Planta (1982)155 : 342-344

P l a n t a © Springer-Verlag 1982

Gas chromatographic-mass spectroscopic identification and quantification of arachidonic acid in wheat-germ oil B.

Janistyn

Institut f/Jr Pharmazeutische Biologic der Universit/it, Sch/inzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany

Gas chromatographic-mass spectroscopic evidence is presented for the presence of arachidonic acid ((all-Z)-5,8,11,14-eicosatetraenoic acid) in virgin wheat-germ oil. The amount of arachidonic acid in wheat-germ oil, determined as methyl ester using gas chromatographic detection, was quantified with reference to a standard methyl ester from authentic arachidonic acid. The presence of arachidonic acid and prostaglandins in higher plants is discussed. Abstract.

K e y w o r d s : Arachidonic acid - P r o s t a g l a n d i n Wheat-germ oil.

Introduction

The recently published findings on cyclic adenosine-3': 5'-monophosphate (c-AMP) (Newton et al. 1980; Janistyn 1981 ; Johnson et al. 1981) calmodulin (Anderson et al. 1980; Cheung 1980; Marm4 1981), and the prostaglandin F2~ (PGF2~) (Janistyn 1982) in higher plants indicate a closer metabolic relationship between animals and plants than was formerly assumed. Whereas the presence of arachidonic acid, a natural substrate for prostaglandin formation, is well known in mammals (Ramwell 1973), arachidonic acid has hitherto not been detected with certainty in higher plants (Catalano 1967; Laskowski and Kulikowska 1967; Dziedzianowicz-Wierzbicka and Krauze 1970; Hitchcock and Nichols 1971 ; Kunau 1976). Here, evidence is presented for the presence of arachidonic acid in the acid fraction of virgin wheat-germ oil. The acid was unquestionable identified using gas chromatography-mass spectrometry. 0032-0935/82/0155/0342/$01.00

M a t e r i a l s and m e t h o d s Taking into account the quantitative aspect of the problem of detecting such a labile lipophilic compound as arachidonic acid, the investigation was started with 10 kg of virgin wheatgerm oil. Because saponification of the oil by preliminary experiments had raised some doubts whether it was possible to detect arachidonic acid using this method, the free acidic compounds of the oil were extracted using an aqueous sodium bicarbonate solution. In 1 kg portions, 10 kg cold pressed (virgin) wheat-germ oil (Keimdi/it GmbH, Augsburg, FRG) was added at 5° C to 2 1 ethanol (99%). The mixture was layered on 3 1 of a saturated aqueous sodium chloride solution. Under constant but not too vigorous stirring a solution of saturated aqueous sodium bicarbonate was added until a pH of 8.0 was reached. The stirring was continued for 12 h at the same temperature. Thereafter, the phases were allowed to separate in a separating funnel at room temperature. The separated aqueous phase was extracted twice with 1 1 diethylether. The organic phases were discarded, the aqueous phase was acidified carefully by adding solid tartaric acid until no further turbidity occurred. Three extractions, each with I 1 diethylether, followed. After separation, the organic phases were dried over sodium sulfate, filtered and preevaporated on a vacuum rotary vaporizer. The residual diethylether was removed under high vacuum to yield a light brown residue of 50.5 g (0.5% weight of the starting material) which was kept under nitrogen. As the residue was dissolved in 250 ml hot absol, ethanol and allowed to stand at - 2 0 ° C for 48 h, a dense flocculation appeared which was removed by filtration through a glass suction frit. The filtrate was concentrated under a stream of nitrogen at room temperature. When the volume of the solution was reduced respectively to 150 ml and further to 100 ml cooling and filtration were repeated. The concentration was continued until a highly viscous oil was obtained. Residual ethanol was removed completly under high vacuum; yield 25.4 g (0.25%). The residue was dissolved in 400 ml absolute, methanol and after 100 mg of p-toluenesulfuric acid had been added, the solution was boiled by reflux under nitrogen for 8 h. The methyl esters obtained in this way were distillated under nitrogen at 1.3 Pa. The middle fraction with the boiling points 130-150°C (6g; 0.06%) was redistilled [b.p. 120132 ° C; 0.13 Pa; 3 g (0.03%)] and used for the search of methyl arachidonate by gas chromatography and gas chromatographymass spectroscopy (GC-MS).

