Toxicants Occurring Naturally in Foods. (1966)

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96 F, M. STRONG with other MAO inhibitors and other osteolathyrogens such as B-mer- captoethylamine.3 That a similar potentiation of amine toxicity occurs in human beings under special circumstances has recently been very forcefully demon- strated by a series of reports of severe complications following the consumption of aged cheese by patients receiving MAO-inhibiting drugs,* particularly the tranquilizer Tranylcypromine (Parnate). The relationship of cheese to this problem was elucidated by Asatoor et al., who drew attention to the high amounts of pressor amines present in aged cheese.! The difficulty reported was of the nature of a hypertensive crisis which in several cases was severe enough to be fatal. Reports of similar complications in patients on tranquilizers following consump- tion of pickled herring liave recently appeared in the popular press. The serotonin intake of many African peoples who use plantains as a major article of diet may reach 100-200 mg per day. It has been sug- gested? 6 that this may be a factor in the high incidence of endomyo- cardial fibrosis among Africans, as the symptoms resemble the lesions of carcinoid heart disease, a condition in which large amounts of serotonin are produced endogenously. In this connection it has been demonstrated that ingestion of 100-160 mg of serotonin per day for several days resulted in greatly increased platelet serotonin levels in human subjects,!4 although the patients showed no obvious ill effects. The toxicity of pressor amines administered intravenously to mice is enhanced by thyroxine.” This observation suggests the possibility of deleterious effects from the combination of hyperthyroidism and con- sumption of foods containing pressor amines. The importance of histamine, catecholamines, and amino indoles to mental health, and some possible nutritional relationships thereto, have recently been pointed out.8 The human body is continuously exposed to these compounds both directly from their presence in common foods and indirectly from bactertal degradation of amino acids in the in- testinal tract. The vital necessity of efficiently functioning bodily mechanisms for coping with this influx of toxic compounds, and the likelihood of trouble from other chemicals that may interfere with these mechanisms, are obvious. * Additional information on the pressor amine content of several foods and their effects on patients receiving MAO-inhibiting drugs has recently appeared in Nutr. Rev., 23, 326 (1965).

PRESSOR AMINES 97 REFERENCES 12. 13. 14. 15. 16. 17. . A. M. Asatoor, A. J. Levi, and M. D. Milne, “‘Tranylcypromine and Cheese,” Lancet, ii, 733 (1963). D. W. Bruce, “‘Carcinoid Tumors and Pineapples,” J. Pharm. Pharmacol., 13, 256 (1961). M. A. Crawford, “Excretion of 5-Hydroxyindoleacetic Acid in East Africans,” Lancet, i, 352 (1962). J. M. Foy and J. R. Parratt, ““A Note on the Presence of Noradrenalin and 5-Hydroxytryptamine in Plantzin (Musa sapientum var. paradisiaca),” J. Pharm. Pharmacol., 12, 360 (1960). J. M. Foy and J. R. Parratt, “‘S-Hydroxytryptamine in Pineapples,” J. Pharm. Pharmacol., 13, 382 (1961). J. M. Foy and J. R. Parratt, “Urinary Excretion of 5-Hydroxyindoleacetic Acid in West Africans,” Lancet, i, 942 (1962). B. N. Halpern, C. Drudi-Baracco, and D. Bessirard, ‘*Exaltation of Toxicity of Sympathomimetic Amines by Thyroxine,” Nature, 204, 387 (1964). M. K. Horwitt, ““Nutrition in Mental Health,” Nutr. Rev., 23, 289 (1965). W. Keil and H. Kritter, “The Chemistry and Pharmacology of Fermented Foods I,” Arch. Exptl. Pathol. Pharmakol., 175, 736 (1934). . §S.H. Lipton, J. J. Lalich, J. T. Garbutt, and F. M. Strong, “Identification of Cyanoacetic Acid as a Urinary Metabolite of 8-Aminopropionitrile,” J. Am. Cher. Soc., 80, 6594 (1958). . P. Marquardt, H. Schmidt, and M. Spath, “The Presence of Histamine in Alcoholic Beverages,”’ Arzneimittel-Forsch., 13, 1100 (1963). P. B. Marshall, ‘““Catechols and Tryptamines in the ‘Matoke’ Banana (Musa paradisiaca),” J. Pharm. Pharmacol., 11,639 (1959). B. E. McDonald, H. R. Bird, and F. M. Strong,‘‘Production of Aortic Aneurysms in Turkeys and Effect of Various Compounds in Potentiating Induced Aortic Ruptures,” Proc. Soc. Exptl. Biol. Med., 113,728 (1963). K. Melmon and A. Sjoerdsma, “‘Serotonin in Platelets. Its Uptake and Disap- pearance after Administration of the Amine by Mouth,” Lancet, ii, 316 (1963). I. Stewart and T. A. Wheaton, ‘“1-Octopamine in Citrous: Isolation and Identification,” Science, 145, 60 (1964). T. P. Waalkes, A. Sjoerdsma, C. R. Creveling, H. Weissbach, and S. Udenfriend, “Serotonin, Norepinephrine, and Related Compounds in Bananas,” Science, 127, 648 (1958). G. B. West, “Tryptamine in Tomatoes,” J. Pharm. Pharmacol., 11,319 (1959).

SAMUEL LEPKOVSKY Antivitamins in Foods Adequate intake of vitamins with food is no assurance that the animal is free of marginal or outright vitamin-deficiency states. Vitamins may exist in food in bound forms that are unavailable to animals. Species differences exist so that dietary vitamins may be available to some animals, but not to others. Some foods contain factors that destroy vitamins before they are eaten, and they may continue their destructive activity in the intestinal tract and in the body after the vitamins have been absorbed from the gut. Imbalances of nutrients increase vitamin requirements, as do drugs that are administered. Rachitogenic Factors A rachitogenic factor was discovered in yeast! when large amounts of it were used as a source of protein for pigs. No information on the chemical nature of this factor was obtained. Such a factor effective in sheep? and rats? was also found in green oats. Evidence was adduced that the rachitogenic factor was carotene ;34 this was based on studies using the McCollum 3143 rachitogenic ration. However, Coates et al.5 have failed to demonstrate an antagonism between carotene and vitamin D. Much of the evidence pointed to B-carotene® as a rachitogenic factor in grass, though in all likelihood it is not the only such factor. It is of interest in this connection that heavy doses of vitamin A were found to induce rickets in rats on the rachitogenic McCollum diet 3143, but not on normal diets.67 Cereals have been shown to be rachitogenic, and Harrison and Mellanby® showed that the rachitogenic factor is phytic acid. 98

