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u T, Zein AZ, Spencer PS. leurotoxic disorder. Int J Epid . Addis Ababa: Commercial )r the determination of a leurotoxin.

. Anal Biochem ^Oxalvlamino-L-alanine, the i e d )-rass-pea: Threat and tion, 1989:128-132. ^-aminopropionic acid, and >;15:1257-1259. alyl-L-a, -diaminopropionic lytochemistry 1977,16:1211xicities of a- and -N-oxalylett 1985;55:89-94. - oxalyl derivatives of neurotoxin in this and other H, Yeshanew A. Grass Pea :thiopia. (A paper presented lgladesh, 30 November- 3 es. Central Statistic Office, new dimensions. Nutrition oundation of India. 1984:19Khesari {Lathyrus sativus) ia Nutr; 1987 Mar;37(1 ):101 ures for the removal of toxic 6;54:410-9. the Toxicity of processed al 1987:36:851-855. )AA and tannins in legumes 989;34:229-238.

ITRITION RESEARCH, Vol. 13, pp. 1113-1126,1993 71-5317/93 $6.00 + .00 Printed in the USA. pyright (c) 1993 Pergamon Press Ltd. All rights reserved.

PATTERN OF LATHYRUS SATIVUS (GRASS PEA) CONSUMPTION AND BETA-Af-OXALYL-a--DIAMINOPROPRIONIC ACID (-ODAP) CONTENT OF FOOD SAMPLES IN THE LATHYRISM ENDEMIC REGION OF NORTHWEST ETHIOPIA Redda Tekle-Haimanot*, Berhanu M. Abegaz", Elizabeth Wuhib*", Angelina Kassina*", Yemane Kidane*", Naod Kebede", Tadesse Alemu* and Peter S. Spencer*"* Faculty of Medicine, Addis Ababa University, P.O.Box, 4147, Addis Ababa, Ethiopia*: Faculty of Science, Addis Ababa University, P.O.Box, 1176, Addis Ababa"; Ethiopian Nutrition Institute, Addis Ababa"; and Oregon Health Sciences University, Portland, Oregon 97201, USA"**.

ABSTRACT Grass pea, a legume whose consumption is associated with human lathyrism, is an important food crop among the poorer sectors of society in many parts of Ethiopia. A nutritional survey, focussing on the preparation and consumption of grass pea, in 224 families of two villages of a lathyrism endemic area in northwestern Ethiopia has been undertaken. Grass pea is grown in only one of the two villages. Raw as well as cooked food samples were collected and the levels of -ODAP determined by the OPT method. Grass pea foods were also prepared in the laboratory following methods used in the villages and analyzed for their toxin level to derive information if certain procedures were capable of reducing the level of toxin. Steeping grass pea in excess water leaches out ca 30% of the -ODAP. Grass pea bread (kitta) and roasted seeds (kollo) showed elevated levels of ODAP compared to the seeds used in the preparation of these foods. Lower levels of -ODAP were found in boiled snacks {nifro) and the flour form {shird).

KEY WORDS: Grass pea, Lathyrus sativus, Lathyrism, Paraparesis, -N-oxalyl-La,-diaminopropionic acid (-ODAP) INTRODUCTION iLathyrism, which is characterized by spastic paraparesis, is a neurotoxic disorder caused jby excessive consumption of chickling or grass pea {Lathyrus sativus) (1,2). Corresponding author: Redda Tekle-Haimanot, P.O. Box 4147, Addis Ababa, Ethiopia. 1113

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Epidemics of the disease have been reported following acute food shortages during drought or after floods (3-5). Recent studies have confirmed that -N-oxalyl-L-a, diaminopropionic acid (-ODAP), [synonym: -N-oxalylamino-L-alanine (BOAA)] is the neurotoxic principle in L sativus responsible for the neurodegenerativo condition (6). Although lathyrism has in the past been reported from other parts of the world, including Europe, it is at present known to be endemic in India (7), Bangladesh (8) and Ethiopia (9). In Ethiopia, the disease occurs in the northern and central parts of the country with recurrent outbreaks reported from the Lake Tana basin of the north-west Administrative Region of Gondar. As part of a multidiscipilinary project for the improvement of Lathyrus sativus and the control of lathyrism, we undertook an epidemiolqgical survey of the Dembia and Fogera subdistricts of the Lake Tana basin (10). A nutritional survey was also carried out in two peasant associations within the project area to find out the feeding habits of the inhabitants particularly in relation to the preparation and consumption of grass pea. Food samples were collected and assayed for (-ODAP) levels. This paper reports on our findings.

