New Technologies for Toxin Analyses in Food Legumes.
Peter C.H. Eichinger' ,Ncil E. Rothnie', Ian Dclaerc
2
and Max E. Tate
2
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-Oxalyl-diamino-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.
686
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.
(1)
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.
687
inAu
83
40 30
20
10
0
Kippunc Acid
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
L. Cicera
L.
Sa:.u.s I Ochius
L.
Psejdocice'a
I Seti'olms
L. Annu jg
[%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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