13 Toxic Substances Associated with Seed Proteins IRVIN E. LIENER Department of Biochemistry, University of Minnesota, St. Paul, Minn.
The trypsin inhibitors and hemagglutinins of legumes are examples of proteins implicated in the poor nutritive value of unheated plant material. A number of amino acid derivatives are the active principles responsible for various forms of lathyrism which accompany the ingestion of peas belonging to genus Lathyrus. Among the glycosides, goiterogenic agents have been isolated from various cruciferous oil seeds, and cyanogenetic glycosides have been demonstrated in lima beans, linseed meal, and other legumes. Glycosides in soybeans include the saponins, which have hemolytic properties, and the isoflavones, with esterogenic activity. Among the miscellaneous toxic factors are gossypol (cottonseed), the causative agent of favism, the metal– binding constituent of soybean protein, and antivitamin factors in some legumes.
I
t is ironic that while Nature has generously provided man with a liberal supply of plant protein foods, she has at the same time seen fit to contaminate these foods with a variety of substances which may be con sidered " t o x i c " to the animal body. I n our present discussion I do not wish to restrict myself to a definition of "toxic" that a toxicologist might use—a substance which when administered to an animal produces death at a certain level which is expressed i n terms of an LD . Rather I would prefer, for want of a better term, the phrase "antinutritional" to describe any adverse physiological response which is produced by the ingestion of a particular plant protein, whether i t be an acute lethal effect resulting i n death, or a chronic effect resulting i n poor growth or some glandular disorder. It would manifestly be impossible to include a detailed coverage of all of the deleterious substances that are known to be present i n plant materials. I concern myself here only with plants whose seeds have present or potential 50
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value as a source of protein for human feeding. This will include such important oil-bearing seeds as the soybean, peanut, cottonseed, and a number of beans and peas which form an important part of the diet i n India, Africa, Central and South America, and the Mediterranean countries. Although the subject of toxic substances in legumes has been reviewed (31), the present paper brings the subject matter up to date and em phasizes the chemical aspects of the problem. From the point of the chemist, these toxic substances may be classified into three main categories as shown in Table I : proteins or protein deriva tives, glycosides, and a miscellaneous group of substances of diverse but, in most cases, unknown chemical structure. Table I. I.
II.
III.
Antinutritional Factors in Seed Proteins
Proteins or amino acid derivatives 1. Trypsin inhibitors 2. Hemagglutinins 3. Osteolathyrogens 4. Neurolathyrogens Glycosides 5. Goiterogens 6. Cyanogens 7. Saponins 8. Isoflavone glycosides Miscellaneous 9. Gossypol 10. Causative principle of favism 11. Metal-binding factor 12. Antivitamin factors
Proteins and Protein Derivatives Trypsin Inhibitors. Perhaps the best known and certainly the most studied of all of the antinutritional factors is a trypsin inhibitor first isolated from the soybean by K u n i t z i n 1945 (28). This is a protein having a molecular weight of about 20,000, and it forms an inactive com plex with trypsin. Since Osborne and Mendel i n 1917 (47) had noted that raw soybeans supported growth poorly unless subjected to heat treatment, it was logical to assume that the trypsin inhibitor was the substance responsible for the poor nutritive value of the unheated bean. The hypothesis that its effect could be readily explained on the basis of its ability to inhibit intestinal proteolysis was an appealing one. The fact that methionine also markedly improved the nutritive value of raw soybeans (Table II) was taken to indicate that the trypsin inhibitor some how interfered with the availability or utilization of methionine from the raw legume.
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Table II.
Effect of Heat and Methionine on Nutritive Value of Soybeans (34)
Diet Raw soybean meal Autoclaved soybean meal Raw soybean meal + 0 . 6 % methionine
Protein Efficiency 1.33 2.62 2.42
It now appears that the explanation for the growth-inhibiting property of the trypsin inhibitor is not a simple one, and, although 20 years have elapsed since K u n i t z first isolated his trypsin inhibitor, there is still a decided lack of agreement as to the mechanism whereby the trypsin inhibitor exerts its effect. Furthermore, there are at least four trypsin inhibitors (62), and it is not certain whether all four produce the same physiological effects. There is little doubt that pancreatic hypertrophy is one of the primary physiological effects of feeding raw soybean (12). Booth et al. (7) are of the opinion that pancreatic hypertrophy leads to an excessive loss of endogenous protein i n the form of exocrine protein secreted by the pancreas. Since this protein is rich i n cystine, this re presents a net loss of cystine from the body. This increased need for cystine for protein biosynthesis during pancreatic hypertrophy is reflected by an increase in the conversion of methionine to cystine in the pancreas (4). This would explain why the need for methionine is particularly acute in diets containing raw soybeans. Trypsin inhibitors have also been found in a large number of other legumes including the peanut, navy bean, lima bean, and the various grams and pulses eaten i n India, but the exact nutritional significance of these inhibitors is not clear. I n fact there appears to be no clear-cut correlation between the trypsin inhibitor content of legumes and the beneficial effect of heat on their nutritional value (8). Hemagglutinins. I t was this lack of correlation that led our own laboratory to search for some other factor in legumes which might account for the poor growth-promoting quality of most legumes which are not heated. We succeeded in isolating from raw soybeans a protein which had the rather peculiar property of being able to cause red blood cells to agglutinate (85). Such substances have been known for some time to be present in many plants and are referred to as phytohemagglutinins (32). Some of these are highly toxic, such as ricin from the castor bean (62). Another fairly well known hemagglutinin is concanavalin A , which was first crystallized from the jack bean by Sumner (61). Landsteiner had reported many years ago that even such common edible legumes as the navy bean, lentil, and garden pea also contained these hemagglutinins (32). U n t i l we got interested in the hemagglutinins over 10 years ago, the nutritional significance of these substances was largely overlooked.
