This presentation was given at the Konzo - lathyrism workshop 21-22.9.2009 at the University of Ghent, Belgium.

1. A SUITABLE BIOASSAY IS NEEDED TO MODEL LATHYRISM IN MONO-GASTRIC ANIMALS Dirk Enneking School of Food and Agriculture, University of Adelaide, Waite Agricultural Research Institute, Waite Rd. Glen Osmond , 5064 South Australia (Australia).

5. Global Production 1-1,5 Mio ha 0.7 - 1 Mio t of grain +/- forage Also in Chile Canada China Australia Food & Feed No problem with toxicity

6. Mitchell, R. D. (1971). Ass Pacific Coast Geogr Yearbook 33: 29-46 % poor 53 57 62 66 62 63 51

9. Neurolathyrism is intricately linked to famine (due to drought or conflict), poverty and malnutrition .

13. Ortiz de Landazuri (1944) “ in the presence of a protein poor diet (2000 kilo calories/day), 70 g/day of Lathyrus sativus do not produce lathyrism. Up to 300 g of L. sativus can be consumed/day in the presence of protecting factors derived from better quality food (meat, cheese, milk) without provoking lathyrism. A higher dose of L. sativus leads to lathyrism even in the presence of protecting factors.”

14. Nutritional neuropathies Hong-Kong POW camp, 756 cases of peripheral neuropathy (Smith, 1946) B-complex vitamins helped to alleviate symptoms Smith, D. (1946). Nutritional neuropathies in the civilian internment camp, Hong Kong, January 1942—August 1945. Brain 69:209–22 Spillane, J. D. and Scott, G. (1945). Obscure neuropathy in the Middle East. Report on 112 cases in prisoners-of-war. The Lancet (6366)261-264

15. Malnutrition is also an important factor associated with other neuropathies, Konzo, optic neuropathy etc.

16. “ It demonstrates the sensibility of certain structures of the nervous system to various agressions when there exists a basic level of undernutrition ” (Moya et al. Rev. Hyg. San. Hig. Publ. 1967)

17. Chronic undernutrition Wasting: Mobilisation of nutrient reserves Stunting: lower height for age due to prolonged wasting Marasmus: severe malnutrition, no reserves left

19. Which nutrients are in short supply when a monotonous diets of grasspea or incompletely processed cassava is consumed for extended periods of time ? Which animal bioassay allows us to model neurolathyrism and Konzo?

20. Lathyrism in animals Neurolathyrism in animals Lathyrism in the horse hindleg paralysis and and hoaring. Death occurs due to asphyxiation. Large animals such as the horse are more frequently affected than small animals (Maleval, 1927)

21. Neurolathyrism in animals. Numerous studies feeding grasspea to a variety of animals have produced mixed results (Fumarola and Zanelli, 1914)*. Geiger et al. (1933) suggested that the differences in susceptibility of individual animals is related to the quality of the base diet used in these experiments. =>a poor quality base diet is likely to succeed better than an optimally balanced one to produce neurodegenerative symptoms *Fumarola, G. and Zanelli, C. F. (1914). Anatomische experimentelle Forschungen ueber den Lathyrismus. Archiv Fuer Psychiatrie Und Nervenkrankheiten 54 (2)489-536

23. Deficiencies in B vitamins B1: polyneuritis, beriberi B6: not well understood; convulsions, peripheral neuropathy, secondary pellagra B6 Sources: whole grains, potatoes, green vegetables, maize, liver, red meat

24. B12 Important for all cells, particularly the nervous system. Deficiencies lead to subacute degeneration of the spinal chord. It is not found in significant amounts in plant foods but can be sourced from animal products (eggs, dairy, meat, liver, kidney, heart) The B complex in general is involved in red blood cell formation , the normal functioning of the gastrointestinal tract and the metabolism of protein . Sources: dark-green vegetables, legumes , whole grain, yeast, heart and pancreas.

