70 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. I, NO.2

87 Romer FK. Sarcoidosis and Cancer: A critical review. In: Jones 100 Eisenberg H, Terasaki P, Sharma OP. HLA association studies in Williams W, Davis BH (eds), Eighth International Conference on black patients with sarcoidosis. Tissue Antigens 1978;11:484. Sarcoidosis and Other Granulomatous Diseases. Alpha Omega 101 Newill CA, John CJ, Cohen BH, et al. Sarcoidosis, HLA and im- Publishing Ltd, Cardiff, UK, 1980;567-71. munoglobulin markers in Baltimore Blacks. In: Chretien 1, Mar- 88 James DG. Is sarcoidosis a precursor of lung cancer? Cancer Con- sac J, Saltiel JC (eds), Sarcoidosis and Other Granulomatous Dis- sultation 1985;1(2):19. orders. Pergamon Press, Paris, 1981;253-6. 89 Kyle RA, Bayrd ED. Amyloidosis: Review of236 cases. Medicine 102 Tachibana T, Shirakura R, Yamazaki Y. HLA-DR antigens in sar- (Baltimore) 1975;54:271. coidosis. Sarcoidosis 1985;2:83. 90 Gordonson JS, Sargent J, Jacobson G, et al. Roentgenographic 103 Thunell M, Soundell K, Stjerberg N. HLA-antigens in patients manifestations of pulmonary amyloidosis. J Can Assoc Radiol with sarcoidosis from Northern Sweden. Sarcoidosis 1985;2:48. 1972;23:269. 104 Nowack D, Goebel K. Genetic Aspects of Sarcoidosis: Class II his- 91 Cole SR, McCormick, Sulavik SB. Granulomatous lymph node in- tocompatibility antigens and family study. Arch Intern Med volvement in amyloidosis. Sarcoidosis 1985;2:78. 1987;147:481. 92 Fresko D, Lazarus SS. Reactive systemic amyloidosis complicating 105 Crystal R, Roberts WC, Hunninghake GW, et at.' Pulmonary sar- longstanding sarcoidosis. NY State J Med 1982;82:232. coidosis: A disease characterized and perpetuated by activated 93 Sharma OP, Koss M, Buck F. Sarcoidosis and Amyloidosis: Is the lung T lymphocytes. Ann Intern Med 1980;94:73. association casual or co-incidental? Sarcoidosis 1987;4:139. 106 Crystal R, Gadek JK, Ferrans VJ, et al. Interstitial lung disease: 94 Sack GH Jr, Lease 11. Human amyloid-A gene isolation and struc- Current concepts of pathogenesis, staging and therapy. Am J Med tive studies. Sarcoidosis 1985;2:68. 1981;70:542. 95 Harf RA, Ethevenaux C, Gleize J, Perrin-Fayolle M, Geurin JC, 107 Lem VM, Lipscomb MF, Weissler lC, et al. Bronchoalveolar cells Ollagnier C. Reduced prevalence of smokers in sarcoidosis. Re- from sarcoid patients demonstrate antigen presentation. J Im- sults of a case control study. Ann NY Acad Sci 1986;465:625-31. munoI1985;135:1766. 96 Lawrence EC, Fox TB, Teague RB, Bloom K, Wilson RK. 108 Daniele RP, McMillan LJ, Dauber lH. Immune complexes in sar- Cigarette smoking and bronchoalveolar T-cell populations in sar- coidosis: A correlation with activity and duration of disease. Chest coidosis. Ann NY Acad Sci 1986;465:657-64. 1978;74:261. 97 Moilers E, Hedfors E, Wiman LG. HLA genotypes and MLR in 109 Quismorio F, Sharma OP, Chandor SB. Immunopathological familial sarcoidosis. Tissue Antigens 1974;4:299. studies on the cutaneous lesions in sarcoidosis. Br J Dermatol 98 Kueppers F, Mueller-Eckhardt G, Heinrich D. HLA Antigens of 1977;97:635. patients with sarcoidosis. Tissue Antigens 1974;4:56. 110 Okabe T, Suzuki A, Ishikawa H, Watanabe I, Takaku F. 99 Brewerton D, Cockburn G, James DG. HLA antigens in sar- Chromosomal aneuploidy in sarcoid granuloma cells. Am Rev coidosis. Clin Exp ImmunoI1977;27:277. Respir Dis 1986;134:300-4.