ANTIVITAMINS 99 Liver has been reported to contain a rachitogenic factor that impairs the absorption of calcium from the gut of the chicken.? The chemical nature of the compound has not been established. Antivitamins for Carotene and Vitamin E Complicated antagonism exists between unsaturated fatty acids and carotene.!° Tests with methyl! linoleate and methyl linolenate revealed an antagonistic action when low levels of carotene were fed. This antagonism was overcome when the diet contained sufficiently high levels of carotene. Unsaturated fatty acids in foods are readily oxidized before they are eaten, during their stay in the intestinal tract, and in the tissues after they are absorbed and deposited there. Vitamin E protects unsaturated fatty acids against oxidation in vitro and in vivo. In carrying out this function, vitamin E is used up. To be effective in vivo, vitamin E must be absorbed from the gut, and fat facilitates this process. Linoleic acid acts as an antivitamin to d/-alpha-tocopherol, by decreasing the amount available for absorption as a consequence of the destruction of the vitamin in the intestine. Linoleic acid also in- creases the requirements of d/-alpha-tocopherol under conditions that exclude any influence on the uptake of vitamin E from the intestine.!! The highly unsaturated fish oils, e.g., cod liver oil, are very destruc- tive of vitamin E, and they have been exhaustively studied. The litera- ture has been reviewed by Dam.!2 Singsen et al.!3 found a discrepancy between the tocopherol content of alfalfa and its biological activity for the chick. It was traced to an ethanol-soluble compound that is antagonistic to tocopherol, increasing its excretion and decreasing its availability.14 An antagonist for vitamin E has also been found in yeast.!5 It has been located in the ash, but the compound has not been identified. An antagonistic relationship exists between excessive thyroid activity and vitamin E. Tocopherol protects chicks!® and rats!7 against the symptoms produced by feeding thyroid. Antivitamin A Raw soybeans contain lipoxidase which oxidizes and destroys carotene.'8 Large amounts of yeast decreased the amount of vitamin A in the liver of the pig,!9 indicating increased requirements. Thyroxine aggravates vitamin A deficiency, and increased levels of vitamin A are needed to antagonize the toxic effects of increased intake of thyroxine.2°

100 SAMUEL LEPKOVSKY Citral, found in oranges, acts as an antagonist to vitamin A, and its damaging effects can be prevented by additional vitamin A in the diet.! Antivitamin K Spoiled sweet clover hay has furnished us with a true antimetabolite of vitamin K. This is dicoumarol (3,3’-methylenebis[4-hydroxycou- marin]).22 The compound causes hypoprothrombinemia and hemor- rhagic conditions in animals,22 and these are reversed by feeding vitamin K. Vitamin A in large doses seems also to be an antagonist of vitamin K, causing hemorrhages in rats that can be prevented by additional intake of vitamin K.23:24 Raw soybeans show anticoagulant properties® that are not reversed by vitamin K. The anticoagulant properties of raw soybeans were attributed to the antitrypsin of the raw soybeans. Antithiamine Raw carp flesh contains an enzyme, thiaminase, that destroys thia- mine.26 In addition to fish, many crustacea contain thiaminase.?’ Heating destroys the enzyme. Bracken fern fed to rats causes symptoms of thiamine deficiency, which can be cured by the administration of the vitamin.28 Heating destroys the factor responsible. High levels of carbohydrates in the diet increase the requirements for thiamine and hence have an antivitamin effect. Fat decreases the re- quirements for this vitamin.29 Pellagragenic Factor Most or all of the antipellagra factor (nicotinic acid) of cereals, includ- ing corn, is present in a “bound” form and is unavailable to animals, including human beings. It is made readily available by treatment of the grain with dilute alkali3°-33 This pellagragenic property of corn (maize) has been the subject of much investigation, and it is commonly thought that the relative freedom from pellagra of the maize-eating populations of Central America stems from the custom in that area of treating maize with lime water during its preparation as food.

ANTIVITAMINS 101 Antivitamin By? High-protein diets increase requirements for vitamin By.*4 Ingestion of raw soybeans rich in antiproteolytic activity>5 also increases require- ment of By, as do diets containing large amounts of lard.>6 Vitamin By usually occurs in food in bound forms, and differences have been ob- served in the availability to different species of bound vitamin By. The vitamin By, in sow’s milk is available to the suckling pig, but is only poorly utilized by chicks and rats. Similarly, vitamin By given by mouth with a preparation from pig pylorus had low activity for chicks and rats, but was fully active for the pig.37 _ Glycine is toxic to chicks fed a vitamin By-deficient diet. This toxicity is overcome by folic acid and vitamin By.38 Imbalance of amino acids depresses growth. Growth depression caused by leucine and glycine fed at 4 percent levels or zein at 8 percent in the diet was corrected by vitamin By. The growth-depressing action of zein was attributed to its leucine content. L-Tyrosine and DL-aspartic acids fed at the level of 4 percent also depress growth, but vitamin Bp is only partially active in overcoming the growth depression. B,2 is not effective in correcting the growth depression caused by excesses of DL-alanine, DL-methionine, or L-cystine.39 Large doses of thyroid-active materials decrease growth of rats fed low-fat soybeans*° or corn meal‘? as a source of protein. Vitamin By is effective in overcoming the effects of the thyroactive material? in rats on diets containing these sources of protein, but not if they are on diets containing sucrose as the carbohydrate.‘? Antipyridoxine Factors Linseed oil meal in diets containing adequate amounts of pyridoxine for growth precipitates a pyridoxine deficiency when fed to chicks. The deficiency can be prevented by synthetic pyridoxine. The pyridoxine antagonist disappears when the linseed meal is treated with water.*! Antifolic Acid and Anticholine Excessive intake of nicotinamide by rats results in the depletion of labile methyl groups due to obligatory methylation of nicotinamide, causing growth depression and fatty livers. This does not occur in guinea pigs and rabbits since they do not methylate nicotinamide before its excretion. As a consequence, excessive intake is not dele- terious, as it is in rats.43 The effects of nicotinamide toxicity are corrected by choline or by folic acid.4