B. Janistyn : Arachidonic acid in wheat-germ oil

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Fig. 1. Gas chromatogram of the methyl ester fraction (120-132 ° C; 0.13 Pa) isolated from wheat-germ oil. Conditions: Finnigan G C (9510); 10% F F A P on Gas Chrom Q, 100/120 mesh; 2 m - 2 mm i.d., glass column; N2, 50 ml min -1, 240 ° C, column temp. 220 ° C, FID 200 ° C. The peak with a retention time of 26 rain corresponds to methyl arachidonate (see Fig. 2)

Fig. 3. Mass spectrum of the methyl ester of natural arachidonic acid isolated from wheat-germ oil. The spectrum was recorded from the peak with a retention time of 26 min (see Fig. J). Ordinate: Relative abundance (%). Abscissa: m/e. Conditions: The mass spectra were recorded on a Finnigan G C (96/0)/MSModel 4000 instrument with sample introduction through the gas chromatograph inlet. 10% F F A P on Gas Chrom Q, 100/120 mesh; 2 m - 2 m m i.d., glass column; He, 50 ml min -1, 250 ° C, column temp. 230 ° C; glass-jet-seperator 260 ° C, ion source temp. 270 ° C; accelerating voltage - 2 kV and I.E. 70 eV

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Fig. 4. Mass spectrum of authentic methyl arachidonate. Ordinate: Relative abundance (%). Abscissa: m/e. Conditions: see legend of Fig. 3

344

B. Janistyn: Arachidonic acid in wheat-germ oil

Results and discussion

As shown in Figs. 1 and 2, a peak ocurred at the same retention time of 26 rain in the gas chromatograms of the methyl esters from the wheat-germ oil and from authentic methyl arachidonate. Furthermore, no difference could be found between the peaks, when the authentic methyl arachidonate was added to the methyl ester mixture. Gas chromatography-mass specroscopy analysis of the substance with the retention time of 26 min showed the characteristic fragment ions [m/e 318 (M), 150, 119, 105, 91, 79, 67, 55, 41 and 29] of methyl arachidonate (Myher et al. 1974). This result demonstrates the presence of arachidonic acid in virgin wheat-germ oil. The content of arachidonic acid in the wheatgerm oil, determined as its methyl ester using gas chromatographic detection, was quantified with reference to a standard of methyl arachidonate. Based on a content of 12% oil in the wheat-germ and the amount of arachidonic acid found in this oil, the content of arachidonic acid was calculated (Table 1). The mild production method for the oil, as well as the gentle isolation steps used, strongly indicate, that the wheat-germ itself contains arachidonic acid. The calculated content does not support the idea that this fatty acid may be a reserve compound of the wheat-germ. It is well established that arachidonic acid is the predominant physiological precursor of the prostaglandins E 2 and F2= in the animal kingdom (Ramwell 1973). Some questions about the occurrence and the possible role of prostaglandins and their precursors in plants have been reviewed (Saniewski 1979). If the presence of arachidonic acid could be demonstrated in higher plants in general, a biosynthesis of the prostaglandins E 2 and F2~ via arachidonic acid could be postulated. At this time arachidonic acid has been found only in certain algae, mosses, ferns and in Ginkgo biloba (Hitchcock and Nichols 1971; Kunau 1976). In the red alga, Gracilaria lichenoides, the pros t a g l a n d i n s E 2 and F2~ were found for the first Table 1

Arachidonic acid - content of

Amount (pmol g -

Wheat-germ oil Wheat-germ a (fresh weight) Wheat-germ" (dry weight)

