This paper presents a protocol for routine assays of human blood cholinesterase activities which separates erythrocytes from plasma by centrifugation and measures acetylcholinesterase activity in unwashed erythrocytes and butyrylcholinesterase activity in the plasma. The recommended substrate for both enzymes is 1.0 mM acetylthiocholine. The protocol is compared with other two recommended protocols for the activity measurements of the two enzymes using the Ellman method. The paper discusses the advantages and disadvantages of each and concludes with a proposal for an international agreement between laboratories for the evaluation of a standardized protocol.Mjerenjem aktivnosti acetilkolinesteraze i butirilkolinesteraze u krvi može se u osoba izloženih organofosfornim spojevima i karbamatima odrediti apsorpcija tih spojeva. Za mjerenje aktivnosti butirilkolinesteraze u ljudskoj plazmi postoji standardizirani protokol usvojen u velikom broju kliničkih i toksikoloških laboratorija, dok za mjerenje aktivnosti eritrocitne acetilkolinesteraze ima nekoliko predloženih protokola, ali ni jedan još nije opće privaćen. Zajednički princip svih predloženih protokola je spektrofotometrijska Ellmanova metoda, koja za mjerenje aktivosti kolinesteraza rabi tiokolinske supstrate. Autori ovoga rada predlažu jednostavni protokol u kojem se zasebno mjere aktivnosti u plazmi i u eritrocitima, koji nisu prani, a koji se odvoje centrifugiranjem. Aktivnost u eritrocitima mjeri se nakon hemolize eritrocita smrzavanjem. MjerI se samo s 1,0 mM acetiltiokolinom. Drugi autori mjere aktivnost u punoj krvi koristeći se dvama supstratima (acetiltiokolinom i butiriltiokolinom), koji se u hemoliziranu krv dodaju sukcesivno i nakon svakog dodavanja supstrata mjeri se aktivnost. Neki autori predlažu uporabu etopropazina, specifičnog inhibitora butirilkolinesteraze, pa se mjerenje vrši u prisutnosti i odsutnosti toga inhibitora. U ovome su radu opisane prednosti i nedostaci pojedinog protokola te izvori mogućih pogrešaka u predloženim postupcima. Za usvajanje standardiziranog protokola za mjerenje aktivnosti eritrocitne kolinesteraze potreban je dogovor autora predloženih protokola nakon čega bi trebala uslijediti međulaboratorijska provjera toga protokola

Elsa Reiner

Goran Šinko

Mira Škrinjarić-Špoljar

Vera Simeon-Rudolf

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13Arh Hig Rada Toksikol 2000;51:13–18COMPARISON OFPROTOCOLS FORMEASURING ACTIVITIESOF HUMAN BLOODCHOLINESTERASES BYTHE ELLMAN METHODElsa REINER, Goran [INKO, Mira[KRINJARI]-[POLJAR, and VeraSIMEON-RUDOLFInstitute for Medical Research andOccupational Health, Zagreb,CroatiaReceived February 2000ORIGINAL SCIENTIFIC PAPERThis paper presents a protocol for routine assaysof human blood cholinesterase activities whichseparates erythrocytes from plasma bycentrifugation and measures acetylcholinesteraseactivity in unwashed erythrocytes and butyrylcho-linesterase activity in the plasma. Therecommended substrate for both enzymes is 1.0 mMacetylthiocholine. The protocol is compared withother two recommended protocols for the activitymeasurements of the two enzymes using theEllman method. The paper discusses theadvantages and disadvantages of each andconcludes with a proposal for an internationalagreement between laboratories for the evaluationof a standardized protocol.Key words:acetylcholinesterase, butyrylcholinesterase,human erythrocytes, human plasmaAbsorption of organophosphorus compounds or carbamates is commonly assessedby measuring the decrease in acetylcholinesterase (AChE; EC 3.1.1.7) or butyrylcho-linesterase (BChE; EC 3.1.1.8) activities in human blood. The primary toxic effect oforganophosphates and carbamates is inhibition of AChE. However, many compoundsbetter inhibit BChE than AChE , in which case BChE is a better indicator of absorp-tion. As there is no general rule to predict which enzyme will be more inhibited by agiven compound, one should measure the activity of both cholinesterases in humanblood in order to detect the absorption of inhibitors more reliably.Erythrocyte AChE activities are usually measured in whole blood, and the mostcommonly used substrate is acetylthiocholine (ATCh). As whole blood also containsBChE, the rate of substrate hydrolysis in whole blood depends on the activity of bothenzymes. The procedure