Minimizing occupational exposure to pesticides: Cholinesterase determination and

organophosphorus poisoning

By

M. VANDEKARo

Contents

I. Introduction ...................................................... 67 II. Inhibition and reactivation of cholinesterase .......................... 68

III. Cholinesterase monitoring in WHO field trials of new insecticides ...... 69 a) Methods employed including development

of spectrophotometric kit ....................................... 69 b) Review of results obtained ........................... . . . . . . . . . . . 73 c) Interpretation of results ........................................ 74

IV. Some problems related to sampling and transportation of samples from the field .................................................... . 76

Summary............................................................. 77 References ........................................................... 78

I. Introduction

In most instances cholinesterase determination in people is carried out either: (1) as part of a surveillance procedure to prevent poisoning in people exposed to organophosphorus compounds or (2) as a diagnostic tool to exclude or confirm that symptoms or signs observed in people are caused by anticholinesterase compounds.

Two types of cholinesterases have been distinguished using a number of criteria such as substrate specificity and reaction of enzyme with so­called selective inhibitors: acetylcholinesterase (acetycholine acetyl­hydrolase, 3.1.1.7), also called specific or true cholinesterase, which uses acetylcholine as its natural substrate and cholinesterase (acetylcholine acyl-hydrolase, 3.1.1.8), also called butyrylcholinesterase, nonspecific cholinesterase, or pseudocholinesterase, for which physiological sub­strate ( s) are still unknown. Both of these are present in blood. While their function in blood remains unknown, both the erythrocyte acetyl-

" Pesticide Development and Safe Use, Division of Vector Biology and Control, World Health Organization, Geneva, Switzerland.

© 1980 by Springer-Verlag New York Inc. Residue Reviews, Volume 75.

68 M. VANDEKAR

cholinesterase and the plasma cholinesterase have been used widely as indices of the degree of absorption of anticholinesterase insecticides. It is important to note that the level of erythrocyte acetylcholinesterase is a better indicator of acetylcholinesterase activity at nerve synapses.

II. Inhibition and reactivation of cholinesterase

Organophosphorus compounds react with cholinesterase to produce a relatively stable phosphorylated enzyme. This reaction is progressive and temperature dependent. It is now a well established fact that this reaction is the same basic process as that which occurs when an enzyme catalyses the hydrolysis of its substrate (ALDRIDGE and REINER 1972).

This process can be described by the following equation:

k+l k+2 k+3 EH + AB~EHAB~BH + EA----+EH + AOH Eq. 1

k-l +H20

where EH is the free enzyme, AB is either substrate or inhibitor, EHAB is a Michaelis complex which breaks down to give BH and EA as first products, and AOH together with EH are the second products. The acylated enzyme (EA) could be either acetylated cholinesterase formed during the hydrolysis of acetylcholine or a phosphorylated (or car­bamylated) cholinesterase produced by interaction with their respective inhibitors.

The difference between an inhibitor and acetylcholine (substrate) is only in the rates of the reaction. The acetylated e'nzyrne formed from acetylcholine hydrolyses very quickly with a half-life of 2.3 X 10-6 min or less, whereas the phosphorylated enzyme hydrolyses very slowly: the half-life for the hydrolysis of dimethylphosphorylated human acetyl­cholinesterase is about 50 min and that of diethylphosphorylated enzyme is about 60 hr. The reason why acetylcholine is a substrate and methyl paraoxon is an inhibitor can be readily appreciated by comparing the turnover rates for these two substances which are 300,000 and 0.0085/ min, respectively (ALDRIDGE and REINER 1972).

This reaction, when the activity of acylated (inhibited) cholinesterase is restored by reaction with water, is known under several names: spon­taneous reactivation, de acylation, dephosphorylation in the case of in­hibition by organophosphorus compounds, and decarbamylation in case of inhibition by carbamates. Extensive data on the rates of spontaneous reactivation of phosphorylated cholinesterases from a number of animal species have been summarized by REINER (1971).

