Studies on neurolathyrism in Ethiopia: dietary habits, perception of risks and prevention.

This study describes the correlation of traditional perceptions and dietary habits with the incidence of neurolathyrism to propose preventive measures. Therefore, 118 households of South Wollo and North Gondar (Amhara Regional State, Ethiopia), of which one third had at least one neurolathyrism affected member, were interviewed. Most of the affected families in this study had one neurolathyrism victim, being predominantly male and of younger age. The incidence among youngsters (boys and girls) was significantly correlated with the consumption of green unripe seeds (eshet), confirming this as a risk factor for developing neurolathyrism. The consumption of other popular grass pea preparations was not age related. Neurolathyrism patients did not attempt any medication as most people knew that neurolathyrism is incurable, but the consumption of grass pea was abandoned after developing the disease. The minority 'Woito' tribe was virtually unaffected. They were the only people reporting to consume fish which is rich in amino acids such as methionine and using metallic kitchen utensils in addition to clay pots. This observation points to the correlation between low neurolathyrism incidence and a better balanced diet as well as metallic kitchen utensils, suggesting a new approach for neurolathyrism prevention.

Unraveling the mechanism of ß-N-oxalyl-α,ß-diaminopropionic acid (ß-ODAP) induced excitotoxicity and oxidative stress, relevance for neurolathyrism prevention.

ß-N-Oxalyl-α,ß-diaminopropionic acid (ß-ODAP) is a plant metabolite present in Lathyrus sativus (L. Sativus) seeds that is proposed to be responsible for the neurodegenerative disease neurolathyrism. This excitatory amino acid binds to α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and several lines of evidence indicate that ß-ODAP triggers motor neuron degeneration by inducing excitotoxic cell death and increasing oxidative stress. In addition, this toxin is known to disturb the mitochondrial respiration chain and recent data indicate that ß-ODAP may inhibit the uptake of cystine thereby compromising the cells' abilities to cope with oxidative stress. Recent work from our group furthermore suggests that ß-ODAP disturbs the cellular Ca(2+) homeostasis machinery with increased Ca(2+) loading in the endoplasmic reticulum (ER)-mitochondrial axis. In this review, we aim to integrate the various mechanistic levels of ß-ODAP toxicity into a consistent pathophysiological picture. Interestingly, the proposed cascade contains several aspects that are common with other neurodegenerative diseases, for example amyotrophic lateral sclerosis (ALS). Based on these mechanistic insights, we conclude that dietary supplementation with methionine (Met) and cysteine (Cys) may significantly lower the risk for neurolathyrism and can thus be considered, in line with epidemiological data, as a preventive measure for neurolathyrism.

Konzo and dietary pattern in cassava-consuming populations of Popokabaka, Democratic Republic of Congo.

Food frequencies and 24h diet recall were registered from 487 randomly selected heads of household in Popokabaka (Bandundu Province, DRC) where the first cases of konzo were reported more than seventy years ago. Konzo is still occurring in this area with a prevalence of 1.4%. High prevalence of konzo was found to be associated with female gender (P = 0.0024), unmarried status (P = 0.030), illiteracy (P = 0.021), farmer as main occupation (P < 0.05) and with consumption of cassava from own farm land (P = 0.045). The diet was largely dominated by cassava. Luku, cassava flour stiff porridge, was consumed at least once during the day in 99.2% of households. A median of 304 g (max. 592 g; min. 120 g) of cassava flour providing 1070 kcal (max. 2085 kcal; min. 422 kcal)/day/person was used to prepare luku. Saka-saka (pounded cassava leaves) (40%), cowpeas (30%), sesame (23.2%), mbondi (Salacia pynaertii) (18.1%), mushrooms (17.7%) and mfumbwa (Gnetum africanum) (11.3%) were consumed as side-dishes with luku. These results showed that major foods consumed are of poor quality in protein, especially in sulphur containing amino acids.

Cassava cyanogens and free amino acids in raw and cooked leaves.

