Indian J Med Res 140, July 2014, pp 96-101 grass pea consumption & present scenario of neurolathyrism in Maharashtra state of india Arjun L. Khandare, J.J Babu*, M. Ankulu, N. Aparna, Amol shirfule & G. Shankar Rao Food & Drug Toxicology Centre (ICMR) & *Department of clinical studies, National Institute of Nutrition (ICMR), Hyderabad, India Received February 15, 2013 Background & objectives: Neurolathyrism is a non progressive motor neuron disorder engendered by the prolonged over-consumption of Lathyrus sativus (grass pea) seeds which contain a neurotoxic amino acid, β-N oxalyl- L-α, β-diaminopropionic acid (β-ODAP). It is characterized by spastic paraparesis in the hind limbs. The present study was conducted in 105 households (HHs) of Gondia district in Maharashtra, India, where grass pea is cultivated and consumed to assess the health implication of its consumption. Methods: a cross-sectional survey was carried out in 105 HHS in five villages and grass pea samples were collected for β-ODAP estimation. Amino acid analysis was also done, neurolathyrism cases were identified by snowball sampling method and neurological examination was carried out. Results: The study revealed that 61 per cent of population was consuming this pulse as a part of diet. β-ODAP concentration in grass pea was high in Bora village (1254.5 ± 528.21 mg %) and less in Malgaon village (413.6±415.79 mg %). The nutritional status of the people was within the normal range (BMI 18± 3.40 kg/m2) in the surveyed households. Consumption of grass pea was observed to be less than 25g. Conclusions: The cases of neurolathyrism declined in all the studied villages due to reduced β-ODAP exposure through Lathyrus sativus consumption, however, the grass pea was cultivated and consumed in Gondia district of Maharashtra State. key words β-ODAP - grass pea - khesri dal - Lathyrus sativus - neurolathyrism Lathyrus sativus (grass pea/khesari dal) is a high yielding; drought-tolerant legume crop containing 31 per cent protein, 41 per cent carbohydrate, 17 per cent total dietary fiber1,2. The protein content of grass pea seeds is higher compared to other legume seeds3. Grass pea is cultivated and consumed in India, Nepal, Bangladesh and in many parts of Africa that are prone to recurrent droughts4,5. It is often considered a lifesaver crop6 or as insurance crop7. It is an abundant source of protein for human and animal consumption7. Presence of homoarginine (a source of potent vasodilator nitric oxide) in grass pea can be used to explore its inherent potential to treat cardiovascular disorders associated with vascular endothelial function8. The major grass pea growing states in India are Bihar, Madhya Pradesh, Maharashtra, West Bengal and Chhattisgarh. Other states of Uttar Pradesh, Rajasthan, 96 Khandare et al: Assessing diet in populations at risk for Neurolathyrism 97 Gujarat, Andhra Pradesh and Karnataka produce this pulse in a limited quantity. People from lower economic sections consumed this legume crop as a staple food during famines and floods. Grass pea samples were collected from all the available households in five villages. The samples were labelled and transported to the NIN, Hyderabad, to measure the β-ODAP content. Excessive consumption of grass pea containing β -N oxalyl- L-α, β-diaminopropionic acid (β-ODAP)9, analogue of glutamate for 2-3 months as a monotonous diet, leads to an irreversible motor neuron disorder, neurolathyrism10. Pathological symptoms are caused by symmetrical degeneration of pyramidal tracts in the spinal cord11, and loss of pyramidal cells in the motor cortex of the brain leads to the impairment of the hind limb movements12. reports suggest that β-ODAP is metabolized in human beings13. However, metabolic pathway of   β-ODAP in human beings is not exclusively known. Earlier epidemiological studies have depicted illiteracy, poverty and young age as major risk factors for neurolathyrism14,15. Estimation of β-ODAP in L. sativus seeds: Grass pea seed samples were made into a fine powder and sieved