for determination of BChE activity in human plasma orserum is well established, while this is not the case with the erythrocyte AChE.In this paper we propose a procedure based upon separation of plasma fromerythrocytes, followed by separate measurements of BChE in plasma and AChE inunwashed erythrocytes. The procedure has been prepared for presentation at theThird Chemical and Biological Medical Treatment Symposium (1, 2).14 Reiner E, et al.: PROTOCOLS FOR CHOLINESTERASE ACTIVITY MEASUREMENTSArh Hig Rada Toksikol 2000;51:13–18EXPERIMENTAL PROCEDURE AND CONDITIONSThe activities of AChE and BChE were measured by the spectrophotometric methodof Ellman and co-workers (3).Venous blood was collected into dried heparinised tubes. After blood centrifuga-tion, plasma was separated from the erythrocytes. In that step it was essential towithdraw all plasma without withdrawing any erythrocytes. The erythrocytes were thensuspended in deionised water in a volume corresponding to the initial volume of thewhole blood, diluted 60-fold with a buffer (0.1 M phosphate buffer, pH=7.4), andthen frozen for haemolysis. After thawing, the suspension was further diluted with thebuffer (9-fold) and the thiol reagent 5,5'-dithio-bis(2-nitro benzoic acid) (DTNB) wasadded. The final DTNB concentration during the enzyme assay was 0.33 mM. Tenminutes after the addition of DTNB, the substrate ATCh was added (the final ATChconcentration during the enzyme assay was 1.0 mM) to the suspension and theincrease in absorbance was read at 412 nm against a blank containing haemolysederythrocytes suspended in buffer. The BChE activity in plasma was also measuredwith 1.0 mM ATCh using the same buffer and DTNB concentration. The final assayvolume for both enzymes was 3.0 ml. Stock solutions of ATCh (100 mM) were pre-pared in deionised water whereas further dilutions were made in the buffer. Stocksolutions of DTNB (10 mM) were prepared in the buffer. During the enzyme assay,the final dilution of erythrocytes was 600-fold and that of plasma 150-fold. The en-zyme activities were measured at 25 oC or 37 oC.The concentration of haemoglobin in the erythrocyte suspension was determinedspectrophotometrically with a modified Zijlstra reagent containing potassium ferricya-nide, potassium cyanide, sodium bicarbonate, and Triton X-100 (4). The final reac-tion volume was 1.5 ml and the final erythrocyte dilution was 120-fold. The absor-bance was read at 546 nm against buffer at room temperature.The activities of AChE were expressed as micromoles of hydrolysed ATCh perminute and per milliliter of whole blood, or per milligram haemoglobin or per micro-mole iron. The activities of BChE were expressed as micromoles of hydrolysed ATChper minute and per milliliter of plasma. The molar coefficient of absorbance used in thisstudy for the thiolate anion of the 5-thio-2-nitro benzoic acid was 13600 M-1 cm-1 (3)and for haemoglobin 10800 M-1 cm-1 (4 ).RESULTS AND DISCUSSIONEvaluation of the suggested protocolThe described experimental conditions concerning the choice of the buffer and sub-strate were evaluated in an earlier study concerning routine measurements of cho-linesterase activities in the human whole blood and plasma (5). The phosphate bufferwas chosen because the effect of temperature on the pH of that buffer is small andthe buffer can therefore be prepared at the room temperature and used at a temper-ature ranging from 10 oC to 40 oC. ATCh is a suitable substrate for both AChE and15Reiner E, et al.: PROTOCOLS FOR CHOLINESTERASE ACTIVITY MEASUREMENTSArh Hig Rada Toksikol 2000;51:13–18BChE. For routine purposes it is convenient to use the same substrate concentration(1.0 mM) for both enzymes. At 1.0 mM of ATCh, AChE has its optimal activity andBChE about half of its maximum activity. The rate of spontaneous hydrolysis of 1.0 mMATCh at pH=7.4 is slow and corrections for the non-enzymic substrate hydrolysis arerequired only at 37 oC.The optical density of the haemolysed unwashed erythrocytes measured at 412 nmis not stable. The absorbance measured against buffer decreases about 0.006 absor-bance units over 10 min at 25 oC or 37 oC (mean value of 12 measurements onerythrocyte samples from 7 individuals). That decrease is very small and need not betaken into account, particularly if the activities are measured against