WendyGruber Independent Research

Presences of Neurotoxin ODAP in Hedysarum Alpinum and Hedvsarum Mackenzii seeds

Introduction The goal of this research is to determine if ODAP (p-N-Oxalyl-L-a,pDiaminopropionicacid) or BOAA (p-N-Oxalylamino-L-alanine) is present in the Lathynrsspecies of Hedysarum Alpinum and Hedysarum mackenzii.The presence of ODAP could be causingtheseseeds to be toxic. ODAP is a known toxin in Lathynrs sativusseeds. It was thoughtthat it could also be presentin Hedysarum alpinum and Hedysarum mackenzii. Therewas onetest that was conducted looking for ODAP if the geniusof Hedysanrm for four differentspecies but they studydid not state which species it examined They also found no suchODAP in the 4 species of Hedysarum that they tested(Quereshi, 1977). The first stepin this process was research based on Lathyrusseeds andthe amino acid in question, ODAP. I lookedto seeif it was possiblefor our lab to purify ODAP from Lathynrssativusseeds. This was donebecause we were unsureif we could get a pure sample of ODAP. I alsolookedto seeif othercompanies had ODAP in which we could purchase.We found that therewas no company that sold theseseeds, but one of the main researchers that dealswith ODAP, Dr Rao, has'a small companythat makes ODAP so we boughta smallsample from him. Therefore, we no longerhadto purify the ODAP from seeds.From the research dealingwith purifying ODAP from seeds I found that manyof the procedures calledfor a largenumberof seeds to makethe ODAP. The underlying theorybeingthat thereis not muchODAP in eachseed.

The next thing I researched was the separation of amino acidsvia differentTLC methods.I combined oneof Rao's separation techniques with a standard technique found in Basic BiochemicalMethods. I ran two separate gelsthat dealt with different amino acids and comparedthese to our standardODAP sample. I found that there was no commonaminoacidthat appeared at the same spotasODAP on the siliconplate. I looked for different meansto separate the seedsand look for the presence of ODAP. Duringthis process with the mechanisms I hadavailable to me TLC experiments look to be the bestoption. Therewere two differentmethods that lookedplausible. The first was the extractionof s and p-N-Oxalyldiaminopropionic acid via Harrison,Nunn, and Hill methodand an extractionmethodfrom Dr. Rao. Both of theseprocesses are described in the experimentationsection of my lab report. Over the time of

experimentation I foundthe Harrison,Nunn, andHill methodto give the bestresults. So that method was used with the unknowns Hedysarum alpinum and Hedysarum mackenzii. I havestarted by taking a purchased sample of ODAP andrunningTLC platesto seeif it hasthe sameRf as other commonlyknown amino acids. I attempted to extract the aminoacidsfrom the two seeds in question andthenI compared these to the seed that is knownto haveODAP,Lathynrssatiws, asmy positivecontrol. I alsousedseeds, such as Ferry Morse Peaswandeseeds as my negativecontrol'to makesureno such'ODAP' spotis present in them. Conclusion From my methods of analysisit appears that thereis ODAP presentin Lathyrus sativus. It also appears that there is ODAP in both of the unknownsI was testing,

Hedysarum alpinumandHedysarum mackenzii. This is controversial if the test downby testedthe species of Hedysarum that I tested. It appears that Rao's methodof Quereshi, purificationof ODAP from the LathynrsSativusseeds is not quite as definitive as the Harrison,Nunn, andHill methodin my analysis.In the ODAP standard I usedNaHCO: to make it more soluble.So in my Lathynrs sativussampleI used NalICOr in both preparations to seeif it offeredany difference in Rfvalues. To be ableto saythat ODAP is definitely presentin the seeds we would needto u$e anotherdimensionof analysis probablyby HPLC-MS so we couldlook at the actualmassof the aminoacidsthat were eluting out. This way we could say with more confidence that ODAP is presentin Hedysarum alpinumandHedysarum mackenzii.

LiteratureCited: l. Quereshi, M. Yasin,David J. Pilbeanq ChristineS. Evans,andE. Arthur Bell.Ihe Neurolathyrogen, a-Amino-B-Oxalylaminopropionicacid in legume seeds. Phytochemistry,1977, Vol. 16,pp. 477-479. tr' ( rtn) Ht,-,u{ | Pftyro<+Jeh E-TfLi 16 rul l- / Btoctlftm,sTFi it 93L- </34

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