The neurotoxic disease

DWIJENDRAN. ROY

INTRODUCTION ditions, when L. sativus constitutes approximately two- Human lathyrism, or , is caused by the in- thirds of the daily diet, humans and domestic animals de- gestion of certain species, namely L. sativus velop lathyrism. 1-7 (chickling vetch), L. cicera (flat-podded vetch) and L. The search for a causative factor or group of factors in clymenum (Spanish vetchling). Lathyrus presumed to be responsible for the onset of the In certain areas of the world, L. sativus provides a com- disease has resulted in the identification of various chemi- paratively nutritious and inexpensive diet among poor cals having potential toxic properties, all of which have people. During natural calamities such as flood and been generally termed 'lathyrogens'. drought, when other general food crops are damaged or destroyed, the hardy crop, L. sativus becomes a survival THE DISEASE LATHYRISM food for humans and domestic animals. Under these con- Historical and General Background The ancient Hindu treatise, Bhavaprakasa, mentions that Institute of Neurotoxicology, Department of Neuroscience, 'the triputa pulse causes a man to become lame and it crip- Albert Einstein College of Medicine, Bronx, New York, USA ples and irritates nerves.s Hippocrates (460-377 B.C.) © The National Medical Journal of India, 1988 ROY: THE NEUROTOXIC DISEASE LATHYRISM 71 noted that certain peas were found to be toxic to human documented by General Sleeman, of an epidemic of beings. In the seventeenth century, the Duke of Wurtem- lathyrism in India which took place between 1829 and berg prohibited the use of Lathyrus flour in the making of 1831:6 bread, recognizing its paralysing effects upon the legs." In 1829 the wheat and other spring crops in Saugor and sur- Contani, in Italy, first coined the term 'lathyrism' in 1873 rounding villages were destroyed by severe hailstorms and rains to identify the unique neurological disorder which re- and in 1831 they were destroyed by blight. During these three sulted from ingestion of Lathyrus seeds.? years the 'teori' or what in other parts of India is called Khesari Throughout the eighteenth and twentieth centuries, (L. sativus), a kind of wild vetch which, though not sown itself, outbreaks of lathyrism occurred in certain parts of is left carelessly to grow among the wheat and other grains and given in the green and dry state to cattle, remained uninjured Europe, Central India, North Africa, Middle East, Af- I and thrived with great luxuriance. In 1831 they reaped a rich ghanistan and Russia. ,4.5.IOMajor epidemics were re- crop of it from the blighted wheat fields and subsisted on its grain ported in France (1700-1; 1836) and Spain (1940s).6.IO.11 during that and the following years giving the stalks and leaves Lathyrism has been reported in many districts of India in- only to the cattle. In 1833, the sad effects of this food began to cluding Jammu and Kashmir, the Punjab, Bihar and West manifest themselves. The younger part of the population of this Bengal. Madhya Pradesh experienced a high incidence of and the surrounding villages, from the age of thirty downwards, lathyrism with about 60 000 cases reported in 1922. A sur- began to be deprived of the use of their limbs below the waist by paralytic strokes, in all cases sudden, but in some cases more se- vey carried out between 1956 and 1961 revealed 25000 vere than others. About half of the youths of this village of both cases in Rewa and 7000 cases in Satna districts. The oc- sexes became affected during the year 1833-34 and many of currence of lathyrism was also reported in Orissa and them have lost the use of their lower limbs entirely and are un- Maharashtra. able to move. The youth of the surrounding villages in which the In India, the pulse of L. sativus is known by various teori from the same sources formed the chief article of diet dur- names including khesari (in Hindi, Bengali, Nepali, ing the years 1831-32 have suffered to an equal degree. Since the year 1834 no new case has occurred but no person once at- Oriya), teora, batra and teora-dal (in Hindi), gharas(in tacked has been found to recover the use of limbs affected, and Kashmiri), vattu-parippu and khesari (in Malayalam), my tent was surrounded by great numbers of youth, in different lakh (in Marathi), khesari-chural (in Punjabi), lamka- stages of the disease, imploring my advice and assistance against pappu (in Telugu) and khesari-parappu (in Tamil). the dreadful visitations. Some of them were very fine young men The pulse, known to be a native of Southern Europe of good caste and respectable families and all stated that their and West Asia, is grown in India, Bangladesh and pains and infirmities were confined entirely to the part below the Ethiopia. In India, four per cent of the land used for pulse waist. They described the attack as coming on suddenly, often while the person was asleep, and without any warning symptoms cultivation is occupied by L. sativus, and its production whatever, and stated that a greater portion of young men were constitutes about three per cent of the total pulse pro- attacked than of young women. It is the prevailing opinion of duced. The Lathyrus growing states are Madhya Pradesh, natives throughout the country that both horses and bullocks, Uttar Pradesh, Bihar, West Bengal and Assam. which have been fed on teori, are liable to lose the use of their L. sativus (Fig. 1) belongs to the botanical family of limbs, but if the poisonous qualities abound more in grain than leguminosae. It is an annual herb, with creepers 45-60 em the-stalk of leaves, a man who eats nothing but the grain must be long which produce hairy pods. The seeds are greyish- more liable to suffer from the use of food than beasts, which eat it merely as they eat grass or hay. brown and black in colour. The following was taken from the first description, Epidemics of neurolathyrism were reported in Europe and Asia in the eighteenth and nineteenth centuries and were associated with famine and the consequent use of certain Lathyrus species in the preparation of flours and cereals.8.12.13A clinically similar disease, thought to be potentiated by vitamin deficiency, occurred in concentra- tion and prisoner-of-war camps during World War 11.12.15 Based on observations reported in Israel and Asia, it is concluded that lathyrism is a stereotyped, self-limiting neurotoxic disorder found in nourished and under- nourished individuals who consume 200-400g per day of L. sativus.»

Disease Characteristics Lathyrism generally follows famine conditions caused by adverse weather such as drought, flood and other natural calamities, although sporadic cases have been reported during normal climes. Early reports also indicate that domestic animals subsisting on Lathyrus fodder displayed typical signs of the disease. In humans, the disease gener- ally appears when Lathyrus constitutes more than two- Fig. 1 seeds thirds of the diet for periods of 3 to 6 months. The disease 72 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. I, NO.2

fig. 2 a and b-Lathyrism patients affects all ages, but is especially common in young men. Three modes of manifestation of spastic paraparesis Women are infrequently affected, and to a lesser degree. have been described. 16 Most common is a sudden onset in In many cases, the onset of the disease is sudden and with- which the affected individual complains of heavy out warning; the subjects develop stiffness of the leg mus- weakened legs after falling down or awakening from cles and partial paralysis or loss of control of lower limbs. sleep, Some subjects report a subacute onset of walking The earliest sign is calf muscle spasm locally known as disability, while others experience an insidious progres- 'lodakas' in India, which generally disappears if the intake sion of spastic paraparesis spanning over a period of of Lathyrus is discontinued soon after the mild attack. 16.17 months. In Asia, the degree of neurological involvement Stockman, who reported extensively on lathyrism from is classified according to the extent of walking difficulty 1917 to 1934, describes the clinical syndrome in man as and the need of physical support. The large majority of in- follows: 1,5 dividuals walk abnormally, but without aid (No-stick stage); less often, a pair of sticks for bilateral support are In man the symptoms usually begin suddenly and seem often to needed (Two-stick stage). Very severely affected indi- be precipitated by exposure to cold and wet and fatigue, this viduals with advanced spastic paraparesis are forced to being the reason assigned for the comparative immunity of women and children and elderly men. Sometimes there are pro- move about on hands and knees (Crawler stage). Figure 2 dromal sensory symptoms of pain, prickling, numbness and (a and b) shows typical stages of the affected individuals. cramps, but most commonly the victim quite suddenly feels the Symptoms generally consist of muscle cramping and legs and loins weak and heavy, the muscles are tremulous when weakness of legs, but occasionally include tremulousness weight is put on them, there is a dragging of the legs, increased or tremor, and coarse involuntary movements of upper reflexes and more or less inability to walk. This may go on to a extremities. Most of these clinical manifestations of appa- fuller stage of extreme spasticity and rigidity of the leg muscles: the gait becomes jerky with short steps taken' on the balls of the rent central nervous system overactivity disappear after feet and with the knees slightly flexed; ankle clonus and adduc- the onset of walking disability and the individual is left tor spasm of the thigh muscles are easily excited and account for with varying degrees of spastic paraparesis with minor the jerky crossed-legged walk, in which the limb is raised up with weakness and greatly increased muscle tone in the legs, an effort and a heave of the corresponding shoulder; the foot is brisk knee jerks, ankle clonus and extensor plantar refle- turned in with the great toe tending to scrape on the ground and xes indicative of corticospinal involvement. The degree of the heel drawn up by gastrocnemius spasm. In the slighter cases disability varies, but in many it progresses if consumption the patient can get about with or without the aid of a stick, but often the disability is very severe and he finds himself unable to of Lathyrus is continued. Individuals least affected run walk or stand, or only able to do so with the support of one or with difficulty, due to thigh adductor spasm and walk with two long sticks which he grasps high up. In extreme cases he is a stiff-legged gait. More severely affected subjects have driven to progress on his hands and feet in a sitting posture. additional gastrocnemius spasm and walk on the balls of There may be no sensory disturbances and no muscular wasting their feet with a scissoring gait. The most severely dis- and almost invariably the lower limbs are only affected. The le- abled exhibit total spastic paraplegia and severe leg weak- sion in chronic is evidently a partial degeneration of the motor tracts in the cord. Babinski's sign is present as well as ness and crawl on their knees or buttocks. In summary, the exaggerated deep reflexes. But in many cases other the clinical picture of lathyrism is dominated by signs of symptoms have been reported which points to a wider implica- irreversible pyramidal dysfunction. tion of the spinal cord. This clinical picture is supported by sparse ROY: THE NEUROTOXIC DISEASE LATHYRISM 73

