The effect on cholinesterase activity of storage of undiluted whole blood sampled from men exposed to o-isopropoxyphenyl methylcarbamate (OMS-33)

Bull. Org. mond. Sant 1969, 40, 91-96 Bull. Wld Hlth Org. The Effect on Cholinesterase Activity of Storage of Undiluted Whole Blood Sampled from Men Exposed to o-Isopropoxyphenyl Methylcarbamate (OMS-33) M. VANDEKAR 1 & K. WILFORD 2 An observation made during an operational field trial with o-isopropoxyphenyl methyl- carbamate (OMS-33) suggested that the cholinesterase in undiluted blood samples from workers exposed to that insecticide was inhibited during the storage of those samples before assay. A laboratory investigation has now shown that rapidfalls of whole-blood cholinesterase activity do indeed occur during the storage of undiluted samples collectedfrom workers at different times after exposure to OMS-33, but that no such change occurs during the same period of time in circulating blood. Several possible explanations are put forward to account for the fact that, although the carbamate persists in the circulating blood for several hours after cessation of exposure, it does not appreciably inhibit cholinesterase in vivo but only under storage conditions in vitro. The implications of this phenomenon, in sofar as methods for determining cholin- esterase activity in persons exposed to carbamate insecticides are concerned, are discussed; and the authors point out that, whenever storage of undiluted samples precedes the assay, the spectrophotometric method should be regarded as giving results reflecting daily fluctuations of inhibitor concentration rather than as measuring the actual whole-blood cholinesterase in the body. An observation indicating possible inhibition of cholinesterase during storage of undiluted blood samples subsequent to their collection from workers exposed to o-isopropoxyphenyl methylcarbamate (OMS-33) 3 was made in the course of an operational field trial with that insecticide in Iran (Vandekar et al., 1968). Assays of whole-blood cholinesterase activity performed 21/2 hours after sampling revealed a mean change of -5 % as compared with assays performed within 5 minutes of sampling, the dif- ference being statistically significant at the 1% level. Since this comparison was made on samples obtained from workers who had not had any contact with the insecticide for 1½/2 days, the possibility was pointed out that a more pronounced change in cholinesterase activity might have occurred during storage in sam- 1 Scientist/Toxicologist, Vector Biology and Control, World Health Organization, Geneva, Switzerland. s WHO Consultant; Biochemist, Technical Officer, Toxicology Research Unit, Medical Research Council Laboratories, Carshalton, Surrey, England. 3Other designations for this compound include Baygon and IMPc; " arprocarb " has also been used but has been applied to another carbamate as well. ples collected immediately after exposure to the insecticide. During the present studies, carried out at the WHO Anopheles Control Research Unit No. 1 in Kaduna, Nigeria, use was made of the laboratory facilities available there to investigate the above observation further. The results obtained have clearly shown that a pronounced inhibition of cholinesterase does take place during the storage of undiluted blood samples collected from workers exposed to OMS-33, although no change in cholin- esterase activity occurs during the same period of time in circulating blood. MATERIALS AND METHODS Whole-blood cholinesterase activity was deter- mined by the spectrophotometric method of Ellman et al. (1961). As a rule 6-7 blood samples of 20-,u each were collected fromn a single finger-prick into Sahli haemoglobin pipettes rinsed with 2% heparin immediately before use. After the sampling and after the tip of the pipette had been wiped, the 2278 -91 M. VANDEKAR & K. WILFORD FIG. I WHOLE-BLOOD CHOLINESTERASE ACTIVITY a IN SAMPLES FROM 2 SPRAYMEN, THE SAMPLES BEING COLLECTED (A) BEFORE, AND (B) 1 HOUR, (C) 3 X HOURS AND (D) 20 HOURS AFTER SPRAYING OMS-33 AND ASSAYED AT DIFFERENT TIMES AFTER COLLECTION 120 100 80- I> 60- w= 4 - 20- 0 15' 30' Ih 100 - 80 - =~ 60-- C.) 40- i f 0 15' 30' 44' lh 100- ,,80 =~' 60- cc40 C.)20 0 15' 30' lh 100 a 80 *, 601 ,,, 40 C.2 B I ~~II[K1I 2h 4h 30' 8h 12h 16h 18h C FL1 2h 2h 15' 4h 8h 12h 16h D Time (h) of storage before assay WtO 81878 a Expressed as percentage of mean pre-exposure values obtained for each sprayman. 