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The Nitrilosides in Plants and Animals
Since the principal objective of this presentation is a study of the clinical use of the Laetriles (nitrilosides), because these substances yield nascent HCN [hydrogen cyanide/prussic acid] when they undergo enzymatic hydrolysis in vivo, it will be helpful if one begins with a general study of the nitrilosides in plants and animals.
A nitriloside is a naturally occurring or synthetic compound which, upon hydrolysis by a beta-glucosidase, yields a molecule of a non-sugar, or aglycone, a molecule of free hydrogen cyanide, and one or more molecules of a sugar or its acid. There are approximately 14 naturally occurring nitrilosides distributed in over 1200 species of plants. Nitrilosides are found in all plant phyla from Thallopliyta to Sperimatophyta.
The nitrilosides specifically considered in this paper are 1-mandelonitrile-beta-diglucoside (amygdalin) and its hydrolytic products; l-para-hydroxymandelonitrile-beta-glucoside (dhurrin); methylethyl-ketone-cyanohydrin-beta-glucoside (lotaustralin); and acetone- cyanohydrin-beta-glucoside (linamarin). All of these compounds are hydrolyzed to free HCN, one or more sugars and a non-sugar or aglycone. For the purposes of this study, they may be considered as physiologically and pharmacologically identical and varying essentially only in the percent of free HCN they produce upon hydrolysis by beta-glucosidase.
The concentration of nitrilosides in plants varies widely and ranges from small traces to as much as 30,000 mg/kg in some of the common pasture grasses (in the dry state). There is no evidence that animals synthesize nitrilosides under normal conditions. The metabolism of all the higher animals, and most of the invertebrates as well, involves the hydrolysis of plant-derived nitrilosides ingested in the plant components of the diet. This hydrolysis is produced by beta-glucosidase occurring in the gastro-intestinal tract and produced in various tissues of the animal. The enzyme occurring in the intestinal tract is produced by various bacteria or microflora. When the enzyme so produced or that enzyme existing in the organs acts to hydrolyze the nitrilosides to free HCN, sugar and a non-sugar moiety, the CN ion released is detoxified or converted by an enzyme normally occurring in the organism and known as rhodanese or thiosulfate transulfurase. The product of such conversion is thiocyanate, a compound found in the tissues of all vertebrates, many invertebrates and a number of plants.
It is one of the objectives of this report to survey extensively but not intensively the indispensable but long-overlooked role of the nitrilosides in the plant and animal kingdoms. The material utilized for this paper comprises, to a large extent, an abstract of a book now in preparation on the subject. The latter carries a bibliography in excess of 3,000 titles. It is not possible in this report to supply an adequate bibliography. We have therefore limited the references in this paper, as a rule, to isolated or specific experimental observations; and we have omitted the citation of reference sources for data that are commonplace or unquestioned facts in the universe of the relevant expert. For this reason, statements undocumented here may often appear extraordinary to a reader not intimately acquainted with sophisticated data derived from disciplines often distant from his own. For example, even to experts in animal husbandry, agriculture, pharmacology, and toxicology, it may come as an almost unbelievable statement that cattle, in the course of grazing, may daily ingest grasses containing as much as 30,000 mg/kg of nitriloside (carrying over 2.0 grams of derivable HCN) over a period of years without discernible effect. The grasses involved have, however, been repeatedly assayed by reliable and universally accepted techniques and the quantities ingested by sheep and cattle have been repeatedly and carefully measured. The results have been duly published in acceptable journals over the world.
NITRILOSIDES AND NITRILES
No area of the earth that supports vegetation lacks nitriloside-containing plants. Over 30 per cent of all tropical plants, edible or inedible by man or animals, contain a nitriloside. From the nitriloside-rich salmon-berry, cloud-berry or buffalo-berry (Rubus spectabilis) growing on the Arctic tundra and the arrow-grass growing in arctic marshes and supplying the major fodder for the caribou, to the cassava or manioc - the bread of the tropics - plants extraordinarily rich in nitriloside, and serving as food for man and animals, are found in abundance. All life on earth participates directly or indirectly in the chain of nitriloside metabolism. In terms of living forms, the nitrilosides appear as ubiquitous in time as they do in space. There is some evidence that life on earth commenced in conjunction with hydrogen cyanide.