102 SAMUEL LEPKOVSKY Antibiotin Biotin was shown to be inactivated by raw egg white,45 and the binding factor was shown to be a protein, avidin.4647 Heating releases the biotin from the avidin-biotin complex. Conditioning Factors Hyperthyroidism increases the need for fat-soluble vitamins and for vitamins of the B complex.8 49 Thus, hyperthyroidism acts as an anti- vitamin for these vitamins. Intestinal synthesis of vitamins acts in general to decrease the re- quirements for vitamins in the diet.5° The synthetic activity of intestinal bacteria may be greatly influenced by the diet, as in the case of “‘re- fection,” where the B requirements are markedly reduced.5° Intestinal bacteria may also destroy vitamins. Some intestinal bacteria contain thiaminase and cause hypothiaminosis. This has been shown in human subjects whose feces decompose thiamine.5! REFERENCES 1. R. Braude, S. K. Kon, and E. G. White, “Yeast as a Protein Supplement for Pigs; Further Observations on Its Rachitogenic Effect,” J. Comp. Path., 54, 88 (1944). T. K. Ewer, “Rachitogenicity of Green Oats,” Nature, 166, 732 (1950). A. B. Grant, “Carotene: A Rachitogenic Factor in Green Feeds,” Nature, 172, 627 (1953). 4. J. Weits, “‘A Factor in Hay Inhibiting the Action of Vitamin D,” Nature, 170, 891 (1952). 5. M. E. Coates, S. K. Kon, and J. W. G. Porter, “‘Vitamins in Animal Nutriton,” Brit. Med. Bull., 12, 61 (1956). 6. J. Weits, ““‘The Influence of Carotene and Vitamin A on the Antirachitic Action of Vitamin D,” Z. Vitaminforsch., 30, 399 (1960). New Zealand Dept. Agr., Ann. Rept., 1956, p. 49. D. C. Harrison and E. Mellanby, “‘Phytic Acid and the Rickets-Producing Action of Cereals,”” Biochem. J., 33, 1660 (1939). 9. M. E. Coates, G. F. Harrison, and E. S. Holdsworth, “‘The Effect of a Rachi- togenic Factor on Calcium Metabolism in Chicks,” Brit. J. Nutr., 15, 149 (1961). 10. W. C. Sherman, “The Effect of Certain Fats and Unsaturated Fatty Acids upon the Utilization of Carotene,” J. Nutr., 22, 153 (1941). 11. F. von Weber, H. Weiser, and O. Wiss, “‘Bedarf an Vitamin E in Abhangigkeit von der Zufuhr an Linolsdure,” Z. Erndhrungswiss., 4, 245 (1964). WN g o

ANTIVITAMINS 103 12. 13. 14. 15. 16. 17. 18. 19. 21. 23. 24. 25. 26. 27. . P. H. Weswig, A. M. Freed, and J. R. Haag, “‘Antithiamine Activity of Plant 29. 31. H. Dam, “Influence of Antioxidants and Redox Substances on Signs of Vitamin E Deficiency,” Pharmacol. Rev., 9, 1 (1957). E. P. Singsen, L. M. Potter, R. H. Bunnell, L. D. Matterson, L. Stinson, S. V. Amato, and E. L. Jungherr, “Studies on Encephalomalacia in the Chick. 6. The Utilization of Vitamin E from Alfalfa and Wheat Middlings for the Preven- tion of Encephalomalacia,” Poultry Sci., 34, 1234 (1955). W. J. Pudelkiewicz and L. D. Matterson, “‘A Fat-Soluble Material in Alfalfa that Reduces the Biological Availability of Tocopherol,” J. Nutr., 71, 143 (1960). J. G. Bieri, G. M. Briggs, and C. J. Pollard, ‘‘Acceleration of Vitamin E De- ficiency by Torula Yeast. II. Effect of Torula Yeast Ash and Lipide,” Proc. Soc. Exptl. Biol. Med., 99, 262 (1958). R. S. Wheeler and J. D. Perkinson, “‘Influence of Induced Hypo- and Hyper- thyroidism on Vitamin E Requirement of Chicks,” Am. J. Physiol., 159, 287 (1949). L. Tentori, G. Toschi, and G. Vivaldi, ‘‘L’effetto dell’ ipertiroidismo speri- mentale sulla comparasa di Jesioni muscolari nel ratto mantenuto ad una dieta carente di vitamina E,” Boll. Soc. Ital. Biol. Sper., 29, 90 (1953). J. B. Sumner and A. L. Dounce, “Carotene Oxidase,” Enzymologia, 7, 130 (1939). R. Braude, K. M. Henry, S. K. Kon, and S. Y. Thompson, “The Effect of Yeast on Liver Size and Vitamin A Storage in the Pig,” Brit. J. Nutr., 1, vi (1947). B. Sure and K. S. Buchanan, “‘Infiuence of Hyperthyroidism on Vitamin A Reserves of the Albino Rat,”’ J. Nutr., 13, 521 (1937). E. H. Leach and J. P. F. Lloyd, ‘‘Citral Poisoning,” Proc. Nutr. Soc. (Engl. Scot.), 15, xv (1956). H. A. Campbell and K. P. Link, “Studies on the Hemorrhagic Sweet Clover Disease. IV. The Isolation and Crystallization of the Hemorrhagic Agent,” J. Biol. Chem., 138, 21 (1941). T. Moore and Y. L. Wang, “‘Hypervitaminosis A,’”’ Biochem. J., 39, 222 (1945). R. F. Light, R. P. Alscher, and C. N. Frey, “Vitamin A Toxicity and Hypopro- thrombinemia,” Science, 100, 225 (1944). S. L. Balloun and E. L. Johnson, “Anticoagulant Properties of Unheated Soybean Meal in Chick Diets,” Arch. Biochem. Biophys., 42, 355 (1953). R. G. Green, W. E. Carlson, and C. A. Evans, **The Inactivation of Vitamin B, in Diets Containing Whole Fish,” J. Nutr., 23, 165 (1942). A. Fijita, ““Thiaminase,” Advan. Enzymol., 15, 389 (1954). Materials,” J. Biol. Chem., 165, 737 (1946). L. J. Harris, ‘‘Antivitamins and Other Factors Influencing Vitamin Activity,” Brit. J. Nutr., 2, 362 (1949). D. K. Chaudhuri and E. Kodicek, “The Biological Activity for the Rat of a Bound Form of Nicotinic Acid Present in Bran,” Biochem. J., 47, Proc., XXxiV (1950). D. K. Chaudhuri and E. Kodicek, “The Availability of Bound Nicotinic Acid to the Rat. 4. The Effect of Treating Wheat, Rice and Barley Brans and a Purified Preparation of Bound Nicotinic Acid with Sodium Hydroxide,” Brit. J. Nutr., 14, 35 (1960).