neuropathological evidence which includes degeneration tive viscosity of extractable .ss These os- of Betz cells, and of anterior and dorsolateral corticospi- teolathyrogens show no common chemical structural re- nal tracts in thoracic, lumbar and sacral regions of the spi- lationship, except that they all have, or potentially have, nal cord. 17-21 an available terminal primary group. 1,43 The N-acyl blocking of the terminal amino group of BAPN is toxic CLASSIFICATION OF LATHYRISM provided no cleavage of the acyl-group takes place in The term lathyrogen was initially used to describe natur- vivo. 44 ally occurring that induce skeletal () Generally, osteolathyrogens interfere with the initial and blood vessel changes (angiolathyrism) in experimen- reaction in the formation of cross-links in connective tis- tal animals.? These disorders, found in rats fed a diet of sues. The ability of collagen and elastin fibres to function Lathyrus odoratus (flowering ), exhibited as major structural components of tissues is dependent on changes in collagen and elastin and produced abnor- a system of covalent cross-links between the polypeptide malities in and musculature. First reported in 1933, chains of the respective . Cross-linking of elastin animals with osteolathyrism manifested curvature of long involves two unusual amino acids, demosine and and kyphoscoliosis or gibbus deformity of. the isodemosine, the biosynthesis of which involves the for- thoracic spine.22 The experimental disorder (osteo- or an- mation of intermediates.o Oxidative deamina- giolathyrism) has no known relationship to human tion of peptide-bond and hydroxylysine leads to the lathyrism or neurolathyrism, although minor abnor- production of an aldol- or aldamine-type cross-link malities of bone are occasionally reported in humans with through the intermediate formation of the reactive group. neurological disease.t? The compound, ~-N- 'Y -glutamyl , isolated from L. odoratus and L. TABLE I Compounds having osteolathyritic activities.s PUSillUS23,24 was- the first toxic principle identified in Latyrus Spp. The ~-aminopropionitrile (BAPN) (Fig. 3a) Compounds References moiety of this compound was found to be primarily re- sponsible for the disruption of connective tissues in ex- 1. Organic nitriles: perimental osteo- and angiolathyrism. (a) ~-aminopropionitrile (BAPN), and Bachhuber et 0/.25; ~-( Y-glutamyl) aminopropionitrile Wawzonek et 01.26; Although abnormalities of the vertebral column may Schilling & Strong» lead to secondary neurological changes in animals with os- (b) Aminoacetonitrile (AAN) Starnlerzs; teolathyrism, BAPN is not known to have any effect on Ponseti et of. 29 the nervous system. Excessive consumption of L. sativus, (c) Methyleneaminonitrile (MAAN) Ponseti et 01. 29 L. cieera, or L. clymenum species, which contain the (d) ,6,W -iminodipropionitrile (IDPN)b.c Stamlerss; neurotoxin ~-N-oxalylamino-L-alanine (BOAA), may Syn: aminodipropionitrile, Rosmus et 01. 30; sometimes induce bone changes along with nervous sys- bis-(,6-cyanoethyl) amine Hugon er e/.» (e) ,6-hydrazinopropionitrile Dasler et 01. 32 tem abnormalities. (f) 2-cyanopropylamine (2-CPA) Roy et 0/.33; Roy34 2. Ureides: Osteolathyrism and Osteolathyrogens (a) Semi carbazide (SCA) Roy et 01. 35;Levene» (b) Acetone Levene» After the discovery of the osteolathyrogen in L. odoratus 3. Hydrazides: and L. pusillus, several chemicals have been shown to (a) Isonicotinic acid hydrazide Levene» possess similar osteolathyritic characteristics and fall into (b) Nicotinic acid hydrazide Levene» four major chemical groups: nitriles, urides, hydrazines (c) Cyanoacetic acid hydrazide Leveneie and hydrazides.! Table I presents these compounds ac- (d) Benzhydrazide Levene» cording to diminishing potency based on an index of rela- (e) 'y -l-glutarnyl hydrazide Levene-e (f) Glycine hydrazide Levene» (g) Carbohydrazidev Rosmus et 01. 30 (h) Thiocarbazide Rosmus et 01. 30 (i) hydrazide Rosmus et 01. 30 U) p-nitrobenzine acid hydrazide Rosmus et 01. 30 4. Hydrazines: Beta-aminoprOrionitrile Levene36 (BAPN (a) Hydrazine hydrate (b) Unsymmetrical dimethyl hydrazine Rosmus et 01. 30 (c) Symmetrical dimethyl hydrazine Levene36 Rosmus et 01. 30 HOOC - CO -NH - CH2 - CH - COOH (d) Phenyl hydrazine Dasler et 01. 32 5. Miscellaneous: (a) Cyanide> Rosmus et 01. 30 I (b) Thiosemicarbazide Neuman et al.37 NH2 (c) D-penicillamine Pinnell et 01. 38 (d) ,6-mercaptoethylamine Daslerw; Stamlerts; Beta-N-oxalylamino-L-alanine Royetal.33 (BOAA)