suspended haemolysedunwashed erythrocytes as a blank.DTNB reacts not only with thiocholine, but also with thiol groups in the haemolysederythrocytes. Under the above experimental conditions, the reaction between DTNBand thiol groups in the haemolysed erythrocytes is completed within 10 min, and thisis why it is suggested to add the substrate 10 min after adding DTNB. The reactionbetween DTNB and thiol groups in the erythrocytes reduces the DTNB concentrationby only about 0.5% (mean value of 20 measurements on erythrocyte samples from10 individuals).The suggested procedure is well reproducible. The within-run and between-runimprecision for the activity measurements of AChE in unwashed haemolysed erythro-cytes was 2% each, and for the haemoglobin determination 1% each. The corre-sponding imprecisions for plasma BChE activity measurements were 1–3 and 6–7%(6 and this paper).Comparison with protocols suggested by other authorsSeveral procedures have been suggested so far to measure the erythrocyte AChE inthe whole blood. Worek and co-workers (4) suggest that the AChE activity be mea-sured in the presence of 20 µM ethopropazine in order to inhibit plasma BChE in thewhole blood. Wicki (7) and Portmann (8, 9) suggest that both AChE and BChE bemeasured in the whole blood by successive addition of ATCh and butyrylthiocholine(BTCh). Reiner and co-workers (5, 10) assessed the erythrocyte AChE activity bymeasuring the rate of hydrolysis of 1.0 mM ATCh in the whole blood, as under theseconditions the rate of hydrolysis primarily depends on the erythrocyte AChE. Finally,one can separate erythrocytes from plasma by centrifugation, wash the erythrocyteswith saline or buffer, and measure AChE activities in washed erythrocytes as describedby Ellman and co-workers (3). Our procedure outlined in this paper also separateserythrocytes from plasma, but AChE is measured in unwashed erythrocytes.The following is an assessment of advantages and disadvantages of each of theabove protocols.Unwashed erythrocytes might contain some residual plasma. In order to testwhether BChE in the residual plasma affects the AChE activity in unwashed erythro-cytes, we applied ethopropazine as a potent BChE inhibitor. Ethopropazine (20 µM)inhibited the hydrolysis of 1.0 or 0.5 mM ATCh by 8.8% in unwashed erythrocytes andby 8.5% in washed erythrocytes (mean values of 16 measurements on samples from6 individuals). The difference between inhibition of washed and unwashed erythro-cytes is very small and indicates that BChE in the residual plasma did not show up16 Reiner E, et al.: PROTOCOLS FOR CHOLINESTERASE ACTIVITY MEASUREMENTSArh Hig Rada Toksikol 2000;51:13–18in the assay, because otherwise the inhibition in unwashed erythrocytes would behigher than in washed erythrocytes. We therefore think that it is not necessary towash erythrocytes before the AChE assay, particularly because washing might causehaemolysis. The above result further indicates that the 8–9% inhibition measured inerythrocytes must be attributed to the inhibition of AChE, which is in agreement withthe 5% inhibition of AChE reported by Worek and co-workers (4).We further tested the proposed protocol by measuring the cholinesterase activity ofthe whole blood with 1.0 mM ATCh without separating the erythrocytes from plasma.Knowing the haematocrit, the cholinesterase activity of the whole blood sample wascalculated as the sum of separate measurements of AChE in unwashed erythrocytes andBChE in plasma. The mean values of the measured and calculated activities in samplesfrom 15 individuals were in very good agreement: 7.16 and 6.97 µmol min-1 ml-1, respec-tively.We think that our protocol is suitable for routine assays, particularly because wesuggest to measure both enzymes with the same substrate (ATCh) and with the samesubstrate concentration (1.0 mM). The protocol, however, requires centrifugation ofwhole blood and careful separation of plasma from erythrocytes. This is a disadvan-tage for the separation of stored blood samples which are usually haemolysed.Worek and co-workers (4) suggest an AChE assay in whole blood with ATCh assubstrate, but in the presence of ethopropazine (20 µM) in order to inhibit BChE.When ethopropazine is applied as a selective inhibitor of plasma BChE one shouldbear in mind that the usual BChE phenotype is 98% inhibited