Animal sera and specific enzymes inthe treatment of poisoning

T h e Journal o[ P E D I A T R I C S

711

Animal sera and fioecific enzymes in the treatment of poisoning H a r r y C. Shirkey, M.D., ~ Gilbert C. Schmidt, Ph.D., Ronald G. Miller, M.S., ":~':" Leroy Honkomp, M.S., and G a r y Flarnm, M.S. ~e+ B I R ]VI I N G t-I A M ,

ALA.

T H A T certain rabbits can eat solanaceous plants with impunity has been recognized for approximately 100 years. Heckel, 1 for example, fed rabbits a diet consisting entirely of solanaceous drugs and did not observe ill effects. Although there has been disagreement about the mechanism of this tolerance, most investigators associated it with the occurrence of atropinesterase in the serum of resistant animals. Atropinesterase is present in the sera of only some rabbits. Its presence is genetically determined, and it is presumably an "incompletely dominant" characteristic. 7 This enzyme is not present in normal human serum. However, atropinesterase and related enzymes 2-G are present in sera of different animals. These enzymes will act on This paper was abstracted in part from a thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of requirements [or the degree of Master of Science, The project was supported in part by Research Grant 8447, National Institutes o[ Health. ~:Address, Director, the Children's Hospltal o[ Birmingham, Birmingham, Ala. ~x~United States Public Health Service Pre-Doctoral Fellow, University o[ Cincinnati College o/ Pharmacy. ":'e:'*United States Public Health Service Pre-Doctoral Fellow, University o] Cincinnati, College o[ Medicine, Department o[ Biological Chemistry.

nitrogen-alcohol esters and catalyze degradation of cocaine, tropacocaine, atropine, homatropine, heroin, and benzoylcholine. The degradation appears to be affected by specific serum enzymes. M a n y animals can tolerate large amounts of materials extremely toxic to man. 8 Enzymatic specificity genetically determined, appears to be a major factor in resistance of such animals to otherwise toxic doses of various drugs. Consequently, specific enzymes, derived from the sera and tissues of resistant animals, or from other natural sources such as bacteria, offer a potential armamentarium of highly specific antidotes for m a n y extremely toxic substances. Schinz 9 injected serum from resistant rabbits into cats and into nonresistant rabbits and studied the effects of atropine on the pulse rate of these animals in comparison with animals that had received no serum. Both intensity and duration of vagal response were diminished in the serum-treated group. He also observed, but did not further investigate, a diminished pupillary response to atropine in serum-treated animals. La Mendola ~~ confirmed some of the observations of Schinz but was unable to protect dogs against the lethal effects of atropine.

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Shirkey e~ al.

May 1962

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0

20

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40

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80 [00 (MINUTES)

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120

140

160

Fig. 1. Effect of pooled rabbit serum on response of mouse pupil to atropine. Upper curve, pupillary response to 2 mcg. of atropine sulfate injected intraperitoneally. Lower curve, pupillary response to 2 mcg. of atropine sulfate following intraperitoneal injection of 0.2 ml. of pooled rabbit serum, as described in text. Pupil diameter expressed in scale divisions of the ocular scale. T h e lethal manifestations of toxicity are those of central nervous system stimulation followed by respiratory and circulatory collapse. H e observed that the effects of atropine, pretreated by incubation with rabbit serum were retarded and mitigated as related to the heart and eye, but that other toxic effects were not lost. H e concluded that atropine effects on the eye and heart bore no relationship to its toxicity. T h e need for additional toxicologic work is apparent from these observations. Atropine poisoning in children results from ingestion of jimson weed, nightshade, henbane or other solanaceous plants or medicaments containing atropine or atropinelike compounds. Poisoning also occurs from accidental overdosage by several routes or from abnormal sensitivity to the drugs. CLINICAL

FINDINGS

Six years ago we treated our first severe case of jimson weed poisoning with antipertussis (rabbit) serum. T h e patient was a 4-year-old boy. T h e great majority of accidental poisoning occurs in the 1- to 5-yearold group (toddler). All of our patients treated were of this age group. T h e choice

was made because the antipertussis serum was prepared from rabbit serum and might, therefore, contain atropinesterase. In the absence of previous data or clinical experience in the use of rabbit sera for this purpose, a dose of 8 mI., representing the contents of 2 vials was arbitrarily chosen. T h e vials were from 2 separately numbered lots. Because of the arbitrary nature of the dosage, and since there is not m u c h weight variation in this "toddler" group, no allowances have been made for differences in body weight. The serum was administered by intramuscular injection and the dose was not repeated. I n none of the treated cases was the exact a m o u n t of ingested plant or alkaloidal salt known. Previous and subsequent experience has been gained with a n u m b e r of children and adult patients who have been treated with short-acting barbiturates or otherwise treated symptomatically. Thus, there was some clinical basis for the expected speed of recovery after poisonous, but unknown, dosage. Objective improvement included return to normal in walking (loss of ataxia) and the loss of delirium. T h e return to normal of these functions preceded the return to normal of pupillary di-

