Grass pea (Lathyrus sativus L.): orphan crop, nutraceutical or just plain food?

MAIN CONCLUSION: Although grass pea is an environmentally successful robust legume with major traits of interest for food and nutrition security, the genetic potential of this orphan crop has long been neglected. Grass pea (Lathyrus sativus L.) is a Neolithic plant that has survived millennia of cultivation and has spread over three continents. It is a robust legume crop that is considered one of the most resilient to climate changes and to be survival food during drought-triggered famines. The hardy penetrating root system allows the cultivation of grass pea in various soil types, including marginal ones. As an efficient nitrogen fixer, it meets its own nitrogen requirements and positively benefits subsequent crops. However, already in ancient India and Greece, overconsumption of the seeds and a crippling neurological disorder, later coined neurolathyrism, had been linked. Overemphasis of their suspected toxic properties has led to disregard the plant's exceptionally positive agronomic properties and dietary advantages. In normal socio-economic and environmental situations, in which grass pea is part of a balanced diet, neurolathyrism is virtually non-existent. The etiology of neurolathyrism has been oversimplified and the deficiency in methionine in the diet has been overlooked. In view of the global climate change, this very adaptable and nutritious orphan crop deserves more attention. Grass pea can become a wonder crop if the double stigma on its reputation as a toxic plant and as food of the poor can be disregarded. Additionally, recent research has exposed the potential of grass pea as a health-promoting nutraceutical. Development of varieties with an improved balance in essential amino acids and diet may be relevant to enhance the nutritional value without jeopardizing the multiple stress tolerance of this promising crop.

Vascular insult accompanied by overexpressed heme oxygenase-1 as a pathophysiological mechanism in experimental neurolathyrism with hind-leg paraparesis.

Neurolathyrism (NL) is a motor neuron disease characterized by spastic paraparesis in the hind legs. ß-N-oxalyl-l-α,ß-diaminopropionic acid (l-ß-ODAP), a component amino acid of the grass pea (Lathyrus sativus L.), has been proposed as the cause of this disease. In our NL rat model, we previously reported that transient intra-parenchymal hemorrhage occurred in the lower spinal cord during the early treatment period. We show here a possible pathological role of the hemorrhage in motor neuron damage and paraparesis pathology. In the lumbo-sacral spinal cord, blood vessel integrity was lost with numerous TdT-mediated dUTP nick end-labeling-positive blood vessel-like structures occurring simultaneously with the hemorrhage. We observed a coincident >10-fold increase in heme oxygenase-1 (HO-1) only in the lower spinal cord. The early period of paraparesis in the lower leg was greatly suppressed by pretreatment with zinc protoporphyrin IX, a HO-1 inhibitor. In vitro, l-ß-ODAP was toxic to human umbilical vein endothelial cells compared to l-glutamate. The present data shed light on the role and the mechanism of vascular insult in causing dysfunction and moribund motor neurons in experimental NL.

Stress-related over-enhancement of the hypothalamic-pituitary-adrenal axis causes experimental neurolathyrism in rats.

Neurolathyrism is a motor neuron disease that is caused by the overconsumption of grass peas (Lathyrus sativus L.) under stressful conditions. The neuro-excitatory ß-N-oxalyl-L-α,ß-diaminopropionic acid present in grass peas was proposed the causative agent of spastic paraparesis of the legs. Historical reports of neurolathyrism epidemics, studies of neurolathyrism animal models, and in vitro studies on the mechanism of ß-N-oxalyl-L-α,ß-diaminopropionic acid toxicity support the hypothesis that stress increases susceptibility to neurolathyrism. To elucidate the role of stress in neurolathyrism-induced motor dysfunction, we focused on the hypothalamic-pituitary-adrenal axis in a rodent model of neurolathyrism. Our results implicated increased glucocorticoid and neuroinflammation in the motor dysfunction (paraparesis) exhibited by the stress loaded rat models of neurolathyrism.

