesterase,, . Physoetigznine *15 Acetylchol in., Irreversible ChE Inhibitors, "Ai

rlaaicmytric ChiE Assay Tiatr i U2hE AA*v . =[1fABSTRACT (Coftminu on rwverl if necessary and Idgemtfy by blOck numnber)

The radiomnetric method (I) for measuring ChE activity [Siakotoe et al., Biocheei. Mod. 3, 1,19693] was modif ied to preclude the use of p-dioxane, a hazardous material. Tte modif iedprocedure (II) uses 0.4 N perchioric acid (PCA), instead of p'.dioxane, to denature the ChEand stop hydrolysis of 14C-acetylcholine (ACh). The unreacted substrate (ACh) is renmomvedby cationic exchange resin suspended in W5 r The supernatant (acidic, water solution)containing the product of hydrolysis, L.C-acetic acid, is mixed with nwihazar&es -<scintillation cocktail and counted. The incubation mixture (37 ddagrt.s C) for II is

*similar' to I and is composed of 0.1 ml. of buffer-salt moolution (pH 7.p), 0.1 ml of guineapig whole blood (WB)-water suspension arnd 0.1 ml of 3m ACh solution. I Proceduree I and IIwere curIpared to a titragraphlic assay for ChE activity; specific activity values of NED

* ~ ~lesACh hydrolyzed/ml/hr) were found to be 72.4, 137.6 and 135.0, respectively. ~fC-acetic acid was processed through procedures I "n II, significantiy less (pK0.05) C

was found in the supernatant from I, whereas all df the expected C was f OurK in the

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Block 19. Con't.

supernatant of II, suggesting that resin in dioxane will renove significant a.mts of theproduct of hydrolysis; this finding may explain the observed difference in specific ChEactivity between I and II. In summary, our modified procedure II is sensitive, precise andutilizes aqueous PCA instead of p-dioxane.

FOREWORD

This research was done under protocol 1-02-86-004-A-389, entitled"Measurement of Inhibitory Potency of Novel AnticholinesteraseCompounds using Whole Blood or Erythrocyte Acetylcholinesterasefrom Several Species," under Project No. 3M263002D, Task AreaNo. 995BA. We acknowledge the participation of SPC Glen Jacksonand Karl Ford *in this project.

OBJECTIVES AND LOCATION OF DATA

Objective: The objective of these experiments was to establish asensitive and precise method for measuring cholinesteraseactivity that would preclude the use of the hazardous solventp-dioxane.

Location of data: The data are located in notebook 019-88 and infile folders held by L.W. Harris, D.R. Anderson and W.J. Lennox.

,2A

NTIS CRA&A~OTIC TA EUrdnnoC r "

Justit•cjlor t

PyYOist' D b" ,

i~i

TABLE OF CONTENTS

Page

LIST OF TABLES AND FIGURES ................................. ovii

INTRODUCTION.... o.......... ............................... 1

MATERIALS AND METHODS ........................... ......... 1

RESULTS .............. ................. .................... 4

DISCUSSION .................................. 5...........5

REFERENCES ........................ 11

DISTRIBUTION LIST.. .. ...................................... 13

V

LIST OF TABLES AND FIGURES

Tables Page

1. Effect Cf 0.1 N Perchlc�ric Acid Hydrolysis of 14C IAcetyicholine............................................. 7

2. Comparative Binding of 14C Acetylcholine by IRP-69Cationic Exchatige Resin.............................. 7

3. Comparative Binding of � Acid (14C-Acetylcholine Hydrolysate) by IRP-69 CationicExchange Resin............................................ 8

Three Assay Methods.........................................84. Comparison of Specific Activity Estimates for Guinea

Pig Whole Blood Acetycholinesterase Obtained by

Figures

1. comparative Effects of IRP-69 Resin in Water and inDioxane on Radiometric Assay. Estimates of CholinesteraseActivity of Guinea Pig Blood Inhibited by PhysostigmineSalicylate...................................................9

2. Comparative Effects of IRP-69 Resin in Water and inDioxane on Radiometric Assay Estimates of CholinesteraseActivity of Guinea Pig Blood Inhibited by S-2-NN-diethyl -N-methylammonioethyl-o-pinacolylmethylphcsphor.othioate methylsulfate, (DMP�O4)...............10

vii

INTRODUCTION

Since the early 1960s, several radioisotopic assays have beendescribed for determination of cholinesterase (ChE) activity. 1 "6

In 1968, McCaman et Al.6 described a sensitive radiometricprocedure capable of measuring ChE activity in microliter ormilligram samples of tissue; in the procedure 14C-acetic acidfrom the hydrolysis of the substrate, acetyl- -1C-choline (ACh),is measured and Vie unhydrolyzed substrate is quantitatively,removed ?s an insoluble Reinecke salt. In two of the .arliermetbods, ' ion exchange resin in ethanol was used to remove theunreacted substrate.