A glance at the vegetation about us almost anywhere will disclose nitriloside-containing plants. The common weed and fodder, Johnson-grass, often carries 15,000 mg/kg or more of nitriloside. A similar concentration is found in Sudan-grass, Velvet grass, white clover, the Yetches, buckwheat, the millets, alfalfa or lucerne, lima beans, even some strains of green or garden peas, the quinces, all species of the passion-flower. The seeds as well as the leaves and roots of the peaches and various cherries are but a few of the natural sources of this essentially non-toxic water-soluble factor.
Upon hydrolysis in the intestinal tract of man or animals, the nitriloside exerts a variable antibiotic effect through the action of the freed hydrogen cyanide and, in the case of some nitrilosides such as amygdalin or dhurrin, through the antiseptic action of benzaldehyde or p-hydroxybenzaldehyde aglycone. The latter from Johnson-grass, before and after oxidation to a benzoic acid, is about 30 times more antiseptic (in terms of the phenol coefficient) than ordinary benzaldehyde or benzoic acid. It is now experimentally established that only those nitrile compounds that are hydrolyzed to free hydrogen cyanide lend themselves to the formation, through rhodanese in the presence of utilizable sulfur, of thiocyanate.
In the case of nitrilosides with an acetone aglycone or an ethylmethyl-ketone aglycone, the ketone aglycones as well as the sugar moiety are probably fully metabolized to carbon dioxide and water with the HCN residue contributing to the production of thiocyanate, some of which may be eliminated from the body in the urine and feces with the remainder persisting as part of the normal "cyanide metabolic pool".
EVIDENCE FOR BETA-GLUCOSIDASE
Nitrilosides are also hydrolyzed to free HCN when injected into the peritoneal cavity of the rabbit. The fluid in this area apparently is lacking in rhodanese activity, since free HCN has been observed in the peritoneal fluid of rabbits following injections of large doses of amygdalin. Extensive studies have also been published on the hydrolysis of nitrilosides to free HCN by the rumenal microflora of sheep.
EVIDENCE FOR OCCURRENCE OF
It appears that the concentration or activity of rhodanese in the tissues of animals varies directly with the normal nitriloside content of the general diet characterizing each species. The livers of rats, rabbits and cows appear to be more active than those of monkeys, men, dogs, and cats in descending order. Rhodanese activity is as widely distributed in living forms as are the nitrilosides. Both have been found in forms as diverse as fish, squid, insects and plants. The enzyme has been isolated in crystalline form by Sorbo and a substantial literature on it has developed. The action of rhodanese is highly specific. It is limited not merely to nitriles but only to those nitrilosides which surrender free HCN ions upon hydrolysis.
The administration of rhodanese has been found to protect experimental animals from doses of cyanide or its salts ten times or more in excess of normally lethal doses. The concentration of rhodanese in tissue is generally proportional to that of beta-glucosidase and always functionally in excess of the latter. Rhodanese may also appear in the absence of beta-glucosidase as in the case of the brain just as beta-glucosidase may appear in conjunction with cancer or trophoblast cells in the absence of rhodanese. The high sensitivity of cerebral tissue to hypoxia would tend in the course of natural selection to provide a high rhodanese activity against adventitious HCN and to exclude any enzymatic means by which the cyanide ion could be hydrolyzed in this area. The rationale for the occurrence of a high beta-glucosidase concentration in the absence of rhodanese in the case of trophoblast is associated with the role the trophoblast plays in hemopoiesis, especially as it concerns the nitrilization of hydrocobalamin to active vitamin B12 (cyanocobalamin).
Rhodanese, beta-glucosidase, nitrilosides and thiocyanates are found throughout the phyla of the plant and animal kingdoms from bacteria to giant trees, and from protozoa to man.