a Rosmus et 01.40; Padmanaban-': Barrow et 01.1; Roy42 Fig. 3 a. Chemical structure of the osteolathyrogen, BAPN b Do not produce similar types of osteolathyritic signs h. Chemical structure of the neurolathyrogen, BOAA c Neurological changes (axonopathy) distinct from neurolathyrism 74 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL.!, NO.2

These reactions generate desmosine and isodesmosine. In Three nomenclatures have been suggested-a for the ~- collagen, cross-linking occurs between dehydrohydroxy isomer of the neurotoxin from L. sativus: ~-N- lysine and hydroxy-norleucine.se oxalylamino -L-alanine (BOAA), ~-N-oxalylamino-L- Lysil oxidase requires cupric ions for activity, and a.,~-diaminopropionic acid (Ox-dapro, ODAP), and L-3- activity is not detected in tissue extracts of oxalylamino-2-aminopropionic acid (OAP). The a.-isomer copper-deficient animals. In avian species, BAPN and has been referred to as the «-isomer of N-oxalyl-a.,B- copper deficiency produce a common pattern of deficit diaminopropionic acid, and as L-2-oxalylamino-3- including increasing solubility of collagen, bone defor- aminopropionic acid. It is likely to be produced in the mities and modification of arterial elastin, with plant by an intramolecular rearrangement through a consequent reduction in tensile strength of the aorta. hypothetical cyclic intermediateu (Fig. 5). However, the disorders induced by BAPN and copper The concentration of BOAA in the seed of L: sativus deficiency are potentiated by ascorbic acid, the combined varies considerably depending on the source (which may effect resulting in aortic rupture and aneurysm.' Neither explain earlier conflicting reports on the of L. ascorbic acid nor copper supplementation modifies the sativus seeds). Values range from 0.1 to 2.5% of the dry toxic action of BAPN. 47,48 weight of the seeds.s- and has been confirmed in 21 Experimental osteolathyrism is associated with disrup- species of Lathyrus.» tion of mesenchymal tissues and disorders of growth of Contamination of L. sativus seeds with those of Vicia cartilage and bone. These include changes in endochond- has been an important source of earlier confusion in the ral ossification and the formation of irregular hyperplastic investigation for a possible neurotoxic factor responsible epiphyseal cartilage.w Malformation of long bones, for the causation of the disease. ~-cyano-L-alanine caused by changes in epiphyseal discs and the develop- (BCLA) was first identified in seeds of the common vetch ment of exostoses, occurs at sites of attachment to muscles Vicia sativa» and subsequently, a r -glutamyl-derivative exposed to continuous tension. Affected animals display of BCLA was detected in the seed.58 The presence of this chest deformities, kyphoscoliotic alterations of the spine, toxic nitrile was established in 16 of 48 species of Vicia.» and loosened intervertebral discs resulting in prolapse of Administration of BCLA to weanling male rats by the nucleus pulposus into the spinal canal. Certain avian stomach tube produced reversible hyperexcitability, con- species, such as chickens and turkeys, are more suscepti- vulsions and rigidity; when a higher dose was adminis- ble to the action of osteolathyrogens.34,39 tered subcutaneously, convulsions, rigidity, prostration Experimental angiolathyrism is associated with a low- and eventual death occurred. The naturally occurring ered resistance of the vascular wall to stretches, causing form of BCLA is a r -glutamyl derivative, which consti- aneurysms. Elastin formation in the arterial wall is dis- tutes 0.6%, and 1.7-2.6% of the seeds and the young turbed, fibroblasts proliferate abnormally, and collagen seedlings, respectively, when calculated on a dry weight fibres are arranged irregularly. basis. The dipeptide of BCLA is as potent as the free in rats, and a mixture of the dipeptide and free Neurolathyrism and Neurolathyrogens amino acid (BCLA), at one-half their level in V. sativa The observation of an acute convulsive paralytic effect seeds, induced convulsions and opisthotonos in chicks.57 (Fig. 4) of an alcoholic extract of L. sativus seed adminis- Decarboxylation of BCLA produces BAPN, the osteola- tered to neonatal chicksv initiated studies to isolate an ac- thyrogen from L. odoratus. tive principle of L. sativus seeds, a free non- amino The observation that V. sativa contains BCLA, com- acid, ~-N-oxalylamino-L-alanine (BOAA)5!.52 (Fig. 3b). bined with the suggestion of contamination of V. sativa in Investigationsv.v demonstrated that the ~-isomer of L. sativus in endemic zones of lathyrism.e' led to the con- BOAA exists naturally in an isomeric mixture with the a- clusion that Vicia was responsible for lathyrism.57 The isomer. The ratio of a.- to ~-isomers in L. sativus seeds is role of BCLA in the aetiology of the disease was held in about 5 to 95%, respectively. 54 question for several years until subsequent detailed sur- veys showed that seeds of L. sativus collected from en- demic zones of India were free of Vicia contamination.e- Table II lists the acutely neurotoxic amino acids isolated from Lathyrus and Vicia species. Another neurotoxic amino acid, L-a.- r-diaminobutyric acid (LDBA), the lower homologue of ornithine, was also found in Lathyrus seeds62 and has since been identified in 10 of 13 Lathyrus species.62,63 In 10 of the Lathyrus species, LDBA was found to be accompanied by oxalyl diaminopropionic acid, and the r -oxalyl derivative of diaminobutyric acid. LDBA causes weakness, tremors, and convulsions in rats,62,64 and also acts as an inhibitor of ornithine--carbamyl transferase in the mammalian liver, disturbing ornithine-urea cycle reactions, thereby induc- Fig. 4 Lathyrus extract-administered chicken with neurological ing ammonia toxicity. involvement The toxic nature of BOAA has been demonstrated in ROY: THE NEUROTOXIC DISEASE LATHYRISM 75