were readily accepted by housewives into the kitchens. The activities of the enumei were supervised closely by the three experienced nutritionists of the research ( (March-April 1989). The purpose of the nutritional study was explained and discussed with the elder; local leaders of the peasant associations. After obtaining the consent and support < community, a total census of the two villages was performed initially. Random samples of 110 and 114 families from Shina and Montura peasant assocU respectively were taken into the study. The survey was undertaken during harv( season of grass pea (March-April). The dietary history was obtained by determining the average food intake of an indr during the 24 hours preceding the interview. The survey was conducted for ; consecutive days by daily visits to the families. During the survey the enumerators and nutritionists made close observations recorded the methods used in the preparation and consumption of cereals, legume drinks. The consumption of guests and absence of the family members were take account. In most parts of Ethiopia, particularly northern regions, teff (Eragrotis teff), wheat, b maize and sorghum, either singly or in combination, are used to produce a ferrm sour pancake-like leavened bread called enjera. The dough used for enjera is u fermented for less than three days. The enjera is eaten together with wott, which Ethiopian sauce traditionally prepared by cooking, in a clay pot, a mixture of onion pepper blended with several spices and butter or vegetable oil, meat, vegetable, or legumes. When the hot pepper is omitted from the preparation of the wott, the : so prepared is called alitcha. To the alitcha, green pepper is added for flavou turmeric {Curcuma longa) for colour (11). Shiro is the flour prepared from legume used for the, preparation of wott or alitcha, generally in poor families. Kitta i unleavened bread prepared from the flour of cereals or legumes or their mixture snacks, cereals, legumes or their mixture are served either roasted {kollo), or I {nifro). The raw and cooked food items were weighed or measured. Samples of food cont; grass pea were collected from some families. These samples were refrigerate transported to the Chemistry Department of the Addis Ababa University fc determination of -ODAP. Determination of -ODAP The method employed for the analysis of -ODAP is that of Rao [12], with modification, which involves a neutralization step prior to addition of the chrome reagent (13). Extraction of -ODAP. Samples of bread, nifro, kitta and wott samples were dehy< to constant weight, in an oven at 40C and 120C. Whole and decorticated seeds finely powdered prior to extraction. Solutions obtained by boiling grass pea seeds

MATERIALS AND METHODS In the subdistrict of Fogera of the Gondar Administrative Region, two peasant associations were selected for the nutritional survey. Shina (pop. 6,142) is a village in a grass pea cultivating area, while Montura (pop. 2,560) situated 50 km north of Shina is a non-grass pea growing village. Both villages are about 600 km north of Addis Ababa. In Shina, most of the farmland invariably gets flooded during and after the heavy rainy months of July to September. Farmers therefore resort to the cultivation of the hardy crop, grass pea. On the other hand, Montura which is situated on the highlands, has a more fertile and productive land where cereals like teff (Eragrotis teff), wheat, barley and millet are cultivated. For the nutritional survey of the two villages, a questionnaire was developed to get the following essential information: supply and preparation of food, distribution and consumption of food and infant feeding, with particular emphasis on grass pea. The questionnaire was carefully designed to obtain the comparative dietary pattern of the target populations of the two villages. The questionnaire, initially prepared in English, was translated into Amharic, the local language spoken in the region. Based on pilot studies, the questionnaire was further modified to formulate the questions in accordance with the cultural background and educational levels of the population. Ten female research assistants were recruited from among the secondary school graduates of Woreta, a town situated between the two villages. These enumerators were trained for two weeks by nutritionists of the research team on the concept and techniques of dietary field survey. As the enumerators were recruited locally, a fairly reliable rapport and confidence was developed between them and the respondents. As females, they