104 32. 33. 34. 35. 36. 37. 38. 39. 41. 42. 43. 45. 47. 49. 51. SAMUEL LEPKOVSKY R. Braude, S. K. Kon, K. G. Mitchell, and E. Kodicek, ‘“‘Maize and Pellagra,” Lancet, 268, 898 (1955). H. Chick, ““The Causation of Pellagra,” Nutr. Abstr. Rev., 20, 523 (1951). H. Yacowitz, R. F. Miller, L. C. Norris, and G. F. Heuser, ““Vitamin B,2 Studies with the Hen,” Poultry Sci., 31, 89 (1952). A. Frdlich, “Relation Between the Quality of Soybean Oil Meal and the Re- quirements of Vitamin B,: for Chicks,”’ Nature, 173, 132 (1954). M. R. Spivey, G. M. Briggs, and L. O. Ortiz, “Effects of Diets High in Fat or Protein on Vitamin B;2 Deficiency in Non-depleted Chicks,” Proc. Soc. Exptl. Biol. Med., 85, 451 (1954). M. E. Coates, M. E. Gregory, G. F. Harrison, K. M. Henry, E. S. Holdsworth, and S. K. Kon, “The Availability to Animals of Vitamin B,, Bound with Their Own or with Foreign Binding Factors,” Proc. Nutr. Soc. (Engl. Scot.), 14, xiv (1955). L. J. Macklin, A. H. Lankenau, C. A. Denton, and H. R. Bird, “Effect of Vitamin By and Folic Acid on Growth and Uricemia of Chickens Fed High Levels of Glycine,” J. Nutr., 46, 389 (1952). P. Hsu and G. F. Combs, “Effect of Vitamin B,, and Amino Acid Imbalances on Growth and Levels of Certain Blood Constituents in the Chick,” J. Nurr., 47, 73 (1952). B. H. Ershoff, ‘Protective Effects of Soybean Meal for the Immature Hyper- thyroid Rat,” J. Nutr., 39, 259 (1949). F. H. Kratzer and D. E. Williams, ‘The Relation of Pyridoxine to the Growth of Chicks Fed Rations Containing Linseed Oil Meal,” J. Nutr., 36, 297 (1948). U. J. Lewis, D. V. Tappan, V. D. Register, and C. A. Elvehjem, “Effect of Carbohydrate on Growth Response to Vitamin B,: in the Hyperthyroid Rat,” Proc. Soc. Exptl. Biol. Med., 74, 568 (1950). P. Handler, “The Effect of Excessive Nicotinamide Feeding on Rabbits and Guinea Pigs,” J. Biol. Chem., 154, 203 (1944). . P. Fatterpaker, U. Marfatia, and A. Sreenivasan, “Observations on the Relation- ship Between Pteroylglutamic Acid and Nicotinamide Metabolism,”’ Biochem. J., 59, 470 (1955). R. E. Eakin, E. E. Snell, and R. J. Willams, “‘A Constituent of Raw Egg White Capable of Inactivating Biotin Jn Vitro,” J. Biol. Chem., 136, 801 (1940). . D. Pennington, E. E. Snell, and R. E. Eakin, “Crystalline Avidin,” J. Am. Chem. Soc., 64, 469 (1942). D. W. Wooley and L. G. Longsworth, “Isolation of an Antibiotin Factor from Egg White,” J. Biol. Chem., 142, 285 (1942). . V. A. Drill, “‘Interrelations between Thyroid Function and Vitamin Meta- bolism,” Physiol. Rev., 23, 355 (1943). B. H. Ershoff, ‘‘Conditioning Factors in Nutritional Disease,” Physiol. Rev., 28, 107 (1948). | . R.J. Williams, R. E. Eakin, E. Beerstecker, Jr., and W. Shive, The Biochemistry of the Vitamins, Reinhold, New York (1950), pp. 297-300. Anon., ““Thiaminase Disease,” Nutr. Rev., 14, 166 (1956).

ANTHONY M. AMBROSE Naturally Occurring Antienzymes (Inhibitors) This review is restricted to the proteolytic enzyme inhibitors that occur naturally in foods and feeds normally consumed by man and animals. NATURALLY OCCURRING ENZYME INHIBITORS OF PLANT ORIGIN Seed proteins (cereal grains, legumes, and oil seeds) are important sources of dietary protein in many areas of the world, particularly in those areas where for religious or economic reasons animal proteins are limited or unavailable. Some of them contain enzyme inhibitors. Perhaps the best known and most extensively studied of these toxic factors in seeds used as foods and feeds for man and animals are the trypsin inhibitors, so called because of their ability to interfere with tryptic digestion in the digestive tract. However, under conditions of controlled processing, the antitryptic factor in the seeds can be partially or completely altered or eliminated and the nutritional value improved. Since the report of Ham and Sandstedt! on the presence of a trypsin inhibitor in raw soybeans, considerable interest has been aroused con- cerning the possible role of trypsin inhibitors in altering the nutritive value of proteins. Ham ef al.2 subsequently demonstrated that an acetone-insoluble fraction from unheated soybean meal inhibited the growth of chickens. Similar growth-depressing effects have been noted with active antitryptic factors from unheated soybeans in rats?-5 and mice.® It is interesting to note that the harmful effects of the active soybean extract were not associated with decreased food intake. 105