The contents of the neuro-excitatory amino acid ß-ODAP (ß-N-oxalyl-l-α,ß-diaminopropionic acid), and other free and protein amino acids in the seeds of different genotypes of grass pea (Lathyrus sativus L.).

The free and protein amino acids of nine different genotypes of grass pea (Lathyrus sativus L.) seeds were analysed by HPLC with pre-column PITC (phenyl isothiocyanate) derivatisation. Among the free amino acids, homoarginine was quantitatively the most important (up to 0.8% seed weight) and stable while the neuro-excitatory amino acid ß-ODAP (ß-N-oxalyl-l-α,ß-diaminopropionic acid) showed highest variation (0.02-0.54%) in the nine genotypes examined. Among protein amino acids, glutamic acid was quantitatively most significant, followed by aspartic acid, arginine, leucine, lysine and proline. The sulphur amino acid, methionine, showed the lowest concentration in all the L. sativus genotypes, and also in lentil (Lens culinaris) and in soybean (Glycine max) seeds analysed at the same time.

Comparison of urinary amino acids and trace elements (copper, zinc and manganese) of recent neurolathyrism patients and healthy controls from Ethiopia.

OBJECTIVE: The irreversibly crippling disease neurolathyrism is caused by prolonged over-consumption of Lathyrus sativus seed. The molecular mechanism of toxicity is unclear and more biochemical information is needed. METHODS: The urinary amino acids from 5 recent patients and 9 healthy subjects in Ethiopia were analysed by HPLC after PITC (phenyl isothiocyanate) derivatisation. The trace elements Cu, Zn and Mn of urine and seeds were determined by atomic absorption spectroscopy. RESULTS: The free amino acids aspartic acid, glycine, beta-aminoisobutyric acid, arginine, alpha-aminoadipic acid and phenylalanine were statistically higher (p<0.05) in urine from patients than in urine from control subjects. The trace element Cu was also statistically higher (p<0.05) in patient urine. CONCLUSION: The amino acid metabolism of the neurolathyrism patients is disturbed by over-consumption of grass pea seeds. The high concentrations of Cu found in the patient urine might indicate the involvement of trace elements in the aetiology of neurolathyrism.

Plant toxin ß-ODAP activates integrin ß1 and focal adhesion: A critical pathway to cause neurolathyrism.

Neurolathyrism is a unique neurodegeneration disease caused by ß-N-oxalyl-L-α, ß- diaminopropionic (ß-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, ß-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of ß1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

Food-aid cereals to reduce neurolathyrism related to grass-pea preparations during famine.

Neurolathyrism is a spastic paraparesis that can be caused by excessive consumption of the drought-resistant grass pea (Lathyrus sativus). Devastating neurolathyrism epidemics have occurred during major famine crises in various parts of the world. We investigated in a case-control study the effects of food aid on risk of paralysis. Risk increased with consumption of boiled grass pea (adjusted odds ratio 2.78, 95% CI 1.09-7.13 with cereals; 5.22, 2.01-13.55 without cereal) and raw unripe green grass pea (1.96, 1.16-3.31; p=0.011), but not with the fermented pancake, unleavened bread, and gravy preparations. In a correlational study there was an inverse relation between the number of new cases and the amount of food-aid cereals distributed per person. During famine, cereals and nutritional information should reach people before they have grass pea as the only food.

Neuroactive and other free amino acids in seed and young plants of Panax ginseng.

The seeds and one to three years old plants of Asian ginseng (Panax ginseng C.A. Meyer) were analyzed for their free amino acid contents. The neuro-excitatory beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid), suggested to be the cause of the crippling neurolathyrism, was the major component in the seed extract (70% of the total free amino acids detected) and showed the highest concentration (0.43% by wt) compared to that in the different parts of young plants. beta-ODAP concentration was higher in the shoots as compared to roots and declined in older plants. The amount of beta-ODAP in the roots may be considered as an indirect measure of age and quality. Another neuro-active non-protein amino acid, GABA (gamma-aminobutyric acid), increased dramatically after germination and reached highest concentration in different parts of 3 year-old plants. Glutamine and arginine were the two major free proteinogenic amino acids in the ginseng plants and together they constituted over 50% of all the free amino acids detected in the root.