in a 0.125 mm sieve. Twenty mg of the powder was taken in different test tubes (duplicates were maintained), 2 ml of distilled water was added, the test tubes were kept in boiling water bath for 15 min, and care was taken to prevent the formation of any precipitate at the bottom of the tubes. The tubes were allowed to cool and were centrifuged at 605 g for 10 min. The supernatant was collected for β-ODAP estimation16 in aliquots without disturbing the sediment. The harmful effects depend on quantity of the pulse consumed, its β-ODAP content, method of cooking; and nutritional status of the individual. Susceptibility to neurolathyrism varies from person to person even within a family. The National Institute of Nutrition (NIN), Hyderabad, Andhra Pradesh, India, has undertaken this study to assess the grass pea cultivation and its health implications in Gondia district of Maharashtra state in India with the following objectives. (i) quantity of the pulse consumed and exposure of β-ODAP per day, (ii) its health implications, (iii) nutritional status of the consumers, and (iv) to determine the level of β-ODAP in the grass pea samples. Material & methods A cross-sectional survey was carried out in the Gondia district of Maharashtra state, India, in 2012, where cultivation of grass pea is common. A total of 105 households (HHs) in five villages with a population of 496 were surveyed. Of 907 total villages in the district, the five villages were selected based on the highest cultivation of lathyrus sativus. this study was approved by the Ethics Committee of the National Institute of Nutrition, Hyderabad, India. Diet survey was carried out by food frequency questionnaire in 105 households using random sampling method. Nutrition status of the individuals was assessed by measuring height and weight. BMI was calculated using the formula weight/height in meter2. Amino acid analysis: Amino acid analysis was done based on seed weight by hydrolyzing the samples with 6 N HCl in sealed ampoules in an oven at 110 ◦C for 22 h17. Excess acid was removed by continuous flash evaporation at 45 °C under reduced pressure. The dried sample was dissolved in citrate buffer (pH 2.2) and aliquot of the sample was loaded into an automatic amino acid analyzer (Biochrom-30, Cambridge, UK). Cysteine and methionine were determined separately after performic acid oxidation. Each amino acid was identified and quantified using externally calibrated standards (50, 100 and 150 pmol/µl) and validated using authentic amino acid standard mixture (National Institute of Standards and Technology, USA, SRM 2389). Identification of neurolathyrism cases by ‘Snowball sampling’method: Snowball sampling of neurolathyrism patients was opted and done by investigating and identifying the subjects from support groups of patients obtained from different households, health authorities (district hospital, community health centre and primary health centre), and through interviews with important people within the community (e.g. village panchayat members). Neurological examination of all subjects was carried out. A pilot study was done in 10 villages to enquire about the cultivation and consumption of lathyrus, on the basis of results of pilot study five villages were selected by taking 20 households/village for main study. the consumption of Khesari dal was found to be in 60 per cent household; assuming 95 per cent confidence interval, 20 per cent relative precision, the 98 INDIAN J MED RES, july 2014 required number of households was 64. Sample size for β-ODAP was calculated to be five. Statistical analysis: Simple, descriptive statistical tools were used to analyze the survey data. Computer software, statistical package for social sciences (SPSS) - version 10 (SPSS Inc., USA) was used to analyze the data. Results & Discussion The literacy rate among the HHs was found to be high (84%). Most of them depended on agriculture as an occupation. The average land holding was less with ST (Scheduled tribe) community when compared with other