Phosphorylated cholinesterase can be reactivated not only by water but also by a variety of compounds, including nucleophilic reagents such as bis-pyridinium aldoximes. This reactivation can proceed to completion, but sometimes this is not so, the main reason for this being the aging of the inhibited enzyme. It has been shown that when a phosphorylated

Cholinesterase and OP poisoning 69

enzyme loses one of the groups attached to the phosphorus atom, it can­not reactivate spontaneously or be reactivated by oximes or other nucleo­philic reagents.

k+l k+2 k+3 EH + AB~EHAB~BH + EA~EH + AOH Eq. 2

k-l ~~. k+4 ~

EA'

Since the inhibited enzyme reactivates spontaneously as well as ages (see Eq. 2), the extent of aging will depend upon the relative rates of re­activation (k+3) and aging (k+1) and also on the continued presence of inhibitor. If the inhibitor (AB) is removed by any means, the relative proportion of active (EH) and irreversibly inhibited enzyme (EA') will depend on the above rate constants. If the inhibitor is not removed all the enzyme will eventually become irreversibly inhibited (EA').

Most of the organophosphorus insecticides currently used in public health are dimethyl derivatives which produce an unstable dimethyl phosphorylated enzyme. The half-life for the aging reaction of the dimethyl phosphorylated human acetylcholinesterase is 4 hr (SKRINJ ARIC­

SPOLJAR et al. 1973). Thus, it may be predicted that 50% inhibition on one day will yield on the next 41 % of the original active enzyme and 9% of the aged enzyme (World Health Organization 1979). One of the toxicological criteria which should be satisfied if a pesticide is to be acceptable for vector control is that any effects of absorption of sprayed material during one day's work should be toxicologically insignificant at the beginning of the next day's operation (World Health Organization 1979).

III. Cholinesterase monitoring in WHO field trials of new insecticides

a) Methods employed including development of spectrophotometric kit

Many laboratory and field methods for measuring cholinesterase activity have been developed. The principles of these methods and their usefulness and limitations under different conditions have been described in several review article (e.g., WrITER 1963, SIMEON 1967, AUGUSTINSSON 1971, HOLMSTEDT 1971, and LONG 1975). Only a few of these methods are suitable for work under true field conditions since such methods should not require complicated equipment (and preferably not elec­tricity) and should be simple enough to be used by nonspecialists. Several methods used in WHO field trials have been reviewed by the WHO Expert Committee on Safe Use of Pesticides and in its report (World Health Organization 1967) recommendations were made on their suit­ability with suggestions for their improvement. The results of both

70 M. VANDEKAR

research carried out in the WHO collaborating laboratories and field studies on cholinesterase monitoring will be discussed below.

Six methods have been used in WHO field trials. As their charac­teristics differ to a great extent (Table I), their choice depended on the type of compound sprayed and circumstances under which the trial was carried out.

The Ellman spectrophotometric method (ELLMAN et al. 1961) has been found adequate for determining whole blood, erythrocyte, and plasma (WILHELM 1968) cholinesterase activity. It was originally intro­duced as a laboratory (reference) method to obtain reliable results when cholinesterase inhibition by carbamates was to be detected; due to its quick assay time (2 min) no corrections are required for spontaneous reactivation of carbamylated enzyme. Subsequently this method was used in trials with organophosphorus compounds, replacing the eIectrometric method (MICHEL 1949, STUBBS and FALES 1960) previously used. As a rule, only whole blood and plasma cholinesterase activity are measured, the determination of erythrocyte cholinesterase having been discontinued at an early stage due to the difficulty in the field of handling erythrocytes packed in capillaries. When whole blood cholinesterase is assayed by the spectrophotometric method, assuming a haematocrit of 50, the erythrocyte cholinesterase contributes 92% to the total activity (WILHELM et al. 1973).

While both the tintometric method (EDSON 1958) and acholest method (SAILER and BRAUNSTEINER 1959) have been found suitable, the former has proved faster and more convenient; it does not require blood cen­trifugation. When whole blood is assayed tintometrically, assuming a haematocrit of 50, 82% of the total activity is contributed by the erythrocyte cholinesterase (WILHELM et al. 1973). Slight modification of the procedure, with the substrate being added to the reaction mixture immediately after addition of the blood sample, renders the method suitable for measuring carbamate induced inhibition.

The acholest method was used in a number of trials satisfactorily. It has been thoroughly investigated in the field (HOLMSTEDT and OUDART 1966, OUDART and HOLMSTEDT 1970) and under laboratory conditions (PLESTINA 1966). A simple hand-driven micro centrifuge for separating plasma has been developed (HOLMSTEDT 1965) and is commercially avail­able.

