The interference by erythrocyte “acetylthiocholinesterase” in the estimation of the blood cholinesterase activity of the chicken

TOXICOLOGY

The

AND

APPLIED

PHARMACOLOGY

39,229-231(1977)

Interference by Erythrocyte “Acetylthiocholinesterase” the Estimation of the Blood Cholinesterase Activity of the Chicken

in

C. E. PICKERINGAND R. G. PICKERING Shell Research Limited, Tunstall Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, England Received August 20,1976; accepted October 8,1976

The Interference by Erythrocyte “Acetylthiocholinesterase” in the Estimation of the Blood CholinesteraseActivity of the Chicken. PICKERING, C. E. AND PICKERING, R. G. (1977). Toxicol. Appl. Pharmacol. 39, 229-237.In deducingpossibleexposureof chickensto organophosphorus compoundsfrom the measurementof blood cholinesteraseactivity, it is preferableto useplasmaseparatedfrom the whole blood rather than the whole blood. Whole blood must not be usedin methodswhich involve hemolysisand useacetylthiocholine as substrate.This is becausethere is acetylthiocholinesterasebut not acetylcholinesteraseactivity within the chicken erythrocytes. When only small samplesof unhemolyzed whole blood are available (e.g., under field conditions), it is possibleto measure plasmacholinesteraseactivity by the Voss and Sachsse(1970, Toxicol. Appl. Pharmacol. 16,764772) method,whichavoidshemolysis,but doesuse acetylthiocholineassubstrate.

The method of Voss and Sachsse (1970), which uses small samples of whole blood, has been used in field trials of esterase inhibitors involving man and other animals (Sachsse and Hess, 1972). This method employs thiocholine derivatives as substrate. The blood cholinesterase activity of chickens exposed for a short time to organophosphorus compounds can also be of interest in field studies. This report examinesthe suitability of extending the Voss and Sachssemethod to the estimation of cholinesteraseactivity in chicken blood.

The main activity of chicken blood against acetylcholine is widely reported to be found in the plasma, while there is virtually no activity against acetylcholine in erythrocytes (Stedman and Stedman, 1935; Augustinsson, 1948; Blaber and Cuthbert, 1962). Recently, Abou-Donia and Preissig(1976) published a report which included the effect of feeding Leptophos, 0-(4-bromo-2,5-dichlorophenyl)O-methylphenylphos-

phonothioate,

upon the erythrocyte and plasma cholinesterase of chickens. Their

method of assay used hemolyzed erythrocytes and acetylthiocholine as substrate. We have found that birds and some other specieshave an “acetylthiocholinesterase” present in their erythrocytes. This enzyme, as its trivial name implies, reacts with acetylthiocholine, but is not inhibited by lo-’ M eserineand several organophosphorus compounds. There would seem, then, to be a real possibility of erroneous results if Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

229 ISSN

004-008X

230

PICKERING

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PICKERING

acetylthiocholine is mistakenly used to study the inhibition of the erythrocyte acetylcholinesterase. We report some investigations of erythrocyte acetylthiocholinesterase which show that this enzyme is liberated by hemolysis. The Voss and Sachsse method uses a buffer which does not hemolyze the erythrocytes, so we used this method to measure the blood cholinesterase activity of chickens inhibited in vitro and in vivo with organophosphorus compounds and compared the results with estimations made by two other methods which use acetylcholine as substrate.

METHODS

Materials

Adult Sykes tinted crotophos, Azodrin’ technical 82.9 % (Z); sulphoxide (DMSO)

or Babcock hens about l-year-old and in lay were used. Monopure (TSL/62/70/P > 99 %); chlorfenvinphos, Birlane undiluted dicrotophos, Bidrin undiluted technical 86.2 % (E); and dimethylwere used.