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diluted one hundred fold prior to analysis. TABLE 1 Absorbances were measured on a Beckman model 24 spectrophotometer. Diaminopropionic acid hydrochloride (DAP.HCI) and o/tftophthalaldehyde (99% and 97% respectively) were purchased from Aldrich; mercaptoethane was acquired from Hopkin and Williams. Water was redistilled from an all-glass set-up. Ethanol (60% aq.) was used for extraction. Preparation of reagent. Ort/70-phthalaldhyde (OPT) reagent was prepared by dissolving 100 mg of the aldehyde in 1 ml ethanol and 0.1 ml mercaptoethane. This was added to 99 ml of 0.1 M sodium tetraborate buffer. In all cases a freshly prepared reagent was used. -ODAP analysis. The extracts were filtered and taken in duplicate (0.1 ml each), treated with 3N KOH (0.2 ml) and kept in boiling water bath for 30 min. After cooling to room temperature, 3N HCI (0.2 ml) was added and the final volume made to 1 ml with water. A sample blank was prepared with the extract (0.1 ml) and water (0.9 ml) to account for any interference from the reaction between the OPT reagent and other constituents of the extract. The OPT reagent (2 ml for each sample) was added to the hydrolysed and control samples and absorbance measured at i^^ - 476 nm after 30 min. Reagent blank was prepared by diluting the OPT reagent (2 ml) with water (1 ml). -ODAP concentration was calculated from a calibration curve prepared by using commercial DAP.HCI and assuming 100% conversion of -ODAP to DAP at the given conditions of hydrolysis [12]. Statistical method The t-test was employed for comparing two means. RESULTS Numbers and types of meals (Table 1 ) Most families (95%) in both peasant associations had two main meals during the day. The meals were served in the middle of the day and in the evening. The main dishes in both communities were enjera and wott. The enjera was most often prepared from finger millet alone and sometimes millet was mixed with teff, corn, and sorghum. The wott made of shiro was mainly prepared from grass pea alone or mixed with chickpea (Cicer anietinum) in both villages. In some families, lentil was also used for wott. In Montura, potato was added to wott (in addition to legumes). Other foods like nifro and kollo were prepared from grass pea and consumed as snacks. Kitta was also consumed by a few of the families studied. Dietary Habits of the Inhabitants of the L Sativus Growing Village of Shina and Montura Type of Food Enjera Millet Teff Sorgum Corn Barley L sativus Nifro Kollo Kitta Shiro Vegetables ' Daily consumption gm/person/day (% of total) SHINA 578.3(50.8) 283.0 (24.8) 160.5(14.1) 117.5(10.3) MONTURA 375(23.1) 320 (19.6) 231 (14.2) 372(22.8) 332 (20.4) 38(100) same

91.3(49.5) 72.1 (39.1) 21.0(11.4) consumed with every meal

Potato Kale

139(51.1) 133(48.9)

Different methods are used in the preparation of grass pea for shiro. The most comme methods used are as follows: 1. Aftr removal of foreign particles, grass pea is lightly toasted on a clay or mei pan, transferred into cold water in a wooden trough or clay pot and thorougr washed. The wet grass pea is toasted until browned and set aside for cooling. The husk is then removed by the use of a stone handmill {woftcho). The split gra pea is again washed, set in the sun for a while, and then mixed with peeled onic garlic, ginger and basil. The mixture is pounded in a wooden mortar and set in tl sun for final drying. It is then ground into flour. This method is mostly used Montura. Grass pea, free of all foreign particles, is added to boiling water on the sto\ removed from heat after a few minutes and kept aside overnight. The followi day, the liquid is discarded and the grass pea toasted until browned. After cooli for a while, the toasted grass pea is dehusked and thoroughly washed. T washed split grass pea is then set in the sun for a while. The rest of t preparation until the final flour product is as described above (under 1).

2.

1118 3.

R. TEKLE-HAIMANOT et al. Cleaned grass pea is toasted and dehusked. The split grass pea is washed and set in the sun to dry. It is then ground into shiro.