A method in which whole blood is absorbed on filter-paper, dried, and shipped to a laboratory for a separate determination of erythrocyte and plasma cholinesterase activity mannometrically has been investigated and used extensively by AUGUSTINSSON (1955) and adapted to tropical conditions by AUGUSTINSSON and HOLMSTEDT (1965). The difficulty of extracting the erythrocyte enzyme quantitatively from the dried blood spot on filter paper (AUGUSTINSSON et al. 1978) limits this method to plasma cholinesterase only. Its use in monitoring workers' exposure dur­ing insecticide field trials has been rather limited since results cannot be

Tab

le I

. M

ain

char

acte

rist

ics

of m

etho

ds u

sed

for

mea

suri

ng c

holin

este

rase

act

ivit

y in

WH

O fi

eld

tria

ls.

Tim

e of

N

o. s

peci

men

s C

ompl

exit

y of

S

uita

bili

ty f

or

reac

tion

w

hich

can

be

proc

edur

e an

d ca

rbam

ate

Met

hod

(Ref

eren

ce)

Cho

line

ster

ase

(min

) pr

oces

sed

in 6

h

fiel

d us

e ex

posu

re

Acc

urac

y

Spe

ctro

phot

omet

ric

Pla

sma

2 30

-40

Mod

erat

ely

Yes

A

ccur

ate

(ELL

MA

N e

t al.

1961

, E

ryth

rocy

te

com

plex

; fi

eld

WIL

HEL

M 1

968,

W

hole

blo

od

kit

avai

labl

e W

orld

Hea

lth O

rgan

-iz

atio

n 19

78)

Ele

ctro

met

ric

Pla

sma

60

40

-60

F

ield

sam

plin

g N

o A

ccur

ate

(MIC

HE

L 1

949,

E

ryth

rocy

te

met

hod

deve

l-ST

UBB

S &

FA

LES

oped

; as

say

1960

) co

mpl

ex

Tin

tom

etri

c (E

DSO

N

Who

le b

lood

20

60

-100

V

ery

sim

ple;

Y

es,

by s

ligh

tly

±

12.5

%

1958

, WA

TSO

N &

fi

eld

kit

avai

l-m

odif

ied

EDSO

N 1

964)

ab

le a

nd

pr

oced

ure

com

mon

ly u

sed

Ach

olt

t (S

AIL

ER &

P

lasm

a 1

5-3

0

50

V

ery

sim

ple;

Y

es

Fai

rly

BRA

UN

STEI

NER

195

9,

used

fre

quen

tly

accu

rate

R

ICH

TER

ICH

196

2)

in t

he

fiel

d

Fil

ter-

pape

r m

etho

d P

lasm

a 30

3

0-4

0

Com

plex

ass

ay;

No

Acc

urat

e of

sam

plin

g fo

r E

ryth

rocy

te

(ass

ay)

fiel

d sa

mpl

ing

for

plas

ma

man

nom

etri

c as

say

itse

lf v

ery

chol

ines

tera

se

sim

ple

(AU

GU

STIN

SSO

N &

H

OLM

STED

T 19

65)

Rad

iom

etri

c W

hole

blo

od

0.33

4

0-5

0

Mod

erat

ely

Yes

A

ccur

ate

(WIN

TER

ING

HA

M &

co

mpl

ex;

unde

r D

ISN

EY 1

964)

un

suit

able

for

la

bora

tory

fi

eld

use

cond

itio

ns

72 M. VANDEKAR

obtained quickly unless a Warburg apparatus is available near the area of sampling.

A radiometric method developed by WINTERlNGHAM and DISNEY (1964) was used on two occasions under field conditions. In both cases technical difficulties precluded analysis of the results (V ANDEKAR and SVETLICIC 1966).

It should be noted that the ambient temperature under field condi­tions frequently varies between 20° and 40° C. Thus temperature con­version tables are indispensable. For the tintometric method, a modified procedure has been worked out (WATSON and EDSON 1964) which en­ables the user to regulate the time of assay according to the ambient temperature. Correction factors for a range of temperature are available for the acholest method (RICHTERICH 1962), the range being expanded subsequently (PLESTINA 1966, HOLMSTEDT and OUDART 1966). As part of the development of a spectrophotometric field kit described below, tem­perature conversion tables for human whole blood and plasma cholin­esterases have also been worked out (REINER et al. 1974).

