New Technologies for Toxin Analyses in Food Legumes.

Peter C.H. Eichinger' ,Ncil E. Rothnie', Ian Dclaerc2 and Max E. Tate2
1. Agricultural Chemistry Laboratory, Chemistry Centre (WA), 125 Hay St., East Perth, WA, 6004; 2. Department of Plant Science, University of Adelaide, Waite Campus, P.O. Box I,Glen Osmond, South Australia, 5064

Abstract

A key requirement for agricultural research is access to rapid, accurate and inexpensive analyses. This is particularly the case, once desirable crop traits are identified and plant breeders attempt to translate these results into high yielding crops having favourable characteristics. The utility of capillary electrophoresis and diffuse reflectance mid-infrared spectroscopy as tools for rapid analyses of value to plant breeding projects will be described.

INTRODUCTION
In conjunction with the Cooperative Research Centre for Legumes in Mediterranean Agriculture (CLIMA), and the University of Adelaide, we have been involved in screening biologically active components of grain legumes. A range of Vicia and Lathyrus species has been evaluated for anti-nutritional factors (ANF's), with particular emphasis on those crops likely to have potential in the dry marginal areas of the Australian wheat belf which typically has poor soils. Vicia and Ijzthyrus spccies have evolved a wide range of antiprcdator ANF's in their seeds. Hie low Mwt amphiphilic analytes discussed in this paper are: p-Oxalyl-diamino-propionic acid (p-ODAP), [synonym (3-Oxalyl-amino-L-alanine (BOAA)] L-Homoarginine, a homologue of L-arginine Lathyrine, an aromatic guanidine, structurally related to L-homoarginine Y Glutamyl-S-Fthenyl-cysteine (GEC) - a dipeptide L-Canavanine, an analogue of L-arginine y-glutamyl-p-cyano-L-alanine and p-cyano-L-alanine Vicine and Convicine (pyrimidine glycosides) These polar analytes, usually analysed by high performance liquid chromatography (HPLC), are also amenable to quantification using a range of capillary electrophoretic techniques. The advantages of capillary electrophoresis for the quantification of these Lathyrus and Vicia analytes, over HPI.C, will be described. The application diffuse reflectance mid-infrared spectroscopy to'the rapid quantification of nitrile toxins in Vicia saliva will be outlined. Lathyrus Species Lathyrus spccies have been consumed for thousands of years in many areas of the world, being considered in early times as an aphrodisiac - its Latin name being 'a stimulant'. Unfortunately, many Lathyrus species are by no means harmless and contain the known neurotoxin, P-Oxalyldiamino-propionic acid (P-ODAP). f We have now used CE analysis for a range of Lathyrus species, most notably Uithyrus sativus (grass pea), lxithyrus ochrus (cypress vetch), Lathyrus cicera (flat pod pea vine) and Lathyrus clymenitm (Spanish vetch). All these species are able to thrive under conditions of waterlogging
685 R. Knight ted.). Linking Research and Marketing Opportunities for Pulses in the 21st Century. 635-692. (c) 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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such as rice paddies or duplex soils and are also drought tolerant. They arc resistant to insects, fungal diseases and in general they are exceedingly hardy plants. However, prolonged ingestion of the seed by humans, especially when it is the sole food sourcc under famine situations, has been associated with the degenerative neurological disease known as Lathyrism. This disease is characterised by a type of paralysis, which leaves the individual (usually male) crippled, with devastating consequences for both the victim and the immediate family. The problem of lathyrism is well documented. The analytical data of bodily fluids, obtained from a group of volunteers consuming cooked Lathyrus, was reported by Nunn et al (1995). Breeding progams require a simple, accurate and rapid analytical protocol if they arc to produce locally adapted cultivars of Lathyrus sativus without significant levels of the lathyrogenic agent (3-ODAP (1) in their seed.