Procedure

The Voss and Sachsse (1970) method was used and is conveniently referred to hereafter as the V/S method. Reagents. These were used as described. Distilled water which had been redistilled in an all-glass still was used throughout. Standardsolution. Prepared immediately prior to use was 63.05 mg of cysteine hydrcchloride/liter cf distilled water, i.e., 0.4 pmol of cysteine/ml. Methcd. Each sample was assayed in duplicate as follows. Thirty microliters of heparinized chicken blood was pipetted into 10 ml of DTNB buffer solution in a Pyrex round-bottom centrifuge tube and mixed thoroughly; 4 ml was removed into a second centrifuge tube (W). The residual 6 ml was centrifuged for 3-4 min at 18OOg and 4 ml of the resultant supernatant was transferred to a third tube (P). The tubes P and W were placed in a water bath at 30°C. Included with each batch of duplicate assays were two tubes (B) containing 4 ml of DTNB buffer. After all of the tubes had been equilibrated to 30°C 1 ml of substrate solution was added by automatic pipet to all tubes (P, W, and B) at 5-set interva!s. Exactly 10 min after the addition of substrate, 50 ~1 of inhibitor solution was added to each tube, the tubes were removed from the bath, and their contents were mixed. The tubes W were centrifuged as before. The contents of tubes P and B and the supernatant from tubes W were placed in glass cuvettes (light path, 1 cm) and the absorbance was measured at 420 nm using a Unicam SP1800 or SP600 spectrophotometer. The simultaneous incubation of tubes P, W, and B and the concurrent measurement of absorbance were preferred to the technique outlined by Voss and Sachsse (1970). Preparation of standard curoe. Standard solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1.O ml (equivalent to 0,0.08,0.16,0.24,0.32, and 0.40 pmol of cysteine) made up to 1 ml with distilled water were each mixed with 4 ml of DTNB buffer solution and their absorbances were measured at 420 nm. 1 Azodrin, Birlane, and Bidrin are registered trade names of Shell.

CHOLINESTERASE

IN CHICKEN

231

BLOOD

Calculation. Each 4-ml aliquot of blood-DTNB buffer solution contained 12 ~1 of whole blood. Absorbance of W is a measure of the whole-blood cholinesterase, absorbance of P is a measure of the cholinesterase in the plasma derived from 12 jtl of blood, and S is the absorbance produced by 0.2 pmol of cysteine.

P-B Plasma cholinesterase = s

0.2 1 x ~ x = pmol min-’ 10 min 0.012

W-P erythrocyte cholinesterase = S

0.2 1 = pmol min-’ x iii x 0.012

ml-’ of whole blood; ml-’ of whole blood.

Two additional methods were used: the Michel method (Pickering and Martin, and the pH stat method (Pickering and Pickering, 1974). Inhibitor

1970)

Studies

In vitro studies. Chicken blood (0.3 ml) was incubated at room temperature with 5 ~1 of DMSO containing amounts of Birlane varying from O-7 pg. After 15 min, two aliquots of 30 ,ul were removed for assay by the V/S method and the remaining blood was centrifuged to provide plasma which was assayed simultaneously by the pH stat method. A similar experiment was carried out in which the V/S and Michel methods were compared. In vivo studies. The chickens were divided into groups of three. Blood was taken from the wing vein into heparinized syringes and transferred to tubes containing heparin. All cf the chickens were bled prior to dosing them orally with capsules: Group 1, control empty capsules; Group 2,2 mg/kg of pure Azodrin (LD50,6.7 mg/kg); Group 3,2 mg/kg of Bidrin (86.2 %) (LD50,8-9 mglkg); Group 4,lO mg/kg of Birlane (82.9 %) (LD50, 44-62.5 mg/kg). Approximately 2 hr after dosing, blood was taken from all birds. Two aliquots of each sample of heparinized blood were assayed by the V/S method. The remaining blood was centrifuged for 5 min at 18OOg and the plasma was assayed by the Michel methcd. Blood from Group 4 birds was reassayed after incubation for 15 min with DMSO (16.7pl/ml of blood). Substrcte Specijicity S&dies

The erythrocytes remaining after centrifuging the Group 1 control blood samples were washed with 0.9% saline and recentrifuged. The washed packed erythrocytes were assayed by the pH stat method using 0.002 M acetylcholine iodide. The assay was repeated using 0.002 M acetylthiocholine iodide as substrate. The pH stat method was then modified to omit saponin and maintain the erythrocytes in an isotonic medium of 0.9 % saline and thus prevent hemolysis. The activity of the unhemolyzed erythrocytes was measured with the two substrates. RESULTS Methodology

A comparison of the individual results (Tables 1 and 2) obtained by the V/S method with those obtained by the Michel method for the chicken plasma cholinesterase is