Although several field methods for measuring cholinesterase activity are available, none of these are entirely satisfactory. The need for an alternative, more exact field method for determining both erythrocyte and plasma cholinesterase was stressed by a WHO Expert Committee (W orId Health Organization 1973). Through collaborative work with several laboratories, a field kit based on the Ellman spectrophotometric method has been developed. The kit consists of basic equipment, acces­sories, and preweighed chemicals sufficient for about 1,000 whole-blood and 1,000 plasma cholinesterase determinations. A document describing the kit and assay procedures ( World Health Organization 1978) contains details of the kit, which is available from WHO if ordered by govern­mental laboratories or institutions.!

By making use of a portable spectrophotometer, and pre-weighed reagents, whole blood and plasma cholinesterase activity can be deter­mined in the field on blood taken from a finger prick. A Single experienced operator can carry out 40 whole blood and 40 plasma cholinesterase assays/day. For practical purposes regular replenishment of reagents and equipment is required at a base which should consist of a room con­taining a clean working surface, clean water, electric outlets, and a re­frigerator. The operator should have some experience in a laboratory.

The automatic method of pipetting reduces experimental error in pipetting reaction components. Since the miniaturized spectrophotometer is used in the field, the reproducibility of the method is somewhat less than with a standard laboratory spectrophotometer. This is mainly due to the diminished read-out accuracy associated with the small size of the

1 The document is available from, and orders should be sent to, the Chief, Pesti­cides Development and Safe Use Unit, World Health Organization, 1211 Geneva, 27, Switzerland.

Cholinesterase and OP poisoning 73

scale. Under normal conditions of work in the field, the reproducibility of the method is within -+ 5 %. It can be increased to some extent by the use of the optional recorder.

When a prototype kit was evaluated under field conditions more than 1,000 analyses were carried out during a 2-mon round of spraying with fenitrothion. After a few minor modifications to the procedure the per­formance of the prototype kit was found to be very satisfactory.

The technical difficulties of separating and measuring cholinesterase activity in erythrocytes under field conditions limit the ability of the spectrophotometric kit to measuring enzyme activity in whole blood and plasma only. In theory it should be possible to calculate reliable values for erythrocyte cholinesterase activity from the results obtained for whole blood and plasma cholinesterase activity, providing that the haematocrit value is known and by applying the following formula:

E = ~O[ W - p( 1 - 1~) ] Eq.3

where E, W, and P are respectively erythrocyte, whole blood, and plasma cholinesterase activities (expressed as .6.A/min/ml of erythrocytes, whole blood, or plasma at the same temperature) and H is the haematocrit value. Laboratory experiments to validate this equation have been carried out recently (WILHELM 1980). In a group of 16 normal subjects with a haematocrit range between 37 and 49, whole blood erythrocyte and plasma cholinesterase activity was determined spectrophotometrically and the ranges of activity (.6.A/min/ml) were 15.5 to 31.0 (whole blood), 1.3 to 7.8 (plasma), and 32.0 to 58.0 (erythrocytes). A good agreement between the calculated and experimentally obtained values for erythro­cyte cholinesterase activity was found, the correlation coefficient being 0.92. These results demonstrated the suitability of the spectrophoto­metric kit for determining indirectly the erythrocyte cholinesterase under field conditions.

b) Review of results obtained

Monitoring of cholinesterase in malaria spraymen has been carried out in a number of field trials in Africa, Central America, and Southeast Asia concurrently with the evaluation of the efficacy and safety of candi­date anticholinesterase insecticides in village-scale or larger field trials. It has proved invaluable for assessing the cumulation of inhibitory action of a compound, for deciding when an operator should be withdrawn from further exposure to insecticides, and for making comparisons with other compounds. The incidence of adverse reactions in people exposed has been low (VANDEKAR 1965, World Health Organization 1967, ARNAN

1971) all occurring in trials in early years. A good agreement was observed between the degree of cholinesterase inhibition and the fre­quency of signs and symptoms (VANDEKAR 1975). There was also good

74 M. VANDEKAR

correlation between oral acute toxicity data determined in rats and observed effects on cholinesterase in exposed humans.