/co-eOOH HOOC_co""

NH NH2 NH -. /NH2

eH2-eH '" eH2--eH /

""eOOH ""eOOH

~-(N)-oxalyl-L-a,~-diamino- a-(N)-oxalyl-L-a,~-diamino- propionic acid propionic acid (~-isomer) (a-isomer)

about95% about 5%

/CO-CO""

NH NH

"'C 2- / H CH

""COOH

Possible intermediate

Fig. 5 Isomers of the oxalyl derivative of ~,~-diaminopropionic acid with a possible intermediate

several experimental systems, exhibiting acute activity in nificant degree.68,69 To test this, BOAA was administered micro-organisms such as Neurospora crassa, Staphylococ- to monkeys via the lumbar route, thus bypassing the cus aureus, Escherichia coli and Candida albicans» and in BBB; a generally flaccid hind limb paralysis developed.w chicks, rats, pigeons, young squirrel monkeys (Saimiri sci- Further, adult animals (rats, mice, chicks and monkeys) ureus), and also mice when administered via the intra- pretreated with chemicals, such as calcium chloride, or peritoneal route. The intraperitoneal dose required to drugs known to induce an 'acidotic condition' (such as produce a convulsive neurological disorder in neonatal Diamox, salicylic acid and sulphonamide), responded to chicks is 20-30 mg (500/750 mg/kg body weight), while the intraperitoneal administration of BOAA in a manner oral dose which produces a similar neurological involve- similar to those administered via the lumbar route." ment is approximately three times greater.e« In general, Studies on the function of the BBB in the presence of avian species intoxicated with BOAA exhibit opisthotonos, BOAA revealed that a tracer dose of BOAA could enter wry neck, head retraction and convulsions (Fig. 4). the central nervous system of primates and the radioactiv- BOAA, an excitotoxic glutamate analogue, induces ity was detected which might not be due to residual convulsions in rodents and primates and in rats, vascular blood." Subsequently, administration of radioactive degeneration of dendrites and cell bodies of circumven- BOAA to adult rats, chicks and rhesus monkeys de- tricular organs (areas where the blood-brain barrier monstrated that BOAA entered the central nervous sys- (BBB) is normally absent) including the area postrema, tem irrespective of species susceptibility. 72 subcommissural organs, subfornical organs, organum The route of entry of BOAA, either across the BBB or vasculosum of the lamina forminalis and the arcuate- circumventricularly, is unclear. Undernourished rhesus median eminence region of the hypothalamus.s? monkeys (Macaca mulata) fed L.sativus diet containing In early experiments, adult animals of any species dis- 0.68% BOAA plus a daily oral dose of L. sativus extract played some resistance to the onset of neurological signs, containing 41 mg of BOAA, displayed visible signs of and always required higher doses. Young animals exhi- paraparesis.t' bited a greater vulnerability to lower doses of BOAA, A model of lathyrism in well-nourished primates was perhaps because the BBB is not yet developed to a sig- developed to compare the effects of repeated, subconvul- 76 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. I, NO.2

TABLE II Naturally occurring neurotoxic amino acids and derivatives in Lathyrus and Vida species and related synthetic compounds.s

Toxic Amino Acid Source References and Derivative

~-cyano-L-alanine and Vicia sativa (common vetch); Resslers"; Y-glutamyl-B-cyano-Lalanlne V. angustifolia and other Ressleretal.58; species of Vicia Bell & Tirimanna59

L-a,Y -diaminobutyric acid, L. latifolius (perennial pea); BellBO L-2, 4-diaminobutyric acid and L. sylvestris (flat pea); Y.N-oxalyl derivative V. aurantiea and 10species of Lathyrus

~~N-oxalyl-L-Gt,~-diaminopropionic acid (ODAP) L. sativus (chickling vetch); Murti et al. 51; or ~-N-oxalylamino-L-alanine (BOAA), L. cieera (flat -podded vetch); Rao et al.a: or L-32-oxalylamino-2-aminopropionic acid (OAP) L. latifolius; L. sylvestris and 18other Roy42 species of Lathyrus

Substituted oxamic acid derivatives of a-alanine, Synthetic Rao & Sarmasi ~.alanine, glycine.e- Y-diaminobutyric acid and lysine