Cassava leaves (Manihot esculenta Crantz) constitute the main daily source of protein as supplement to the major staple food, the processed cassava roots in remote rural areas of Africa. Konzo, an upper motoneurone disease with permanent spastic paralysis of both legs, has been reported among populations consuming this unbalanced diet. In commercial pounded cassava leaves residual cyanogens and the presence of inherent potentially toxic non-protein amino acids were analysed to check their safety. The initial total cyanogens before cooking ranged from 35.9+/-0.4 to 107.5+/-0.8 mg HCN (hydrogen cyanide) equivalent kg(-1) dry weight. After cooking, the residual cyanogens were significantly reduced (P<0.05) ranging from 0.30+/-0.04 to 1.9+/-0.2 mg HCN equivalent kg(-1) dry weight, and were below the recommended FAO/WHO safe limit set at 10 mg HCN equivalent kg(-1) dry weight. The total free amino acids and trigonelline (N-methyl-nicotinic acid) detected varied from 10.8 g kg(-1) dry weight to 38.2 g kg(-1) dry weight in the raw and from 7.4 g kg(-1) dry weight to 25.6 g kg(-1) dry weight in the cooked cassava leaves. The non-protein amino acids gamma-amino butyric acid (GABA) and alpha-amino butyric acid (alpha-ABA) were detected. No known potentially toxic non-protein amino acid was found. In konzo-affected areas, cassava leaves with inadequate preparation and cooking can be a non-negligible source of dietary exposure to cyanogens apart from the cassava roots that are suggested to be involved in the aetiology of konzo.

Amino acids in seeds and seedlings of the genus Lens.

The amino acid content of seeds and 4-day-old seedlings were studied in five species of lentil: Lens culinaris, L. orientalis, L. ervoides, L. nigricans and L. odemensis. Free amino acid and also total protein amino acid content after HCl hydrolysis were determined by HPLC. The nonprotein UV-absorbing amino acids were determined by capillary zone electrophoresis (CZE). The content of free protein amino acids in seeds varied among species and increased dramatically after germination. Asparagine is quantitatively most important in both seed and seedling. The content of free nonprotein amino acids is variable in seeds and seedlings. gamma-Hydroxyarginine, gamma-hydroxyornithine, alpha-aminobutyric acid and taurine were found in both seeds and seedlings. Homoarginine was found in four species but not in L. orientalis while gamma-aminobutyric acid (GABA), alpha-aminoadipic acid (alpha-aaa) and three isoxazolinone derivatives: beta-(isoxazolin-5-on-2-yl)-alanine (BIA), gamma-glutamyl-BIA (gamma-glu-BIA) and 2-carboxymethyl-isoxazolin-5-one (CMI) were found exclusively in the seedlings. CMI was identified for the first time in lentil species. Lathyrine, beta-(2-amino-pyrimidine-4-yl)-alanine, which was reported to be in the seeds of some Lathyrus species was confirmed to be present also in the seedling of L. culinaris (trace amount), L. nigricans and L. odemensis. Trigonelline (N-methyl-nicotinic acid), a plant hormone, is present both in seeds and seedlings in different concentrations except in L. ervoides. The different combination of nonprotein amino acids among the species gives indication of their genetic relationship and might partly explain the varying compatibility for interspecies crossing.

Nonprotein amino acids in edible lentil and garden pea seedlings.

Commercial edible seedlings of garden pea (Pisum sativum L.) and lentil (Lens culinaris L.) contain high concentration of nonprotein amino acids and trigonelline. Both seedlings grown in the laboratory or purchased in a supermarket were studied by HPLC. Samples from both origins contained trigonelline, alpha-aminoadipic acid, homoserine, beta-(isoxazolin-5-on-2-yl)-alanine (BIA), and gamma-glutamyl-BIA. Garden pea seedlings also contained a uracil-alanine derivative (isowillardiine) in substantial amount. Some of these compounds such as BIA and alpha-aminoadipic acid have neurotoxic activity.