ABO blood groups, grass pea preparation, and neurolathyrism in Ethiopia.

An exploratory study was conducted in the rural Estie district of Ethiopia in 1997 to identify the role of ABO blood group, rhesus factor, and type of grass pea (Lathyrus sativus) diet in the susceptibility to neurolathyrism. Five-hundred study subjects (250 cases and 250 controls) were examined and interviewed, and had their ABO and rhesus blood groups determined. The majority (86%) of the cases were males. Blood group O was the most common in the patients and controls followed by groups A, B, and AB. The vast majority of the study subjects were rhesus-positive. The gravy (Shiro) grass pea preparation was consumed by 91.6% of the study population, boiled (Nifiro) by 86%, and roasted (Kollo) by 56.4%. Almost half (48%) of the cases had consumed grass pea for > 4 months compared to 8% of controls (P < 0.001). There was a significant association between the risk for neurolathyrism and the consumption of boiled (adjusted odds ratio [AOR] = 98.4) and roasted (AOR = 55.62) forms of grass pea. There was no risk of paralysis associated with consumption of the gravy form of grass pea (AOR = 0.40, 95% confidence interval 0.1-2.0). Blood group O remained significantly associated with the disease after adjusting for age, type of grass pea preparation consumed, and duration of consumption (AOR = 2.90).

Neurolathyrism in Ethiopia: assessment and comparison of knowledge and attitude of health workers and rural inhabitants.

A cross sectional community based study was done in the Amhara Regional State of Ethiopia in 1999-2000 to assess and compare knowledge and attitude towards neurolathyrism among health workers and the rural community. A sample of 217 health workers selected by probability proportional to size and randomly selected 589 heads of household from a rural district were interviewed using pre-tested questionnaires. Neurolathyrism was widely known among the health workers and the community. More than half of community respondents associated the disorder with walking or lying on the straw and the stalks of grass pea. In a multivariate analysis. poor neurolathyrism knowledge among the community was associated with illiteracy and with presence of a neurolathyrism patient at home. Among health workers, contact with vapour or steam of grass pea foods was the commonest cause cited. In a multivariate analysis nurses had the poorest knowledge among the health workers. Depending on the subject, health workers and community respondents had more or less knowledge than the other. The prevailing recurrent adverse climatic conditions might promote grass pea as a 'friendly' crop to the poor peasants in marginal areas who otherwise rely on it only during times of food shortages and could increase the incidence of neurolathyrism. The poor knowledge among health workers and the community and the general neglect of neurolathyrism requires urgent intervention. Appropriate strategies for the dissemination of information education, and communication (IEC) are needed.

Food safety and amino acid balance in processed cassava "Cossettes".

Processed cassava (Manihot esculenta Crantz) roots provide more than 60% of the daily energy intake for the population of the Democratic Republic of Congo. Insufficiently processed cassava roots in a diet deficient in sulfur amino acid have been reported to cause the irreversible paralytic disease konzo, afflicting thousands of women and children in the remote rural areas of Bandundu Province. "Cossettes" (processed cassava roots) purchased in several markets of Kinshasa were analyzed for their content of cyanogens, free amino acids, and total protein amino acids. Residual cyanogen levels were below the safe limit recommended by the codex FAO/WHO for cassava flour (10 mg kg(-1)). The amino acid score was evaluated. Lysine and leucine were the limiting amino acids. Methionine content was very low and contributed about 13% of the total sulfur amino acids. Dietary requirements for sulfur amino acids need to be adjusted for the loss caused by cyanogen detoxification.

Research on motor neuron diseases konzo and neurolathyrism: trends from 1990 to 2010.