a Roy42 sive doses of BOAA. These studies demonstrated that LDBA causes chronic ammonia toxicity in adult rats.83 signs of myoclonus and corticospinal dysfunction ap- Neurotoxic effects develop in animals 12-20 hr after injec- peared in cyanomolgus monkeys fed for 3-10 months with tion, and death occurs after 3-8 days. Also, secondary an analysed diet of L. sativus seed that had been brain damage was observed. LDBA inhibits ornithine supplemented above the minimum nutritional require- trans-carbamylase activity in the liver resulting in a low- ments for the species. Morphological examination of ered urea synthesis in liver slices. motor cortex taken from these primates revealed mild The following is an overview of results obtained from chromatolytic changes of motor neurons. Clinical and studies on BOAA: neurophysiological signs of corticomotoneuronal dys- 1. BOAA in young rats produced a small, but signific- function were also found in animals fed L. sativus plus ant increase of free ammonia concentrations of blood and BOAA, or BOAA alone. Cessation of dosing resulted in brain, thereby resulting in a striking accumulation of the disappearance of characteristic hind limb extensor glutamate in brain. The production of ammonia may be posturing, indicating the successful modelling of the due to the increased catabolism of proteins. 84 early, reversible stage of human neurolathyrism. 74,75 2. BOAA intoxication induced biochemical changes Moreover, elevation of blood calcium-phosphate ratio comparable to those induced by an excitant amino acid. and increased alkaline phosphatase activity in animals fed Convulsive activity was associated with decreased levels fortified L. sativus (but not BOAA) suggested the pre- of glycogen and high energy phosphate, and an increase sence of additional changes in bone function.te in inorganic phosphate and lactic acid levels. The a-isomer of BOAA has low acute toxicity in 3. A growth inhibitory effect of BOAA in Neurospora chicks,"? and is neither acutely nor chronically toxic in crassa can be counteracted by glutamic acid, glutamine, rats. Both IX-and ~-isomers produce membrane depolari- and asparigine.ss BOAA is a potent antagonist of L- zation and an increase in input conductance, the [j.-isomer glutamic acid transport in resting yeast cells, and acts simi- being 10 times more potent than the (%-isomerJ8 The (J,- larly at the synaptic leve1.85 BOAA inhibits glutamate isomer is inactive in tissue culture. 79 transport in isolated mitochondria.86 BOAA in vitro in- In connection with BOAA, r -N-oxalyl-L-IX, r- hibits the high affinity uptake of glutamate by isolated diaminobutyric acid, N-oxalyl-glycine, and N-oxalyl-~- synaptosomes. Raised concentrations of BOAA were alanine are potent acute neurotoxins.n The N-substituted detected in nerve terminals isolated from rat brain and oxamic acids, in which the substituent carries a free car- adult monkey spinal cord from animals exhibiting boxyl group, are generally found to be neurotoxic in neurotoxic effects, and synaptosomes obtained from neonatal chicks (Table II). these animals displayed a significant alteration in gluta- mate transport and a considerable decrease in high affin- Mechanism of Action of Neurolathyrogens ity uptake of glutamate. Neurolathyrogens have various modes of action in diffe- 4. BOAA administered intraperitoneally to young rent biological systems. Pretreatment with pyridoxal hyd- mice induced lesions in the retina, hypothalamus and rochloride can delay the onset and alleviate the symptoms lower medulla, a pattern of damage somewhat compara- of BCLA toxicity, decreasing the mortality of lethally ble to that demonstrated in animals after oral or sub- dosed rats.82 However, pyridoxal does not protect when cutaneous administration of glutamate.s? The conforma- given simultaneously with BCLA. IX, r -diaminobutyric tional constraints of the BOAA molecule may cause it to acid and its oxalyl derivatives may work in a similar fash- interact more selectively than glutamate at central excitat- ion. ory receptor sites. ROY: THE NEUROTOXIC DISEASE LATHYRISM 77

5. The pharmacological action of BOAA, examined in TABLE III Analytical values of components in Lathyrussativu,s93,94 organotypic cord-ganglia muscle tissue cultures and iso- lated synaptosomes,79,88revealed focal vacuolation in the g ormg/l00 gseeds cord component within minutes of application. Vacuoles Protein 28.2 were initially restricted to post-synaptic regions of Fat 0.6-1.0% dendrites. Kainic acid and aspartic acid, established Carbohydrate 58.2-61% neuron excitants, induced comparable changes. Moisture 10% 6. Intrathecal administration of BOAA into the lum- Fibre 15% bar region of rats induced changes in the spinal cordsimi- Ash 3% lar to those seen in vitro after repeated exposure. Calcium 1l0mg Biochemical analysis of tissues and of synaptosomes from Iron 5.6mg Vitamin A 70IU brain and spinal cord, demonstrated that BOAA inter- Thiamine O.lmg fered with high-affinity transport of aspartic acid and Riboflavin O.4mg glutamic acid. These data suggest that BOAA is a potent glutamate analogue capable of inducing changes in selec- Amino Acids g/16gN tive pathways of the spinal cord.e' Arginine 7.85 Histidine 2.51 6.57 ANALYSIS OF BOAA Leucine Isoleucine 6.59 Quantitative estimation of BOAA in L. sativus is deter- Lysine 6.94 mined by two methods: Methionine 0.38 Phenylalanine 4.41 Electrophoretic method» Threonine 2.34 Tryptophan 0.40 L sativus seed extract of known concentration is applied Valine 4.68 on Whatman No.1 filter paper strips. Separation is per- formed in an electric field using a Beckman paper elec- tissues. The results obtained by the two methods are com- trophoretic cell (Durrum type), Model R with a buffer parable. system, acetic acid, pyridine, water (5.0: 0.5 : 95.5 by vol- ume) at pH 3.6, voltage, 250 V for 4-5hr. The strips are FUNDAMENTAL ISSUES AND then dried in an air-oven at 80°C, sprayed with 0.1 % RECOMMENDATIONS ninhydrin solution in acetone, air dried and heated at 80 °C, sprayed and dried again, and heated at the same temp- In 1956-7, about 40 lakh (4 million) acres of land in India erature for another 10min. Standard BOAA solutions are were under cultivation with over 50 varieties of Lathyrus. spotted concurrently and run along with the sample. The Madhya Pradesh is the major L. sativus (Khesari) produc- BOAA spots are eluted individually with 9 ml ofa copper ing state in India. L. sativus occupies 15% of the total land under pulse cultivation and also constitutes 15% of the solution in ethanol (300 mg of CuS04.5 H20 and 50 ml distilled water, mixed with distilled ethanol). The eluted total pulse production in the states. Lathyrus was culti- solutions are read in a Klett-Summerson colorimeter vated on 15.8 lakh acres in 1963-4 and 7.7 lakh acres in using a green filter (No. 54), and calculated using standard 1977-8. The produce during these two periods was 1.9 BOAA curves. lakh tonnes and 3 lakh tonnes, respectively. Of all the Lathyrus species, L. sativus is the most widely used. L. sativus grows in different colours and sizes, and the level O-phthaldehyde (OPT) methodn of BOAA content varies considerably with no correlation BOAA and a, ~-diaminopropionic acid (Dapro) stan- between the physical properties of the seeds and BOAA dards (1 ,umole/ml) are prepared and stored at 4 "C. The content." The whole seed has been analysed=-" (Table III) following reagents are made fresh before use: potassium and has a high percentage of protein with satisfactory borate buffer: 0.5 M in glass-distilled water. OPT reagent: levels of essential amino acids. A potent antinutritional 100 mg of OPT in 1ml of 95% ethanol and 0.2 ml mercap- factor, trypsin inhibitor, comprised of five isoproteins toethanol, added to 99 ml of potassium borate buffer. with a molecular weight of22 000 daltons, was found to be L. sativus extract (50-100 ,ul) is added to two sets of 13 unrelated to BOAA toxicity; this group of proteins is X100 mm test tubes. To one set of tubes, 0.2 ml of 3N thermolabile.95,96BOAA content can be as high as 2.5% KOH is added and the tubes are kept in a boiling water particularly in Lathyrus plants from drought affected bath for 30 min. Tubes containing 10-100 nmol of BOAA areas. are similarly treated for standard. After cooling to room The major findings from epidemiological and laborat- temperature, samples are made up to 1ml with water, and ory studies on Lathyrus and lathyrism conducted over sev- 2 ml of OPT reagent is added. The absorbance of the yel- eral years are as follows: low solution thus formed is measured after 30 min at 400 1. The disease occurs in populations where L. sativus nm against a blank made with or without an equivalent is consumed regularly in large amounts and is rarely seen amount of alkali. The absorbance readings with and with- in individuals of populations that consume -Lathyrus in out hydrolysis give an estimate of BOAA. The OPT small quantities. It is roughly estimated that the risk arises method can also be used for the estimation of BOAA in when two-thirds of the daily diet consists of Lathyrus. 78 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 1, NO.2