106 ANTHONY M. AMBROSE Increased nutritional value of soybean protein resulting from the destruction of the trypsin inhibitor by heat treatment has been sug- gested by a number of investigators. Westfall and Hauge’ have observed an excellent correlation between nutritive value and trypsin-inhibitor content in soybean preparations heated under varying conditions. These authors have concluded that the trypsin inhibitor was the major cause of poor utilization of raw soybean protein. The addition of trypsin powder to the diets of chicks? and rats? counteracted the growth-inhibiting effect of raw soybeans. Pancreatic hypertrophy has also been observed in chicks! and rats!! fed raw soybean meal. Sup- plementing the raw soybean meal diet of rats with tyrosine, methionine, threonine, and valine corrected poor growth and food efficiency but did not prevent pancreatic hypertrophy. Trypsin inhibitors have also been found in a large number of other legumes. Of eleven species of legumes that were fed to rats as the sole source of dietary protein, the nutritive value of five (jack bean, velvet bean, horse bean, lentil, and black-eyed pea) was improved by auto- claving and six (peanut, partridge pea, guar bean, lespedeza, mung bean, and common vetch) were not.!2 Of a total of 17 legume seeds studied in these and other investigations, eight were improved by autoclaving; five of these contained the trypsin inhibitor and three did not. Nine of the 17 legumes were not improved by autoclaving; six contained the trypsin inhibitor and three did not. Thus, in these studies no correlation was observed between the effect of autoclaving on nutritive value and the presence or absence of the trypsin inhibitor in the raw legume seed. Similar results were observed by Jaffé!3 in a study of eight legumes (black and red kidney beans; hyacinth, soy, and lima beans; pigeon and cow peas; and lentils). Kidney, soy, lima, and hyacinth beans, which had the highest in vitro trypsin-inhibitor ac- tivity, were also those in which digestibility was most improved by cooking, as determined in growing rats. According to Lyman and Lepkovsky!* and Lyman,!5 when either raw soybean meal or soybean trypsin concentrates are fed to rats, excessive amounts of pancreatic enzymes are found in the intestines and excreted in the feces. These workers concluded that the growth- depressing effects caused by trypsin inhibitors may be the result of a loss of essential amino acids from endogenous sources, as from a hyperactive pancreas, rather than from depression of normal intestinal proteolysis. A trypsin inhibitor extracted from lima beans was found to depress the growth of rats fed diets containing either casein or heated soybean

ANTIENZY MES 107 meal as the principal source of protein.'6 To a large extent, the in- hibitor was destroyed by steam heating and counteracted by the addition of methionine to the diet. The lima bean inhibitor is very similar to that in soybeans. Recently, the presence of a chick growth inhibitor in guar gum meal, which appears to be proteolytic in nature,!” has been reported. Trypsin activity was inhibited by an extract of guar gum meal. Heating the extract or the meal destroyed its activity. Trypsin inhibitors, which for the most part are completely destroyed during baking or heating in a water bath for 20 minutes, have been reported in wheat flour!’ and in whole wheat flour.!9 Trypsin inhibitors have been reported in a number of foodstuffs of India. In a study of 65 vegetables, 11 were found to contain trypsin inhibitors.2° In another study,?! involving tests on 26 pulses, trypsin inhibitors were found in 23. In both of these studies, the authors report that most of the trypsin inhibitors are partially or completely destroyed by autoclaving. According to these authors, the inhibitor in leguminous vegetables is located only in the seeds and is absent in legumes in which the seeds are not developed. In turnips, the inhibitor shows seasonal variations and is present in only some months of the year. Trypsin inhibitors that are relatively thermolabile are destroyed partially or completely on storage at room temperature for 1 month, while at 15°C destruction is less marked. Pepsin and papain did not affect any of the trypsin inhibitors examined. Extracts from sweet potato completely inhibit the activity of trypsin; activity of the extracts was destroyed by heating for 1 hour at 100°C.22 The isolation from white potatoes of a powerful chymotryptic in- hibitor of protein-like nature, unusually resistant to heat, acid, and alkali, has been reported.* The substance inhibits proteolysis, esteroly- sis, and milk clotting by chymotrypsin through the formation of an inactive enzyme-inhibitor complex. The inhibitor also forms a complex with trypsin and inhibits the proteolysis of casein. This inhibitor was found to be nontoxic to mice on intravenous or intraperitoneal injec- tion and nonirritating to the conjunctiva of rabbits, and it did not change the antigenicity of chymotrypsin.“ NATURALLY OCCURRING ENZYME INHIBITORS OF ANIMAL ORIGIN Ovomucoid, one of the many constituents of egg white, has been identified by Lineweaver and Murray® as an inhibitor of trypsin.

108 ANTHONY M. AMBROSE Ovomucoid apparently inhibits trypsin by the promotion of a relatively stable enzyme-inhibitor complex. In addition to ovomucoid, Matsu- shima?6 has reported the presence of another inhibitor in eggs, “ovo- inhibitor,” that is more effective than ovomucoid against fungal proteinase. ‘“‘Ovoinhibitor,”’ like ovomucoid, inhibits trypsin stoichio- metrically and is more effective than ovomucoid. Commercial egg white preparations contain most of the antitryptic principle of raw egg white, which is not readily destroyed by ordinary cooking. According to Almquist,2” the inhibitory activity of the anti- enzyme in raw egg white on in vivo digestion cou'd be an important limiting factor in the dietary protein ingested with the inhibitor. How- ever, the feeding of powdered commercial egg white, raw or heated, as a supplemental source of dietary protein with chemically isolated ovomucoid of known antitrypsin activity was found to have no effect upon nitrogen retention in human subjects.”8 In young rats, the feeding of the purified trypsin inhibitor from egg white at levels of 2.5 percent in a casein diet was found to have no effect on growth or protein efficiency.29 In contrast, the dog appears to be sensitive to the anti- tryptic factor in egg white that was well assimilated by man and rats.3° Recently, the ovomucoids from eleven different avian species (in- cluding chicken, duck, turkey, guinea, goose, pheasant, and quail) have been isolated and studied in detail.3! The authors have divided the biological properties into four classes: (1) ovomucoids that primarily inhibit trypsin (goose, quail, and chicken); (2) ovomucoids that primarily inhibit chymotrypsin (pheasant); (3) ovomucoids that inhibit equal molar amounts of trypsin and chymotrypsin separately or simultaneously (turkey and guinea); and (4) ovomucoids that inhibit twice as much trypsin as chymotrypsin (duck and quail). Proteolytic enzyme inhibitors have also been reported in beef pancreas,?233 blood,34 and bovine colostrum.35 SUMMARY Reference has been made to toxic hazards associated with the ingestion of certain vegetable proteins (cereal grains, oil seed legumes, and legumes) as the sole source of dietary protein for man and animals. The toxic effects have been attributed to (1) the presence of trypsin inhibitors, (2) unknown toxic principles, and (3) unbalanced amino- grams of the various vegetable proteins studied. It is well recognized that adequate processing frequently improves the nutritional qualities of vegetable proteins. However, inactivation or