Fenitrothion has undergone several large trials as an insecticide for use in malaria Gontrol programmes. In these trials the tintometric method was used routinely for weekly determination of cholinesterase activity. In a trial near Kisumu, Kenya, 8 rounds of spraying, each lasting 2 mon, were carried out during a 2-yr period. In each round, 2 to 3 out of 35 to 40 spraymen had to be withdrawn from spraying because of lowered cholinesterase activity. In this way, except for one unconfirmed case of illness due to unexplained circumstances, no complaints attributable to exposure to the insecticide were recorded in a total of 1,500 man-days of work.

Since 1960, a total of 10 organophosphorus and 8 carbamate insecti­cides have undergone one or more field trials under carefully controlled conditions.2 Since these compounds were applied indoors by the same technique of application and at the same target dosage and spray con­centration, the results obtained were comparable and allowed for a number of conclusions to be drawn with regard to their relative safety when used as residual sprays in malaria control programmes ( World Health Organization 1979). Thus fenitrothion (oral LD"o to rats 500-700 mg/kg) appears to be at the limit of acceptable toxicity, and its relatively narrow margin of safety calls for strict precautionary measures and regular cholinesterase monitoring in exposed operators throughout the spraying cycle. On the other hand, organophosphorus compounds with toxicity similar to or lower than that of malathion (LDso > 1000 mg/kg) should be safe enough to be applied operationally without requiring routine cholinesterase monitoring, provided that suitable precautions are taken. Among carbamate insecticides, propoxur (LDso 116 mg/kg) repre­sents the acceptable limit of toxicity. In view of its relatively low margin of safety, strict adherence to recommended precautionary measures is required. No cholinesterase monitoring is indicated when carbamate insecticides are applied since the inhibited enzyme reactivates too rapidly for this to be of any value in preventing overexposure (V ANDEKAR et al., 1971 ).

c) Interpretation o/results

In interpreting results one should bear in mind that the severity of poisoning is determined not only by the degree of reduction of cholin­esterase activity but also by other factors such as the rate of inhibition of cholinesterase and the type of inhibitory action of the insecticide. The rate at which different insecticides are absorbed and transported varies

2 Full details of the conditions of work and precautionary measures can be obtained from the Chief, Pesticides Development and Safe Use Unit, World Health Organization, 1211 Geneva 27, Switzerland.

Cholinesterase and OP poisoning 75

depending on the nature of the compounds, the vehicle, and the charac­teristics of the routes of entry. A substantial proportion of the absorbed compound does not reach nerve synapses because it is bound to non­synaptic cholinesterase, or other proteins, or transiently stored in fat, and is detoxified in the liver or other sites. Most organophosphorus insecti­cides used today are esters of thiophosphoric acid which are converted to more toxic derivatives with a significantly higher inhibitory activity. It is generally accepted that during repeated exposures to subtoxic doses of organophosphorus compounds, signs and symptoms of poisoning occur when more than 50% of erythrocyte cholinesterase is inhibited. Accord­ing to NAMBA (1971) the threshold level of synaptic acetylcholinesterase, the activity of which can be measured only experimentally, is probably about 50%.

Although determination of plasma cholinesterase is frequently used because of its simplicity, the determination of erythrocyte cholinesterase is preferable as it represents a more specific biological response to ex­posure to anticholinesterase compounds. As mentioned above, the whole blood cholinesterase activity as measured tintometrically or spectro­photometrically is to a great extent composed of the erythrocyte cholin­esterase activity.

As erythrocyte and plasma cholinesterase activity, as well as whole blood cholinesterase activity varies from person to person (AUGUSTINS­SON 1955) the determination of a pre-exposure value is an important prerequisite to assess later the degree of inhibition for a given subject. It is the proportionate reduction of the activity from the normal for the individual that is important rather than the numerical value of the test.

