Yearul Kabir ( Department of Biochemistry, University of Dhaka, Dhaka-2, Bangladesh. )
Kamaluddin Ahmad ( Department of Biochemistry, University of Dhaka, Dhaka-2, Bangladesh. )
Guinea-pigs maintained on a vitamin C deficient diet for 30 days lost body weight (13.3%) and had reduced level of vitamin C (48.9%) in the serum. These guinea-pigs demonstrated a consistent pattern of neurological signs of neurolathyrism when administered intraperitoneally with an extract of Lathyrus sativus seeds, equivalent to 31.5 rng of active toxin, B - N-oxalyl- L-a, B-diarninopropionic acid (ODAP). On the other hand, animals in the control group which received the vitamin (15 mg/day/head) throughout the experimental period were resistant to these symptoms after the same level of the toxin administration. The results therefore demonstrate that vitamin C has a protective role against neurolathyrism in adult guinea-pigs (JPMA 35:2. 1985).
Neurolathyrism is a nervous disease of the humans caused by excessive consumption of Lathynus sativus (Khesari dhal) (200 g or more per day) for prolonged periods1-4 During drought when most other crops fail, the poor eat L. sativus seeds rather than starve. Although epidemiological studies clearly link onset of paralysis to prolonged excessive consumption of L.sativus seeds, the failure of the toxin, B-Noxalyl-L-a, B-diaminopropionic acid (ODAP), present in L. sativus seeds to affect adult experimental animals has raised questions regarding the role of this neurotoxin in the etiology of neurolathyrism4.
Little information is available on the dietary deficiency in animals prone to neurolathyrism. Dietary surveys1 carried out in the epidemic areas have shown that there is no evidence of any dietary protein or calorie deficiency although the diets of the neurolathyrism victims usually lack in vitamin C and A, and fat1. Diets of lathyrism patients were deficient in vitamin C5 which was thought to be playing some role in the causation of lathyrism. It was therefore considered worthwhile to study the effects of
dietary vitamin C depletion and supplementation in producing the symptoms of neurolathyrism in an animal susceptible to this disease e.g. the guinea-pig.
We undertook this experiment on the acute neurotoxicity of L. Sativus seeds in the guinea pig after observing that human subjects afflicted with the disease consumed a nutritionally inadequate diet specially in vitamin C6. A population well-nourished in respect of the vitamin C would not develop neurolathyrism in spite of the use of Khesari as a principal item in the diet.
Material and Methods
Animals: Twenty healthy adult white guinea-pigs of both sexes (weighing between 240 and 350 g) and of various ages collected from the International Center for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B), were used for the study. The animals were divided into two groups: one received a diet supplemented with vitamin C (15 mg/day! head for 30 days) and the other received a diet without vitamin C. Both groups were fed on boiled acer arietinum (deshi chana) seeds and a vitamin-salt mixture7 and housed two per cage under proper hygienic conditions. The diets and water were given ad libijum. Body weights of the animals were recorded every 6 days. Serum vitamin C levels8 were also measured at these intervals.
Preparation of seed extract: L. sativus seed toxin was extracted by steeping the seed powdeE (200 g) in 800 ml 75% ethanol and then dissolved in 10 ml distilled water after evaporating the alcohol present in the filtrates9. The content of the toxin in the seed extract was determined by a specific sensitive colour reaction10
Toxin administration The animals in each group were divided into two subgroups on the 30th day from the beginning of the experiment. The subgroups then were intraperitoneally injected with either 3m1 of normal saline or 3 ml of L. sat’ivus seed extract (equivalent to 31.5 mg toxin) freshly prepared as above. The animals were observed for any symptoms of neurolathyrism or other clinical changes following the injection.
Results and Discussion
Guinea-pigs maintained on a diet supplemented with vitamin C showed an increase (30%) in body weight during the 30 days of feeding, whereas those given no vitamin C registered a significant loss of body weight (13.31%) during the same period (Fig I).
The mean serum vitamin C level kn the vitamin depleted animals (0.23 mg/100 ml) was also significantly lower (P <0.001) than that in the supplemented group (0.59 mg/ 1 00ml) after 30 days (Fig 2).
The present results clearly demonstrate that dietary depletion of vitamin C in adult guinea-pigs results in decline in serum level of the vitamin, concomitant with failure in maintenance of body weight.
Clinical observations showed that the crude alcoholic extract of L. sativus seeds produced all symptoms related to neurolathyrism in the guinea-pigs given a diet without vitamin C, whereas those given the vitamin supplement were not affected (Table).
The affected animals became dull and showed convulsions, tremors and ataxic gait. Paralysis of the legs developed within 3 hours after the toxin administration.
Incidence of neurolathyrism as a clinical disorder after intraperitoneal injection of L. sativus seed extract in the adult guinea-pigs has not been previously reported. The present experiment thus demonstrates that L. sativus seed extract is toxic to adult guinea-pigs and produced neural symptoms comparable tO those seen in human cases of lathyrism, when serum vitamin C dèficiency is prominent. Animals maintained on a vitamin C supplemented diet and thus with normal serum vitamin C level were resistant to the development of such symptoms. Not a single animal of group A (Table ) which received vitamin C showed any neuropathological symptoms, while all animals in group B (which did not have vitamin C) progressively developed symptoms, while all animals in group B (which did not have vitamin C) progressively developed the symptoms of neurolathyrism within 3 hours after the extract administration.
The idea that primates (including man) which are incapable of synthesizing vitamin C and therefore depend on dietary sources of the vitamin might have metabolic differences which make them particularly vulnerable to ODAP intoxication has been implied by the extensive use of monkeys in recent work on the lathyrus toxin10-14 The present study shows that the guinea-pigs are similar to the monkey in susceptibility to the toxin. Because the guinea-pigs are less expensive and easy to breed in the laboratory, its use permits studies such as those described here or other biochemical studies, which would be more inconvenient or expensive with monkey.