In 1969, Siakotos A al.; reported further improvement of theradiometric procedure. In this improvement, unreacted substrate,ACh, is removed on Amberlite CG-120 resin suspended in p-dioxane,and according to the authors, the blanks are lower and the methodis more precise than earlier methods. As in the earlier methods,the hydrolysis product (free, 1 'C-labeled acetic acid in thesupernatant solution) is measured in a liquid scintillationcounting system.

The method of Siakotos et Al.7 has been used extensively byour institute° and by scientists elsewhere. 11' 12 We have foLndthe procedure to be simple. precise in estimating relative ChTinhibition and reproducible among assayists. However, the use ofp-dioxane in the method poses some problems in storage, hanalingand disposal. Foremost, p-dioxan 1e is considered a carcinogen anda hazardous material by the EPA.'14 As a result, procedures fordisposal of radioactive waste materials contaminated with p-dioxane are becoming complex and expensive. For these reasons,we have recently developed a radioretric ChE assay using water assolvent and a nontoxic, nonhazardous, nonflammable andbiodegradable cocktail for scintillation counting. The purposeof the study reported herein was to compare our radiomatric ChEassay to the radiometric method of Siakotos "• A.17 usingAmberiite IRP-69 resin, and each of these radiometric methods tothe titragraphic method1 5 (TG).

MATERIALS AND METHODS

Materials. Physostigmine salicylate (Phy) was supplied by theWalter Reed Army Institute of Research, Washington, DC. Theanticholinesterase S-2-N,N-Diethyl-N-methylammonioethyl-O-pinacolyl methylphosphonothioate methylsulfate (DMPMM) wassynthesized under contract by Ash-Stevens Inc., IRP-69 resinwas purchased from Rohm & Haas, Philadelphia, PA; chemically itis identical to CG-120 resin, but the mean particle size islarcer. The scintillation cocktail (Ecolume') was purchased fromICN Radiocnemicals, Irvine, CA. The substrate acetyl-l- C-choline iodide (2.4 mCi/mmole) was obtained from New England

•'I:-'L• • ° •.• : • :, - • ,:r-•• ': ' ,•• :) .• • • ,,,,w,. .,..• • 1 III • I • ,,, . . ...... 1.

Nuclear Cop., Boston, XA, while p-dioxane was obtained from J.T.Baker Chemical Co., Phillipsburg, NJ. Perchioric acid (70% w/w)was purchased from EM Science, Cherry Hill, NJ. Anticoagulant-treated guinea pig whole blood, erythrocytes therefrom, and eelAChE were used as the sources Af ChE. Blood was obtained fromguinea pigs under protocol. -02-86-004-A-389;ethe blood was drawnvia cardiac puncture, under ether anesthesia, as per DF, dated 27April 1986,t to chairoan, Laboratory Animal Care and UseCommittee. The seLuice was performed by veterinarians of theVeterinary Medicine and Laboratory Resources Division. The eelAChE in solution (1000 units/mg protein) was obtained from SigmaChemical Co., St Louis, MO; Hartley strain guinea pigs of bothsexes were obtained from Charles River Laboratories, Kingston,NY.

Mthods. In preparation for the assays, ansmlurry of the IRP-69resin was prepared by mixing 20 g with sufficient water ordioxane to make 100 ml. The 4 tC-ACh was dissolved in 1 mMacetate buffer at pH 4.5; sufficient unlabeled ACh chloride wasadded to give a total ACh concentration of 3 mM and an activityof 1 gCi/ml of solution. The stock solution of ACh wassubdivided into 20 ml aliquots and stored at -200C. A sample Wasthawed and stored on ice for use during an enzyme assay; the AChwas returned to the freezer at the end of the assay. Eachsampling of enzyme activity was performed in duplicate, and themean of the pair of values was calculated; then value in eachtable and figure herein indicates the number of such duplicatesamplings performed.