Identification and quantification of natural isoxazolinone compounds by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method that is specific, simple, rapid and also cheap was developed to analyse some natural UV-absorbing isoxazolinone compounds with toxic potential present in legume seedlings. The six most common natural isoxazolinone compounds were separated within 10 min with 25 mM potassium phosphate (pH 7.5) containing 8% 1-propanol as running buffer. A 60 cm coated fused-silica capillary (52.6 cm effective length x 75 microm I.D.), with an electric field of 375 V/cm at 30 degrees C was used. The limit of detection ranged from 0.01 mM (3.0 microg/ml) to 0.03 mM (7.7 microg/ml). Linearity between peak areas and concentrations ranging from 0.05 mM to 1.75 mM were determined for each isoxazolinone. The correlation coefficient was 0.9954 or greater. Both relative migration time and peak area were reproducible. The RSD of relative migration time is between 0.44 and 1.94% and RSD of peak area is between 1.26 and 6.86%. The concentrations of isoxazolinones in Lathyrus odoratus and L. sativus seedlings obtained by CZE were in agreement with the previous results from HPLC.

Free amino acids present in commercially available seedlings sold for human consumption. A potential hazard for consumers.

The importance of fresh seedlings for human consumption on European markets continues to increase. Although the contents of free amino acids and potentially toxic free nonprotein amino acids in these fresh and supposedly healthy seedlings is very different from those of the seeds, the crude composition is never mentioned on commercial packages. A commercial product containing seven different kinds of fresh seedlings including kamut, adzuki bean, chickpea, mungbean, pinto bean, garden pea, and lentil has been analyzed by HPLC. Per 100 g of fresh product, 548.2 mg of total free amino acids was found, of which 56.7 mg is free nonprotein amino acids including beta-(isoxazolin-5-on-2-yl)alanine, homoserine, and isowillardiine and the plant hormone trigonelline (N-methylnicotinic acid). The highest amounts of free nonprotein amino acids and trigonelline are found in garden pea (28.3 mg/100 g), mungbean (9.59 mg/100 g), and lentil (7.50 mg/100 g) seedlings. Trigonelline is present in all legume seedlings examined.

Enzymatic formation of 2,3-diaminopropionic acid, the direct precursor of the neurotoxin beta-ODAP, in Lathyrus sativus.

The enzymatic breakdown of beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA) with formation of 2,3-diaminopropionic acid (DAPA), the direct precursor of the neurotoxin beta-ODAP in Lathyrus sativus, was confirmed in vitro. Some properties of the enzyme responsible for the biosynthesis of DAPA are described.

Epidemic of neurolathyrism in Ethiopia.

After a drought and famine, overconsumption of the drought-tolerant grasspea triggered an epidemic of neurodegenerative neurolathyrism in Northeast Ethiopia. Environmental, nutritional, and medical factors seem to affect the susceptibility.

Evidence of osteolathyrism among patients suffering from neurolathyrism in Bangladesh.

In a study of 500 patients suffering from neurolathyrism in Bangladesh it was found that 60 (all male) complained of bone pain and showed skeletal deformities suggestive of osteolathyrism. On X-ray examination a failure of fusion in both vertebral and iliac epiphyses was found in two patients. At the age of these patients (30 and 37 years) such failure was considered a clear evidence of osteolathyrism. All 60 patients were accustomed to eating the green parts of Lathyrus sativus, which contain 2-cyanoethyl-isoxazolin-5-one, a compound that chemically and metabolically can produce the osteolathyrogen beta-aminopropionitrile (BAPN), as well as foods made from the seeds of the same plant which contain the neurotoxin 3-N-oxalyl-2,3-diaminopropanoic acid (beta-ODAP).

Inhibitory activity of a naturally occurring heterocyclic beta-substituted alanine, beta-(isoxazolin-5-on-4-yl)-L-alanine, on the L-glutamate/L-aspartate transporter (GLAST) expressed in Xenopus oocytes.