The mechanism of vitamin C protection against neurolathyrism is not clear at present. It has previously been observed that ODAP produces toxic effects in young animals e.g. one-day old chick9,15 or squirrel monkey15 and not in the adult. It was also observed that neurolathyrism can be developed in adult primates when the neurotoxin is injected intrathecally i.e. directly into the central nervous system10,14,17,18 These observation led to the suggestion that ODAP intoxication is related to the blood-brain barrier (EBB) and not the result of unique species sensitivity. However, the results of the present experiments do not agree with the above view, because in adult guinea-pigs the BBB is considered to be mature19 BBB is in fact known to be mature in this species prenatally). The results, on the other hand, seem to be more related to the vitamin C status of the animal. It is possible that vitamin C helps in the quick detoxification of ODAP, by increasing the rate of its hydroxylation. It is also possible that vitamin C directly or indirectly prevents the toxin from binding with its specific receptor sites in the post-synaptic membrane. Studies with labelled ODAP could clarify these possibilities. It is also necessary that detailed biochemical studies be carried out, before a comprehensive picture of the neurotoxin effect and its prevention by vitamin C is obtained.
The authors are grateful to Dr. H.K.M. Yoüsuf Associate Professor, Department of Biochemistry, University of Dhaka, for his help in the preparation of the manuscript.
1. Ganapathy, K.T. and Dwivedi, M.P. Studies on clinical epidemiology of lathyrism. Indian Council of Medical Research, Rewa, 1961, p. 29. Gandhi Memorial Hospital.
2. Dwivedi, M.P. and Prasad, B.G. An epidemiological study of lathyrism in the district of Rewa, Madhya Pradesh. Indian J. Med. Res., 1964; 52 : 81.
3. Sarma, P.S. and Padmanaban, G. Lathyrogens, in toxic Constituents of plant foodstuffs. Edited by I.E. Liener, 2nd ed. Newyork, Academic Press, 1969, p.267.
4. Bell, E.A. Aminonitriles and amino acids not derived from proteins in toxicants occurring naturally in foods, Washington, National Academy of Sciences, 1973, p.153.
5. Govil, K.K., Gupta, B.M., Chakravarty, N.L., Bhatnagar, D.P. and Pant, K.C. Field investigations of lathyrism in Uttar Pradesh. .j. Indian Med. Assoc., 1959; 33: 499.
6. Ahmad, K. Lathyrism, in adverse effects of foods. Edited by E.F. P. Jeiliffe and D.B. Jelliffe. New, York Plenum Press, 1982, p.71.
7. Oser, B.L. In : Hawk’s Physiological Chemistry (Bernard L. Oser, ad.) Fourteenth Edition, Tata McGraw-Hill Publishing Company Ltd, Bombay, New Delhi.
8. Lowry, O.H., Lopez, J.A. and Bessey, O.A. The determination of ascorbic acid in small amounts of blood serum. J. Biol. Chem., 1945 ; 160 : 609.
9. Roy, D.N., Nagarajan, V. and Gopalan, C. Production of neurolathyrism in chicks by the injection of Lathyrus sativus concentrates. Curr. ScL, 1963;32: 116.
10. Rao, S.L.N. A sensitive and specific colorimetric method for the determination of a, j3 - diamino propionic acid and the Lathyrus sativus neurotoxin. Anal. Biochem., 1978; 86 : 386.
11. Mani, K.S., Sriramachari, S., Rao, S.L.N. and Sarma, P.S. Experimental neurolathyrism in monkeys. Indian J. Med. Res., 1971; 59
12. Lakshmanan, J. and Padmanaban, G. Effect of B - N - oxalyl-L- a, B - diaminopropionic acid on glutamate uptake by synaptosomes. Nature, 1974 ;249:469.
13. Mehta, T., Zaighami, N.S., Cusick, P.K., Parker, A.J. and Haskell, B.E. Tissue distribution and metabolism of the Lathyrus sativus neurotoxin L-3-oxalylamino-2-aminopropionic acid in the squirrel monkey. J. Neurochem., 1976; 27 : 1327.
14. Lakshmanan, J. and Padmanaban, G. Studies on the tissue and subcellular distribution of B-N oxalyl-L-a; B-diaminopropionic acid, the Lathyrus sativus neurotoxin. J. Neurochem. 1977; 291121.
15. Nagarajan, V., Mohan, V.S. and Gopalan, C. Toxic factors in Lathyrus sativus. Indian J. Med. Res., 1965;53: 269.
16. Parker, A. J., Mehta, T., Zarghami, N.S., Cusick, P.K. and Huskell, BE. Acute neurotoxicity of the Lathyrus sativus neurotoxin, L-3-oxalylamino-2aminopropionic acid, in the squirrel monkey. Toxicol. Appl. Pharmacol., 1979;47: 135.
17. Rao, S.L. Adiga, P.R. and Saxma, P.S. The isolation and characterization of beta-N-oxalyl.a-alpha, betadiaminopropionic acid; a neurotoxin from the seeds of Lathyrus sativus concentrates. Curr. Sci.,1963;32: 116.
18. Rao, S.L. and Sarma, P.S. Neurotoxic action of B-N-oxaiyl-L-a; B-.diaminopropionic acid. Biochem. Pharmacol., 1967; 16 : 218.
19. Davson, H. The blood-brain barrier, in the structure and function of nervous tissue. Edited by G.H. Bourne. New york., Academic Press, 1972, p. 323.