36 38 40

31 37 39

ControP

Bidri# (2 mg/W

0.127 0.020 0.124

0.107 0.072 0.134

0.071 Nil

0.048 Nil 0.016 0.048

BLEND

Pre

0.107 0.103 0.110

0.106 0.068 0.087 0.103 0.083

0.882 0.973 0.999

0.116

0.882 0.972 1.025 1.123 0.859

0.106 0.187 0.154

0.147 0.147 0.150

0.164 0.216 0.887 1.120 0.857

0.190

0.952 0.840 0.910

Plasma

of whole blood)

Post

ml-’

Erythrocytes

0.869

0.943 0.946 1.023

Plasma

1

BEFORE AND AFTER A SINGLE

V/S method (pm01 min-’

IN CHICKEN

RPre-dose bleeding was carried out 2 hr prior to dosing. b Pre-dose bleeding was carried out 24 hr prior to dosing.

32 33

30 31 34 29

Erythrocytes

Chicken number

(2 mdb)

Azodrin”

Control”

VALUES

CHOLINESTERASE

TABLE

17 15 15

19 22 87 100 100

22

101 89 89

Plasma activity remaining (%I

ORAL

DOSE

0.71 0.73 0.82

0.28 0.23 0.26

0.18 0.18 0.76 0.96 0.79

0.80 0.76 0.74 0.23

0.91 0.83 0.88 0.74 0.80 0.83 0.77 0.88 0.74

Post

Plasma

39 32 32

23 22 99 109 107

88 92 84 31

Plasma activity remaining (%)

Michel method hr-’ 0.1 ml-l of plasma)

OR BIDRIN

Pre

(dpH

OF AZODRIN

E Q

z c, ?I!

CHOLINESTERASEIN TABLE

233

CHICKENBLOOD 2

PLASMA CHOLINESTERASE VALUES IN THE BLEND OF CHICKENS BEFORE AND AFTER A SINGLE ORALDOSEOFBIRLANE V/S method pmol min-’ ml-’ whole blood)

Michel method (dpH hr-’ 0.1 ml-’ of plasma)

Activity remaining (%I

Activity remaining (%I

-Chicken number

Pre”

Post

36 38 40

0.848 1.092 1.011

0.572 0.774 0.666

67 71 66

36b 3Sb 40b

0.978 1.167 1.141

0.725 0.900 0.848

74 77 74

Pre”

Post

0.66 0.86 0.77

0.43 0.60 0.47

-

65 70 61

0 Pre-dose bleeding was carried out 24 hr prior to dosing. b Assays were carried out on blood preincubated for 15 min with DMSO (16.7 pi/ml).

FIG. 1. Comparison cholinesterase.

of the V/S method with the Michel method for the assay of chicken plasma

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PICKERING AND PICKERING

shown in Fig. 1. The regressionequation obtained from these data (n = 30) was Voss = 1.30 (Michel) - 0.08. The correlation coefficient, r, was 0.97. In Vitro Studies There was a linear relationship between the percentage plasma cholinesterase inhibition and the log of the concentration of Birlane added to the blood (Fig. 2). This

FIG. 2. The relationship between the percentage inhibition of plasma cholinesterase as measured by the V/S, Michel, and pH stat methods, and the concentration of Birlane added to the blood.

relationship was observed between 6 and 24 pg of Birlane/ml of blood for the V/S and pH stat methods and between2 and 24 pg of Birlane/ml of blood for the Michel method. The V/S method gave a lower inhibition than did the Michel method over the whole range and a lower inhibition than did the pH stat method at concentrations greater than 10 pg/mi of blood. Up to 10 fig of Birlanejml of blood (40% inhibition), the agreement between the V/S and pH stat methods was good.