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In 1953 we showed that the purified soybean hemagglutinin was capable of significantly inhibiting the growth of rats (Table III). It would appear, however, from the work of B i r k and Gertler (6) that the growth impair ment of rats and chicks fed raw soybean meal may be due largely to factors Table III.
Growth-Inhibitory Effect of the Soybean Hemagglutinin (SBH) (33)
Protein Component of Diet
Wt. Gain in 2 Weeks, G.
25% heated soybean meal 25 % raw soybean meal 25 % heated soybean meal + 0 . 8 % S B H
% Growth Inhibition
60.0 28.0 45.0
0 43.2 25.6
other than the soybean hemagglutinin. These authors reported that the insoluble residue which remained after extraction of the raw meal with acid at p H 4.2 retained the capacity 'to inhibit growth, despite the fact that this fraction exhibited little hemagglutinating activity. Table I V compares some of the physical properties of the soybean hemagglutinin (SBH) with ricin and concanavalin A . S B H has been shown to be a mucoprotein containing about 5 to 6 % carbohydrate, made up largely of mannose and glucosamine (87, 64). It also appears to be composed of two chains with alanine residues at each of the iV-termini and serine and alanine at the C-termini (#4). Table IV.
Hemagglutinin
SBH Ricin Concanavalin A
Comparison of the Physical Properties of SBH with Ricin and Concanavalin A Source
Sedimentation Diffusion Constant, Constant, Sv>, w DM, W
Mol. Isoelectric Ref. Weight Point, pH
Soybean Castor bean
6.4 6.4
5.7 6.0
96,000 98,000
6.1 5.5
(48) (62)
Jack bean
6.0
5.6
96,000
5.5
(61)
When we turned our attention to the hemagglutinins from other legumes, the nutritional significance of these substances became even more apparent. We were particularly interested i n legumes which enjoy popular consump tion i n underdeveloped countries. For this purpose, we were able to obtain a black bean from Guatemala, Bengal and red gram from India, mung bean from the Philippines, and the domestic kidney bean. The effect of heat on the nutritive value of these beans is shown i n Table V and the hema gglutinating and antitryptic activities are shown i n Table V I . Only the growth promoting values of the black bean and kidney bean were
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Table V.
Effect of Heat on
itive Value of Some Legumes (22) Gain in Weight, G./Day
Source of Protein
Raw
a
Heated
Phaseolus vulgaris Black bean K i d n e y bean
- 1 . 9 4 (4-5) - 1 . 0 4 (11-13)
+1.61 +1.48
Bengal gram
+1.25
+1.16
R e d gram
+1.33
+1.74
+1.05
+1.07
Cicer arietinum Cajanus cajan
Phaseolus aureus M u n g bean α
100% mortality observed during ι
Table VI.
(in days) shown in parentheses.
Hemagglutinating and Antitryptic Activities of Crude Extracts of Raw Legumes (22)
Legume
Hemagglutinating Activity 0
HU/Ml.
Antitryptic Activity 0
TIU/Ml.
Phaseolus vulgans Black bean K i d n e y bean
Cicer arietinum Cajanus cajan Phaseolus aureus
2450 3560
0 0 0
2050 1552
220 418 260
a u u = hemagglutinating units, T I U = trypsin inhibitor units as defined by Honavarei. aZ. (**).