12,560 1,507 1,718

1)

a The calculation of the arachiconic acid content of the fresh and dry weight of the wheat-germ is based on 12% total oil content of the wheat-germ and 14% water content data from Keimdifit GmbH, Augsburg, F R G

time in the plant kingdom (Gregson et al. 1979). Recently the prostaglandin Fz~ was detected in flowering Kalanchoe blossfeldiana (Janistyn 1982). In this connection it is noteworthy that the lipoxygenase-2 enzyme from soybeans can transform arachidonic acid into prostaglandin F2~ (Bild et al. 1978). The technical assistance of Mrs. M. Weber is gratefully acknowledged. I am also obliged to Dr. R. Haas for critically reading the manuscript. I am indebted to Keimdifit GmbH, D-8900 Augsburg, for l0 kg virgin wheat-germ oil.

References Anderson, J.M., Charbonneau, H., Jones, H.P., McCann, R.O., Corrnier, M.J. (1980) Characterization of the plant nicotinamide adenine dinucleotide kinase activator protein and it's identification as calmodulin. Biochemistry 19, 3113-3120 Bild, G.S., Bhat, S.G., Ramadoss, C.S., Axelrod, B. (1978) Biosynthesis of a prostaglandin by a plant enzyme. J. Biol. Chem. 253, 21-23 Catalano, N. (1967) Glyceride composition of olive oil. I. Oils from various areas. Ind. Agr. 5, 529-540 Cheung, W.Y. (1980) Calcium and cell function, vol. 1 : Calmodulin. Academic Press, New York London Dziedzianowiez-Wierzbicka, W., Krauze, St. (1970) Determination of the composition of fatty acids in beech (Fagus silvatica) seed oil. Roez. Panstw. Zakl. Hig. 2l, 653-664 Gregson, R.P., Marwood, J.F., Quinn, R.J. (1979) The occurrence of prostaglandins PGE2 and P G F ~ in a plant - the red alga Graeilaria liehenoides. Tetrahedron Lett. 46, 4505-4506 Hitchcock, C., Nichols, B.W. (1971) Plant lipids biochemistry. Academic Press, New York London Janistyn, B. (1981) Gas chromatographic, mass- and infraredspectrometric identification of cyclic adenosine-3' : 5'-monophosphate (c-AMP) in maize seedlings (Zea mays). Z. Naturforsch. 36e, 193 196 Janistyn, B. (1982) Gas chromatographic-mass spectroscopic identification of prostaglandin F2~ in flowering Kalanchoe blossfeldiana. Planta 154, 485-487 Johnson, L.P., MacLeod, J.K., Parker, C.W., Letham, D.S., Hunt, N.H. (1981) Identification and quantitation of adenosine-3':5'-cyclic monophosphate in plants using gas chromatography-mass spectrometry and high-performance liquid chromatography. Planta 152, 195-201 Kunau, W.H. (i 976) Chemic und Biochemie unges/ittigter Fetts/lurch. Angew. Chem. 88, 97-111 Laskowski, K., Kulikowska, A. (1967) Physicochemical properties of walnut oil. Rocz. Panstw. Zakl. Hig. 18, 483-486 Marm6, D. (1981) Calcium, Calmodulin und ihre zellul/ire Funktion. Biologic in unserer Zeit 3, 71-77 Myher, J.J., Marai, L., Kuksis, A. (1974) Identification of fatty acids by GC-MS using polar siloxance liquid phases. Anal. Biochem. 62, 188-203 Newton, R.P., Gibbs, N., Moyse, C.D., Wiebers, J.L., Brown, E.G. (1980) Mass spectrometric identification of adenosine3' : Y-cyclic monophosphate isolated from a higher plant tissue. Phytochemistry 19, 1909-1911 Ramwell, W.P. (1973) The prostaglandins, vol. 1. Plenum Press, New York Saniewski, M. (1979) Questions about occurrence and possible roles of prostaglandins in the plant kingdom. Acta Hortie. 91, 73-81 Received 22 February; accepted 20 May 1982