THIOCYANATES IN PLANTS
In soils carrying normal concentrations of iodine, no such effects have been observed in sheep or cattle despite the fact that some of these animals may ingest as much as 300 grams of nitriloside a day through dry arrow-grass, Johnson-grass, clovers, or other fodder.
It will also be recalled that Wilder Bancroft, Professor of Physical Chemistry at Cornell University, ingested 1,000 mg. of thiocyanate a day for a period of 23 years in the process of studying the cumulative properties of this chemical. He reported no untoward result from the experiment. To the contrary, he associated it with some suspected positive benefits that need not be considered at this time.
While prolonged excessive ingestion or development of thiocyanate in the presence of a severe iodine deficiency has apparently been associated with a goitrogenic effect in both human and animal populations, there has never been anything to suggest the possibility of any cumulative toxicity arising from the cyanide ion itself.
It is apparently impossible to develop cumulative toxicity to HCN in animals. The reason for this is that the biological experience with the cyanide ion in metabolism is almost as ancient and extensive as the biological experience with water, oxygen, nitrogen, salt, or the like. All can prove fatal to animals if administered in excessive quantities or in an improper way. As a result of an almost archetypical ignorance of, or superstition towards HCN engendered by observations of the swiftness of its lethality made in days when chemistry had barely emerged as a science, a powerful cultural antipathy toward cyanide developed.
Cyanide was indiscriminately and falsely classified, because of its toxic potentiality, with protoplasmic poisons utterly foreign to the biological experience of the organism. Unfortunately, this ancient misapprehension has been perpetuated among botanists, physiologists, toxicologists and even pharmacologists. And, in their culturally induced fear or antipathy toward cyanide as a poison, they have unwittingly foreclosed adequate attention to, and study of, the critically important factors in the physiology of plants and animals. An atmosphere of pure nitrogen or pure carbon dioxide is just as lethal as one of hydrogen cyanide. The major differences among these compounds possessing almost equal biological experience are those of concentrations and rates, and none are capable of producing chronic or cumulative toxicity. As we shall study in a subsequent section, sheep have received as much as 460 mg of HCN in the course of an hour without any evidence of acute toxicity and as much as 210 mg of HCN a day for two years without any evidence of cumulative toxicity or resistance or immunity of any kind to HCN. This biological experience qualitatively parallels that for water, salt, sodium chloride and compounds with similar biological experience.
Though in our early studies on the nitrilosides we attempted because of our then limited knowledge of their basic significance in terms of biological experience to ascertain some evidence of cumulative toxicity for them, we now agree with such students of the problem as Coop and Blakely that it is impossible for compounds that have, through nutrition, been a part of the biological experience of plants, animals and man and an inherent part of his physiology since his appearance, to produce any cumulative toxic effects. Whether we are dealing with the first nitriloside to be discovered, amygdalin, or with linamarin or lotaustralin, it would seem vain to expect to find from their hydrolytic products of glucose and HCN and their aglycone of benzaldehyde or benzonic acid in the case of the first, or acetone or methylethylktone, respectively, in the case of the latter, any possibility of cumulative effect. Glucose, thiocyanate, benzoic acid, and even acetone, are components normal to the metabolic pathways of the organism, which would have to be susceptible to a development of a cumulative toxicity to itself in order to sustain one to the components which comprise the organism.
If the obvious is belabored to reductio ad absurdum, it is because even at this late date there are apparently some unacquainted with the fact that the hydrolysis in vivo of a nitriloside by one or more endogenous beta-glucosidases with the production of free HCN, detoxified as thiocyanate by the enzyme rhodanese in order to protect the organism, or sometimes left undetoxified by cells or organisms lacking or deficient in rhodanese, comprises biological phenomena that were commonplace in organisms as old as man himself. As a result of a deficient rhodanese mechanism, some organisms have been destroyed by the HCN emitted by other organisms rich in beta-glucosidase and rhodanese.