0 First sp,aynran. I Second sprayman. r - w - f /92 chE INHIBMON IN STORED BLOOD FROM MEN EXPOSED TO A CARBAMATE 93 column of blood was drawn 1 cm-2 cm up into the pipette. Except for the first sample, which was assayed immediately (the assay began within 20-40 seconds of the blood being drawn), the samples were stored on ice in a vacuum-flask at about 10°C for differing lengths of time. Before storage the pipettes were sealed with rubber bands of a suitable size and dilution of each sample was not carried out until immediately before the assay. The assays were per- formed at 25°C in an air-conditioned laboratory. A Unicam SP600 spectrophotometer was used, the cholinesterase activity being measured as the change of extinction during a 2-minute period.' One pre-exposure and 3 post-exposure batches of samples were collected from 2 spraymen who each sprayed 4 pump-charges during the course of 2 hours, the post-exposure samplings being performed 1, 31/4 and 20 hours after cessation of spraying. Addi- tional investigations were carried out on a mixer after he had completed the preparation of 16 pump- charges and at given intervals after work. All the precautionary measures recommended for OMS-33 spraying were observed: the spraymen wore sou'westers, clean overalls and canvas ankle shoes, and the mixer rubber gloves in addition. Spraymen- washed their hands and face after each pump-charge and all operators took a shower immediately after returning from the field. RESULTS The results obtained with the samples from the 2 spraymen were in general agreement (Fig. 1). No significant change in cholinesterase activity was observed in the samples collected before exposure during the 2-hour storage on ice (Fig. IA). (This point was confirmed by a separate experiment in which blood samples from two unexposed persons were stored on ice for more than 7 hours-see the table.) The samples collected 1 and 31/4 hours after exposure to OMS-33 showed a slight drop in cholinesterase activity when assayed immediately after collection. However, a very pronounced depression in cholinesterase activity was observed in the corresponding samples stored at about 10°C for 15-120 minutes, with a relatively slow subsequent rise after longer storage (Fig. lB and 1C). In the samples collected 20 hours after spraying, only a slight, although progressive, drop in cholin- I It has recently been shown (Wilhelm, Vandekar & Reiner-unpublished data) that about 92% of the enzyme activity measured by this method is contributed by the ery- throcyte cholinesterase. WHOLE-BLOOD CHOLINESTERASE ACTIVITY OF SAMPLES COLLECTED FROM 2 UNEXPOSED PERSONS AND ASSAYED AFTER STORAGE UNDILUTED AT 10°C Time of storage Cholinesterase activity a (min) Subject I Subject II 30 95 91 75 91 101 120 92 102 300 102 105 430 95 109 a Expressed as percentage of the mean values of 25 deter- minations (subject 1) and 10 determinations (subject 11), made during a 3-week period. esterase activity was observed during storage in one of the spraymen (down to 86% activity), and hardly any depression in the other (Fig. ID). A similar effect of storage on cholinesterase activity was observed in the samples from the mixer exposed to OMS-33. While the samples collected 5, 60 and 120 minutes after exposure and diluted within 40 seconds for the assay showed no inhibition of cholinesterase (the activities determined being 103%, 99% and 102% of the pre-exposure value respectively), the cholinesterase in the corresponding samples stored on ice showed a progressive inhibi- tion as time passed (Fig. 2). FIG. 2 WHOLE-BLOOD CHOLINESTERASE ACTIVITY a IN SAMPLES FROM A MIXER, THE SAMPLES BEING COLLECTED (A) 5 MINUTES, (B) 1 HOUR AND (C) 2 HOURS AFTER EXPOSURE TO OMS-33 AND ASSAYED AT DIFFERENT TIMES AFTER COLLECTION A.B.C T d0w~~ 20 0 WIT 371 lb 21 2Mh :31 Tim(h)dstompragbu1srmon" a Expressed as percentage of the mean of 9 pre-exposure values. 7 M. VANDEKAR & K. WILFORD DISCUSSION There is no doubt that the observed effect of storage on cholinesterase activity is associated with the exposure to OMS-33, since it only occurs in samples obtained from-workers after contact with the insecticide. Thus, the fall in cholinesterase is due to inhibition by the carbamate during storage. Since practically no change in cholinesterase activity occurred during the same period of time in circulating blood (compare the first columns in Fig. lB and IC), the possibility can be ruled out that subsequent inhibition of cholinesterase during storage is due to fact that, at the moment of blood collection, the process of enzyme inhibition was in a " pre-steady state" (i.e., had not yet levelled off). The possibility that contamination of the sample by insecticide occurred while blood was withdrawn can also be excluded. Apart from the fact that the spraymen had a