The formulation of guidelines on the interpretation of results from the surveillance of exposed workers will be influenced by several factors which include: (1) type of compound( s) /formulation( s); (2) antici­pated level of exposure; (3) level of supervision; (4) method employed for determining cholinesterase activity; and (5) frequency of cholin­esterase determination. In the case of indoor spraying of fenitrothion water dispersible powder in malaria control programmes the t~ntometric method is most commonly used for weekly monitoring of cholinesterase activity in exposed operators, and instructions regarding the interpreta­tion of the results obtained by this method have been prepared and distributed to the field. They call for (1) enforcement of better adher­ence to precautionary measures when about 25% reduction of activity is found, (2) thorough investigation to find out the most likely reason leading to over exposure and allocation of a lighter spraying schedule for the following wk at 37.5% reduction and (3) withdrawal from spray­ing and any other exposure to insecticide when 50% reduction in activity is found. In a well-organized spraying operation the latter degree of inhibition should occur only exceptionally. The withdrawal period should last until cholinesterase activity returns to normal which may sometimes take more than 2 wk. While 12.5% reduction of activity

76 M. VANDEKAR

observed in an individual worker may reflect normal fluctuations in activity or the experimental error of the method, if the same reduction is observed in several men in a group, field supervisors should be advised to ensure better adherence to precautionary measures. Whenever complaints of HI health are received, the worker should be referred to the medical officer associated with the project.

The FAO/WHO data sheets on pesticidesB in referring to surveillance tests give 70% as action level for erythrocyte cholinesterase. The same level has been recommended by two international workshops (Permanent Commission and International Association on Occupational Health Sub­committee on Pesticides 1972 and 1979).

IV. Some problems related to sampling and transportation of samples from the field

A number of factors which can interfere in obtaining correct results have to be borne in mind. Probably one of the most important is the contamination of the blood sample by direct inhibitors of cholinesterase such as some insecticides or their derivatives. The amounts of blood used in micro-techniques are small enough that significant contamination may occur by the traces of inhibitors present on the finger tip of the blood donor. RASMUSSEN et al. (1963) have shown that it was not possible to remove traces of insecticide from the skin by ordinary cleansing before samples were taken by finger prick from volunteers exposed to 0040 to 0.55 }log of dichlorvos/L of air. The possibility of the presence of traces of direct inhibiting derivatives in finger-prick samples collected from workers during reentry interval studies should be borne in mind in view of the findings reported by POPENDORF and LEFFINGWELL (1978). These authors found a significant production of available paraoxon in parathion foliar residues on orange trees, with peak levels generally occurring 1 to 3 days following application. The inhibition of blood cholinesterases by contaminants, which is progressive with time, can be prevented to great extent by immediate dilution of the sample (WILHELM and REINER 1973) or circumvented altogether by venepuncture.

Contamination of reagents by either alkali or acid will obviously cause erroneous results, particularly in methods which are based on formation of acid. Thus in the tintometric method a number of precautions have to be taken to avoid the introduction of CO2 from the air into the reaction mixture.

When samples are transported from the field to the laboratory or stored before analysis, adequate precautions should be taken to ensure that enzyme activity does not change in either direction due to spon­taneous reactivation, reactivation induced by the presence of an oxime in the blood of a treated patient, progressive inhibition if a steady state

3 Obtainable on request from the Chief, Pesticides Development and Safe Use Unit, World Health Organization, 1211 Geneva 27, Switzerland.

Cholinesterase and OP poisoning 77

has not been reached, or decrease in enzyme activity due to denaturation of protein by elevated temperatures and/or prolonged storage.

In clinical and other adequately equipped laboratories the most commonly used manual method is the electrometric method of MICHEL (1949), while most automated methods (e.g., GROFF et al. 1976) use the spectrophotometric procedure described by EllMAN et al. (1961). As a rule, automated methods are more precise, are less dependent on the capability of the investigator and reduce errors due to temperature eHects and other conditions associated with a field method. Nevertheless, the accuracy of results obtained in a sophisticated laboratory by either manual or automated methods will greatly depend on the way samples are collected and transported from the field.

Summary

The nature of the reaction of organophosphorus and carbamate insecticides with cholinesterase is outlined. The rates of both reactivation and aging of the inhibited enzyme differ for diHerent groups of organo­phosphorus compounds and are the main determining factors on which depend the proportion of the irreversibly inhibited enzyme formed dur­ing prolonged exposure to insecticide.