communities (Table I). Due to meager land holding, most of the population in SC, ST communities are working as agricultural labourers. The most commonly cultivated crop in the study area is paddy (Oryza sativa) which is sown in July to October season, and is a major part of diet. Apart from paddy, they are cultivating the flax seed (Linum usitatissimum) as a commercial crop. Grass pea is mainly cultivated during rabbi season as it can withstand extreme temperatures in summer and gives a good yield (Table I). Consumption of grass pea and exposure of β-ODAP: It was found that 61 per cent of the HH surveyed were consuming grass pea in different forms, and OBC (other backward casts) community was the major consumer as they were cultivating grass pea in their respective land holdings. Six HHs were found to be consuming grass pea above 25  g/day due to their low socio-economic status and lack of land holding. However, in other communities, the quantity of grass pea consumed was less (12-25 g), which was in the form of gravy (cooked grass pea mixed with water until it assumes a semisolid state) alone or in combination with other pulses. Vada (soaked and deep-fried grass pea flour) was found to be the most commonly prepared food stuff with grass pea. Majority of the HH (61%) were aware of the toxic effect of grass pea (Table II). Of all the villages surveyed, Bora village was found to be consuming 25 g grass pea/person daily. In other villages, it was less than the 25 g and the quantity of consumption was associated with prices of other legume pulses in the market. The exposure of daily β-ODAP was calculated based on average β-ODAP content in grass pea collected from the different villages. The results showed β-ODAP exposure was 266 mg in Beradipar A which was much less when compared to the high exposure during famine and floods. A few households reported gastrointestinal problems like gas formation and abdominal pain apart from that there were no side effects and health complications. β-ODAP content in grass pea samples: A total of 32 grass pea samples were collected. Maximum concentration of β-ODAP (1254.5+528.21 mg %) was found in samples collected from Bora village, and minimum (102.6±248.38 mg %) was in Menda village (Table III). The variability in the β-oDAP content could be due to various environmental factors or their combination18. Nutrition status: All subjects surveyed were healthy, and no side effects were reported from any household. Table I. Demographic information of population studied in Gondia block, Maharashtra Total number of households – 105 Community SC - 28 (26.7%) Total - 105 ST - 15 (14.3%) Education Illiterate - 70 (14.1%) Read/Write - 6 (1.2%) Literate - 420 (84.7%) Total - 496 Other labourer - 23 (4.6%) Land holding Own cultivation - 50 (10.1%) Total - 105 Yes - 75 (71.4%) No - 30 (28.6%) Land holding community-wise SC - 14 (18.6%) Others - 3 (2.9%) Total - 496 Occupation Agriculture labourer - 144 (29.0%) OBC - 59 (56.2%) Total - 75 ST - 13 (17.4%) OBC - 46 (61.3%) Cultivation of KD Total - 75 Yes - 27 (36.0%) No - 48 (64.0%) Artisans - 1(0.2%) Khandare et al: Assessing diet in populations at risk for Neurolathyrism 99 Table II. Consumption pattern of grass pea in the studied area Consumption of grass pea n=105 Total - 105 Yes - 63 (60.0%) No - 42 (40.0%) Consumption of grass pea community-wise SC - 13 (20.6%) ST - 11 (17.5%) OBC - 37 (58.7%) Consumption of grass pea education-wise Total - 105 Illiterate - 12 (11.4%) Read and Write - 1 (1.0%) Cost of grass pea (57 respondents) Maximum - 50 Minimum - 20 Consumption of grass pea pattern Total - 105 Daily - 6 (5.7%) Weekly - 15 (14.3%) Consumption of grass pea in the form Total - 63 Gravy - 27 (42.9%) Vada - 5 (7.9%) Gravy & vada -23 (36.5%) Gravy & gram flour -5 (7.9%) Others - 2 (3.2%) Literate-92 (87.6%) Mean - 29.44 Weekly twice - 18 (17.1%) Gram flour - 3 (4.8%) Population know about adverse effects of grass pea consumption Yes - 64 (61.0%) No – 41 (39.0%) There was no significant decrease in BMI range in