25. Trace elements Copper Zinc Iron

26. López Aydillo, N. R. and Toledano Jiménez Castellanos, A. (1968) Trab. Inst. Cajal. Invest. Biol 60157-190 L. Sativus| missing

28. López Aydillo, N. R. and Toledano Jiménez Castellanos, A. (1968) Trab. Inst. Cajal. Invest. Biol 60157-190 Totally Adjusted diet Per 100g L. sativus flour fed as pellets

29. López Aydillo, N. R. and Toledano Jiménez Castellanos, A. (1968) Trab. Inst. Cajal. Invest. Biol 60157-190 % survival Male Female Control 100 100 Mixed L.s. (control + L. s.) 93.3 100 Total Adj L.s. (100 %L.s. + olive oil, salt, vitamins, casein, aminoacids+ 40 80 Total Adj L.s. No olive oil 0 0 Total Adj L.s. No salt 20 0 Total Adj L.s. No vitamins 40 0 Total Adj L.s. No casein 0 40 Total Adj L.s. No aminoacids 40 80

31. Legume seeds are generally deficient in sulfur amino acids Exceptions: Vigna (cowpea) & Acacia spp. glutamyl-S-methyl-cysteine sulphoxide Vicia (V. narbonensis et al.) glutamyl-S-ethenyl-cysteine

32. Legume defence strategy: Deplete reduced thiols

33. Sulfur hypothesis: Concerted action of legume antinutritional factors leads to depletion of reduced thiols in their predators

34. Example: Vicia sativa Beta-cyanoalanine inhibits conversion of methionine to cysteine (source of reduced thiols). Gamma-glutamyl-betacyanoalanine ends up in the brain as analog of glutathione (glu-cys-gly) Impairment of glutathione metabolism!

35. Vicine oxidises reduced thiols in red blood cells. Lack of sufficient reduced thiols –> hemolysis (favism in individuals lacking glucose 6-P-dehydrogenase in their red blood cells) = genetically susceptible individuals are affected!

36. Protease inhibitors [high S aa content]* bind with proteases in the digestive tract. Increased biosynthesis of proteases by the pancreas leads to pancreatic hypertrophy. => Depletion of sulfur amino acids + other aa Other enzyme inhibitors (alpha amylase i., lipase i. etc.) are likely to have similar effects. *TI in lima and navy beans 2.5% of total, 32-40% of total cystine (cys-cys) Lajolo & Genovese (2002) J. Sci.Fd.Chem. 50, 6592-6598

37. Vicia sativa continued: Tannins also bind digestive enzymes Cyanogenic glycosides vicianine and prunasine release cyanide Ressler & Tatake, (2001) J. Agric. Food Chem. 49, 5075-5080 Cyanide is detoxified by thiosulphate derived from sulfur amino acids -> A concerted attack on reduced thiols

38. Chowdhury, D.; Tate, M. E.; McDonald, G. K., and Hughes, R. (2001). Towards reducing seed toxin levels in common vetch (Vicia sativa L.). Proceedings of the 10th Australian Agronomy Conference, in Hobart Australian Society of Agronomy. http://www.regional.org.au/au/asa/2001/5/c/chowdury.htm Vetch % 12.5 25 12.5% vetch + 12.5 % L. cicera 25% Lathyrus sativus

39. Their anti-nutritional factors deplete sulfur amino acids in predators Many target glutathione (glu-cys-gly) dependent systems (reduced thiols)

40. Lathyrus sativus seeds contain: Low levels of sulfur amino acids Trypsin inhibitors [high SAA] Tannins Lectins ODAP Homoarginine Phytate

42. ODAP inhibits tyrosine amino transferase (SLN Rao) -> L-DOPA and its metabolites accumulate, These are O-methylated This requires S-adenosylmethionine => Depletion of sulfur amino acids

43. Hypothesis: A lack of reduced thiols predisposes to lathyrism This is caused through depletion by antinutrients, low intake of SAA, oxidative stress and/or genetic defect (s) in thiol metabolism

44. Animal model of toxic nutritional neuropathy Poor base diet Toxin Oxidative stressors Sulfur amino acid deficiency/ depletion by inhibitors B complex vitamin deficiencies Energy depletion Lipids, Micronutrients +/- genetic knockout models

45. Conclusion Neurolathyrism and Konzo are intricately linked to nutritional deficiencies. To model these and other nutritional neuropathies, a malnutrition animal model is needed. Legumes deplete sulfur amino acids (saa) in their predators, suggesting that adequate saa supplementation protects against neurolathyrism. Additional neuroprotective nutrients ie. olive oil may also play a role. Genetic animal models may help to dissect the underlying mechanisms.

46. Eradicate lathyrism ! Reduce toxicity (ODAP) in cultivars and provide adapted non-toxic varieties to grasspea eaters Manage nutritional stress (famines, poverty) & excessive grasspea consumption

47. Identify undernourished grasspea eaters during times of hardship Monitor grasspea production, consumption and prices Educate about processing options (detoxification) + supplements