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Sera and enzymes in treatment of poisoning

ameter. The diminution or absence of sweating was used as a diagnostic aid but was not followed. Striking improvement was obtained in all early trials, however, during the last 2 or 3 years our results have shown no clinical success from the rabbit serum. No patients died; however, none were expected to die. In the interval between the period of successful and unsuccessful clinical results (as related to rabbit serum use), a change was made in the rabbit colony from which serum was obtained as well as a change in the method of fraetionation of the pooled serum. Since the value of antipertussis (rabbit) serum in atropine poisoning could not be determined from clinical data alone, we designed biochemical and pharmacologic experiments. It was anticipated that data from the laboratory would supplement those from the bedside. MANOMETRIC ASSAYS FOR ATROPINESTERASE

All clinically ineffective sera were free of atropinesterase on manometric assay, 11 but sera used in the successful trials were not available for esterase determinations. As a result, no relationship between clinical response and esterase content can be established at the present time. Seven different samples of sterile, pooled, commercially available rabbit serum* were manometrically assayed for atropinesterase content, and all showed good activity. This known source of atropinesterase was employed in all pharmacologic studies described. PROTECTION AGAINST L E T H A L D O S E S OF A T R O P I N E

The protective action of atropinesterasecontaining sera against lethal doses of atropine sulfate was demonstrated in white mice with the use of the null hypothesis and contingency based on chi square? 2 One hundred to 200 male white mice, nSterile, pooled rabbit serum (S-6) was pmchased from Cappel Laboratories, West Chester, Pennsylvania. It is not intended for human use.

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weighing 10 to 12 grams, were randomly distributed into 2 equal groups. The control group was intraperitoneally injected with 0.8 ml. of distilled water, immediately followed by 250 mg. per kilogram of atropine sulfate dissolved in 0.2 ml. of distilled water. The test group was treated in the same way, except that 0.8 ml. of atropinesterase-containing serum replaced distilled water as the initial injection. Deaths and survivals were recorded after 1 hour, and the results were statistically analyzed. Results of a typical experiment are shown in Table I. A highly significant protective action against the lethal effects of atropine is apparent from the data. We have ascribed this protective action to the injected serum. Several repetitions of this experiment, with the use of horse serum, which does not contain atropinesterase, gave no significant protection. We, therefore, concluded that the protective action of rabbit serum was not due to adsorption of atropine onto injected protein in the peritoneal cavity. EFFECT

OF R A B B I T

SERUM

ON P U P I L L A R Y D I L A T I O N Protection against lethal effects of atropine appeared to be accompanied by objective improvement in the test animals. One phase of this improvement was demonstrated by a qualitative modification of Pulewka's mouse eye assay procedure, 13 with the use of sublethal doses of atropine sulfate. Twenty male white mice, weighing 15 to 16 grams were randomly divided into 2 equal groups. Mean pupil diameter was determined for each group. The control group was then intraperitoneally injected with 0.2 ml. of distilled water, immediately followed by 2 mcg. of atropine sulfate dissolved in

Table I. Effect of pooled rabbit serum on LDso of atropine*

] Alive I Dead I Totals Control Test Totals

38 62 100

52 28 80

90 90 180

~x2o) = 13.00. Critical value = 10.83 at the 0.00l level.

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Shirkey et al.