Blum & Woodring (Science, 138:513, 1962), in a paper on "Secretion of Benzaldehyde and Hydrogen Cyanide by the Millipede Pachydesmus crassicutis", describe how this large millipede, whose known distribution is limited to Louisiana and southern Mississippi, protects itself against its natural prey, the imported fire ant (Solenopsis raevissima v. richteri Forel) by secreting a mixture of benzaldehyde and hydrogen cyanide against the predator when disturbed by it. The millipede is equipped with paired glands located on eleven of the notal projections; from these glands, benzaldehyde and HCN are ejected. The water-clear secretion of Pachydesmus was collected by touching the dorsal surfaces of the notal projections with a small square of filter paper which rapidly absorbed the liquid discharge. This discharge was then analyzed by gas chromatography and infra red photospectroscopy. The major component was found to be benzaldehyde. HCN and glucose were also found together with a disaccharide which appears to be the sugar moiety of the nitriloside amygdalin. The millipede secretes its own beta-glucosidase, which hydrolyses the nitriloside in the notal glands to free HCN, benzaldehyde and sugar. While the millipede protects itself from the HCN through its endogenous rhodanese, this HCN is emitted against a predator relatively deficient in rhodanese.
David A. Jones, Department of Genetics, and John Parsons, Department of Pharmacology, Oxford University, in a paper on "Release of Hydrocyanic Acid from Crushed Tissues in All Stage of the Life-Cycle of Species of the Zygaeninae (Lepidoptera)" Nature, 193 (4810), p.52, 1962) reported that 50 crushed eggs (weight of about 50 eggs 2.6 mg - 4.0 mg) of this moth release up to 150 microgram of HCN, which HCN thus accounts for about 5 per cent of the weight of such eggs.
The foregoing examples were selected from a comprehensive body of similar data for the purpose of adumbrating the ubiquity of the biological occurrence and experience among all forms of life, not only in terms of nitriloside, but also in terms of beta-glucosidase, rhodanese, thiocyanate, and the selective susceptibility of rhodanese-deficient cells to the noxious effect of adventitious HCN. Some of the data briefly reviewed in the two papers just cited concern the occurrence of rhodanese in the parasites of the gastro-intestinal tract of animals ingesting nitriloside-rich foods. Such rhodanese is, of course, necessary as a protection against the free HCN released from the ingested nitrilosides by the beta-glucosidase produced by the intestinal flora and possibly also by the intestinal mucosa of the host.
Civilized, Westernized or Europeanized man, on the other hand, relies on a diet that probably provides an average of less than 2 mg of nitriloside a day.
It is noteworthy that no case of cancer has ever been reported among the peoples of one tribe in the Karakorums over a period of about 60 years of medical observation. For a period of at least 80 years the Eskimaux have been observed with even greater scrutiny by medical men, missionaries, teachers, traders and others for the specific purpose of attempting to discover the possible incidence of cancer among them. Despite such observations, no case of cancer has yet been reported among these two native populations while they lived on their native diet; however, in the case of the Eskimaux, a number of cancer victims have been found among those who left their original dietary habits for a Westernized diet.
The medical scrutiny by which such cancer cases were noted was no less intense than that given a large proportion of the natives not having access to modern foods.
The observations made of the Eskimaux on this subject are recorded in Vilhjalmur Stefansson's book on "CANCER: Disease of Civilization? An Anthropological and Historical Study" (Hill and Wang, N.Y. 1960). Philip R. White, M.D., has written an interesting preface to the book, while Rene Dubos' introductory chapter is most instructive.
The remarkable freedom primitive populations show to dental caries is, of course, a commonplace to students of anthropology. Many of the nutritional reasons for such freedom from caries among these people are not difficult to find in terms of the food that they eat, and especially of the food that they do not eat. In the similar freedom of these populations from cancer, the possible role of nutrition has been at best vague and general - as it was in the case of pellagra and the anemias prior to the discovery of the specific factor involved in the deficiency.