The two radiometric methods and the TG method were run inparallel using guinea pig whole blood to compare specific ChEactivities; Also, two inhibitors of ChE were used to test the

capability of each radiometric procedure to quantitate ChE

inhibition; the inhibitors were the carbamate Phy and DMP!, an&organophosphorat anticholinesterase. Fresh stock solutions ofDMPMM (0, 0.1, 0.25, 0.5, 1, 1.5 and '2 x 10',M) or Phy (0, 0.025,0.05, 0.1, 0.25, 0.5,and 2 x 10-"M) were prepared daily in salineand stored on ice; ten Ml of each concentration .of inhibitorsoluition was added to 1 ml por'tions of whole blood, mixed, andincubated for 30 min at 37*C. The samples were then removed fromthe incubator and placed on ice. Immediately thereafter, fouraliquuts (25 Al each) of inhibited blood wvere removed from each,sam~ple and placed into tubes, each containing 75 Al of twicedistilled water, 100 4l of 0.1 M PO, buffer (pH 7.8, containing0.3 M NaCl) and 100 gl of 14 C-ACh stock solution, for ChE assayby the two radiometric methods. Following addition of the'.Labeled ACh, each tub-! was jincubated for .aither 5 (perchloricacid p'xicedure) or 7 (dioxane -rocedure) min at 37*C; theseincubation t-,'~es yielded equal hydrolysate activity,approximately 10,000 dpm. Hydrolysis of substrate was then;topped by the addlition of either 100 gl of 0.4 N perchloric acid(?CA) foli,ýwed by sufficient water/IRP-69 resin to make 5 ml or

2

dioxane/IRP-69 resin to make 5 ml. Tubes were then brought to 10ml with additional water or dioxane and centrifuged; two ml (PCAmethod) or 5 ml (dioxane method) was transferred into 10 ml ofscintillation fluor for counting. Parallel blanks (no enzyme)were run to correct fr spontaneous hydrolysis of substrateduring the incubation period.

In preparing erythrocytes for enzyme assay, inhibited wholeblood samples were e&ch washed with 10 ml of cold saline,followed by centrifugation for 5 min at 2,300 x q (3,000 rpm inan IEC, DPR-6000 centrifuge). After removing the supernatant, 15pl aliquots of sedimented erythrocytes were removed, combinedwith 85 gl of twice distilled water, and assayed for ChE activityusing the two radiometric methods described above.

Preliminary comparisons of the two methods. revealed thathigher dpm values were found in supernatant solutions fromsamples treated with PCA, suggesting that PCA might behydrolyzing the ACh. This possibility was tested, at roomtemperature, by preparing 20 tubes, each with 0.3 ml ofbuffer/water/ 1C-ACh stock substrate (100 Alof each component).At 20-sec intezcvals, 100 Ml of 0.4 N PCA (0.1 N finalconcentration) or water was added. Immediately after treatingthe last sample, 5 ml of water/resin was added to each of thefirst four samples, and they were further processed for countingas described above. The process was repeated on additional setsof four, at 5-min intervals, until all 20 samples were processed.'

Another possible reason for higher counts in PCA-treatedsamples is that the functional moieties of the resin might beinteracting differently with the 1C-labeled ions depending uponthe suspending medium, dioxane or water. To investigate thishypothesis, two 6-ml preparations of water/ "C-ACh were made.The first preparation contained 4 ml of water and 2 ml of the

4C-ACh stock solution; it was incubated for 30 min at 37"C.Each of five 100 oil aliquots was transferred to a scintillationvial containing 10 ml of fluor for measurement of total dpm. Ten0.3 ml aliquots were placed into tubes; five ml of dioxaneiresinslurry was added to each of 5 tubes, while 100 pl of 0.4 N PCAsolution followed by enough water/resin slurzy to make 5 ml wasadded to each of the other 5 tubes. Each of the 10 tubes wasbrought to 10 m! with the appropriate vehiclG, dioxane or water,mixed and centrifuged as before; two ml (each PCA tubs) or 5 ml(each dioxane tube) of supernatant solution w-ma put into 10 ml1 offluor and counted.

The second 6-ml preparation was made by adding 50 al of eelAChE solution to the tube, adding sufficient water to make 4 mland then adding 2 ml of 14C-ACh stock solution; this tube wasalso incubated for 30 min at 37"C. (The eel AChE hydrolyzesvirtually all of the ACh.) The contents were then subdivided andfurther treated as stated above for the first preparation.

3t

The specific activitý of ChE can be accurately measured bythe titragraphic method; the automated titragraphic equipmentmeasures and adds the amount of KOH (0.01 M) solution required tomaintain a constant pH durinn the course of the reaction. Thismethod was used as the standard for comparison of ChE activities.Briefly, the system was ca1irated at 370C using National Bureauof Standards buffers of pH 4100 and pH 7.00. The set point wasmaintained at pH 7.8 by the addition of 0.01 N KOH solution. The *normality of the base was de ermined by titration of 100 ;&1 of0.01 N potassium phthalate s lution. The substrate was I;acetylcholine Cl (1 mM) in s line. The substrate solution wasadmitted to the reaction ves el, and 45 M1 of 0.01 N KOH solutionwas added to bring the subst aate closer to the set pH of 7.8,followed immediately by the ddition of 20 g1 of guinea pig wholeblood. The rate of hydrolys s was measured for 7 min, but thefirst 4 min were used to sta ilize the instrument at the set pH;only the last 3 min of the h rolysis curve, which was linear,were used to determine the s ecific AChE activity.