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In the past, the most convenient method has been to derivatise a milled seed extract with fluorenylmethoxy chloroformatc (FMOC-C1) and quantify the [i-ODAP (1) adduct using HPLC. At CLIMA, the method of Geda et al. 1993, required a total run time of 50 minutes. Furthermore, re-equilibration of the column, required an additional 15-20 minutes. So although the procedure was accurate, such a slow throughput time was unacceptable for a preliminary screening of the large ICARDA Lathyrus germplasm collection, as also was the cost of the acelonilrile solvent. Bccause (3-ODAP is a polar non-protein amino acid derivative, whose charge is pH dependent, it was recognised (Arentoft et al, 1995) that it was ideally suited for capillary electrophoresis (Figure 1). Injection to injection time was reduced to 10 minutes and between batch reproducibility was 3.2% (c.f. 8.2% reported for IIPLC). With CE it was possible to increase the sample throughput six-fold! Most importantly, the labour intensive FMOC derivatisalion was no longer required.

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Kippunc Acid

40 30

20
10
0
bcta-ODAP

Jvji^wT
4 Min.

Figure I. Electropherogram of Lathyrus sativus. Conditions: pH = 7.80 0.05 ; 20mM sodium phosphate, bare fused silica 46mm x 50mm (effective length 40 cm), Voltage = +25kV, Inject 3.0sec (50mBar). The pH conditions used for the capillary electrophoresis are important and poor peak shape is encountered if the pH is not buffered within narrow limits. Furthermore, in this mode, preconditioning of the capillary with I .ON sodium hydroxide should be avoided, because the peak shape is distorted.

Variation in p-ODAP amongst Lathyrus Accessions (ICARDA)

No. of Accessions

Lathyrus Species

I Seti'olms L. A n n u j g

L. Sa:.u.s I Ochius L. Psejdocice'a

L. Cicera

[%Beta-ODAP] Figure 2. Frcqucncy distribution of [5-ODAP (I) concentrations amongst Lathyrus species ex ICARDA collection (Aleppo, Syria)

L-Homoarginine Yusuf et al (1995) have reported that L-homoarginine (2), a homologue of L-arginine confers some protection against the debilitating effects of Lathyrism in chickens.

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COOH The amino acids in Lathyrus spp. have previously been separated by conventional ion exchange chromatography HPLC and then derivatised on-line using ninhydrin. However, in both speed and resolution, CE in the micellar clectrokinetic chromatography (MEKC) mode has distinct advantages over HPLC. In this technique for L-homoarginine, very similar conditions are used to the electrophorctic method described for p-ODAP, but a surfactant, namely sodium cholate is added (data not shown). The results of the homoarginine analyses are plotted against the results for p-ODAP (Figure 3) for the Lathyrus species from the ICARDA germplasm.

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o L . annuus L. setifolius I . clymenum x L. pseudocicera L. ochrus o L. cicera - L. sativus

20

30

40

50

60

I%Homoarginine(x100)]
Figure 3. The Variation of % p-ODAP (xlOO) against % L-Homoarginine (xlOO) tor selected Lathyrus spccics from (he ICARDA Germplasm Bank.

While we have yet to compare the data for ail ICARDA accessions, it is clear from Fig 3, that in addition to having lower mean levels of p-ODAP than L. clymenum, L ochrus,and /,. setifolius, the mean levels of homoarginine are particularly high for L. cicera and higher still for L. pseudocicera. In light of the suggestions of Yusuf et al (1995) concerning the protective effect of L- homoarginine against the toxicity of P-ODAP, it would seem that the potential of L. cicera and L. pseudocicera for animal nutrition are worthy of further investigation. What is the source of L-homoarginine? In L. tingitanus, biochemical pathways have been identified (Brown and Al-Baldawi 1977), which confirm the reversible intcrconversion of lathyrine (3) and homoarginine (2) and its 4-hydroxy derivative. However, the evidence provided by these workers suggests that homoarginine arises from the ring opening of lathyrine derived via the orotate pathway from a preformed pyrimidine rather than Lhomoarginine acting as a precursor for lathyrine (3)

^ (3)

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Vicia Specics
Narbon bean (Vicia narbonensis) is unique amongst grain legumes in being high in sulfur. Unfortunately, much of this sulfur is bound in an anti-palatibility factor recently identified as y-glutamyl-S-ethenylcysteine (GEC: 4) Enneking et al (1998).