ANTIENZYMES 109 destruction of the trypsin inhibitors by cooking or autoclaving does not always improve nutritional value, possibly due to unbalanced amino- grams or to the presence of other toxic principles. Although the addi- tion of certain essential amino acids to a raw soybean diet of chicks and rats improves growth and food efficiency, it does not prevent pancreatic hypertrophy. The suggestion has been made!4!5 that the effects of feeding raw soybean meal or soybean trypsin concentrates are due to pancreatic stimulation resulting in excessive loss of essential amino acids from endogenous sources rather than to depression of normal intestinal proteolysis. With other vegetable proteins containing trypsin inhibitors, the cause and effect may be essentially the same as that observed with soybeans. In conclusion it should be emphasized that there are problems that are yet to be solved in the use of vegetable proteins as the sole source of dietary proteins for man and animals. Aside from the fact that they must be cooked to improve their nutritive value and digestibility, toxic materials that may be contained in them must be detoxified or removed. REFERENCES 1. W. E. Ham and R. M. Sandstedt, “‘A Proteolytic Inhibiting Substance in the Extract from Unheated Soybean Meal,” J. Biol. Chem., 154, 505 (1944). 2. W. E. Ham, R. M. Sandstedt, and F. E. Mussehl, “‘The Proteolytic Inhibiting Substance in the Extract from Unheated Soybean Meal and Its Effect upon Growth in Chicks,”’ J. Biol. Chem., 161, 635 (1945). 3. A. A. Klose, B. Hill, and H. L. Fevold, “‘Presence of a Growth Inhibiting Substance in Raw Soybeans,” Proc. Soc. Exptl. Biol. Med., 62, 10 (1946). 4. R. Borchers, C. W. Ackerson, F. E. Mussehl, and A. Moehl, ‘Trypsin Inhibitor. VIII. Growth Inhibiting Properties of a Soybean Trypsin Inhibitor,” Arch. Biochem., 19, 317 (1948). 5. I. E. Liener, H. J. Deuel, Jr., and H. L. Fevold, “The Effect of Supplemental Methionine on the Nutritive Value of Diets Containing Concentrates of the Soybean Trypsin Inhibitor,” J. Nutr., 39, 325 (1949). 6. R.J. Westfall, D. K. Bosshardt, and R. H. Barnes, “‘Influence of Crude Trypsin Inhibitor on Utilization of Hydrolyzed Protein,” Proc. Soc. Exptl. Biol. Med., 68, 498 (1948). 7. R. J. Westfall and S. M. Hauge, “The Nutritive Quality and the Trypsin Inhibitor Content of Soybean Flour Heated at Various Temperatures,” J. Nutr., 35, 379 (1948). 8. H. J. Almquist and J. B. Merritt, ‘““Effect of Crystalline Trypsin on the Raw Soybean Growth Inhibitor,” Proc. Soc. Exptl. Biol. Med., 83, 269 (1953). 9. R. Borchers and C. W. Ackerson, “‘Nutritive Value of Legume Seeds. XI. Counteracting the Growth Inhibitor of Raw Soybeans,” Proc. Soc. Expil. Biol. Med., 78, 81 (1951).

110 ANTHONY M. AMBROSB 10. S.S. Chernick, S. Lepkovsky, and I. L. Chaikoff, “‘A Dietary Factor Regulating the Enzyme Content of the Pancreas. Changes Induced in Size and Proteolytic Activity of the Chick Pancreas by the Ingestion of Raw Soybean Meal,’’ Am. J. Physiol., 155, 33 (1948). 11. A. N. Booth, D. J. Robbins, W. E. Ribelin, and F. De Eds, “Effect of Raw Soybean Meal and Amino Acids on Pancreatic Hypertrophy in Rats,’ Proc. Soc. Exptl. Biol. Med., 104, 681 (1960). 12. R. Borchers and C. W. Ackerson, “The Nutritive Value of Legume Seeds. X. Effect of Autoclaving and the Trypsin Test for 17 Species,” J. Nutr., 41, 339 (1950). 13. W. G. Jaffé, “Protein Digestibility and Trypsin Inhibitor Activity of Legume Seeds,” Proc. Soc. Exptl. Biol. Med., 75, 219 (1950). 14. R.L. Lyman and S. Lepkovsky, “The Effect of Raw Soybean Meal and Trypsin Inhibitor Diets on Pancreatic Enzyme Secretion in the Rat,” J. Nutr., 62, 269 (1957). 15. R. L. Lyman, “The Effect of Raw Soybean Meal and Trypsin Inhibitor Diets on the Intestinal and Pancreatic Nitrogen in the Rat,” J. Nutr., 62, 285 (1957). 16. A. A. Klose, B. Hill, J. D. Greaves, and H. L. Fevold, ‘““Growth-Depressing Fractions in Raw Lima Beans,” Arch. Biochem., 22, 215 (1949). 17. J. R. Couch, Y. N. Bakshi, J. M. Prescott, and C. R. Creger, “Trypsin Inhibitor in Guar,” Federation Proc., 24, 687 (Abstr. No. 3085) (1965). 18. E. M. Learmonth and J. C. Wood, “A Trypsin Inhibitor in Wheat Flour,” Chem. Ind. (London), 1969 (1960). 19. G. Shyamala, B. M. Kennedy, and R. L. Lyman, “Trypsin Inhibitor in Whole Wheat Flour,” Nature, 192, 360 (1961). 20. K. Sohonie and A. P. Bhandarkar, “Trypsin Inhibitors in Indian Foodstuffs. I. Inhibitors in Vegetables,” J. Sci. Ind. Res. (India), 13B, 500 (1954). From Chem. Abstr., 49, 534a (1955). 21. K. Sohonie and A. P. Bhandarkar, “‘Trypsin Inhibitors in Indian Foodstuffs. II. Inhibitors in Pulses,” J. Sci. Ind. Res. (India), 14C, 100 (1955). From Chem. Abstr., 50, 10303a (1956). 22. K. Sohonie and P. M. Honawar, “Trypsin Inhibitors of Sweet Potato (Ipomea batata),” Sci. Culture (India), 21, 538 (1956). From Chem. Abstr., 50, 16924h (1956). 23. C. A. Ryan and A. K. Balls, “An Inhibitor of Chymotrypsin from Solanum tuberosum and Its Behavior Toward Trypsin,” Proc. Natl. Acad. Sci. U.S., 48, 1839 (1962). 24. F. De Eds, C. A. Ryan, and A. K. Balls, ““Pharmacological Observations on a Chymotrysin Inhibitor from Potatoes,”’ Proc. Soc. Exptl. Biol. Med., 115, 772 (1964). 25. H. Lineweaver and C. A. Murray, “Identification of the Trypsin Inhibitor of Egg White with Ovomucoid,” J. Biol. Chem., 171, 565 (1947). 26. K. Matsushima, ‘An Undescribed Trypsin Inhibitor in Egg White,” Science, 127, 1178 (1958). 27. H.J. Almquist, “Nutrition,” Ann. Rev. Biochem., 20, 305 (1951). 28. H. H. Scudamore, G. R. Morey, C. F. Consolazio, G. H. Berryman, L. E. Gordon, H. D. Lightbody, and H. L. Fevold, “Nitrogen Balance in Men Consuming Raw or Heated Egg White as a Supplemental Source of Dietary Protein,” J. Nutr., 39, 555 (1949).