2. BOAA, the in Lathyrus, can be leached out by practical methods have yet to be documented. Ideally, de- the simple process of soaking the pulse in hot water. velopment of a toxin-free strain of Lathyrus spp. would Methods recommended for domestic purposes are: (a) eliminate the need for additional precautionary methods cooking the pulse in excess amount of water followed by which are now critical for human use. draining, (b) soaking the pulse in cold water overnight, ACKNOWLEDGEMENTS and (c) steeping the dehusked seeds in hot water. The re- moval of BOAA is greatest from dehusked seeds than The author is greatly indebted to Ms Monica Bischoff- whole seeds, although complete removal is not possible in Fenton for her valuable suggestions and editorial and any known process. It is claimed that steeping and boiling technical assistance in the preparation of this manuscript. methods do not result in a marked change in the protein The author thanks Ms Christine Esposito-Torres for typ- profile of the dehusked seeds, but there is a significant re- ing the manuscript. duction of water-soluble vitamins and minerals after the REFERENCES treatment. The steeped dehusked seeds can be easily sun- 1 Barrow MY, Simpson CF, Miller EJ. Lathyrism: A review. Q Rev dried, and the grain thus obtained can be pulverized to a Bioi 1974;49:101-28. flour.s? A maximum of 80 per cent of BOAA can be re- 2 Gebre-ab T, Gabriel ZO, Maffi M, Ahmed Z, Ayele T, Fanta H. 'moved from the pulse by any of the above methods. Neurolathyrism-a review and a report of an epidemic. Ethiop Med Roasting the whole seed at 150°C for 20 min destroys 75% ] 1978;16:1-11. of BOAA.41 3 Roy DN, Spencer PS, Nunn PB. Toxic components of Lathyrus, Colloquium on Lathyrus, Pau, France, 1985, Lathyrus and 3. Low-toxin: or no-toxin strain of L. sativus: Agricul- lathyrism. Proc Colloque Lathyrus 1985; 287-96. tural scientists have not yet evolved strains of L. sativus 4 Stockman R. Lathyrism. I Pharmacol Exp Ther 1929;37:43-53. completely free of the toxin, BOAA, although it has been 5 Selye H. Lathyrism. Rev Cand Bioi 1957;16:1-82 reduced to a minimum level.98 Since the arbitrary 'one- 6 Sleeman WH. Rambles and Recollections of an Indian Official, Vol 1. Hatchard, London, 1844. third' limit on consumption had been suggested as permis- 7 Rao SLN, Malathi K, Sarma PS. Lathyrism. World Rev Nutr Diet sible on the basis of epidemiological evidence, the reduc- 1969;10:214-38. tion of over 65% of BOAA content could be considered 8 Chopra RN. British Encyclopaedia of Medical Practice, VII. Butter- safe. It must be emphasized, however, that the 'safe' limit worths, London, 1938; 615-57. is not based on indisputable evidence. Unanswered ques- 9 Contani A. Latirismo (Lathyrismus) illustrata de tre casi clinici. II Morgni 1873;15:747. tions remain with regard to different locations, rainfall 10 Griffin JW, Price DL. Proximal axonopathies induced by toxic and seasons, and whether the low-toxin trait is maintained chemicals. In: Spencer PS, Schaumburg HH (eds), Experimental over successive generations. Considering the story of gos- and Clinical Neurotoxicology, Williams and Wilkins, Baltimore, sypol in cotton seeds, which is retained over successive 1980; 161. generations and different locations, a 'low-toxin strain' of 11 Spencer PS, Schaumburg HH. Lathyrism: A neurotoxic disease. Neurobehav Toxicol TeratoI1983;5:625-9. Lathyrus may not be the solution. I? 12 Martens HG, Zur Klinik des Lathyrismus. Nervenarzt 1947;18:493-9. 13 Gardner AF, Sakiewiez N. A review of neurolathyrism including SUMMARY the Russian and Polish literature. Exp Med Surg 1963;21:164-91. 14 Kessler A. Lathyrismus. Monatsschriff fUr Psychiatrie und The neurotoxic disease lathyrism is a disorder dominated Neurologie 1947;113:345-75. by signs of irreversible pyramidal dysfunction in humans 15 Ludolph AC, Hugon J, Dwivedi MP, Schaumburg HH, Spencer PS. and domestic animals that subsist on a Lathyrus diet, par- Studies on the aetiology and pathogenesis of motor neuron disease. ticularly when L. sativus or its plant products represent a Brain 1987;110:149-