A repeated measures anal sis of variance was used to testfor significant differences 1etween the two radiometric methods,between the two tissues and among the six concentrations ofinhibitor, as well as for thl interactions among these factors.A one-way analysis of variance followed by Newman-Keuls testingof the means was used to evaluate differences among all threemethods used to measure ChE activity.

RESULTS

Figure 1 illustrates the effects of various concentrations ofPhy on guinea pig whole blood and erythrocyte ChE activity usingthe water/resin and dioxane/ esin systems for the removal ofunhydrolyzed ACh; Figure 2 atso represents a similir study inwhich DMPMM, also an irreversible, but reactivatable inhibitor of I!ChE, "las used. Analysis of .he data revealed no significantdiffe:rence between tne two methods for either inhibitor. Thedata :'n Figure 2 also reveallthat DMPMM inhibited erythrocyte ChEmore than whole blood ChE. I

The data in Table 1 demolstrate that, when compared withwater, 0.1 N PCA does not significantly hydrolyze ACh during the20-min test period. This te~t period was chosen because it isexpected that in future enzyme assays all samples will receiveresin ro later than 20 sim a ' ter addition of PCA.

Table 2 shows that both .he water/resin and dioxape/resinsyvtems were quite efficient in binding unhydrolyzed 4C-ACh.

'Asuming the same totai dpm with and without resin, percentbound - (dpm without resin - dpm with resin) + (dpm withoutresin) x 100.) There wag only a slight difference, 3.4, inpercent of added radioactivity bound by resin. However, under

4

the reasonable assumption that the radioactivity of unboundmaterial in supernatant solution is that of C-acetic acid fromthe spontaneous hydrolysis of labeled ACh, such radioactivityfrom the dioxane/resin system was only 5,958 + 14,400 - 0 41times that from the PCA-water resin system. The date in Table 3may provide an explanation; these data revnal that the. acidfollowed by water/resin bound virtually no 4C, while significant(p<O0.05) binding occuried with dioxane/resin. The ratio of dpmfor dioxane/resin to dpm for acid followed by water/resin inTable 3 is 126,555 + 214,296 - 0.59, which is roughly similar tothat in Table 2, or 0.5 overall.

A comparison of the TG assay with the two radiometric methodsfor determination of specific ChE activity of guinea pig wholeblood is presented in Table 4. The specific ChE activity by theacid radiometric procedure was virtually identical to that by theTG method, whereas the activity using the dioxane/resin methodwas significantly lower (p<0.05).

DISCUSSION

The radiometric procedure for determination of ChE activityreported here differs from that of Siakotos At Al.7' in tworespects. First, the reaction is stopped by using perchloricacid before the resin to denature the ChE instead of bycoincident denaturation of ChE by p-dioxane and adsorption ofunreacted substate by rsin. Second, water instead of p-dioxaneis used as the vehicle for suspension of the resin. These stepshave completely eliminated p-dioxane, a hazardous material andpotential carcinogen, from the assay and thereby resolved thedisposal problem.

When the two methods were run in parallel to estimate ChEinhibition caused by various concentrations of Phy or DMPMM, nosignificant differences (p>0.05) in relative inhibition wereobserved (Figures 1 & 2). However, it was found thatdioxane/resin removed a significant amount of the product ofhydrolysis, "C-]abeled acetic acid, frob the supernatant (Table3). because che ratios of the dpm values (resin in dioxane/resinin water) with slight (Table 2) and essentially completehydrolysis of the C-labeled substrate (Table 3) were roughlysimilar, the relative ChE inhibition values determined by the twomethods were similar, as expected, using Phy or DMPMM. However,nonspecific remoal of the acetic acid by dioxane/resin presentsproblems when comparing specific activities by the differentmethods (Table 4). Note that the specific ChE activity usingdioxare/resin is only 53% of that obtained with acid followed bywater/'esin or with the standard TG method.

In summery, a different radiometric method for measuri.ig ChE

actility is presented. The method utilizes perchloric acid to

5

stop the enzymatic reaction and a water slurry of IRP-69, 14cationic exchange resin to remove the unreacted substrate ( C-ACh). The aqueous Isupernatant containing the product ofhydrolysis (free, C-labeled acetic acid) is mixed with anontoxic, nonflammable, biodegradable scintillation cocktail forcounting. The method is recommended for routine assays toobviate the use of dioxane.