Major General Sir Robert McCarrison, before and during his appointment as Director of Nutrition Research in India under the Research Fund Association, treated and studied the people of Karakorum. From the perspective of 20 years of observation he reported that he had failed to find a single case of cancer among this population. Later John Clark, M.D. served in a medical mission to this population. He was properly critical of the tendency of some to romanticize the allegedly perfect health of these long-lived people. He described, as had McCarrison, a relatively high incidence of goiter among these people as well as certain skin diseases and a substantial incidence of dental caries. The nutritional basis for the high incidence of goiter among them is clear in the relative iodine deficiency of their diet. Their incidence of dental caries likewise has a clear nutritional basis. The tendency to goiter though resting on an iodine deficiency is exacerbated by the presence in their diet of an abundant quantity of nitriloside, which contributes a corresponding quantity of thiocyanate that, in the absence of adequate iodine, is goitrogenic, as we have seen in the case of human populations eating vegetables of the thiocyanate-rich Cruciferae, grown in areas deficient in iodine or in the case of ewes grazing on nitriloside-rich (i.e., thiocyanate-producing) clover grown in iodine deficient soil.
At any rate, John Clark, while recognizing and describing the many pathological conditions to which these people, like all others, are subject, did add that he, too, had never observed a single case of cancer among them.
While cancer may elude diagnosis in some cases, early cases ultimately become terminal cases, and when the latter involve the skin, breast, the lymphatic glands, mouth, tongue, lungs, or rectum, they do not go unrecognized even by the medically naïve - certainly not by medical observers.
Over a dozen books and articles we have read on these people are unanimous in the report that the apricot is the major staple in their diet. In view of our work on the nitrilosides in relation to human cancer, the predominance of the apricot in the nutrition of these reportedly cancer-free people was frequently called to our attention over the years. We originally dismissed the matter on the basis of pure coincidence, especially since the meat or flesh of the apricot contains little or no nitriloside, which is concentrated in the seed that resides in the pit. The seed is the size of a small almond and may be mistaken for a shelled almond.
Finally, upon investigating the diet of these people, we found that the seed of the apricot was prized as a delicacy and that every part of the apricot was utilized. We found that the major source of fats used for cooking was the apricot seed, and that the apricot oil was so produced as inadvertently to admit a fair concentration of nitriloside or traces of cyanide into it. The apricot seed is so prized among these people that there are experts chosen among them for the purpose of testing the seeds of new apricot trees for their bitterness, since occasionally there appears strains that produce apricot seeds carrying extraordinary concentrations of nitriloside and beta-glucosidase. These trees are destroyed.
The peoples of the Karakorum share with most western scientists an ignorance of the chemistry, toxicology and physiology of the nitrilosides and nitriles. Empirically, however, they have apparently discovered the value of these factors to nutrition. They prepare a solution of HCN (prussic acid) by allowing the apricot kernel nitriloside to react, in the presence of a little water added to defatted meal, with the endogenous beta-glucosidase (emulsin) to release free HCN. The resulting solution of HCN is then maintained as a form of bitters that is added drop-wise, because of its recognized toxicity, to wines immediately before they are drunk. It is held that this solution is contributory to health and even longevity.
Upon further investigation of the Eskimaux diet we found that one berry grew abundantly in the Arctic areas and that this berry is extraordinarily rich in nitriloside. This is the salmon-berry, cloud-berry, or buffalo-berry (Rubus spectabilis). It is eaten by birds, animals and men. It is also incorporated into pemmican, which is eaten during all seasons of the year. It was noted also that animals such as the caribou are important in the diet of these people. In eating the caribou, the frozen contents of the rumen or paunch are utilized as a salad and considered a delicacy. In view of this we investigated the forage upon which the caribou feeds. Among the grasses that grow in Arctic marshes, arrow-grass (Triglochin maritima) is very common. Studies made by the United States Department of Agriculture on the nitriloside content of arrow-grass (Triglochin maritima) show it to be probably richer in nitrilosides than any common grass. On a dry weight basis, one kilogram of arrow-grass was found to contain over 30,000 milligrams of nitriloside. One teaspoonful of such rumenal salad might be expected to carry 100 mg or more of nitriloside. This nitriloside is p-hydroxymandelonitrile-beta-glucoside; whereas the dominant one among the Karakorum is 1-mandelonitrile-beta-diglucoside, though both nitrilosides occur in the diet of both groups.
