Free Radical Biology & Medicine, Vol. 8, pp. 61-69, 1990

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Review Article ANTIOXIDANT FUNCTIONS OF PHYTIC ACID ERNST GRAF*'t and JOHN W. EATON:~ tThe Pillsbury Company, Technology Center, 311 Second Street S.E., Minneapolis, MN 55414, U.S.A. ; ~tUniversity of Minnesota, Department of Laboratory Medicine and Pathology, Dight Institute, 400 Church Street S.E., Minneapolis, MN 55455, U.S.A. (Received 17 January 1989; Revised and accepted 27 April 1989)

Abstract--Phytic acid is a natural plant antioxidant constituting 1-5% of most cereals, nuts, legumes, oil seeds, pollen and spores. By virtue of forming a unique iron chelate it suppresses iron-catalyzed oxidative reactions and may serve a potent antioxidant function in the preservation of seeds. By the same mechanism dietary phytic acid may lower the incidence of colonic cancer and protect against other inflammatory bowel diseases. Its addition to foods inhibits lipid peroxidation and concomitant oxidative spoilage, such as discoloration, putrefaction, and syneresis. A multitude of other industrial applications are based on the antioxidant function of phytic acid. K e y w o r d s - - F r e e radical, Colonic cancer, Phytic acid, Iron, Chelating agent, Antioxidant, Ferroxidase, Myoinositol phosphate esters

I. INTRODUCTION

Partial protection against o x i d a t i v e d a m a g e is likely conferred by the severe d e h y d r a t i o n o f seeds during storage which decreases the kinetic m o b i l i t y o f reactants and catalysts. Slight increases in the ambient relative h u m i d i t y are k n o w n to increase their v u l n e r a b i l i t y to o x i d a t i v e stress and decrease their survival rate. ~ A d d i t i o n a l p r o t e c t i o n is afforded by natural antioxidants, such as a - t o c o p h e r o l and a wide range of phenolic c o m p o u n d s present in m a n y seeds. 2 A third m e c h a n i s m for extending seed v i a b i l i t y - - a part of the subject of this r e v i e w - - m a y be iron chelation by phytic acid. This recently d e v e l o p e d hypothesis asserts that phytic acid maintains iron in the Fe(III) oxidation state and obstructs generation of h y d r o x y l radical and other activated o x y g e n species by occup y i n g all of the available iron coordination sites. 3-5 In this r e v i e w , we shall discuss the k n o w n antioxidant effects o f phytic acid and, in addition, e x a m i n e the p r o p o s i t i o n that this substance m a y play an important role in the natural and artificial preservation o f o x i d i z a b l e materials and, perhaps, in the prevention and treatment o f a n u m b e r o f human disorders.

The seeds of certain plants m a y remain viable 400 years.~ The reasons for this r e m a r k a b l e l o n g e v i t y are largely unknown. These hardy properties are particulary puzzling considering that seeds contain a potentially reactive mixture of large amounts of highly unsaturated lipids, iron, and o x y g e n . Clearly, elements within seeds must conspire to p r e v e n t the occurrence of extensive o x i d a t i v e injury which w o u l d l o w e r their germinability.

Ernst Graf is a senior scientist in the Technology Center at the Pillsbury Company in Minneapolis, Minnesota. He received his undergraduate education in Switzerland, and in 1981 he earned his PhD degree in Biochemistry from the University of Minnesota. His primary research interests include the chemistry and biochemistry of activated oxygen species, i.e., their formation, reactivity, effects on biological and food systems, and chemical means of mitigating the consequences of these oxidative events. He is particularly interested in the influence of iron and iron chelates, such as Fe(IlI)phytate, on oxygen-mediated reactions. He has participated in several symposia and has authored a book and over a dozen publications on various aspects of phytic acid. In his spare time he likes to camp with his family and run marathons. John Eaton is Professor of Laboratory Medicine/Pathology and Medicine at the University of Minnesota, Minneapolis. He was trained in Anthropology (PhD, University of Michigan, 1969) and his research interests include biological redox chemistry and pathogenhost interactions. The Iron Bolt Award was inflicted on him by the Oxygen Radicals Gordon Conference in 1987. In his spare time he avoids exertion. *Author to whom correspondence should be addressed.

2. STRUCTURE AND CHEMICAL PROPERTIES OF PHYTIC ACID A.

a

61

Occurrence

Phytic acid or m y o - i n o s i t o l h e x a p h o s p h o r i c acid is ubiquitous plant c o m p o n e n t that constitutes 1 - 5 % by

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E. GRAF and J. W. EATON

weight of most cereals, nuts, legumes, oil seeds, spores, needles and pollen (Table 1). It typically accounts for 60-90% of the total seed phosphorus 6 and usually occurs as a mixed calcium-magnesium-potassium salt in discrete regions of the seeds, such as the aleurone layer of wheat and rice. 7 In the past its primary functions during dormancy have been believed to be 1) for the storage of cations 8 and phosphorus, 9 2) a cell wall precursor,I° and 3) as storehouse for potential energy. ~1 In view of its newly discovered antioxidant potential, however, phytic acid has been proposed to serve a vital role in protecting the seeds against the deleterious effects of oxygen and iron. Elsewhere in nature, phytic acid comprises up to 50% of the total weight of the larva of the mesozoan Dicyemid tyous, a 28-cell organism, where it is stored as a hydrated magnesium salt in the 2 apical cells. Its role in these amoebae is unknown, but it is believed to bind cations that stimulate pinocytosis.12 Myo-inositol pentaphosphate, a lower phosphate homologue of phytic acid, is present in both avian and amphibian erythrocytes. This substance binds avidly to hemoglobin, reducing its affinity for oxygen and causing release of oxygen at higher 02 tensions, a function served by diphosphoglycerate in mammalian red blood cells.~3 B. Structure and chemical properties The structure and chemical properties of phytic acid have been extensively reviewed in a prior monograph. ~2X-ray crystallographic analyses of crystalline sodium phytate have revealed its exact structure as the hexaorthophosphate ester of myo-inositol, with the phosphates at positions C1, 3, 4, 5 and 6 in the axial position and that at C2 equatorial. 14 Attempts at crystallizing other metal salts of phytic acid have failed and therefore the conformation of iron phytate is unknown. Several NMR and Raman spectroscopic studies, however, have deduced the structure of phytic acid