with 20 µM ethopro-pazine, while the atypical BChE phenotype is inhibited only 74%, and the heterozygotsof the atypical variant between 94 and 85% (11). The occurrence of BChE pheno-types other than the usual is rare in Caucasian populations and the activities of BChEphenotypes other than UU are much smaller than the UU activities (12–14). Theerrors in estimated AChE activities due to the presence of BChE phenotypes otherthan UU will therefore be small and will rarely occur. Worek and co-workers (4)further suggest that plasma BChE is measured with BTCh as a substrate. Conse-quently, determination of AChE and BChE suggested by Worek and co-workers (4)requires two different substrates, one selective inhibitor, and centrifugation of thewhole blood to obtain the plasma.In the procedure of Wicki (7) and Portmann (8, 9) AChE and BChE are bothmeasured in the whole blood. The activity of both enzymes is first measured with ATCh(1.0 mM) after which BTCh (5.0 mM) is added to the same cuvette and the activitymeasurement continued. The calculation of AChE and BChE activities is based on theassumption that BTCh is hydrolysed only by BChE and that the addition of BTCh stopsthe hydrolysis of ATCh. The advantage of this protocol is that it does not require bloodcentrifugation. One should, however, bear in mind that BTCh is not fully specific forBChE, but is also hydrolysed by AChE, although at a considerably slower rate. Weobtained the following mean activities of washed erythrocytes in samples from 5 indi-viduals: 4.35 µmol min-1 ml-1 measured with 1.0 mM ATCh and 0.31 µmol min-1 ml-1measured with 5.0 mM BTCh.The AChE activities can be expressed either per volume of whole blood, per hae-moglobin content, or per iron content. We measured the AChE activities with 1.0 mMATCh in unwashed erythrocytes obtained from 15 individuals. The mean activities (±SD)were 5.44±0.68 mmol min-1 ml-1BLOOD and 0.661±0.080 µmol min-1 µmol -1IRON.17Reiner E, et al.: PROTOCOLS FOR CHOLINESTERASE ACTIVITY MEASUREMENTSArh Hig Rada Toksikol 2000;51:13–18This means that the relative standard deviations of the mean were 12–13% irrespectiveof how the activities were expressed. However, the mean haemoglobin (iron) con-centrations in these 15 samples were 8.82 µmolIRON ml-1 in the whole blood and8.28 µmolIRON ml-1 in unwashed erythrocytes. The difference is small, but it mightpoint to a slight loss of haemoglobin due to withdrawal of plasma following cen-trifugation of blood.CONCLUSIONA standardised protocol for measuring AChE and BChE activities in human blood isneeded. That protocol should be simple in technical terms and should give reliableand well reproducible results. Each of the above described protocols has its ownadvantages and disadvantages. The protocol of choice should be based on an inter-national agreement between laboratories following an interlaboratory quality controlstudy on samples over a wide range of AChE and BChE activities in the whole blood.REFERENCES1. [krinjari}-[poljar M, [inko G, Reiner E, Simeon-Rudolf V. Acetylcholinesterase and butyrylcho-linesterase activity measurements in human blood by the Ellman method: I. Evaluation ofexperimental conditions. In: Technical Program of the Third Chemical and Biological Treat-ment Symposium; 2000 May 7–12; Spiez, Switzerland. Abstract No. 19.2. Reiner E, [krinjari}-[poljar M, [inko G, Simeon-Rudolf V. Acetylcholinesterase and butyrylcho-linesterase activity measurements in human blood by the Ellman method: II. Procedure proto-col and comparison with procedures suggested by other authors. In: Technical Program of theThird Chemical and Biological Treatment Symposium; 2000 May 7–12; Spiez, Switzerland.Abstract No. 17.3. Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determi-nation of acetylcholinesterase activity. Biochem Pharmacol 1991;7:88–95.4. Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination of acetylcholinest-erase activity in human blood. Clin Chim Acta 1999;288:73–90.5. Reiner E, Bunti} A, Trdak M, Simeon V. Effect of temperature on the activity of human bloodcholinesterases. Archiv Toxicol 1974;32:347–50.6. Simeon V. Mjerenje aktivnosti serumske kolinesteraze: usporedba komercijalnih i vlastitih test-re-agencija, enzimski standardi i numeri~ka obrada rezultata [Measurements of serum cholinesteraseactivity: Comparison of commercially available assay kits and laboratory prepared reagents, enzymequality control standards and the imprecision of the method, in Croatian]. Arh Hig Rada Toksikol1989;40:183–9.7. Wicki, A. Monitoring of acetylcholinesterase and butyrylcholinsterase in human whole blood.In: Proceedings of the Chemical and Biological Medical Treatment Symposium CBMTS; 5–8Dec 1994; Spiez, Switzerland. Portland (ME): Applied Science and Analysis; 1994. p. 1.18–1.24.8. Portmann R, Hofmann W. Instructions for the test kit for the interlaboratory comparison teston measurements of cholinesterase activity. Internal document of NC Laboratory, Spiez (Swit-zerland); 1998. p. 1–7.18 Reiner E, et al.: PROTOCOLS FOR CHOLINESTERASE ACTIVITY MEASUREMENTSArh Hig Rada Toksikol 2000;51:13–189. Portmann R. Preliminary results of the interlaboratory comparison test on measurements ofcholinesterase activity. In: Bokan S, Orehovec Z, Price B, editors. Proceedings of the Chemicaland Biological Medical Treatment Symposium CBMTS-Industry I; 25–31 Oct 1998; Dubrovnik,Croatia. Portland (ME): Applied Science and Analysis; 1999. p. 262.10. Reiner E. Standardization of experimental conditions for blood cholinesterase activity measure-ments. In: Bokan S, Orehovec Z, Price B, editors. Proceedings of the Chemical and BiologicalMedical Treatment Symposium CBMTS-Industry I; 25–31 Oct 1998; Dubrovnik, Croatia. Port-land (ME): Applied Science and Analysis; 1999. p. 296–7.11. Simeon-Rudolf V, [inko G, Reiner E. Inhibition of human serum butyrylcholinesterase pheno-types by ethopropazine. In: Ferjan I, Kr‘an M, Stanovnik L, ^ arman-Kr‘an M, editors. Book ofabstracts and programme of the Life Sciences Conference 1999; 18–22 Sep 1999; GozdMartuljek, Slovenia. Ljubljana: Slovenian Pharmacological Society; 1999. p. 87.12. Evans RT. Cholinesterase phenotyping: Clinical aspects and laboratory applications. CRC CritRev Clin Lab Sci 1986;23:35–64.13. Whittacker M. Cholinesterase. Ethnic distribution. In: Beckmann L, editor. Monographs in Hu-man Genetics. Basel: Karger A.G.; 1986. p. 45–64.14. Simeon V, Bunti} A, [urina B, Flegar-Me{tri} Z. Cholinesterase phenotyping and distribution ofactivity in sera of 346 individuals. Acta Pharm Iugosl 1987;37:107–14.Sa‘etakUSPOREDBA PROTOKOLA ZA MJERENJE AKTIVNOSTIKOLINESTERAZA U LJUDSKOJ KRVI ELLMANOVOM METODOMMjerenjem aktivnosti acetilkolinesteraze i butirilkolinesteraze u krvi mo‘e se u osoba izlo‘enih organofosfornimspojevima i karbamatima odrediti apsorpcija tih spojeva. Za mjerenje aktivnosti butirilkolinesteraze u ljudskojplazmi postoji standardizirani protokol usvojen u velikom broju klini~kih i toksikolo{kih laboratorija, dok za mjerenjeaktivnosti eritrocitne acetilkolinesteraze ima nekoliko predlo‘enih protokola, ali ni jedan jo{ nije op}e priva}en.Zajedni~ki princip svih predlo‘enih protokola je spektrofotometrijska Ellmanova metoda, koja za mjerenje aktivostikolinesteraza rabi tiokolinske supstrate. Autori ovoga rada predla‘u jednostavni protokol u kojem se zasebno mjereaktivnosti u plazmi i u eritrocitima, koji nisu prani, a koji se odvoje centrifugiranjem. Aktivnost u eritrocitima mjerise nakon hemolize eritrocita smrzavanjem. MjerI se samo s 1,0 mM acetiltiokolinom. Drugi autori mjere aktivnost upunoj krvi koriste}i se dvama supstratima (acetiltiokolinom i butiriltiokolinom), koji se u hemoliziranu krv dodajusukcesivno i nakon svakog dodavanja supstrata mjeri se aktivnost. Neki autori predla‘u uporabu etopropazina,specifi~nog inhibitora butirilkolinesteraze, pa se mjerenje vr{i u prisutnosti i odsutnosti toga inhibitora. U ovome suradu opisane prednosti i nedostaci pojedinog protokola te izvori mogu}ih pogre{aka u predlo‘enim postupcima. Zausvajanje standardiziranog protokola za mjerenje aktivnosti eritrocitne kolinesteraze potreban je dogovor autorapredlo‘enih protokola nakon ~ega bi trebala uslijediti me|ulaboratorijska provjera toga protokola.Klju~ne rije~i:acetilkolinesteraza, butirilkolinesteraza, ljudska plazma, ljudski eritrociti, preciznost mjerenja, etopropazinRequests for reprints:Elsa Reiner, Ph.D.Institute for Medical Research and Occupational HealthP. O. Box 291, HR–10001 Zagreb, CroatiaE-mail: ereinerimi.hr

Usporedba protokola za mjerenje aktivnosti kolinesteraza u ljudskoj krvi Ellmanovom metodom

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