A quick glance at native populations in tropical areas, such as South America and South Africa, discloses a great abundance of nitriloside-containing foods. Over one-third of all plants in these areas contain nitrilosides. Cassava or manioc, sometimes described as "the bread of the tropics", is one of the most common as well as richest sources of nitriloside. As eaten by primitive populations, the bitter and nitriloside-rich manioc is preferred. People in the cities on Westernized diets favor the sweet cassava. Even in the case of these the cassava is so processed as to eliminate virtually all nitriloside or nitrile ions. The cassava eaten by those still near a Stone Age culture, on the other hand, retains a large quantity of nitriloside and nitrile ions. When these primitive and relatively cancer-free people move to the cities, the incidence of cancer among them rises, as they assume the nitriloside-free, Westernized diet. Like the rest of civilized mankind, they then show a cancer incidence of one in every three or four individuals if they live for a sufficiently long period.
RELATIVE FREEDOM OF SHEEP,
Domesticated horses, however, may be deprived of a variety of plant foods and be limited more or less to fodder completely deficient in nitriloside. In such animals the incidence of cancer appears to be reasonably high, though no formal statistics are obviously available.
The liver cancer, which killed the captive bears in San Diego, is suggestive of the liver cancer which kills 95 per cent of all Bantus who die from cancer in the hospitals of one area of South Africa. In their native state, liver cancer is virtually unknown among these people. When they migrate to urban areas or to the mines, their diet is changed to one consisting, for economic reasons, almost exclusively of low-grade carbohydrates completely devoid of nitrilosides. A staple of this diet is fermented milk and corn meal in a mixture known as mealie meal. When this ration was fed for a prolonged period to rats, most of the rats developed cirrhosis of the liver and the pre-cancerous changes observed in the male Bantus.
Bears in the wild state eat nitriloside-rich berries, such as choke berries, salmon berries; grasses also rich in this factor; wild fruits - apricots, peaches, apples, cherries, plums - the seeds of which are all rich in nitriloside with often the leaves and roots carrying a high concentration of the factor; and barks, roots, twigs, and flowering plants rich in nitriloside. Since bears are omnivores, they also eat game. Peter Krott, Ph.D. in his "Bears in the Family", (E. P. Dutton & Co., Inc., N.Y., 1962) describes the predatory habits of the bear as follows:
"Isolated footmarks showed the shepherds where to go and it was not long before they found the remains of the sheep in the undergrowth. The body was carefully cleaned out - a butcher could not have done better. While we roasted a leg of mutton I asked the men why they did not leave the carcass in place, as the bear would surely return to finish it."
The significance of the rumenal contents of sheep in terms of nitrilosides and nitrates will become increasingly clear in the next section. The nutritional pattern in civilized man, as well as in omnivores in captivity, is reversed from what obtains in nature: the viscera is largely discarded and that which animals in the wild state treat as second rate is utilized to the exclusion of a rich source of nitrilosides.
Krott also reported the fondness of bears for whole cherries. He describes feeding two bear cubs 20 pounds of cherries. Like all the non-human primates and most primitive men, the bears eat the seeds as well as the meat of cherries.
Cancer is generally considered a chronic disease. So far no chronic or metabolic disease has ever found prophylactic or therapeutic resolution except through normally occurring accessory food factors. Certainly none has ever been known to have a viral or bacterial etiology. Pellagra, scurvy, beri-beri, rickets, the anemias, a wide range of neuropathies, etc., etc. - all have found total prophylactic and therapeutic resolution only in factors accessory to normal food. No chronic or metabolic disease has found any other resolution. It is not probable that cancer will prove the first exception.
© Copyright 2003 Phillip Day