Excitatory amino acid (EAA) transporters are of physiological importance in the regulation of the extracellular concentration of excitatory amino acids and the neuroexcitation in CNS. Among four identified transporters, the Na+-dependent high-affinity L-glutamate/L-aspartate transporter (GLAST) is highly expressed in glial cells. Here, we report a naturally occurring inhibitor of GLAST, derived from bovine retina, using the Xenopus oocyte expression system. Beta-(isoxazolin-5-on-4-yl)-L-alanine (TAN), an antifungal antibiotic, inhibited [14C]L-glutamate (L-Glu) transport into GLAST-expressing oocytes. TAN also served as a substrate for this transporter in voltage-clamp experiments measuring the current coupled to the EAA transport. The maximum current of TAN itself was approximately 1/3 of that of L-glutamate, and its apparent affinity was almost the same as L-Glu. In combination with L-Glu, TAN antagonized L-glutamate transport. In radioisotope experiments, the inhibitory potency of this compound against [14C]L-Glu uptake into oocytes was approximately 1/6 of that of L-(-)-threo-3-hydroxyaspartate (THA). The glucoside of TAN (TANG), occurring in seedlings of the garden pea, the lentil and some Lathyrus species, did not show any electrophysiological activity nor was it transported into oocytes. It is proposed that TAN is a novel type antagonist of natural origin on GLAST. By affecting such transport system, naturally occurring compounds may affect the regulation of the extracellular level of endogenous EAA.

Enzymatic synthesis of two isoxazolylalanine isomers by cysteine synthases in Lathyrus species.

Two isoxazolylalanine isomers, beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA, 1) and beta-(isoxazolin-5-on-4-yl)-L- alanine (TAN-950A, 2) were confirmed to be derived from O-acetyl-L-serine (OAS) and isoxazolin-5-one by cysteine synthases (CSases) with a different ratio in different plant parts. Some properties of this enzyme in the biosynthesis of both isomers are described.

Epidemiological study of lathyrism in northwestern districts of Bangladesh.

Two northwestern districts of Bangladesh with a population of 629,752 were surveyed from June 1991 to March 1993 to detect and follow up lathyrism patients. Out of 2,567 neurological patients, 882 were diagnosed as having lathyrism, giving a prevalence rate of 14.0/10,000. This prevalence was higher among young males: only 12.9% of the patients were female, and only 19.3% of the patients were over 30 years of age at onset of the disease. The average family size was 4.6 members. In the surveyed area, 730 families were evaluated: 622 families had only 1 affected member, and 108 families had 2-8 affected members. Most of the patients were working, while only 4% were not. The majority of the patients had a very low intake of animal protein. A few cases of human T-lymphotropic virus infection and osteolathyrism were found during this study.

Effects of ß-ODAP and its biosynthetic precursor on the electrophysiological activity of cloned glutamate receptors.

3-N-Oxalyl-l-2,3-diaminopropanoic acid (ß-ODAP) induces neurolathyrism, a motor neuron disease. To elucidate the pathogenic mechanism of this process, the action of ß-ODAP on the excitatory amino acid (EAA) receptor-mediated currents was examined using cloned EAA receptors expressed in Xenopus oocytes. On the voltage-clamp recordings of an AMPA receptor (α (1)α (2) heterooligomer), ß-ODAP was a strong agonist on this receptor, the potency being almost the same as l-glutamate. On the other hand, ß-ODAP had little effect on the glutamate-evoked currents through the expressed NMDA receptor (NR1(A)/NR2A), but showed a weak inhibitory effect on the glycine-modulatory site. ß-ODAP may cause the neurodegenerative disease, neurolathyrism, mainly through the excitotoxic interaction with AMPA receptors.

Inhibitory and excitatory amino acids in cerebrospinal fluid of neurolathyrism patients, a highly prevalent motorneurone disease.

Neurolathyrism is caused by overconsumption of seeds containing 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP). Amino acids levels of cerebrospinal fluid (CSF) were studied in 50 patients with neurolathyrism and 12 healthy volunteers. The levels of excitatory amino acids glutamate and aspartate were 281% and 71% respectively of control values. The concentration of inhibitory amino acids glycine and taurine were 277% and 198% respectively of the levels in CSF from control individuals. There was a significant correlation between the level of glycine and the duration of the disease. We also found increased levels of threonine, serine and alanine. In contrast to reports on other motor neurone diseases where an increase of isoleucine was observed we found a significant decrease of isoleucine. The results suggest a disturbance of amino acid metabolism due to excitotoxic damages caused by beta-ODAP, a dietary excitatory amino acid.