The level of erythrocyte cholinesterase is a better indicator of acetyl­cholinesterase activity at nerve synapses than that of plasma. The deter­mination of erythrocyte cholinesterase, or of whole blood cholinesterase using methods in which the activity measured is predominantly con­tributed by erythrocyte cholinesterase, is preferable.

The results of research carried out in WHO collaborating laboratories and of field studies on cholinesterase monitoring are described. At present the tintometric and Ellman spectrophotometric methods are those most used in WHO field trials. The latter has been adapted for use under field conditions when more accurate results are required, and a kit consisting of basic equipment, accessories, and preweighed chemicals is available from WHO if ordered by governmental laboratories or institutions. As with the tintometric method minimal back-up laboratory facilities are required. The recent investigations demonstrated its suitability for deter­mining indirectly the erythrocyte cholinesterase activity.

Monitoring of cholinesterase in malaria spraymen in the course of WHO field trials of new insecticides has proved invaluable for assessing a compound's potential for the cumulation of inhibition, for deciding when an operator should be withdrawn from further exposure to an anticholinesterase insecticide, and for making comparisons with other compounds. Thus, as a result of weekly cholinesterase determination in a 2 yr trial with fenitrothion, 2 to 3 out of 35 to 40 spraymen had to be withdrawn from spraying during each 2 mon round of spraying and in this way no complaints attributable to exposure to the insecticide were recorded in a total of 1,500 man-days of work.

In discussing the interpretation of the results it is stressed that the

78 M. VANDEKAR

severity of poisoning is detennined not only by the degree of reduction of cholinesterase but also by the rate of inhibition and the type of in­hibitor. The detennination of pre-exposure cholinesterase activity is of great importance in view of a wide range of activity in nonnal, unex­posed populations. It is the proportionate reduction of activity from the nonnal for the individual that is important rather than the numerical value of the test. For erythrocyte cholinesterase, 70% activity has been recommended as an action level by both WHO and the Subcommittee On Pesticides, Pennanent Commission and International Association on Occupational Health.

Several factors which can interfere with the accuracy of results are mentioned. The possibility of contamination of the blood sample by a direct inhibitor of cholinesterase is stressed, since it may be impossible to remove all traces from the skin. They can produce artifacts particu­larly in a micro-technique using fingertip capillary blood. Even when samples are analyzed in a laboratory the accuracy of results will depend to a great extent on the conditions in which samples are collected and transported from the field.

Acknowledgments

The author expresses his gratitude to Dr. J. F. Copplestone of the World Health Organization for useful discussions and help with the preparation of this manuscript.

References

ALDRIDGE, W. N., and E. REINER: Enzyme inhibitors as substrates, p. 328. Amsterdam­London: North-Holland (1972).

ARNAN, A.: Experience in the WHO field programme for evaluating the safety of new insecticides. Bull. World Health Org. 44,273 (1971).

AUGUSTINSSON, K.-B.: The normal variation of human blood cholinesterase activity. Acta Physiol. Scand. 35, 40 ( 1955).

-- Determination of activity of cholinesterases. In D. Glick (ed.): Methods of biochemical analysis, suppl. vol. Analysis of biogenic amines and their related enzymes, pp. 217-273. New York: Interscience (l971).

--, H. ERIKSSON, and Y. FAIJERSSON: A new approach to determining cholinesterase activities in samples of whole blood. Clin. Chim. Acta 89, 239 (1978).

--, and B. HOLMSTEDT: Determination of cholinesterase in blood samples dried on filter-paper and its practical application. Scand. J. Clin. Lab. Invest. 17, 573 (1965) .

EDSON, E. F.: Blood tests for users of OP insecticides. World Crops 10, 49 (1958). ELLMAN, G. L., K. D. COURTEY, V. ANDRES, JR., and R. M. FEATHERSTONE: A new

and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88 (1961).

GROFF, w. A., A. KAMlNSKlS, and R. I. ELLIN: Interconversion of cholinesterase enzyme activity units by the manual ApH method and a recommended auto­mated method. Clin. Toxicol. 9, 353 (1976).

Cholinesterase and OP poisoning 79

HOLMSTEDT, B.: Simple microcentrifuge for use in the field. Science 149, 977 (1965). -- Distribution and determination of cholinesterases in mammals. Bull. World

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Manuscript received February 19, 1980; accepted April 1, 1980.