LATHYRISM

Grass pea, shiro wott preparation differed from house to house in the two peasant associations. The most common methods are as follows: 1. Water is poured into a washed clay pot and placed on a stove. When the water starts to boil, grass pea shiro is added a little at a time while stirring the mixture continuously. Salt is added and the sauce cooked until done. When ready the wott is eaten warm or cold with enjera. Peeled and chopped onion is fried in a clay pot by adding a little water at a time until browned. If oil or water from crushed oil seeds is available, it is added while frying. Enough water is then added into the frying onion to boil. When the water starts to boil, shiro is added while stirring continuously. It is then cooked until done. This may take 20 to 30 minutes.

manner: Grass pea is cleaned and set in the sun for sometime. The husk is removed in i stone mill {woftcho). The split grass pea is ground into fine flour. Water is addec a little at a time and the moistened flour kneaded to make a dough. Salt is addec to taste. The thick dough is spread over hot clay or metal griddle by hand am baked on both sides for about 10 minutes. Reliability check on chemical analysis The results of assays obtained at the Addis Ababa University Department of Chemistr on identical samples were in excellent agreement with those of a reference laboratory the Albert Einstein College of Medicine, New York. The Rao method involves an initial hydrolysis of -ODAP to 2,3- diaminopropionic aci< (DAP) which is then reacted with ortAio-phthalaldehyde to generate the derivative whos concentration is determined by measuring its UV absorbance. The limitation of thi method lies in its lack of selectivity to -ODAP in the presence of other species like o ODAP which would give rise to DAP during hydrolysis. This is particularly important sine a-ODAP is claimed to be non-toxic (14-16). However, the natural presence of the o ODAP in the seeds of grass pea is believed to be very small (a 5%). A further concer in dealing with the analysis of processed grass pea foods is the possibility that some c the -ODAP may transform into the a-form (17). However, such transformations are nc expected to occur in solid food samples where the -ODAP exists in solid matrices. A a result it is felt that the analytical data generated for the food items considered in thi study (with the possible exception of the wott samples, and these are only few case; may approximate the actual levels of -ODAP present in them. Levels of -ODAP The -ODAP levels of whole seed samples collected randomly from the study ares ranged from 0.2 - 0.8%. The values of dehusked samples were 0.2-0.7%. The floi contained -ODAP in the range of 0.15 - 0.4%. Although the sample sizes were small.the levels in the shiro sauce made of the flour for and the boiled grass pea were 0.1 - 0.3% and 0.1 - 0.7% respectively. As the preparatic of shiro involved some degree of steeping and later dehusking, the -ODAP level of shi, wott was on the average lower by 40% than the whole seed.. In the preparation of wott, volume dilution by the "addition of various ingredients mi account for the observed lowering of the -ODAP level by 30% compared to the shi used for the sauce preparation (Table 2).

2.

Boiled grass pea (nifro) is most often consumed in Shina. Different methods are used for nifro preparation. The most common methods are as follows: 1. Grass pea is cleaned and washed with cold water. Water is added and the grass pea cooked for a short time. The water is then drained off. Fresh water is added and the legume cooked until tender. Sometimes, chickpea or whole wheat is added to the grass pea before adding water the second time. Grass pea is washed 2-3 times with hot water. Cold water is added and the legume cooked until it is soft enough to eat. Grass pea is soaked in cold water, left overnight, and the liquid discarded the following day. It is then thoroughly washed, water added, and cooked. Cleaned grass pea is thoroughly washed with cold water. Enough water is added and then the legume is cooked until done.

2.

3.

4.

The roasted form of grass pea snack (kollo) was prepared in two alternative ways: 1. Grass pea is added into boiling water and boiled for sometime. Excess water is decanted and the grass pea is roasted over a hot clay or iron griddle. 2. Grass pea is soaked in cold water for some time and excess water is decanted, roasting is carried out as in 1. Very few people consumed grass pea unleavened bread (kitta) during the survey period. Kitta is eaten mainly at times of acute food shortages. It is prepared in the following

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LATHYRISM (Table 5). Although the absolute differences were not statistically significant, neverthless the trend in the increase of ODAP level could not be ignored.