shower and changed to normal clothes before the sampling, their finger-tips had been carefully cleansed with ethanol before being pricked with a disposable lancet. Moreover, in the case of the mixer, long rubber gloves, previously unused, and secured with tight elastic bands at the elbow, were worn, and after work these were removed from his hands by another, unexposed, person. The question which immediately arises is: why is there only slight inhibition of cholinesterase by the carbamate present in the circulation but an increase in that inhibition within a short time after with- drawal of the blood? There are several theoretical explanations: (1) The carbamate might be present in the circulating blood in an inaccessible form (for example, attached to macromolecules such as lipo- proteins); on storage of the blood the carbamate might be liberated so that it was able to inhibit the cholinesterase. (2) The cholinesterase itself might be in a form in vivo which would react only slowly with the carbamate; on withdrawal the protein might change so as to become more reactive. However, the essential similarity of the reactions of carbamate and of acetylcholine with cholinesterase would lead to the conclusion that such non-reactive cholinesterase would hydrolyse acetylcholine more slowly in vivo. The observed phenomenon may also be explained (3) by a greatly increased rate of decarbamylation in vivo (either by a change in the protein or by a specific reactivating substance), or (4) by the pre- sence of a substance in the circulating blood which prevents reaction of cholinesterase with the carba- mate. Acetylcholine is a theoretical possibility, but the published data on its concentration in human circulating blood (Scudamore et al., 1951) make this most unlikely. On the other hand, the presence of natural reactivating agents promoting the dephos- phorylation of inhibited enzyme in vivo has been described in the case of organophosphorus com- pounds (Neubert et al., 1958; Mengle & O'Brien, 1960), and the possibility exists of the in vivo pre- sence of a similar reactive agent in the case of car- bamates. In both possible explanations (3) and (4), however, the substances concerned must be unstable. There is no available information to suggest which of these 4 possibilities is the most likely. Neither can the possibility be excluded that the phenomenon may occur with other carbamates and some organo- phosphorus compounds. Whatever mechanism is responsible for this effect, the results clearly show that the carbamate persists in the circulating blood for several hours after cessa- tion of exposure: although it is unable to inhibit cholinesterase in vivo, or can do so to only a relatively small extent, it produces inhibition, or a more pronounced degree of inhibition, under storage con- ditions in vitro, the degree of inhibition correspond- ing to the actual concentration of inhibitor in the blood. It may be noted from Fig. 1 that a slight degree of inhibition of cholinesterase was found in the samples collected at 1 and 3 hours after exposure, in spite of their being assayed at " zero-time ". It should be pointed out that the actual interval between the moment of emergence of the blood droplet on the fingertip and the moment of dilution was 20-40 seconds. It is therefore impossible to say whether the recorded inhibition was due to inhibition produced in the body or to the " storage effect " of the 20-40 seconds, or to both. While it may be difficult to reduce the time between collection and dilution to a negligible interval, especially in routine analysis, it seems that, at least for samples that have to be transported to the laboratory, it would be more practical to carry out an appropriate dilution of the blood immediately after sampling; in order to diminish the reactivation of the inhibited enzyme, cooling the diluted sample and bringing it tem- porarily to a lower pH value (Reiner & Aldridge, 1967) may prove satisfactory. On the other hand, a method may be developed-making use of the above phenomenon of subsequent inhibition of the un- diluted blood sample in vitro-by which the actual concentration of the carbamate in the bloodstream may be determined. 