Formation of [3H,32P]phytic acid in germinating wheat

ANALYTICAL

131, 351-355 (1983)

BIOCHEMISTRY

Formation

of [3H,32P]Phytic Acid in Germinating

Wheat

ERNSTGRAF' Northern

Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 61604

Received November 29, 1982 Doubiy labeled phytic acid of high specific activity was prepared by incubating whole wheat seeds with [“PIphosphoric acid and [3H]myoinositol for 48 h and purifying by anion-exchange chromatography on AG l-X8 resin. Both degradation and synthesis of phytic acid were inhibited by KF to a similar extent, yet the catabolic and anabolic pathways involved distinctly different enzyme systems, as no [3H,32P]myoinositol tetra- or pentaphosphate could be detected.

Phytic acid (hexaphosphorylated myoinositol) constitutes l-6% by weight of most legume, cereal, and oil seeds (1) where it serves as a major storage form of phosphate that becomes available during germination. Its presence in plant-derived foods lowers the nutritional bioavailability of minerals by forming insoluble complexes with di- and trivalent cations (2). Well-documented examples include human deficiencies in Fe3+ (3) and Zn*+ (4), and several recent reviews also discuss other nutritional ramifications of phytic acid (1 J-7). A variety of methods have been published for the preparation of radioactively labeled phytic acid. [32P]Phytic acid and [‘4C]phytic acid have been prepared by incubating aleurone layers of ripening rice grains with [32P]phosphoric acid and [ 14C]myoinositol, respectively (8). Alternatively, [ “C]phytic acid has been prepared by incubating maturing mustard seeds with [i4C]glucose, [14C]acetate, and [14C]myoinositol (9). Both methods require seeds at a strictly defined developmental stage, thereby restricting their general usefulness for many investigators. Labeled phytic ’ Present address: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minn. 55455. 351

acid formed after administering radioactive precursors to plant stalks (lo-12), but this method suffers from the same limitations as the first two methods. In addition, the method is accompanied by the generation of large quantities of radioactive solid wastes, low yields, and long time intervals between administration of label and isolation of phytic acid. [32P]Phytic acid has also been prepared by incubating mung beans with [32P]phosphoric acid ( 13,14); however, fairly large incubation volumes were required and less than 2% of the total radioactivity became incorporated into phytic acid. Furthermore, a method for specifically labeling the carbohydrate moiety of phytic acid using mature seeds has never been described. In our view, a satisfactory method for preparing [3H]phytic acid, [32P]phytic acid, and [3H,32P]phytic acid does not exist. We report an improved method for the preparation of these compounds and briefly characterize the kinetics and mode of their biosynthetic formation. The ready availability of radioactively labeled phytic acid will enable several important biochemical and nutritional studies and should be of broad utility in view of the recent concern over the safety of soy-substituted foods and renewed interest in the chemistry of phytic acid (15). 0003-2697/83 $3.00 Copyright Q ,983 by Acadermc Press. Inc. All rights of reproduawn in any form reserved

352

ERNST

MATERIALS

AND

METHODS

GRAF

0.3 N HCl, and this resin was placed on top of 4.75 ml fresh resin that had been equilibrated with 0.3 N HCl; final column dimensions were 0.7 X 14 cm. Phytic acid was eluted with a linear HCl gradient consisting of 125 ml0.3NHCland 125ml l.ONHClataflow rate of 0.64 ml/min. Fractions of 1.41 ml/ tube were collected and 3H and 32P were measured simultaneously as described above. The peak was pooled and the identity and purity of phytic acid were established by paper chromatography using 1-propanol:2 N NH3 (2: 1) as the developing solvent ( 16). Authentic phytic acid was detected by dipping the dried chromatogram in HC 1O4: 10% ammonium molybdate:2 N HCkacetone (5: 10:5:80), drying it at 110°C for 3 min, then spraying it with 1% SnC12 in 10% HCl ( 17). Radioactivity was detected by cutting the chromatogram into 0.5-cm strips, soaking each strip in 2 N HCl for 2 h, and counting them as described above. Phytic acid was determined by our previously described HPLC method (18).