11

TABLE 2 -N-Oxalyl, -diaminopropionic acid (-ODAP) content in different grass pea food samples from Shina and Montura peasant associations -ODAP content (mg/100g) Higest 810 690 429 338 704 Lowest 282 239 155 113 70 Average 455 444 270 190 471

Sample type Whole seed Decorlicataed seed Shiro-flour Shiro-sauce Nifro-boiled

Size of samples 63 10 50 13 13

Higher levels of -ODAP were observed in the grass pea bread produced by the kitchen experiment compared with the original grass pea samples. The increase, which was found in 14 out of 20 identical experiments, ranged from 4.1-48.9 % (Table 6)< ' However, due to the high variability in ODAP levels of the bread samples, the differences were not statistically significant. TABLE 4 -ODAP content of boiled grass pea {nifro) samples prepared in the kitchen laboratory by two different methods: boiling directly and after soaking -ODAP content (mg/100 g) Soaked and boiled seed Samples 1 2 3 4 5 6 7 8: 9 10 Mean ; S.d. Dry seeds 901 831 1,042 824 739 796 591 367 475 598 716.4 206.0 Wet seed 739 732 936 697 789 634 380 282 252 343 Discarded water 3.5 8.2 10.9 9.7 11.5 8.7 4.4 1.3 1.7 8.1 Boiled seed 683 676 429 634 704 514 338 334 343 264 491.9 107.9 Boiled seed Discarded water 300 420 598 655 662 535 275 264 387 370 Boiled seed 479 324 697 408 387 331 253 246 317 229 : 367.1 139.8

TABLE 3 -ODAP content of boiled grass pea prepared in the laboratory using different methods of boiling. -ODAP content (mg/100g) Code 36 37 38 39 40 41 42 43 44 45 46 Mean S.d. Dry seed 488 549 387 514 394 514 528 429 359 253 514 488.1 91.8 1 282 211 338 465 380 402 394 275 246 211 387 326.5 86.0 2 239 211 378 444 373 373 429 330 389 218 380 342.2 82.5 3 261 232 465 401 359 373 420 380 450 268 373 362.0 77.3 4 331 553 408 521 331 324 345 401 317 183 408 374.7 101.7

1. Grass pea boiled for a short time, excess water drained. Then boiled with fresh water. 2. Grass pea washed with boiling hot water 3 times and then boiled with fresh water. 3. Grass pea soaked overnight, excess water discarded. Then boiled with fresh water. 4. Grass pea washed with cold water and then boiled with fresh water. Simulated kitchen experiments confirmed that steeping grass pea in a large volume of water for 3 minutes and decanting the excess water leeched out approximately 30% of -ODAP. Improved detoxification was achieved when the boiled snack, nifro, was prepared from grass pea boiled directly with hot water as compared to that prepared from grass pea soaked overnight in cold water. This was statistically verified, (Tables 3 and 4). Roasting grass pea preceded by steeping of the seeds achieved some degree of ODAP reduction. On the other hand, when grass pea roasting was done after only soaking the seeds, there was an increase in the -ODAP level in six of 11 samples

DISCUSSION
i

Food lgumes, including grass pea, form a major part of the protein source and nutrients of the Ethiopian diet. Grass pea, though considered a poor person's food, is cultivated quite extensively in areas of the country subject to adverse agricultural conditions, particularly flooding and drought. Grass pea production is concentrated in the northwest region of Ethiopia, and is also cultivated in northeast, central and southeast regions.

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R. TEKLE-HAIMANOT et al. TABLE 5 -ODAP content of grass pea roasted (kollo) by two different methods

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Raw grass pea -ODAP mg/100gm 704 500 943 789 725 608 436 606 829 681 676 681.6 144.4 Time (min) 1.15 3.15 4.15 5.00 4.05

Roasted Sample 1 -ODAP (mg/100g) Time (min) 4.38 3.33 5.50 5.25 3.40

Roasted sample 2 -ODAP (mg/100g) Discarded water 8.6 6.3 14.6 22.8 8.7 seed 739 584 1133 894 588 770 405 732 613 433 603 681.3 207.4

Code 50 51 52 53 54 1/1 2/1 3/1 4/1 5/1 6/1 Mean S.d.

