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).

Introduction Nutritional neuropathies Malnutrition The need for an animal bioassay to model neurolathyrism Legume defence depletes sulfur amino acids

Introduction

Nutritional neuropathies

Malnutrition

The need for an animal bioassay to model neurolathyrism

Legume defence depletes sulfur amino acids

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

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

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

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.”

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

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

“ 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)

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

grasspea eaters generally appear well nourished are easily missed by conventional nutritional surveys ? Specific form of biochemical wasting

grasspea eaters generally appear well nourished

are easily missed by conventional nutritional surveys

? Specific form of biochemical wasting

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?

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)

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

B group vitamins are all co-enzymes. They comprise: B1 thiamine, aneurine B2 riboflavin B3 niacin, nicotinic acid B6 pyridoxine B12 cobalmin, cyanocobalmin Biotin, folic acid, pantothenic acid B group vitamins and vitamin C are water soluble and not stored in sufficient quantities in organisms Insufficiences Vitamins A,D,E, and K are all fat soluble and stored easily Less likelyhood of insufficiencies.

B group vitamins are all co-enzymes. They comprise:

B1 thiamine, aneurine

B2 riboflavin

B3 niacin, nicotinic acid

B6 pyridoxine

B12 cobalmin, cyanocobalmin

Biotin, folic acid, pantothenic acid

B group vitamins and vitamin C are water soluble and not stored in sufficient quantities in organisms

Insufficiences

Vitamins A,D,E, and K are all fat soluble and stored easily

Less likelyhood of insufficiencies.

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

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.

Trace elements Copper Zinc Iron

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

Normal laboratory diet – this is the test diet (P. B.). oat mixed with milk (or milk soup. Mixed diet: this diet is composed of 2g blue vetch L. sativus flour plus 5g milk soup per rat per day. (milk soup contains approx. 1.33g bread and 3.67g milk). totally adjusted diet (100% L. sativus + fed as pellets: olive oil, minerals, vits, casein and amino acids) Partially adjusted diet Adjusted diet, lacking of olive oil. Adjusted diet, lacking of mineral salts. Adjusted diet, lacking of vitamins. Adjusted diet, lacking of proteins Adjusted diet, lacking of amino acids .

Normal laboratory diet – this is the test diet (P. B.).

oat mixed with milk (or milk soup.

Mixed diet: this diet is composed of 2g blue vetch L. sativus flour

plus 5g milk soup per rat per day.

(milk soup contains approx. 1.33g bread and 3.67g milk).

totally adjusted diet

(100% L. sativus + fed as pellets: olive oil, minerals, vits,

casein and amino acids)

Partially adjusted diet

Adjusted diet, lacking of olive oil.

Adjusted diet, lacking of mineral salts.

Adjusted diet, lacking of vitamins.

Adjusted diet, lacking of proteins

Adjusted diet, lacking of amino acids .

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

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

The results of laboratory diet were normal b) Anatomical hurts were more important and extensive proceeding from exclusive diet flour and total and partial adjusted diets c) They were extensive and steady subarachnoid, pial and subpial hemorrhages (excepting in those with salt depletion); demyelinisation, specially at the Corpus callosum and the white matter of the small cerebellar lobes; both Bergmann's and Alzheimer's ameboid types of gliosis d) Neuronal changes overweighed in Ammon's horn area (Fascia dentata and Stratum pyramidale) e) The hemorrhagic condition as a whole was similar to that of human internal hemorrhagic pachymeningitis f) Morphological changes had no resemblance with those of deficiency diseases , because they were limited to a Nissl's chronic disturbance pattern g) Exclusive daily food of blue vetch peas flour (Lathyrus sativus) as well as added with another complementary food (mixed diet) results in an endemic lathyritic condition. It is in strong nutritional indigence of exceptional times that such exclusive diet of flour originates the classic epidemical lathyrism h) At present, both the clinical observations and the biochemical findings (Adiga's, Murti's, Rao's and Bell's tests) as well as our histopathological data, all support the toxic etiology of human lathyritic disease.

The results of laboratory diet were normal

b) Anatomical hurts were more important and extensive proceeding from exclusive diet flour and total and partial adjusted diets

c) They were extensive and steady subarachnoid, pial and subpial hemorrhages (excepting in those with salt depletion); demyelinisation, specially at the Corpus callosum and the white matter of the small cerebellar lobes; both Bergmann's and Alzheimer's ameboid types of gliosis

d) Neuronal changes overweighed in Ammon's horn area (Fascia dentata and Stratum pyramidale)

e) The hemorrhagic condition as a whole was similar to that of human internal hemorrhagic pachymeningitis

f) Morphological changes had no resemblance with those of deficiency diseases , because they were limited to a Nissl's chronic disturbance pattern

g) Exclusive daily food of blue vetch peas flour (Lathyrus sativus) as well as added with another complementary food (mixed diet) results in an endemic lathyritic condition. It is in strong nutritional indigence of exceptional times that such exclusive diet of flour originates the classic epidemical lathyrism

h) At present, both the clinical observations and the biochemical findings (Adiga's, Murti's, Rao's and Bell's tests) as well as our histopathological data, all support the toxic etiology of human lathyritic disease.

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

Legume defence strategy: Deplete reduced thiols

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

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!

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!

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

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

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

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

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

ODAP Competes with Cysteine for uptake Causes oxidative stress depletion of reduced thiols Inhibition of reduced thiol replenishment

ODAP

Competes with Cysteine for uptake

Causes oxidative stress

depletion of reduced thiols

Inhibition of reduced thiol replenishment

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

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

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

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.

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

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