[2-3H(N)]Myoinositol (12.5 Ci/mmol) was purchased from New England Nuclear. Carrier-free [32P]phosphoric acid in dilute HCl was obtained from Amersham. Sodium phytate was purchased from Sigma Chemical Company. All other chemicals were of reagent grade. Anion-exchange resin AG l-X8 200-400 mesh (Cl- form) was obtained from Bio-Rad Laboratories. HPLC equipment from Waters & Associates included a solvent delivery system Model M-45, automated sample introduction system Model 7 10B WISP, PBondapak C,s column (0.4 X 30 cm), and differential refractometer R40 1. Waldron variety hard red spring wheat seeds ( 198 1 crop) and Eagle variety hard red winter wheat bran (grown and milled in 1978) with phytate contents of 1.O + 0.1% and 3.1 f 0. l%, respectively, were obtained from the Department of Grain Science and Industry, Kansas State University, Manhattan, Kansas. Seven wheat seeds (approximately 250 mg) were incubated with 2 &i [3H]myoinositol RESULTS AND DISCUSSION and/or 2 PCi [32P]phosphoric acid in 150 ~1 We attempted to optimize conditions for tap water at 38°C for the times indicated. At the end of the incubation, these seeds were the preparation of doubly labeled phytic acid washed extensively with water, ground with to meet three criteria: a mortar and pestle, and extracted with 6 ml 1. To achieve maximum uptake of radio0.5 N HCl by vigorous mechanical agitation isotopes by keeping the incubation volumes for 3 h at room temperature. The slurry was to a minimum. As shown in Fig. 1, 96% of centrifuged, and the clear supernatant diluted [32P]phosphoric acid was imbibed during the with 4 volumes of water. An aliquot was re- first 24 h. In comparison, the rate of moved for radioactivity measurement and the [3H]myoinositol uptake was only 57% at 24 remainder passed over 0.65 ml AG l-X8 resin h and the amount of 0.5 N HCl soluble rapacked in disposable polypropylene Minidioactivity declined to 17% after 96 h of inColumns (Kontes Scientific Glassware). The cubation. This decline was presumably due to resin was washed with 25 ml 0.3 N HCl and rapid oxidation of myoinositol and its incorphytic acid was eluted slowly with 10 ml 2 N poration into insoluble polysaccharides and HCI. A l-ml aliquot was added to 10 ml tol- cell wall constituents (19). uene:Triton X-100 (2:l) containing 0.5% PPO 2. To imbibe enough water to activate the and 0.017% POPOP and counted in a Beck- phytate synthesizing enzyme system. Under man LS 7800 scintillation counter. the chosen conditions, both [3H]phytate and To further purify the above 2 N HCl phy- [32P]phytate were formed after an initial lag tate fraction, 30 ml of a dilute 48-h incubation period of 2 h. The yield of labeled phytic acid medium containing both 3H and 32P was per- reached a maximum after 48 h and decreased colated over 0.65 ml AG 1-X8, washed with with longer incubation periods.

DOUBLY

04

w6 0

’

LABELED

353

PHYTIC ACID IN WHEAT

I

I

I

I

20

40

60

80

Incubation

1000

Time lhoursl

FIG. 1. Time course of [32P]phosphoric acid and [3H]myoinositol incorporation into phytic acid. Wheat seeds were incubated with [‘H]myoinositol (upper panel) or [“PIphosphoric acid (lower panel) at 38°C. Percent uptake of radioactivity was measured by counting the radioactivity in the clear acid extract of whole wheat seeds alter centrifugation and does not include the radioactivity retained in the pellet. Each point represents the mean and standard deviation of three separate experiments.

3. To restrict imbibition of water in order to keep the phytase activity at a minimum. Figure 2 clearly demonstrates that no significant hydrolysis occurred during the first 96 h of incubation under the conditions described while the germination of wheat seeds for the same time period resulted in phytate utilization of 33%. This may be due to the lack of active phytase or to the inaccessibility of phytate, since phytic acid probably requires solubilization before undergoing enzymatic hydrolysis. The crude assay for labeled phytic acid as described under Materials and Methods fails to discriminate between phytic acid and myoinositol penta- and tetraphosphate. All three phosphate esters would be retained at least to some extent by the AG l-X8 resin under the conditions described. They can, however, be

fractionated by gradient elution. Figure 3 shows a typical chromatographic profile of [3H,32P]phytic acid that had been prewashed with 0.3 N HCI. A single peak was obtained whose identity and purity was further assayed by paper chromatography with authentic phytic acid. The ratio of 32P counts to 3H counts was constant within experimental error during the entire elution, suggesting that the preparation is pure doubly labeled phytic acid.2 Furthermore, the absence of lower esters of myoinositol suggests a biosynthetic pathway involving acid-insoluble polyphos’ The terms doubly labeled phytic acid and [3H?2P]phytic acid do not imply incorporation of both radioisotopes into the same molecule; the differentiation between [3H,32P]phytic acid and a mixture of [‘Hlphytic acid and [32P]phytic acid is immaterial since they have identical chemical properties.

354

ERNST GRAF

0.2 0 ’

0

I

I

I

I

20

40

60

80

Incubation

100

Time lhoursl

FIG. 2. Time course of phytic acid hydrolysis in wheat seeds. Seven seeds (approximately 250 mg) were incubated in 150 7~1Hz0 at 38°C (full circles) or germinated between wet sheets of filter paper at room temperature (open circles), and phytic acid content was determined by HPLC method described previously (18). Each point represents the mean and standard deviation of three separate experiments.

phorylated intermediates as was first proposed by Asada et al. (10). Both hydrolysis and synthesis of phytic acid were inhibited to the same extent by 10 IIIM KF (Table 1) and raises the possibility that the same enzyme system catalyzes both forward and reverse reactions. However, wheat bran hydrolyzed phytic acid yet failed to synthesize either [3H]phytic acid or [32P]phytic

to 0

'1'