COOH

The narbon bean suppresses feed intake of both pigs and poultry , and when it is fed to dairy cows the milk becomes tainted. The purified crystalline y-glutamyl-S-ethenylcysteine dipcptide (4) produces a most disagreeable taste in the mouth. Furthermore, the content of yglutamyl-S-ethenylcysteine in the narbon bean has recently been correlated with feed intake inhibition in pigs (Enneking ct al 1998, personal communication). Currently, gcnetic and environmental factors contributing to the partitioning of sulphur into storage proteins and GEC are being examined at CLIMA. Studies of sulfur pathways in transformed V. narbonensis have also been reported by Saalbach et al (1995).
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Figure 4. Cli fractionation of GEC(4) and Hippurie acid standards The availability of crystalline GEC (4) has permitted the development of a rapid capillary electrophoresis method (Fig 4). The procedure for extracting the dipeptide, and the electrophoretic conditions arc essentially identical to those used for P-ODAP, cxccpt that a much wider range of pH can be tolerated during the capillary electrophoresis, without loss of peak shape. Under optimal conditions, the resolution efficiencies for GEC and hippurie acid are 185,000 and 109,000 theoretical plates respectively. The UV spectrum of the S-ethenyl moiety of GEC shows a characteristic shoulder at 220nm (Enneking et al 1998) and with a diode array detector, this feature can be used to confirm its eiution time. During a typical batch, the migration time varies < 3 % . The hydrolytic stability of pure GEC in aqueous ethanol is quite high, with <2% decomposition occurring over 36 hours, in a carousel at >28C. [Crude GEC is somewhat less stable]. However, GEC is unstable under acid conditions (0. IN HCI). Observed GEC concentrations in the Narbon bean seed samples have varied from 1.7 - 3.2% DW. For a given narbon bean accession, at a particular site, the amount of GF-C produced shows a good correlation with the amount of added total sulfur (as measured by ICP-MS).

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L-Canavanine: Several vetches, including Vicia ervilia, V. benghalensis and V. villosa have been used as either forage or grain for livestock. However under a variety of circumstances, livestock losses through vetch toxicosis or vetch associated diseases have occurred and the presence of L-canavanine in the ingested green matter or grain has been implicated. L-Canavanine (2.2%) in V. viltosa grain is a potent feed intake inhibitor for pigs (Enneking et al 1993). L-Canavanine (5) is an analogue of arginine, in which the 5 methylene group has been replaced by an oxygen atom. Its close similarity to arginine makes it a suitable substrate for the enzyme arginyl /-RNA synthase and consequently numerous proteins have been identified where this residue has been introduced. However, replacing the methylene group with oxygen causes a dramatic change in the pKa of the guanidine moiety, from pKa = 12.48 in arginine to 7.04 in canavanine (Boyar and Marsh 1982) with significant consequences for the shape and properties of proteins incorporating canavanine in place of arginine. Canavanine(S) undergoes a facile base catalysed cyclisation to deaminocanavanine (6).

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This creates difficulties in derivatisation under basic conditions (eg dansylation and FMOCCl). In HPLC underivatised L-canavanine cannot be quantified by UV absorption in most commercial instruments, because its UV absorption at or above 205nm is negligible. In contrast, fused silica capillaries used for electrophoresis are transparent to 195nm. Quantification of underivatised L-canavanine has been accomplished down to 0.01% using untreated capillaries, with migration times for canavanine (1.05 min) and benzylairiinc standard (1.35 min). This method again meets the plant breeder's goals of being rapid, robust and accurate.

Faba Bean (Vicia Faba) Favism is associated with the ingestion of faba beans. It only affects a small proportion of the population, principly amongst peoples of Mediterranean origin who carry a defective gene for glucose-6-phosphate dehydrogenase. Vicine and convicine (pyrimidine glucosides) in the faba have been associated with the development of the haemoiytic disease known as Favism and the HPLC analysis has been well documented by Marquadt and Frohlich (1981). In the current work, ethanol (60%) extracts of faba bean, with added hippuric acid as an internal standard, have proved satisfactory for separating the pyrimidine glucosides by CE. These components can be readily quantified because of their intense and characteristic UV spectra near 270 nm.
\