•I6

Table 1

Effect of 0.1 N Perchloric Acid on Hydrolysis of "CAcetylcholine

Contact Condition in Aqueous Solution*Time (min) Acid Present Acid Absent

Mean DPM S Mean DPM 6

5 2633 650 2853 7810 2913 64 2981 25415 2977 17 2940 33520 3033 18 3078 49925 2995 20 2872 192

Room temperature, 25"C; 0.75 mM (final) 1'C-ACh (0.25 1sCi/al);for each condition and contact time, a pair of samples wasremoved from thelinitial preparation and counted.6 - within pair difference in DPM.

Table 2

Comparative Binding of ".C Acetylcholine by IRP-69Cationic Exchange Resin

Method DPM (mean + s.d.) Percent Bound(n-5)Perchloric Acid-WaterWith Resin* 14400 + 542 94.1Without Resin 242397 + 30939

DioxaneWith Resin 5958 + 145 97.5Without Resin 242327 + 16908

* n-4

7

Table 3

Comparative Binding of 14C-Acetic Acid (14C-Acetylcholine*Hydrolysate) by IRP-69 Cationic Exchange Resin

Suspending Medium DPMMean ± s.d. (n-5)

None (No Resin) 215660 ± 4992

Perchloric Acid-Water 214496 ± 4981

Dioxane 126555'± 1769

* The ACh was completely hydrolyzed by eel acetylcholinesterase(200 gl eel AChE solution, 15 mmoles/ml/hr) before measurement ofthe radiolabel.

Table 4

Comparison of Specific Activity Estimates for Guinea Pig WholeBlood Acetylcholinesterase Obtained by Three Assay Methods

Specific ActivityMethod mmoles of ACh hydrolyzed/ml WB/hr

mean ± s.d. (n-12)

Radiometric (pH 7.8)Resin in Dioxane 72.4* ± 6.1Resin in Water 137.6 + 12.0

Titragraphic (pH 7.$) 135.0 ± 12.8

* Differs significantly, p < 0.005, from the means of the othertwo methods.

0

*CL0x0

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0-wJ 0

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0 0 0 0 0 0o

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10

REFERENCES

1. Winteringham, F.P.W., and Disney, R.W., Nature 195, 1303(1962).

2. Winteringham, F.P.W., and Disney, R.W., Biochem. J. 91, 506(1964).

3. Reed, D.J., Goto, K., and Wang, C.H., Anal. Biochen. 16, 59(1966).

4. Frady, C.H., and Knapp, S.E., J. Parasit. 53, 298 (1967).5. Potter, L.T., J. Pharmacol. Exp. Therap. 156, 500 (1967).6. McCaman, M.W., Tomey, L.R., and McCaman, R.N., Lift Sci. 7,

233 (1968).7. Siakotos, A.N., Filbert, M., and Hester, R., Biochem. Med. 3,

1 (1969).8. Harris, L., Talbot, B., Anderson, D., Lennox, W., and Green,

M., Proc. West. Pharmacol. Soc. 28, 281 (1985).9. Lennox, W.J., Harris, L.W., Talbot, B.G., and Anderson, D.R.,

Life Sci. 37, 793 (1985).10. Harris, L.W., Talbot, B.G., Anderson, D.R., Lennox, W.J., and

Green, M.D., Life Sci. 40, 577 (1986).11. Clement, J.G., Biochem. Pharmacol. 32, 1411 (1983).12. Clement, J.G., Biochem. Pharmacol. 33, 683 (1984).13. Second Annual Report on Carcinogens. NTP 81-43, 131-133

(1981), in The Merck Index, 10th Ed., 1983, p.482.14. Table 302.4-List of Hazardous Substances and Reportable

Quantities, p.127 in 40 CFR Ch. 1 (7-1-88 Edition).15. Einsel, Jr., O.W., Trurnit, H.J., Silver, S.D., and Steiner,

E.C., Anal. Chem. 28(3), 408 (1956).16. Ash-Stevens Inc., Detroit, MI 48202. U.S. Army Medical

Research and Development Command Contract No. DAMD 17-84-C-4235 (1984).

17. Jennrich, R., Sampson, P., and Frane, J., Analysis ofVariance and Covariance Including Repeated Measures, in BMDPStatistical Software (W.J. Dixon, Chief Ed.), pp. 359-387.University of California Press, Berkeley, CA. (1983).

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