20

I'

40

acid. These results, together with the absence of myoinositol tetra- and pentaphosphates, argue for the existence of two clearly separate enzyme systems for degradation and synthesis of phytic acid, both of which are inhibited by KF and are active in germinating seed. In this paper, we describe the development of an improved method for preparation of doubly labeled phytic acid. Best results were

C""'~'~'~

60

60 Fraction

100 Number

120

140

160

1 160

FIG. 3. Elution profile of [‘H,“P]phytic acid from AC l-X8. Experimental details are given under Materials and Methods. Each fraction was counted for 3H, ‘*P and total cpm. Only total cpm data are shown, since the ‘H and 32P profiles were superimposable.

DOUBLY

LABELED

PHYTIC

TABLE I EFFECT

ACID IN WHEAT

355

labeled phytic acid may aid several other investigators with a variety of experiments.

OF KF ON HYDROLYSIS AND SYNTHESIS OF PHYTIC ACID IN WHEAT’

REFERENCES Synthesis of labeled phytic acid Hydrolysis endogeneous

Sample Seed Seed + KF

Bran Bran + KF

Yield

phytic acid (%) 5f4 4+4 53 + 6 36 k 8

of ‘H

(%) 5.4 3.6 0.01 0.01

2 2 + *

2.0 0.4 0.01 0.01

Yield of 32P (%) 7.7 5.1 0.04 0.09

+ + + f

M. (1980) CRC Crit. Rev. Food Sci. Nutr. 13, 296-335. Vohra, P., Gray, G. A., and Kratzer, F. H. (1965) Proc. Sot. Exp. Biol. Med. 120, 447-449. Davies, N. T., and Nightingale, R. (1975) Brit. J. Nutr. 34, 243-258. Hambidge, K. M., Walravens, P. A., Brown, R. M.. Webster, J., White, S., Anthony, M., and Roth, M. L. (1976) Amer. J. C/in. Nutr. 29, 134-738. Maga, J. A. (I 982) J. Agric. Food Chem. 30, l-9. Erdman, J. W. (1979) J. Amer. Oil Chem. Sot. 56, 736-741. Cosgrove, D. J. (1980) Inositol Phosphates, Elsevier Scientific Publishing Company, New York. Ogawa, M.. Tanaka, K.. and Kasai. Z. (1979) Agric. Biol. Chem. 43,221 l-22 13. Blaicher, F. M., and Mukhejee, K. D. (1981) Z. Naturforsch. 36, 383-384. Asada, K., Tanaka, K.. and Kasai, Z. (1968) Plant Cell Physiol. 9, 185-193. Tanaka, K., Yoshida, T., and Kasai, Z. (1974) Plunt Cell Physiol. 15, 147-151. Nahapetian, A., and Young, V. R. (1980) J. Nutr. 110, 1458-1472. Biswas, S., and Biswas, B. B. (1965) Biochim. Biophys. Acta 108, 710-713. Hauschild-Rogat, P. (1970) Anal. Biochem. 36, 233237. Cook, J. D., Merck, T. A., and Lynch, S. R. (198 1) Amer. J. Clin. Nutr. 34, 2622-2629. Sequi, P., Marchesini. A., and Galante, E. (1966) Ric. Sci. 36, 183-187. Hanes, C. S.. and Isherwood, F. A. (1949) Nature London 164, 1107-l 112. Graf, E., and Dintzis, F. R. (1982) J. Agric. Food Chem. 30, 1094-1097. Loewus, F. A., and Loewus, M. W. (1980) in The Biochemistry of Plants (Preiss. J., ed.), Vol. 3, pp. 43-76, Academic Press, New York. Chetyan,

of

0.8 1.0 0.08 0.06

y Triplicate samples were incubated for 24 h at 38°C in 150 pl Hz0 or 150 pl 10 mM KF containing 2 &i [3H]myoinositol or [“P]phosphotic acid; 7 seeds (approximately 250 mg) of 1981 Waldron wheat or 40 mg of 1978 Eagle wheat bran were used in each experiment. To measure the rate of hydrolysis of endogenous phytic acid under the above conditions, samples were incubated for 24 h in the absence of radioactive precursors and phytic acid content was determined by HPLC method ( 18).

obtained when seven wheat seeds (approximately 250 mg) were incubated with 150 ~1 tap water containing [3H]myoinositol and [32P]phosphoric acid for 48 h at 38°C. Labeled phytic acid was extracted with 0.5 N HCl and purified by anion-exchange chromatography. Due to the absence of myoinosito1 tetra- and pentaphosphates, a gradient elution was unnecessary for preparing pure phytic acid. [3’P]Phytic acid is presently being used to investigate phytate-nucleoside diphosphate phosphotransferases in wheat, [3H]phytic acid will be required to characterize the interactions between phytate and proteins, and [3H,32P]phytic acid will be required to study the nutritional effects of lower myoinositol phosphate esters. The availability of

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