ANTIENZYMES 111 31. 32. 33. 34. 35. A. A. Klose, B. Hill, and H. L. Fevold, “Absence of Growth Inhibiting Activity in Trypsin Inhibitor from Egg White,” Arch. Biochem., 27, 364 (1950). . Cooperative Determinations of the Amino Acid Content, and the Nutritive Value of Six Selected Protein Food Sources, Bur. Biol. Res., Rutgers Univ., New Brunswick, N.J. (1951), p. 114. Abstracted in Nutr. Abstr. Rev., 21, 827 (Abstr. No. 4621) (1951-52). | M. B. Rhodes, N. Bennett, and R. E. Feeney, “The Trypsin and Chymotrypsin Inhibitors from Avian Egg Whites,” J. Biol. Chem., 235, 1686 (1960). M. Kunitz and J. H. Northrop, “Isolation from Beef Pancreas of Crystalline Trypsinogen, Trypsin, a Trypsin Inhibitor and an Inhibitor-Trypsin Com- pound,” J. Gen. Physiol., 19, 991 (1936). L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, “Isolation of a Crystalline Trypsin Inhibitor-Anticoagulant Protein from Pancreas,” J. Am. Chem. Soc., 70, 3034 (1948). P. M. West and J. Hilliard, ‘‘Proteolytic Inhibitors of Human Serum in Health and Disease,” Proc. Soc. Exptl. Biol. Med., 71, 169 (1949). M. Laskowski, Jr., and M. Laskowski, “‘Crystalline Trypsin Inhibitor from Colostrum,” J. Biol. Chem., 190, 563 (1951).

DONALD G. CROSBY Natural Cholinesterase Inhibitors in Food The cholinesterases represent a group of enzymes that are of great significance in both a physiological and economic sense. Their principal characteristic appears to be control of the conduction of nerve impulses, a function that makes them of unique importance to the lives of both higher and lower animals. The intentional inhibition of cholinesterases is one of man’s more powerful chemical weapons against his insect enemies. Two of the three general classes of synthetic organic insecticides—the organophosphates and carbamates—are thought to owe their effectiveness to this mecha- nism. Although the phosphates are the result of laboratory observation, the effective carbamate insecticides such as carbary]l (I) and Zectran (II) are modeled upon the active constituent of the poisonous calabar bean, physostigmine (III). While there is no evidence that cholin- esterases have any function in higher plants, this alkaloid from Physo- stigma venenosum is one of the most potent inhibitors known. If the West African calabar bean can provide powerful cholinesterase inhibitors, it may logically be assumed that more familiar plants might also produce compounds that exhibit this property, and they do. In addition to those from such homely species as the petunia (Petunia hybrida), boxwood (Buxus sempervirens), periwinkle (Vinca minor), and poppy (Papaver orientale), extracts of a number of everyday food items have been shown to inhibit these enzymes. Orgell’ reported the effects of aqueous extracts of 256 plant species on human plasma cholinesterase. Of the 17 edible vegetables and fruits examined, the most active inhibitors were found in members of the Solanaceae (potato family). Aqueous extracts of all parts of the 112

CHOLINESTERASE INHIBITORS as” OO OCONHCH3 Qe Ny HC” CH; I OCONHCH3 ao 113 potato were active; the fruit of eggplant and the root and leaves of tomato also contained inhibitors, although no part of the pepper plant was effective. Table 1 lists foods that yielded active extracts, but it does not include those instances where only nonedible portions of food plants were examined. In this investigation, no nonaqueous extracts were examined. TABLE 1 Vegetables and Fruits Tested for Cholinesterase Inhibition SPECIES COMMON NAME TISSUE REFERENCE Beta vulgaris Beet Leaf 8 Brassica napobrassica Mill. Rutabaga Root 10 Brassica oleraceae L., var. Broccoli Head 10 botrytis Brassica oleracea L., var. Cabbage Head 10 capitata Capsicum frutescens L. Pepper Fruit 10 Citrus sinensis Valencia orange Fruit 6 Cucurbita pepo L. Pumpkin Fruit 10 Cucurbita pepo L., var. Squash Fruit 10 melopepo Daucus carota L. Carrot Root 10 Fragaria chiloensis var. Strawberry Fruit 10 ananassa Duchesne Lycopersicon esculentum Mill. Tomato Fruit 10 Malus sylvestris Mill. Jonathan apple Fruit 10 Malus sylvestris Mill.¢ Stayman apple Fruit 6 Phaseolus limensis Macf. Lima bean Pod 10 Solanum melongena* Eggplant Fruit 10 Solanum tuberosum L.4 White russet potato Whole tuber 10 Solanum tuberosum L.* Irish cobbler potato Whole tuber 10 Solanum tuberosum L.¢ Cherokee potato Whole tuber 10 Solanum tuberosum L.¢ Red lasoda potato Whole tuber 10 Solanum tuberosum L.¢ Red Pontiac potato Whole tuber 10 * Contains cholinesterase inhibitors.