Konzo (caused by consumption of improperly processed cassava, Manihot esculenta) and neurolathyrism (caused by prolonged overconsumption of grass pea, Lathyrus sativus) are two distinct non-infectious upper motor neurone diseases with identical clinical symptoms of spastic paraparesis of the legs. They affect many thousands of people among the poor in the remote rural areas in the central and southern parts of Africa afflicting them with konzo in Ethiopia and in the Indian sub-continent with neurolathyrism. Both diseases are toxico-nutritional problems due to monotonous consumption of starchy cassava roots or protein-rich grass pea seeds as a staple, especially during drought and famine periods. Both foods contain toxic metabolites (cyanogenic glycosides in cassava and the neuro-excitatory amino acid ß-ODAP in grass pea) that are blamed for theses diseases. The etiology is also linked to the deficiency in the essential sulfur amino acids that protect against oxidative stress. The two diseases are not considered reportable by the World Health Organization (WHO) and only estimated numbers can be found. This paper analyzes research performance and determines scientific interest in konzo and neurolathyrism. A literature search of over 21 years (from 1990 to 2010) shows that in terms of scientific publications there is little interest in these neglected motorneurone diseases konzo and neurolathyrism that paralyze the legs. Comparison is made with HTLV-1/TSP, an infectious disease occurring mainly in Latin America of which the clinical manifestation is similar to konzo and neurolathyrism and requires a differential diagnosis. Our findings emphasize the multidisciplinary nature of studies on these neglected diseases, which however have not really captured the attention of decision makers and project planners, especially when compared with the infectious HTLV-1/TSP. Konzo and neurolathyrism can be prevented by a balanced diet.

Effect of environmental factors on the biosynthesis of the neuro-excitatory amino acid ß-ODAP (ß-N-oxalyl-L-α,ß-diaminopropionic acid) in callus tissue of Lathyrus sativus.

Habituated callus tissues derived from leaf explants of Lathyrus sativus L. (grass pea) were cultured under different environmental conditions such as drought, salinity and deficiency or oversupply of micronutrients. The biosynthesis of the neuro-excitatory ß-ODAP (ß-N-oxalyl-L-α,ß-diaminopropionic acid) was induced by feeding the precursor BIA, (ß-isoxazolin-5-on-2-yl)-alanine, to those calli habituated under different stress conditions. Conversion of BIA into ß-ODAP was reduced by Zn(2+) at different levels of Fe(2+) supplements while excess of Fe(2+) enhanced it at different Zn(2+) levels in the media. The biosynthesis of ß-ODAP was increased by both oversupply and deficiency of Mn(2+) manganese while B(3+) as well as Co(2+) increased it significantly by oversupply. Al(3+) enhanced the conversion of BIA into ß-ODAP significantly in a concentration-dependent way. Cu(2+) also reduced the formation of ß-ODAP when increased in the media. Mo(6+) had no apparent effect. NaCl decreased the conversion of BIA into ß-ODAP proportionately with the increase in salinity. ß-ODAP was increased with increasing mannitol concentration till -0.23MPa while at this osmotic potential created with PEG-20,000 the formation of ß-ODAP is completely inhibited in low toxin calli. These experiments demonstrate the importance of environmental factors, especially micronutrients and salinity, on the biosynthesis of ß-ODAP.

Climatic, edaphic and altitudinal factors affecting yield and toxicity of Lathyrus sativus grown at five locations in Ethiopia.

A 2 years (2005-2006) data analysis based on agronomic, qualitative, climatic and edaphic factors was carried out using 10 grass pea (Lathyrus sativus L.) genotypes grown at five eco-divergent locations (Alem Tena, Debre Zeit, Denbi, Akaki, Chefe Donsa) in Ethiopia. Crop yield showed considerable variability among locations, years and genotypes. Path coefficient analysis indicated that rainfall and days to maturity have a large positive influence on yield. High level of micronutrients Mn(2+) and S(2-) negatively affected yield. Path analysis revealed that Zn(2+)/P, days to maturity, yield and K(+) were dominant variables affecting the response variable ß-ODAP (ß-N-oxalyl-L-α,ß-diaminopropionic acid), the neuro-excitatory amino acid in grass pea seeds considered as the cause of neurolathyrism. Linear correlation analysis between ß-ODAP and the 35 factors considered showed that ß-ODAP level was positively correlated (r > 0.70) with K(+) and sunshine hours (ssh) and negatively correlated (r < 0.70) with soil pH, days to maturity and yield. The strongest correlation of ssh with ß-ODAP level was found during the phase of crop maturity. Our results suggest that ß-ODAP biosynthesis and its response to environmental stress are maximized during the post-anthesis stage.