94 chE INHIBITION IN STORED BLOOD FROM MEN EXPOSED TO A CARBAMATE 95 In view of these results, the daily pattern in the fluctuation of whole-blood cholinesterase activity as recorded by the spectrophotometric method in operators exposed to OMS-33 (Vandekar et al., 1968) and analysed some time after withdrawal of the blood should be regarded more as a reflection of the daily pattern of fluctuation of the inhibitor con- centration in the circulating blood than as a measure of actual whole-blood cholinesterase activity in the body. At the same time these findings provide a pos- sible explanation for the puzzling observations that have been made when cholinesterase activity has been determined in workers or villagers exposed to different carbamate insecticides. Blood samples had in most instances been stored on ice during transport and not diluted until immediately before the assay (Vandekar, 1965; Vandekar & Svetlicic, 1966; Vandekar et al., 1968); 1 frequently inexplicable, very pronounced, but symptomless depressions in the I Also unpublished WHO data reported by A. Arnan, J. E. Davies, J. J. Freal, G. E. Quinby, M. Vandekar and K. Wilford. cholinesterase activity had been recorded. No correlation between the frequency of complaints and the degree of cholinesterase inhibition could be found, the complaining workers frequently showing slighter depressions than those without complaints. In the case of the former, the cholinesterase activity was as a rule determined individually, within a short time of sampling, which was usually performed near the laboratory, while otherwise samples, usually 12 or more, were collected in the field and transferred undiluted to the laboratory. It is only owing to the rapidity of the consecutive inhibition which follows the blood collection that this effect remained unnoticed in a relatively large number of field trials. The results obtained in the field by the modified tintometric method (Vandekar et al., 1968; and Vandekar & Wilford-unpublished data, 1967)-in which the blood is immediately diluted by the indicator and substrate solutions, giving a final dilution of 1 : 101-correspond more nearly to the activity of the cholinesterase actually present in the circulating blood. ACKNOWLEDGEMENTS These studies were carried out as a part of a trial conducted by the WHO Anopheles Control Research Unit 1, Kaduna, Nigeria, and we are grateful to the Project Leader and other members of the unit for their invaluable co- operation. RtiSUMti EFFET DE LA CONSERVATION SUR L'ACTIVITE DE LA CHOLINESTERASE DU SANG COMPLET NON DILU, PRELEVt CHEZ DES TRAVAILLEURS EXPOSES AU METHYLCARBAMATE D'O-ISOPROPOXYPHENYLE (OMS-33) Au cours d'un essai destine A verifier I'innocuite d'em- ploi de 1'OMS-33, on a releve des valeurs statistiquement diff6rentes de I'activite de la cholinesterase (ChE) du sang complet non dilue selon que le dosage 6tait effectue peu de temps apres le pr6lvement de I'echantillon chez les sujets expos6s ou pratiqu6 plusieurs heures apres. Des recherches de laboratoire, decrites dans le present article, ont ete entreprises pour tenter d'e1ucider le phenomene. La mesure de I'activite cholinest6rasique a 6te faite par la methode spectrophotom6trique. Des 6chantillons de sang ont ete recueillis chez deux op6rateurs avant et 1, 31/4 et 20 heures apres 1'exposition. La conservation pendant 2 heures des echantillons prelev6s avant l'exposi- tion n'a pas modifie sensiblement les valeurs de I'activite enzymatique. La determination de la ChE dans les 6chantillons pr6leves 1 et 3¼4 heures apres 1'exposition a montre une faible baisse de I'activit6 lorsque le dosage a ete effectue immediatement apr6s le prelevement, mais une forte diminution lorsqu'il a 6te pratiqu6 apres 15 a 120 minutes de conservation du sang A 10°C. La conserva- tion des echantillons pr6leves 20 heures apres l'exposition n'a entrain6 qu'une faible baisse de I'activit6. Aucune variation notable de l'activit6 de la cholinesterase n'a 6te decelee pendant cette meme periode dans le sang circulant des personnes exposees. Les auteurs examinent longuement les diverses explica- tions possibles du ph6nomene. Ils sont d'avis que lorsque le dosage de l'activite de la ChE du sang non dilue par spectrophotometrie a lieu apres un certain d6lai de conservation des echantillons, on mesure les fluctuations de la concentration de l'inhibiteur plutot que l'activite r6elle de la ChE dans le sang circulant. 96 M. VANDEKAR & K. WILFORD REFERENCES Eliman, G. L., Courtney, K. D., Andres, V., Jr & Feather- stone, R. M. (1961) Biochem. Pharmacol., 7, 88-95 Mengle, D. C. & O'Brien, R. D. (1960) Biochem. J., 75, 201 Neubert, D., Schaefer, J. & Kewitz, H. (1958) Naturwis- senschaften, 45, 290 Reiner, E. & Aldridge, W. N. (1967) Biochem. J., 105, 171 Scudamore, H. H., Vorhaus, L. J. & Kark, R. M. (1951) J. Lab. clin. Med., 37, 860 Vandekar, M. (1965) Bull. Wid Hlth Org.,133, 107-115 Vandekar, M., Hedayat, S., Plestina, R. & Ahmady, G. (1968) Bull. Wld Hlth Org., 38, 609-623 Vandekar, M. & SvetliWii, B. (1966) Arh. Hig. Rada, 17 135