different communities feeding on grass pea. All Red BMI in normal range (Table IV). The arginine (8.92±0.19g%) content was high in grass pea and could act as a substrate for nitric oxide production. Among the non-essential amino acids, glutamic acid and aspartic acid were the most representative (17.83±0.14 and 10.63±0.09 g/100 g, respectively). Sulphur amino acids (cysteine and methionine, 0.91±0.01 and 0.99±0.07 g/100 g, respectively) (Table V) were found to be less compared to other amino acids. Neurological examination and presence of neurolathyrism: There was no evidence of the spasticity Table III. β-ODAP content in grass pea samples collected Name of village β-ODAP (mg/100 g) Bora (n=2) 1254.5 ± 528.21 Menda (n=6) 102.6 ± 248.38 Malgom (n=5) 413.6 ± 415.79 Bhanpur (n=5) 994.4 ± 219.12 Malapari (n=8) 895.8 ±197.41 Pindikepar (n=3) 938.7 ± 127.02 Beradipar (n=2) 1034 ± 311.13 Gurde (n=1) 1010.0 in left lower limb during the gait examination. The sensations and cranial nerves were found to be intact. Upper motor lesion with left leg spasticity was not noticed in the lower limbs in the grass pea consuming HHs. Two old cases of neurolathyrism were found (both above 50 yr of age). In the 105 households surveyed, there was no fresh case of neurolathyrism even though they were consuming grass pea. In earlier studies, 136 cases from Bhandara district of Maharastra19 which is next to Gondia district, 2500 cases in Rewa district of Madhya Pradesh20, 1200 cases in central India21, and 41 cases from Unnao district in Uttar Pradesh22 were reported. The deficiency of sulphur containing amino acids methionine and cysteine23 in the grass pea causes a reduction in glutathione synthesis, which is a main antioxidant. However, consumption of grass pea along with other food sources compensated for the lack of sulphur containing amino acids in grass pea. This could also be one of the main reasons for fewer incidences. In conclusion, the present study confirmed that grass pea was being cultivated and consumed in Gondia district of the Maharashtra state. It was also evident from this study that consumption of grass pea in small quantities did not lead to neurolathyrism. The availability of different food sources to even marginal sections of people perhaps increased the nutritional status. If the nutritional value of this pulse can be utilized effectively, it may become a good source of protein. 100 INDIAN J MED RES, july 2014 Table IV. Community-wise BMI in Gondia district, Maharashtra State S.No Village Community SC ST OBC Others 1. Bora 22.46 ± 3.69 (n=5) NA 19.94 ± 3.51 (n=35) 19.5 ± 2.33 (n=9) 2. Menda 18.7 ± 3.40 (n=12) 18.9 ± 3.98 (n=4) 19.1 ± 4.91 (n=28) NA 3. Malpuri 19.4 ± 3.40 (n=10) 19.0 ± 3.59 (n=12) 20.2 ± 3.65 (n=8) NA 4. Beradipar-A 18.3 ± 2.42 (n=11) 20.2 ± 5.61 (n=7) 19.6 ± 1.87 (n=25) 19.6 ± 1.87 (n=3) 5. Beradipar-K 21.1 ± 3.59 (n=22) 18.4 ± 2.16 (n=7) 19.1 ± 2.91 (n=13) NA NA, community not available SC, scheduled caste; ST, scheduled tribe; OBC, other backward classes Table V. Amino acid composition of L. sativus in study area Essential amino acids Content g/l Threonine 3.60 ± 0.10 Valine 5.14 ± 0.03 Methionine 0.99 ± 0.07 Isoleucine 4.25 ± 0.02 Leucine 7.06 ± 0.02 Phenylalanine 4.50 ± 0.11 Histidine 2.41 ± 0.06 Lysine 6.37 ± 0.17 Tryptophan NR References 1. Akalu G, Johansson G, Nair BM. Effect of processing on the content of β-N-oxalyl- α,β-diaminopropionic acid (β-ODAP) in grasspea (Lathyrus sativus) seeds and flour as determined by flow injection analysis. Food Chem 1998; 62 : 233-7. 2. Aletor VA, Abd El-Moneim A, Goodchild AV. Evaluation of the seeds of selected lines of three Lathyrus spp. for β-Noxalylamino-L-alanine (BOAA), tannins, trypsin inhibitor activity and certain in vitro characteristics. J Sci Food Agri 1994; 65 : 143-51. 3. Monsoor MA, Yusuf HK. 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Khandare, Scientist E, FDTRC, National Institute of Nutrition (ICMR) PO Jama-I-Osmania, Hyderabad 500 007, India e-mail: alkhandare@yahoo.com View publication stats 101