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of L-ß-ODAP by increasing the oxidative stress: studies on a motor neuron cell line.

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea (Lathyrus sativus L.) containing L-ß-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of L-ß-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of L-ß-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. L-ß-ODAP further lowered the content in the deprived media to be 32-44% of the controls compared to normal media being 62-74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by L-ß-ODAP under sulfur amino acid-deficient conditions.

L-ß-N-oxalyl-α,ß-diaminopropionic acid toxicity in motor neurons.

The excitatory amino acid L-ß-N-oxalyl-α,ß-diaminopropionic acid (L-ß-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-ß-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-ß-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-ß-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.

Effect of methionine supplement on physical responses and neurological symptoms in broiler chicks fed grass pea (Lathyrus sativus)-based starter ration.

Starter feeding experiments of broiler chicks with raw grass pea (Lathyrus sativus L.) supplemented with different levels of DL-methionine were undertaken for 4 weeks to assess the toxicity of grass pea-based feed and to correlate it with neurological symptoms. Four hundred fifty day-old broiler chicks were divided into two groups and were given formulations containing 35% (ration I) or 98.5% (ration II) grass pea, respectively. Each ration included controls and treatments with added methionine of four different concentrations. Feed intake, weight gain and feed conversion efficiency (FCE) were much higher in ration I than in ration II and these parameters significantly improved by addition of methionine in both rations. Significant increase of neurological signs with higher grass pea intake and significant reduction of acute neurological signs with addition of methionine were observed. Tolerance for grass pea was enhanced with increasing methionine in the diet and with age. Despite a similarity in the initial intake, a significant (p0.05) increase in the final feed intake by the chicks with methionine addition was found in both rations. These results suggest that methionine can improve a grass pea-based diet for broiler chicks and especially can protect young chicks from neurological symptoms.

Hind-limb paraparesis in a rat model for neurolathyrism associated with apoptosis and an impaired vascular endothelial growth factor system in the spinal cord.

Neurolathyrism is a motor neuron disease characterized by lower limb paraparesis. It is associated with ingestion of a plant excitotoxin, beta-N-oxalyl-L-alphabeta-diaminopropionic acid (L-beta-ODAP), an agonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamatergic receptors. Previously, a limited model of neurolathyrism was reported for the rat. To improve upon the model, we stressed rat pups by separation from their mothers, followed by the subcutaneous L-beta-ODAP treatment, resulting in a 4.6-fold higher incidence (14.0-15.6%) of the paraparesis compared with the prior study. The number and size of motor neurons in these rats were decreased only in the lumbar and sacral cord segments, at approximately 13-36 weeks after treatment. Only lumbar and sacral spinal cord tissue revealed pathological insults typical of physical and ischemic spinal cord injury in the surviving motor neurons. In addition, extensive but transient hemorrhage occurred in the ventral spinal cord parenchyma of the rat, and numerous TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were also observed. In parallel, vascular endothelial growth factor receptor (VEGFR)-2 (Flk-1) levels were significantly lowered in the lumbosacral spinal cord of the paraparetic rats compared with their controls, suggesting a failure of the VEGF system to protect neurons against L-beta-ODAP toxicity. We propose, based on these data, a novel pathological process of motor neuron death induced by peripheral L-beta-ODAP. For the first time, we present a model of the early molecular events that occur during chemically induced spinal cord injury, which can potentially be applied to other neurodegenerative disorders.