CHOLINESTERASE

IN CHICKEN

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235

In Viva Studies

In the control group in which the pre-dose bleeding was carried out 2 hr before dosing, there was a slight tendency for the plasma cholinesterase to decrease. In the other control group in which 24 hr had elapsed between the pre-dose bleeding and the dosing, the values were more constant. Treatment with 2 mg/kg of Azodrin (Table 1) produced, within 2 hr, a mean depression in plasma cholinesterase of 79 % by the V/S method or 75 % by the Michel method. The birds were all unsteady on their feet for up to 3 days. Treatment with 2 mg/kg of Bidrin (Table 1) produced, after 2 hr, a mean depression in plasma cholinesterase of 84 % by the V/S method or 66 % by the Michel method. Other than some drowsiness on the first day, no other clinical effects were seen. In the experiment with Birlane (Table 2) only plasma cholinesterase activity was measured 24 hr before and 2 hr after dosing. The mean depressions in plasma cholinesterase measured by the V/S and Michel methods were 32 % and 35 %, respectively. Chicken No. 36 was unable to stand, but squatted with splayed wings and intermittent muscle spasms within 1 hr of dosing. Chicken No. 40 was affected to a lesser degree within 2 hr of dosing, while bird No. 38 was unaffected. The V/S assay was repeated on a 0.3-ml aliquot of each sample of pre- and post-dose chicken blood from this experiment after incubation with 5 ~1 of DMSO (Table 2). The mean depression of the plasma cholinesterase was 25 %. Substrate Specificity Studies

In the 12 pre-dose samples, the erythrocyte activity represented between 0 and 16 % (mean, 6.8 f 5.38) of the plasma cholinesterase activity. In the six control post-dose samples, the erythrocyte activity represented between 9 and 22 % (mean, 13.2 + 5.19) of the plasma cholinesterase activity (Table 1). In contrast to these results obtained by the V/S method, considerable activity was measured by the pH stat method when acetylthiocholine iodide was used as substrate. Values of 1.98-2.90 pmol min-’ ml-’ of packed erythrocytes were obtained for erythrocytes from three control chickens. No detectable activity was measured in the same erythrocytes by the pH stat method if acetylcholine iodide was used as substrate. Similarly, no activity was measured by the pH stat method with either acetylcholine iodide or acetylthiocholine iodide if unhemolyzed erythrocytes were used. DISCUSSION The V/S method was successfully adapted to measure the cholinesterase activity of chicken plasma; 30 ~1of whole blood proved to be a suitable quantity to use for assay by this method. It is essential that no hemolysis has occurred. A small amount of activity, which was not measurable using acetylcholine iodide by either the pH stat or the Michel method, was associated with the erythrocytes. However, in 12 pre-dose blood samples, the erythrocyte activity measured by the V/S method was between 0 and 16 % of the corresponding plasma activity. The erythrocyte activity toward acetylthiocholine iodide in the V/S method contrasted sharply with the activity toward acetylthiocholine iodide in the pH stat method. The explanation would appear to be that the acetylthiocholinesterase is held mainly within the erythrocyte and is

236

PICKERING

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restricted in its ability to act upon the substrate unless the cell is hemolyzed (as occurs in the normal pH stat method). The erythrocyte activity measured by the V/S method is thus not acetylcholinesterase but acetylthiocholinesterase, which possibly enters into reaction with substrate which slowly diffuses through the erythrocyte membrane. If no hemolysis occurs in the V/S method, there is only limited interference with the pasma assay from the acetylthiocholinesterase present within the erythrocyte. There is no merit in measuring the whole blood activity; it is preferable to assay the plasma alone. Under laboratory conditions, as opposed to field conditions, analysis of separated plasma by an acetylthiocholine rate method is perhaps to be preferred (Pickering and Pickering, 1971). In the in vitro studies, the linear relationship between the percentage of plama cholinesterase inhibition and the log of the concentration of Birlane added to the blood indicates that all methods reflect the effect of the compound. However, compared with the other methods, the V/S method underestimates the inhibition. One possible explanation for this is that the preincubation of the blood with DMSO, or just the presence of the DMSO, facilitates the reaction of the acetylthiocholinesterase, since both control and inhibited plasma activities are enhanced. This produces an apparently smaller inhibition (Table 2). In the plasma of the six control chickens (Table l), the percentage activities remaining showed a mean algebraic difference between the Michel and V/S methods of 2.2% (SD f 9.5). Thus, in untreated chickens, a difference of +I9 “/, (i.e., +2 SD) between the remaining activity as measured by the two methods can arise through possible error in the set of four assays involved (i.e., V/S method, pre- and post-dose, and Michel method, pre- and post-dose). The agreement in the in viuo studies (Fig. 1) between the activity of the plasma cholinesterase in the chicken as measured by the V/S method and that as measured by the Michel method was good; the correlation coefficient was 0.97. In the three sets of treated chickens, the agreement between the percentage of remaining activity as measured by the two methods as within the limits outlined above, i.e., +19x. The inhibition caused by the Azodrin was similar by both methods; the V/S method gave a mean remaining activity of 21% while the Michel method gave a mean remaining activity of 25 %, i.e., a difference of 4 % in the mean percentage activity remaining as measured by the two methods. However, with Bidrin, the difference between the mean percentage activity remaining as measured by the two methods was greater: 18 %. In the experiment with Birlane (Table 2), the difference between the mean percentage activity remaining as measured by the two methods was 3 %. It is important in this type of study to carry out the two methods of assay simultaneously to avoid changes that might occur in inhibition of the cholinesterase upon standing. Other distinctions between the methods to be borne in mind when comparing the results are that the V/S method measures at 30°C the plasma activity of whole blood, whereas the Michel and pH stat methods measure at 37°C the activity of separated plasma. It is, of course, necessary to measure the packed cell volume (PCV) of the chicken blood to express the V/S activities in terms of a unit volume of plasma. Clearly, further investigations of this “acetylthiocholinesterase” enzyme are necessary to establish its classification more fully. Evidence so far available indicates that, compared with the acetylcholinesterase present in the erythrocytes of other species, the