26 Wawzonek S, Ponseti IV, Shepard RS, Wiedermann LG. Epiph- 53 Bell EA, O'Donovan JP. Isolation of r-and a.-oxalyl derivatives of yseal plate lesions, degenerative arthritis and dissecting aneurysm a.,r -diaminobutyric acid from seeds of L. latifolius and the detec- of aorta produced by amino nitriles. Science 1955;121:63-5. tion of the a.-oxalyl-isomer of the neurotoxin a.- and ~-oxalylamino- 27 Schilling ED, Strong FM. Isolation structure and synthesis. of propionic acid which occurs together with the neurotoxin in this and Lathyrus factor from L. odoratus. JAm Chem Soc 1955;77:2843-5. other species. Phytochemistry 1966;5:1211-19. 28 Stamler FW. Reproduction in rats fed Lathyrus peas or amino- 54 Roy ON, Narasinga Rao BS. Distribution of a.- and ~-isomers of N- propionitriles. Proc Soc Exp Bioi Med 1955;90:294-8. oxalyl-e.B-diaminopropionic acid in some varieties of Lathyrus 29 Ponseti IV, Wawzonek S, Shepard RS, Evans TC, Steams G. sativus. Current Sci (India) 1968;37:395-6. 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76 Spencer PS. Guam ALSIParkinsonism-Dementia: a long-latency Lathyrus sativus excitotoxin, ~-N-oxaly-L-a,~-diaminopropionic neurotoxic disorder caused by 'slow toxin(s), in food? Canad J Sci acid. Nature (London) 1976;264:659-61. NeuroI1987;347-57. 88 Zagoren JC, Seelig M, Roy DN, Bornstein MB, Spencer PS. 77 Harrison FL, Nunn PB, Hill RR. Synthesis of a- and ~.N-oxalyl-L- Pathological changes in spinal cord induced by Lathyrus neurotoxin a\~rdiaminopropionic acid and their isolation from seeds of (~-N-oxalylamino-L-alanine) in vitro. J Neuropathol Exp Neurol Lathyrus sativus. Phytochemistry 1977;16:1211-15. 1985;44:348. 78 Chase RA, Pearson S, Nunn PB, Lantos PL. Comparative 89 Ross SM, Roy DN, Spencer PS. Beta-N-oxalylamino-L-alanine ac- of a. and ~.N-oxalyl-L-aj~-diaminopropionic acids to rat spinal tion on high affinity transport of neurotransmitters in rat brain and cord. Neurosci Lett 1985;55:89-94. spinal cord synaptosomes. J Neurochem 1985;44:886-92. 79 Nunn PB, Seelig M, Zagoren JC, Spencer PS. Stereospecific acute 90 Roy DN, Bhat RV. Variation in neurotoxin, trypsin inhibitors, and neurotoxicity of 'uncommon' plant amino acids linked to human susceptibility to insect attack in varieties of Lathyrus sativus seeds. motor-system disease. Brain Res 1987;410:375-9. Environ Physiol Biochem 1975;5:172-7. 80 Bell EA. Comparative biochemistry of non-protein amino acids. In: 91 Rao SLN. A sensitive and specific colorimetric method for the de- Harborne JB, Boulter D, Turner BL (eds), Chemotaxonomy of termination of a,~-diaminopropionic acid and the Lathyrus sativus Leguminosae. Academic Press, London and New York, 1971;179. neurotoxin. Anal Biochem 1978;86:386-95. . . 81 Rao SLN, Sarma PS. Neurotoxic properties of N-substituted 92 Roy DN, Visweswara Rao K. Physico-chemical values in different oxamic acids. Indian J Biochem 1966;3:57-8. varieties of Lathyrus sativus and their interrelationships. J Agri 82 Ressler C, Nelson J, Pfeffer M. A pyridoxal-B-cyano-alanine rela- Food Chem 1978;26:687-9. tion in the rat. Nature (London) 1964;203:1286-7. 93 Duke JA. Handbook of Legumes of World Economic Importance. 83 Johnston GAR. Neurotoxic amino acids. In: Simpson LL, Curtis Plenum Press, New York and London, 1981. DR (eds). Neuropoisons, Their Pathophysiological Actions, Vol 2, 94 Sastri BN. Wealth of India, Vol VI, Council for Scientific and In- Plenum Press, New York and London, 1974;179. dustrial Research, New Delhi, 1962;37. 84 Cheema PS, Padmanaban G, Sarma PS.Transamination of ~-N- 95 Roy DN. Biological effects of trypsin inhibitor isolated from oxalylamino-L-a,~diaminopropionic acid, the Lathyrus sativus Lathyrus sativus seeds. Current Sci (India) 1972;41:180-1. neurotoxin, in tissues of rat. Indian J Biochem Biophys 1971;8:16- 96 Roy DN. Trypsin inhibitor from Lathyrus sativus seeds-final 19. purification, separation of protein components, properties and 85 Mehta T, Hsu A-F, Haskell BE. Specificity of the neurotoxin from characterization. J Agri Food Chem 1980;28:48-54. Lathyrus sativus as an amino acid antagonist. Biochemistry 97 Mohan VS, Nagarajan V, Gopalan C. Simple practical procedures 1972;11:4053-63. for the 'removal of toxic factors in L. sativus seeds (Khesari dhal). 86 Doque-Magalhaes MC, Parker L. Action of the neurotoxin ~~N- Indian J Med Res 1966;54:410-14. oxalyl-L-a.~diaminopropionic acid on glutamate metabolism of 98 Swaminathan MS, Austin A, Kaul AK, Naik MS. In: New approach brain mitochondria. FEBS Lett 1972;23:188-90. to breeding for improved plant protein. International Atomic 87 Olney JW, Misra CH, Rhee V. Brain and retinal damage from Energy Agency, Vienna, 1969;71.

AIDS and India

A. N. MALAVIYA, K. K. DATIA

INTRODUCTION developed pneumonia caused by an 'opportunistic' proto- In the summer of 1981 Gottlieb and colleagues, and zoon Pneumocystis carinii along with an 'opportunistic' Friedman-Kien and coworkers, reported independently cancer Kaposi's sarcoma (KS), and mucosal candidiasis.l-' for the first time to the Centers for Disease Control To these workers this new disease appeared to be an (CDC), USA, a unique illness in which previously healthy undescribed acquired form of immunodeficiency. By homosexual young men from New York and California December 1981 well documented reports from New York and Los Angeles were published indicating the nature of this illness, its major clinical features, immunological All India Institute of Medical Sciences, New Delhi 110029, India abnormalities and lethal course.r" Since then this disease A. N. MALAVIYA Clinical Immunology Section, has come to be identified as the acquired immunodefi- Department of Medicine ciency syndrome (AIDS).? In a short span of six years Ministry of Health and Family Welfare, Government of India since its first report, the disease has acquired near pan- K. K. DATTA Directorate General Health Service Office (AIDS Cell) demic proportions." This, along with its high mortality Correspondence to A. N. MALA VIY A and no known cure, has lead to unprecedented concern. © The National Medical Journal of India, 1988