Discarded water 9.9 7.0 21.8 16.9 12.7

seed 651 591 831 866 667 476 488 867 656 503 596 653.8 145.3

different food preparations of grass pea with reference to -ODAP content. Dwivedi and Singh have suggested that the Indian staple, ghotu, prepared by cooking a mixture of Lathyrus sativus and rice in water to form a stiff porridge (paste balls) precipitated lathyrism more rapidly than chapati, the unleavened bread form. This was based on an epidemiological observation. The researchers further suggested that the presence of water in the ghotu permitted the toxic component to be released in solution providing a more rapid absorption (21). To our knowledge -ODAP levels have not been systematically assayed in the different grass pea food preparations, thus this hypothesis has not been tested. Our kitchen experiments have demonstrated that the Ethiopian unleavened bread, kitta, which in its form and preparation is equivalent to the Indian chapati, has higher detectable levels of -ODAP than the dry grass pea seeds from which it is prepared. We have also epidemiological evidence that in the lathyrism outbreaks and epidemics of'northwest Ethiopia, the bread {kitta) form was consumed in large quantities in addition to other types of grass pea-derived foodstuffs. Gebre-ab et al. writing about the 1976/77 lathyrism epidemic in northwest Ethiopia, when over 2500 persons were affected, observed that the commonest form of grass pea consumed by the affected population included kitta, nifro and kollo. They also commented that the kitta form was then a new introduction into the area of the epidemic (5). In normal times, as was the case during our survey, villagers in the lathyrism-endemic area used grass pea commonly as snacks in the boiled and roasted forms and very little as bread. Similarly, we have shown that the roasted form {kollo), if roasted without prior aqueou steeping, may contain higher levels of -ODAP than the native seeds. The conclusion drawn from these experiments, which were designed to simulate the condition in the villages, is that the level of -ODAP is maximal in the bread and roasted forms of grass pea. The level is lower in the boiled snacks, particularly when the process of steeping is employed. The flour form, shiro and the sauce prepared there from, contained relatively lower levels of -ODAP and thus may not be so hazardous. Detoxification of the grass pea through aqueous leeching of the toxic component (i.e. steeping) has been described previously (22). Mohan er al. have suggested the removal of the toxin from L. sativus seeds through two alternative methods: steeping the dehusked seeds in hot water for several hours and boiling'the seeds in water and draining out the supernatant. This removed 70-80% of the neurotoxin (23). Moslehuddin et al. also found that washing the seeds partially removed the neurotoxit while fermentation, steaming or autoclaving appeared to have little effect (24). Ayyagari et ai, based on analysis of a limited number of samples, concluded that the -ODAP content did not decrease much in prepared food. Dry heating, roasting, deep frying, or boiling did not alter the -ODAP levels (25). On the other hand, Rao et al. recorded that the roasting of grass pea for about 15-20 minutes at 140C rendered the seed free of neurotoxin. Howevere, they observed that chapati making made the

It ranks fifth in total area among Ethiopian food legumes with a mean of 60,030 ha of land (8.7%) and 42, 200 t (7.6%) of grain (18). Probably as a reflection of the repeated food shortage situations in Ethiopia, the production of grass pea increased by 20% between 1981 and 1987 (19). Sporadic cases of lathyrism continue to appear in the northwest part of the country (10). In the area studied here, grass pea is mainly used to produce the flour {shiro) used in the preparation of the Ethiopian sauce or gravy, shiro wott. This was the case in both villages studied. The pulse is also used as snacks in the roasted and boiled forms, mainly in Shina, the village where lathyrism is endemic at a prevalence rate of 3% (10). The bread form is consumed widely only in extreme famine situations as was the case during the major lathyrism epidemic of the Fogera and Dembia districts in 1976/77 (5). The consistent view that transpires from interviews of consumers of grass pea is that the pulse has an excellent taste. In the flour form, it possesses the unique water-absorbing and expansive properties which are advantageous during the preparation of the gravy, wott. As a result, in central parts of Ethiopia where grass pea is not cultivated, the pulse is presently popularly used as an admixture in the production of shiro. Food adulteration, with Lathyrus sativus seeds have been reported from India (20). There are only a few studies that have addressed the potential neurotoxicity of

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grass pea foodstuff more toxic (26). However, in the case of the roasting they did not mention whether the seeds were washed or boiled before roasting. TABLE 6 -ODAP content of bread (kitta) prepared in the laboratory from different L sativus samples
Dry grass pea CODE -ODAP mg/100gm 488 549 387 514 394 514 528 429 359 253 514 367 475 452 598 299 416 416 416 416 439.2 85.5 % moisture Bread sample -ODAP mg/100gm 394 426 429 584 521 535 786 583 472 331 401 510 542 590 678 502 493 244 224 227 473.6 145.5 grass pea wet sample (gm) 80 80 80 80 80 80 80 80 80 80 80 Bread preparation water for mixing (ml) 60 60 60 60 60 75 60 60 60 55 60 baking time (min) 7 7 7 7 6 7 7 10 10 8 7