improved by heat, and these beans were the only ones which displayed a significant level of hemagglutinating activity. F o r these reasons, the hemagglutinin was purified from each of these beans and fed at various levels to rats i n a basal ration containing 1 0 % casein (Table V I I ) . A definite inhibition of growth was apparent at levels as low as 0 . 5 % of the diet, the kidney bean hemagglutinin ( K B H ) being much more effective than the black bean hemagglutinin ( B B H ) . I n fact, K B H at a level of 0 . 5 % caused 1 0 0 % mortality after about 2 weeks, whereas 1.2% B B H was necessary to produce a similar rate of mortality. D a t a are also i n cluded which show that the toxicity of the hemagglutinins may be i n activated by heat. A s to the mechanism whereby the hemagglutinin exerts its effect, present evidence would suggest its site of action to be the lining of the intestinal tract. Jaffé (25) noted a definite impairment i n the absorption of protein and fat when rats were fed raw black beans or the hemagglutinin
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purified therefrom. H e was able to show that isolated intestinal loops taken from rats fed the raw bean or the purified hemagglutinin absorbed glucose at about half the rate of loops taken from control animals. These results would indicate that the hemagglutinin might combine with the muco sal cells lining the intestinal wall, thus interfering with the absorption of essential nutrients. Table VII. Effect of Purified Hemagglutinin Fractions from the Black Bean and Kidney Bean on Growth of Rats (26) Source of Hemagglutinin Black bean
Purified Hemagglutinin Av. Gain in Diet, % in Weight, G./Day 0 0.5 0.5 0.75 1.2 2.3 4.6 0 0.5 0.5* 1.0 1.5 6
Kidney bean
+2.51 +1.04 +2.37 +0.20 -0.91 -1.61 -1.72 +2.31 -0.60 +2.29 -0.87 -1.22
Mortality" Days
t
15-19 12-17 5-7 13-16 11-13 4-7
100% mortality observed during period recorded. Blank space indicates no deaths observed. Solution of hemagglutinin boiled for 30 minutes and dried coagulum fed at level indicated. Hemagglutinating activity was completely destroyed by this treatment. a
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Amino Acid Derivatives Lathyrism. Lathyrism is a disease associated with the consumption of certain species of peas belonging to the genus Lathyrus. A graphic description of this syndrome i n India i n the 1830's is provided by Sleeman (2, 68). I n 1829 and i n 1831 the wheat crop failed i n Sangor, and the inhabitants subsisted mainly on L. sativus which grew wild i n the blighted fields. B y 1833 the younger part of the population of this area began to be deprived of the use of their limbs below the waist by paralytic strokes. None attacked recovered the use of their limbs. I n more recent times this disease has affllicted some segments of the population i n India and the Mediterranean area, and is generally associated with the consumption of L. sativus (chickling vetch), L. cicera (flat-podded vetch), and L. clymenum (Spanish vetch). The precise causative agent of human lathyrism has not been easy to elucidate because of the difficulty with which this disease can be produced in animals. The aforementioned species of Lathyrus which have been implicated i n human lathyrism are relatively nontoxic to most animals (28), although two other species of
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Lathyrus—L. latifolius (perennial sweet pea) and L. sylvestris (flat pea)— produce neurological symptoms i n rats which resemble those of human lathyrism (30, 54). Three other peas, however—L. odoratus (sweet pea), L. pusillus (singletary pea), and L. hirsutus (Caley pea)—produce skeletal deformities i n rats, symptoms which are different from human lathyrism (29). It is evident, therefore, that the concept of what actually constitutes lathyrism is not a simple one. Some clarity is achieved if one accepts the conclusion of Selye (56) that the consumption of Lathyrus can produce two distinctly different types of diseases, a "neurolathyrism" which involves damage to the central nervous system, and an "osteolathyrism" which affects bone and connective tissue. This permits the classification of lathyrogenic seeds as shown i n Table V I I I . The whole problem of Table VIII.
Classification of Lathyrogenic Seeds According to Their Physiological Effects (Neurolathyism)
In man L. sativus (chickling vetch) L. cicera (flat-podded vetch) L. clymenum (Spanish vetch)
In rats L. latifolius (perennial sweat pea) L. sylvestris (flat pea) V. saliva (common vetch)
(Osteolathyrism) In rats only L. odoratus (sweet pea) L. pusillus (singletary pea) L. hirsutus (Caley pea) lathyrism has been considerably clarified i n recent years by the isolation and characterization of what appear to be the causative principles of these two forms of lathyrism. I n 1954 two groups of workers, D u p u y and Lee (16) and M c K a y et al. (89), isolated a compound from L. odoratus and L. pusillus which was capable of reproducing all of the symptoms associated with osteo lathyrism. This proved to be j8-(i\T-7-glutamyl)-aminopropionitrile, although i t was subsequently shown that 0-aminopropionitrile ( B A P N ) was the active portion of the molecule (see Figure 1). This compound was notably absent from L. sativus and the other species of lathyrus known to cause human lathyrism. I n 1961, Ressler et al. (63) reported the isolation of a, γ-diaminobutyric acid (Figure 2) from L. latifolius and L. sylvestris which had previously been found to reproduce the neurotoxic symptoms similar to that noted i n man.