0.2 ml. of distilled water. T h e test group was treated in the same way, except that the initial water injection was replaced by 0.2 ml. of atropinesterase-containing serum. M e a n pupil diameter was determined for each group of animals at 5-minute intervals. Pupil diameter was then plotted as a function of time. Fig. 1 shows a representative experiment. It is readily apparent from Fig. 1 that atropinesterase-containing serum exerted a protective effect against atropine-induced mydriasis. These experiments have been repeated, with the employment of tail vein serum injections. T h e same general pattern of protection was obtained. Thus, the protective action cannot be assigned to hydrolysis of the alkaloid in the peritoneal cavity prior to absorption, nor can it be due to adsorption of arug onto the injected protein. It has also been shown that the effect of the serum lasts for approximately 1 week if mydriasis is used as the criterion. SUMMARY

Certain samples of antipertussis serum (rabbit) exerted a favorable effect in atropine-poisoned children, while other sampIes are inactive. No relationship between clinical effectiveness and atropinesterase content has been established although it is tempting to assume that effectiveness requires the presence of the esterase. Sterile, pooled rabbit serum of known esterase content was not employed in clinical studies because of the crude nature of the serum. It has been shown, however, that such sera exert a protective effect against lethal doses of atropine sulfate in white mice. It has also been shown that atropineesterase-containing sera prevent, or sharply diminish, the pupillary response of mice to sublethal doses of atropine sulfate. This protective action lasts approximately 1 week. T h e protective effects against lethal oi' sublethal doses of atropine are not due to adsorption of alkaloid onto injected protein, nor are they due to hydrolysis in the peritoneal cavity prior to absorption of the alkaloid. These observations are in general agree-

May 1962

ment with the observations of Schinz 9 who found that resistant serum protected other animals against the vagal effects of atropine. There is partial agreement with L a Mendola t~ who observed protection against mydriatic and vagal effects. Although the data are not presented here, we have also found that protection against mydriasis and protection against the lethal central nervous system effects do not always parallel one another. This was the case with other sera and with certain other alkaloids. This investigation establishes the potential value of atropinesterase in atropine poisoning and emphasizes the need for further study. We intend to study further the resistance of animals to poisonous plants indigenous to their natural habitat, to constituents of these plants (alkaloids, glycosides, etc.), and to similar synthetic chemicals. We are, also, interested in the study of microorganisms which grow in solutions of toxic chemicals. Either of these sources m a y yield substances of specific nature valuable in the treatment of h u m a n poisoning. We wish to thank Dr. George E. Farrar and Dr. Daniel L. Shaw, Jr., Wyeth Laboratories, for their interest and for generous supplies of antipertussis serum.

REFERENCES

1. Heckel, C. R.: De l'influence des solanes en general et de la belladonna en particular, Aead. de Paris 80: 1608, 1875. 2. Glick, D.: Specificity Studies on Enzymes Hydrolyzing Esters of Substituted Amino Acid and Nitrogen Heterocyclic Alcohols, J. Am. Chem. Soc. 64: 564, 1942. 3. Wright, C. I.: The Enzymatic Deactylation of Heroin and Related Morphine Derivatives by Blood Serum, J. Pharmacol. & Exper. Therap. 71: 164, 1941. 4. Ellis, S.: Benzoylcholine and Atropine Esterases, J. Pharmacol. & Exper. Therap. 91: 370, 1947. 5. Glick, D., and Glaubach, S.: The Occurrence and Distribution of Atropinesterase, and the Specificity of the Trop{nesterases, J. Gen. Physiol. 25: 197, 1941, 6. Glick, D., Glaubach, S., and Moore, D. H.: Azolesterase Activities of Electrophoretlcally Separated Proteins of Serum, J. Biol. Chem. 14i: 525, 1942.

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7. Sawin, P. B., and Glick, D.: Atropinesterase, Genetically Determined Enzyme in the Rabbit, Proc. Nat. Acad. Sc. 29: 55, 1943. 8. Gunn, J. A.: Cellular Immunity: Congenital and Acquired Tolerance to NonProtein Substances, Physiol. Rev. 3" 41, 1923. 9. Schinz, It. R.: Zur angebornen und erworbenen Atropinresistenz des Kaninchen, Arch. exper. Path. u. Pharmacol. 89: 193, 19t8. 10. La Mendola, S.: Resistance to Atropine of Dogs Treated With Serum of Normal Rabbits, Ann. Chim. Med. e di Med. Speriment. 14: 336, 1924.

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11. Bernheim, F., and Bernheim M. L. C.: The Hydrolysis of Homatropine and Atropine by Various Tissues, J. Pharmacol. & Exper. Therap. 64: 209, 1938. 12. Hill, Bradford A.: Principles of Medical Statistics, ed. 6, Oxford University Press~ New York, 1956, pp. 139-143. 13. Pulewka, P.: Das Auge der weissen Maus als pharmakologisches Testobjekt. Eine Methode zur quantitativen Bestimmung kleinster Mengen Atropin und anderer Mydriatika, Arch. exper. Path. u. Pharmaeol. 168: 307, 1932.