114 DONALD G. CROSBY Menn et al. later found that benzene-extractable inhibitors were present in sugar beet root, Valencia orange fruit, and Stayman apple fruit, as well as in the tuber of the Irish potato. Through the use of paper chromatography, they demonstrated that each specimen con- tained at least two separable inhibitors, but the relative activity was not measured. Still another vegetable, asparagus, has been reported to produce an “anticholinesterase” which was thought to be a glycoside.!! Another investigation indicated that while aqueous extracts frequently were difficult to examine with high sensitivity, ethyl acetate extracts of turnip, radish, celery, and carrot all appeared to contain inhibitors (Crosby and Aharonson, unpublished). Several plant species that are of food significance, although not themselves used as human food, have been examined for cholinesterase inhibitors (Table 2). White clover (Trifolium repens), in particular, was found to contain a chloroform-soluble irreversible inhibitor,4 and Orgell’s data also indicate that aqueous extracts of this species appear about equal to those of potato in their effect. Although the very ex- tensive investigations of Orgell and his co-workers provide clear evi- dence that potent cholinesterase inhibitors do occur in many plant families and species, a number of variables may be shown to have a strong influence on their detection and quantitative comparison, particularly in crude extracts. For instance, varietal as well as species differences are most important. Comparison of aqueous extracts of thirty species of Solanum revealed a range of Iso values (concentration of leaf extract producing a 50 percent enzyme inhibition) of 0.01 g to greater than 8.2 g, while the Iso of 12 varieties of S. tuberosum ranged from 0.06 g to 0.76 g.9 The ability to inhibit cholinesterase also depends upon the plant part under examination. For example, although ex- tracts of the ripe and green tomato fruit were inactive, leaves caused a 9 percent inhibition and roots caused a 21 percent inhibition during comparable tests.!° TABLE 2 Other Plants Tested for Cholinesterase Inhibition SPECIES COMMON NAME TISSUE REFERENCE Beta vulgaris L.* Sugar beet Root 6 Glycine max L. Soybean Leaf 8 Medicago sativa L. Alfalfa Leaf 10 Nicotiana tabacum L.* Tobacco Leaf 10 Trifolium repens L.* White clover Leaf 4, 10 « Contains cholinesterase inhibitors.

CHOLINESTERASE INHIBITORS 115 The type and origin of the assay cholinesterase are important to both qualitative and quantitative interpretations. By far the most common enzyme source in the search for natural inhibitors has been outdated human plasma, which contains principally pseudocholinesterases. Enzyme preparations from horse serum and homogenates from several insect species are of a similar type, while red cells (erythrocytes) contain principally the “true” acetylcholinesterases.> It 1s apparent that, in general, variations in concentration, purity, and activity of the prepara- tion affect the assay. The method of plant extraction and resolution of the constituents also are important, and the recent advent of enzymatic detection methods based on the powerful technique of thin-layer chromatography represents a major advance in the detection and isolation of inhibitors.? At present, very little is known about the chemistry of the natural cholinesterase inhibitors in food. Only the potato glycoalkaloid solanine (IV) and its aglycone, solanidine, have definitely been shown to exhibit significant activity. Although several other alkaloids are R =L -Rhamnosy!-pd-galactosyi-b -glucosyl- known to inhibit cholinesterases in vitro, tomatine, a close structural analog of solanine is virtually inactive.67.12 The amount of solanine in a new potato may be as much as 180 mg/kg (180 ppm), while potatoes suspected of human poisoning were found to contain as much as 840 mg/kg (840 ppm).'! On the other hand, solubility and stability tests indicate that perhaps a major part of the enzyme inhibition by potato extracts is due to substances other than the known glycoalkaloids, and these unknown compounds also have been implicated in cases of poisoning by potatoes. Study of the chemistry and toxicology of natural cholinesterase inhibitors is important for a number of reasons. For one thing, they may represent a real hazard under certain conditions—a number of instances of poisoning have been attributed to human consumption of “‘green’’ potatoes high in solanine.5 It has been suggested that bloat in

116 DONALD G. CROSBY cattle may be connected with the natural inhibitor in white clover.‘ A large number of wild plants that undoubtedly are eaten on occasion by domestic animals (as well as by children) have been shown to exhibit a high degree of activity.’ However, among these inhibitory substances, it is quite possible that types of chemical structures may be found that could suggest new synthetic insecticides of low mammalian toxicity. The Colorado potato beetle can exist solely on potato sprouts, the most concentrated natural source of solanine known, without the ill effects visited upon man— perhaps the fact that most people eat natural anticholinesterases each day without apparent harm indicates that the reverse case also is to be found, REFERENCES 1. D.C. Abbott, K. Field, and E. I. Johnson, “Observations on the Correlation of Anticholinesterase Effect with Solanine Content of Potatoes,” Analyst, 85, 375 (1960). 2. D. G. Crosby, E. Leitis, and W. L. Winterlin, ‘“‘Photodecomposition of Carba- mate Insecticides,” J. Agr. Food Chem., 13, 204 (1965). 3. D.V. Heath, Organophosphorus Poisons, Pergamon Press, New York (1961). 4. D. F. Heath and P. O. Park, ‘*An Irreversible Cholinesterase Inhibitor in White Clover,” Nature, 172, 206 (1953). 5. J. M. Kingsbury, Poisonous Plants of the United States and Canada, Prentice- Hall, Englewood Cliffs, N.J. (1964). 6. J.J. Menn, J. B. McBain, and M. J. Dennis, ‘‘Detection of Naturally Occurring Cholinesterase Inhibitors in Several Crops by Paper Chromatography,” Nature, 202, 697 (1964). 7. W. H. Orgell, “Inhibition of Human Plasma Cholinesterase In Vitro by Alka- loids, Glycosides, and Other Natural Substances,”’ Lloydia, 26, 36 (1963). 8. W. H. Orgell, Inhibition of Human Plasma Cholinesterase Jn Vitro by Plant Extracts,” Lloydia, 26, 59 (1963). 9. W. H. Orgell and E. T. Hibbs, ““Human Plasma Cholinesterase Inhibition Jn Vitro by Extracts from Tuber-Bearing Solanum Species,” Proc. Am. Soc. Hort. Sci., 83, 651 (1963). 10. W. H. Orgell, K. A. Vaidya, and E. W. Hamilton, “*A Preliminary Survey of Some Midwestern Plants for Substances Inhibiting Human Plasma Cholines- terase In Vitro,” Proc. Iowa Acad. Sci., 66, 149 (1959). 11. R. A. Rohde, “AcetyJcholinesterase in Plant-Parasitic Nematodes and an Anticholinesterase from Asparagus,” Proc. Helminthol. Soc. Wash., D.C., 27, 121 (1960). 12. E. K. Zsigmond, V. M. Foldes, and F. F. Foldes,“*In Vitro Inhibitory Effect of Psilocybin and Related Compounds on Human Cholinesterases,” Psycho- pharmacologia, 4, 232 (1963).