in solution as the chair conformation of hexaphosphorylated myoinositol as shown in Figure 1.J5-~7 Despite the tremendous potential energy inherent in the six phosphoric ester linkages, phytic acid is inert and very stable. It can be stored as a solid for years and in neutral or alkaline 50% aqueous solutions at 5°C for several months before generating any decomposition products. The release of 50% of the phosphorus requires acid hydrolysis in 5N HC1 at 100°C for at least 6 h, while refluxing at 100°C for 6 h at pH 12 releases no measurable phosphorus at all. 12Both acid hydrolysis and enzymatic treatment with phytase result in a mixture of myo-inositol, inorganic phosphate and myoinositol mono-, di-, tri-, tetra-, penta- and hexaphosphate. 12 Similarly, during germination of seeds phytic acid becomes successively dephosphorylated to yield myoinositol and inorganic phosphate.I° The energetically unfavorable biosynthesis of this compound requires 5 sequential phosphorylation steps of myo-inositol monophosphate. In suspension-cultured rice cells each successively phosphorylated intermediate was produced until, at 7 days, phytic acid was the major form. 9.18 A number of distinct synthetic pathways have been identified in different seeds.l° Most qualitative and quantitative analyses for phytic acid are based on the extreme insolubility of Fe(llI)4phytate and the determination of either residual or complexed iron as described in a previous review. 19A more sensitive and specific method employs reverse phase HPLC separation and refractive index detection. 2° C. Interaction with proteins Phytic acid forms strong electrostatic linkages with basic amino acyl residues at low pH and thereby pre-

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Table 1. Phytic Acid Content of Various Seeds Sample

Phytic Acid (% w/w) 3

Wheat Wheat bran Corn Corn bran Corn germ Soy beans Sesame seeds Peanuts Dehydrated peas Lima beans Barley Oats Wild rice Sunflower seeds

1.1 4.8 0.9 0 6.4 1.4 5.3 1.9 0.9 2.5 1.0 0.8 2.2 1.9

5

I

0 U

® - -o-P-OM OH Fig. 1. Structure of phytic acid in solution? S

Antioxidant functions of phytic acid cipitates most proteins below pH 5.0. At neutral and alkaline pH both phytate and proteins have a net negative charge which leads to their dissociation from each other. Polyvalent cations, however, form metal bridges between phytic acid and proteins and promote their association at neutral pH. By virtue of binding to proteins, phytic acid has been found to inhibit polyphenol oxidase, 5 e~-amylase, 2~ alcohol dehydrogenase, 22 trypsin,23 and other enzymes. These protein-phytate interactions were reviewed previously in detail] 4 A unique type of protein-phytate interaction is the high affinity of phytic acid for the 2,3-diphosphoglycerate site in hemoglobin. In human hemoglobin A, eight basic amino acyl residues form electrostatic bridges and two hydrogen bonds with the six phosphate moieties of phytic acid. The dissociation of this complex is 6 x 10 -8 M for deoxygenated and 1 x 10 -6 M for oxygenated hemoglobin. Therefore, the binding of phytic acid modifies the heme iron-O2 interaction which facilitates dissociation of oxygen from hemoglobin. The chemistry and physiological implications of this sitespecific protein binding are discussed in a previous review. 25 Phytic acid can be incorporated into erythrocytes irreversibly to give functionally intact cells with improved 02 transport capabilities. 26 These phytateladen erythrocytes may prove useful in the treatment of organ ischemia, hemolytic anemia, pulmonary insufficiency and hypererythropoiesis. 27

D. Chelation of metals The unique structure of phytic acid suggests tremendous chelation potential. Indeed, phytic acid exhibits a high affinity for all polyvalent cations in the following decreasing order of stability: Cu 2÷ > Zn 2+ > Ni 2+ > Co 2+ > Mn 2÷ > Fe 3+ > Ca 2+. By virtue of its high calcium affinity, phytic acid also adsorbs tightly to hydroxyapatite, a complex crystalline calcium phosphate (Cas[POa]3OH), which is the chief structural element of vertebrate bone and tooth. 28 However, dissociation constants (Ka's) are not known due to the complexity of these electrostatic interactions, that is, 1 phytate molecule can bind up to 6 divalent cations and the metal could possibly bridge at least 2 phytate molecules depending on the redox state. All of these complexes are coexistent and have different Ka's. Furthermore, metal chelates of high metal-to-phytate ratios precipitate due to their pronounced insolubility. Potentiometric measurements at 20°C estimated the Ka's of the first 2 calcium ions bound to phytate to be 44 x 10-6 M at pH 7.2 and

Antioxidant functions of phytic acid.

Phytic acid is a natural plant antioxidant constituting 1-5% of most cereals, nuts, legumes, oil seeds, pollen and spores. By virtue of forming a uniq...
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