CHOLINESTERASE

IN CHICKEN

BLOOD

237

acetylthiocholinesterase present in chicken erythrocytes is not inhibited by 10e5 M eserine, is not inhibited by some organophosphorus compounds (e.g., Azodrin) at concentrations which inhibit acetylcholinesterase, and is more stable to heat. It exhibits similar activity toward butyryl- and acetylthiocholine, whereas acetylcholinesterase generally exhibits more activity toward acetylcholine than toward butyrylcholine. We have observed a similar enzyme present in the erythrocytes of the duck (Mallard). It would also appear that the erythrocytes of man, dog, and rat have acetylthiocholinesterase activity in addition to acetylcholinesterase activity. Compared to the high acetylcholinesterase activity, the acetylthiocholinesterase activity of human erythrocytes is small. It is probable that this enzyme has not caused greater problems in the determination of erythrocyte acetylcholinesterase because, in species which have a low erythrocyte acetylcholinesterase activity and in which per force larger volumes of erythrocytes are assayed, if hemoglobin is liberated, this would prevent the spectrophotometric measurement at 420 nm of the 5-sulfido-2-nitrobenzoate ion. REFERENCES M. B. AND PREISSIG, S. H. (1976). Delayed neurotoxicity of Leptophos: Toxic effects on the nervous system of hens. Toxicol. Appl. Pharmacol. 35,269-282. AUGUSTINSSON, K. B. (1948). Cholinesterase:A study in comparative enzymology. Acta Physiol. Stand. 15, Suppl. 52, 1. BLABER, L. C., AND CUTHEIERT, A. W. (1962). Cholinesterases in the domesticfowl and the specificity of somereversibleinhibitors. Biochem. Pharmacol. 11,113-124. PICKERING, C. E., AND PICKERING, R. G. (1971).Methods for the estimationof acetylcholinesteraseactivity in the plasmaand brain of laboratory animalsgiven carbamatesor organophosphoruscompounds.Arch. Toxicol. 27,292-310. PICKERING, R. G., AND MARTIN, J. G. (1970).Modification of the Michel A pH methodfor the estimation of plasma,erythrocyte and brain cholinesteraseactivities of various speciesof laboratory animals.Arch. ToxicoE. 26, 179-195. PICKERING, R. G., AND PICKERING, C. E. (1974).Methods for the estimationof acetylcholinesteraseactivity in the erythrocytes of laboratory animals given carbamatesor organophosphoruscompounds.Arch. Toxicol. 31 197-216. SACHSSE, K., AND HESS, R. (1972).Exposure of volunteers and animalsin aerial application test of Phosphamidon(DimecronRIOO)in India. Proc. Eur. Sot. Study Drug Tox. (Wreck) ABOU-DONIA,

14,247-253.

E., AND STEDMAN, E. (1935). The relative cholineesteraseactivities of serumand corpusclesfrom the blood of certain species.Biochem f. 29,2107-211 I. Voss, G., AND SACHSSE, K. (1970). Red cell and plasmacholinesterase activities in microsamplesof human and animal blood determined simultaneouslyby a modified acetylthiocholine/DTNB procedure.Toxicol. Appl. Pharmacol. 16,764-772.

STEDMAN,