Common Vetch (Vicia sativa)

Vicia sativa extracts contain the neurotoxins P-cyanoalanine (7) usually <0.1% and its yglutamyl derivative (8) near 1.1%. These can readily be identified cither by HPLC (Ressler

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et al 1997) or by capillary electrophoresis in borate buffer as their FMOC derivatives in the MEK.C mode. However for screening seeds for toxin content, the most effective quantitative procedure takes advantage of the nitrile (-CN) absorbance band at 2251cm which can readily be detected using diffuse reflectance mid infrared spectroscopy. As little as 3 mg from the drillings of a single seed is all that is required. Using this technique we have screened over 3000 accessions including the entire Vicia sativa collection from ICARDA. It has also been used to demonstrate the unconscionable substitution of Blanche flew vetch for lentils (fig 5) in the markets of Bangladesh. Full details of these techniques for Vicia sativa,

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NITRILE (-CN) ABSORBANCE

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Absorbance (V.sativa) Absorbance (Lens culinaris)

W a v e n u m b e r (cm-1)

Fig 5 Comparison of the infrared nitrile absorbance al 2251cm'1 from a sample (morphologically indistinguishable fom Australian Blanche fleur vetch) containing 1.34% y-jlutarnyl P-cyanoalaninc(S) which was sold as "Brazil lentils" in a market (Dec 1996) in Bangladesh and a genuine lentil sample.

CONCLUSIONS
Capillars' Electrophoresis and diffuse reflectance mid infrared spectroscopy (not to be confuscd with NIR), have proven to be powerful techniques which provide rapid analyses for a wide variety of biologically important components (NPAA's, ANF's) in legumes. The analyses are robust, require only limited sample preparation and time consuming dcrivatisation procedures are usually not required. The cost of consumables is low and the use of hazardous HPLC solvents is avoided, making these "green" technologies. For the plant physiologist, we arc able to measure analytes in small volumes (<0.2mL) of phloem (or xylcm) sap thereby aiding an understanding of ion transport processes. Potential future, applications include single-seed varietal identification and various food "quality" indicators for correlation of analyte profiles with cooking quality and/or metabolisable energy, etc. Clearly, the power of these techniques remains to be fully exploited by the plant breeding community.
Acknowledgements: We wish to acknowledge financial support (in part) from CLIMA. GRDC and Agriculture (WA). B.N. Grierson, J.M. Dimasi and M.L. Brown have also contributed their technical expertise to this work. MET is indebted to Professor Anisul Haque for the sample of "Brazil lentils"

Rcfcrences:
Arentoft, A. M. and Greirson, B.N. 1995. Journal of Agriculture and Food Chemistry 43: 942-945

692 Boyar. A.; and Marsh, R.E. 1982. Journal American Chemical Society 104: 1995-8 Enncking, D.; Giles, L.C.; Tate.M.E.; and Davies, R.L 1993. Journal of the Science of Food and Agriculture 61: 315 Enncking, D.; Dclacrc, I.M.; and Tale. M.E. 1998 Phytochemistry. In Press Gcda, A., Briggs C.J. and Venkataram S. 1993. Journal Chromatography 635: 338 -341 Marquadt, R.R : and Frohljch, A.A. 1981. Journal Chromatography 208(2): 373-379 Nunn. P.B.; Pcrera, K.P W.C.; Bell, E.A.; Campbell, C.O.; Lainbein. F. 1995. International Conference on Lathyrus and Lathyrism: A Decade of Progress, Addis Ababa, Ethiopia. Nov 27-29 pp 13 - 17 Rcssler. C.; Talake. J.G.: Kaiser, E.; Putnam, D.H. 1997. Journal of Agriculture and Food Chemistry 45: 3311-12 Saalbach, I.; Waddel, D.; Pickard.T.; Schieder.O.; MUntz.K. 1995. Journal of Plant Physiology 145: 674-81 Yusuf. II.K M.; Haque, M.K.; Uddtn M.A.; Roy, B.C. and l.,ambein, F. 1995. International Conference on Lathyrus and Lathyrism: /4 Decade of Progress. Addis Ababa, Ethiopia, Nov 27-29 pp 9-12