Study of HTLV-I antibodies in CSF and serum of neurolathyrism patients in Bangladesh.

Neurolathyrism is a form of human spastic paraparesis related to the overconsumption of the legume Lathyrus sativus or grass pea (Khesari in Bangladesh) containing the neurotoxin 3-N-oxalyl-2,3-diaminopropanoic acid (beta-ODAP). The clinical symptoms of neurolathyrism are similar to those of Tropical Spastic Paraparesis. In order to eliminate the proposed causative agent of TSP (HTLV-I) as a potential cause of the symptoms ascribed to neurolathyrism, a total of 444 diagnosed lathyrism patients were screened for HTLV-I antibodies. 50 CSF and 394 serum samples were collected from male (415) and female (29) patients. Only 4 serum samples were found sero-positive for HTLV-I. This agrees with the assumption that overconsumption of beta-ODAP containing Lathyrus seeds, and not HTLV infection, is the causative agent for neurolathyrism.

Interaction of some plant heterocyclic beta-substituted alanines with rat brain N-methyl-D-aspartate (NMDA) receptors.

The neuropharmacological actions of some plant heterocyclic beta-substituted alanines on rat brain N-methyl-D-aspartate (NMDA) receptors were studied. Of the compounds tested, 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP), the causal agent of human neurolathyrism, exhibited an inhibitory activity on the NMDA receptor binding assay at a relatively high concentration (IC50: 4.7 x 10(-5)M). The biochemical precursor of beta-ODAP, beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA) was inactive in this assay. These results suggest that beta-ODAP, the neurotoxin of Lathyrus sativus, in addition to its excitatory action on alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptors, also has neurotoxic potential through its action on NMDA receptors.

In vitro toxicological investigations of isoxazolinone amino acids of Lathyrus sativus.

Two non-protein amino acids of Lathyrus sativus, beta-(isoxazoline-5-on-2-yl)-alanine (BIA) and its higher homologue alpha-amino-gamma-(isoxazoline-5-on-2-yl)-alanine (ACI) were tested for excitotoxic potential. BIA (0.5-2.0 mM) but not ACI (2.0 mM) produced a concentration-dependent neurodegeneration in mouse cortical explants. The neuronal damage was prevented by the prior and simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), indicating that it was mediated by non-N-methyl-D-aspartate type receptors. BIA (0.5-2.0 mM) activated CNQX-sensitive currents which were significantly smaller than those activated by 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the majority of neurons. In a small number of cells, BIA (2 mM) produced currents which were similar in amplitude to those activated by beta-ODAP (50 microM). These results suggest that Lathyrus sativus plants engineered to block the synthesis of beta-ODAP may accumulate a neurotoxic precursor and therefore must be tested for the presence of both BIA and beta-ODAP.

From soil to brain: zinc deficiency increases the neurotoxicity of Lathyrus sativus and may affect the susceptibility for the motorneurone disease neurolathyrism.

Zinc deficiency and oversupply of iron to the roots of grass pea (Lathyrus sativus) induce increases in the content of the neurotoxin beta-L-ODAP (3-oxalyl-L-2,3-diaminopropanoic acid) in the ripe seeds. The transport of zinc to the shoots is enhanced by the addition of beta-L-ODAP. The neurotoxin of L. sativus is proposed to function as a carrier molecule for zinc ions. Soils, depleted in micronutrients from flooding by monsoon rains (Indian subcontinent) or otherwise poor in available zinc and with high iron content (Ethiopian vertisols), may be responsible for higher incidence of human lathyrism, one of the oldest neurotoxic diseases known to man. A role for brain zinc deficiency in the susceptibility for lathyrism is postulated.