instance, the potassium salt of -ODAP is more extractable than the sodium salt. Little or no information is available on the relative extractabilities of other divalent metal salts of the toxin. Although there have been suggestions that the reduction of over 65% in -ODAP content could be considered safe, the non-toxic levels of -ODAP have not yet been established (20). In fact, it will not be easy to set such a cut of standard because of the different concentration of -ODAP in germplasm lines of L. sativus. For instance, in the field samples we collected the -ODAP contents varied from 128 to 985mg/100 gm of dry seeds. Thirty or thirty-five per cent of the most toxic sample may still contain a significant amount of -ODAP. ACKNOWLEDGEMENTS These studies were made possible by the grant from Band Aid which was obtained through Third World Medical Research Foundation (TWMRF). BA is grateful for additional support from the Swedish Agency for Research Cooperation with Developing Countries (SAREC). Aregay Waktola, Principal Investigator of the Lathyrism Project in Ethiopia, Valerie Palmer of TWMRF and Penny Jenden of Band-Aid are thanked for their interest and support. We are grateful to Asmerom Kidane, Professor of Statistics, AAU, for helpful comments. We are grateful to the enumerators that participated in field surveys.

36 37 38 39 40 41 42 43 44 45 46 67 68 69 70 71 1/1 1/2 1/3 1/4 Mean S.d.

70.8 76.5 63.1 65.4 62.8 70.8 62.7 69.8 67.0 70.8 71.4 66.7 65.3 64.3 77.5 '65.3

REFERENCES
1. Prasad LS, Sharan RK. Lathyrism. In: Vinken P J, Bruyen G W. (eds). Handbook of Clinical Neurology: Intoxication of the Nervous System Amsterdam: North Holland Publishing Co., 979: 505-14. Spencer PS, Schaumburg HH. Lathyrism: A neurotoxic disease. Toxicology and Teratology 1983;5:625-9. Neurobehavioral

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Dwivedi MP and Mishra SS. Recent outbreak of lathyrism and experience with propagation of detoxified L sativus. Proc Nat Soc Ind 1974;19:23-30. Kulkarni SW, Attal HC, Choubey BS. An epidemiologlc study of lathyrism in Amgaon Block, Bahandra district. Ind Med J Res 1977;66:602-10. Gebre-Ab T, Wolde-Gabriel Z, Maffi M, Ahmed Z, Ayele TM, Fanta H. Neurolathyrism- a review and a report of an epidemic. Ethiopian Med J 1978;16:1-11. Spencer PS, Roy DN, Ludolph A, Hugon J, Schaumburg HH. Lathyrism: Evidence for role of the neuroexcitatory amino acid BOAA. Lancet 1986li:1066-67. Dwivedi MP. Epidemiological aspects of lathyrism in India- a changing scenario. In: Spencer P S (ed). Grass-pea: Threat and Promise. New York: Third world Medical Research Foundation, 1980:1-26. Haque A, Mannan MA. The problems of lathyrism in Bangladesh. In: Spencer PS (ed). Grass-pea: Threat and Promise. New York: Third World Medical Research Foundation, 1989:27-35. Tekle Haimanot R. Lathyrism in Ethiopia. In: Spencer PS (ed). Grass-pea: Threat and Promise. New York: Third World Medical Research Foundation, 1989:36-40.

However, a serious and challenging question that will arise out of our findings will be why have higher -ODAP levels been recorded in the bread and roasted forms of grass pea? How does direct heat increase -ODAP content of the grass pea? A plausible explanation relates to changes in the extractability of the -ODAP by thermally introduced processes. Other workers who have been studying the extractability of the toxin by imbibing with water over prolonged periods and determining the levels of -ODAP in the imbibed seeds, as well as in the fluid, have found that the two values when added up differ from the level of toxin found by direct analysis of seeds [Lambien, F. personal communication]. Heat treatment may induce enzymatic changes, damage lipid membranes or even alter the nature of the counterions that may be associated with the toxin. It Is possible to speculate changes in the composition and availability of counterions, K+, Na+, Mg++ that may be induced by heat. For

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9.

1126 10.

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19. 20. 21. 22.

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Accepted for publication June 16, 1993.