Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Renal Secondary Hyperparathyroidism

0028 When renal function becomes compromised to such an

extent that creatinine, other nitrogenous metabolic

products, and Pi are retained abnormally and exces-

sively by the body, then several pathophysiological

adaptations occur that have serious effects on health.

One of the important adverse effects of the retention of

Pi is the rapid and progressive loss of mineral mass. The

chronic elevation of serum Pi causes a decline in serum

2þ

, which triggers PTH secretion. The net effect is a

constantly elevated PTH concentration that continues

to act on bone tissue, i.e., resorption, to try to raise

[Ca

2þ

] to its homeostatic set level. Since Pi is also

released from bone along with Ca

2þ

during resorption,

the serum Pi concentration also increases. Because the

kidneys cannot eliminate Pi adequately, [Ca

2þ

] can

never be raised to its set level, and bone tissue continues

to be degraded as part of an unending vicious cycle.

0029 Various dietary manipulations have been tried to

control the loss of bone mass, but no one regimen has

been very successful. Reductions of both dietary pro-

tein, especially animal protein, and phosphorus have

been moderately successful in slowing the progress of

chronic renal disease, but the diets are not very palat-

able or satisfying.

Conclusions

0030 Pi metabolism is much more complex than that of

calcium because of the many intracellular pathways

that utilize Pi ions at one stage or another. The cyto-

solic utilization of Pi is closely linked with that of

glucose for the formation of glucose 6-phosphate

and for triglyceride synthesis through glycerol

3-phosphate formation, as well as with other mol-

ecules, during the postprandial period. Pi is utilized

by cells for many diverse molecules, including regula-

tory peptides and phospholipids. Extracellular regu-

lation of Pi is closely associated with that of calcium

through PTH and other calcium-regulating hor-

mones. Under typical dietary conditions of excessive

phosphorus intake compared with calcium, i.e., low

Ca:P ratio, nutritional secondary hyperparathyroid-

ism and the long-term development of osteopenia are

likely to result. Food fortification with calcium and

calcium supplementation are common ways in which

the low Ca:P ratio can be minimized, but individual

behaviors aimed at selecting a diet higher in calcium

will be needed to overcome the adverse ratio (0.5),

despite calcium fortification and/or supplementation.

Renal secondary hyperparathyroidism, a serious con-

sequence of renal functional impairment, produces

severe bone loss because of altered homeostatic regu-

lation of Pi.

See also: Aging – Nutritional Aspects; Bone; Calcium:

Physiology; Carbohydrates: Requirements and Dietary

Importance; Cells; Cholecalciferol: Physiology; Dietary

Requirements of Adults; Energy: Measurement of Food

Energy; Hormones: Thyroid Hormones; Steroid

Hormones; Osteoporosis

Further Reading

Akesson K, Lau K-H, Johnston P, Iperio E and Baylink DJ

(1998) Effects of short-term calcium depletion and

repletion on biochemical markers of bone turnover in

young adult women. Journal of Clinical Endocrinology

and Metabolism 83: 1921–1927.

Anderson JJB and Garner SC (eds) (1996) Calcium and

Phosphorus in Health and Disease. Boca Raton, FL:

CRC Press.

Anderson JJB, Sell ML, Garner SC and Calvo MS (2000)

Phosphorus. In: Russell R et al. Present Knowledge in

Nutrition, 7th edn. Washington, DC: International Life

Sciences Institute.

Barger-Lux J and Heaney RP (1993) Effects of calcium

restriction on metabolic characteristics of premeno-

pausal women. Journal of Clinical Endocrinology and

Metabolism 76: 103–107.

Bringhurst FR (1989) Calcium and phosphate distribution,

turnover, and metabolic actions. In: DeGroot LJ (ed.)

Endocrinology, 2nd edn, vol. 2. Philadelphia, PA: WB

Saunders.

Brot C, Jorgensen N, Jensen LB and Sorensen OH (1999)

Relationships between bone mineral density, serum

vitamin D metabolites and calcium:phosphorus intake

in healthy perimenopausal women. Journal of Internal

Medicine 245: 509–516.

Calvo MS, Kumar R and Heath H III (1990) Persistently

elevated parathyroid hormone secretion and action in

young women after four weeks of ingesting high phos-

phorus, low calcium diets. Journal of Clinical Endocrin-

ology and Metabolism, 70: 1340–1344.

Calvo MS and Park YM (1996) Changing phosphorus

content of the U.S. diet: Potential for adverse effects on

bone. Journal of Nutrition 126: 1168S–1180S.

Harnack L, Stang J and Story M (1999) Soft drink

consumption among US children and adolescents: Nu-

tritional consequences. Journal of the American Dietetic

Association 99: 436–441.

Institute of Medicine, Food and Nutrition Board (1997)

Dietary Reference Intakes: Calcium, Phosphorus, Mag-

nesium, Vitamin D and Fluoride. Washington, DC:

National Academy Press.

Karkkainen M and Lamberg-Allardt C (1996) An acute

intake of phosphate increases parathyroid hormone se-

cretion and inhibits bone formation in young women.

Journal of Bone and Mineral Research 11: 1905–1912.

McKane WR, Khosla S, Egan KS et al. (1996) Role of

calcium intake in modulating age-related increases

in parathyroid function and bone resorption. Journal

of Clinical Endocrinology and Metabolism 81:

1699–1703.

PHOSPHORUS/Physiology 4545

National Research Council (1989) Recommended Dietary

Allowances, 10th edn, pp. 184–187. Washington, DC:

National Research Council, National Academy Press.

Slatopolsky E, Dusso A and Brown A (1999) The role of

phosphorus in the development of secondary hyperpar-

athyroidism and parathyroid cell proliferation in chronic

renal failure. American Journal of Medical Sciences 317:

370–376.

USDA (1978) Nutritive Value of Foods. Home and Garden

Bulletin No. 72. Washington, DC: US Department of

Agriculture.

Phylloquinone See Vitamin K: Properties and Determination; Physiology

Physical Properties of Food See Rheological Properties of Food Materials

PHYTIC ACID

Contents

Properties and Determination

Nutritional Impact

Properties and Determination

U Konietzny, Karlsruhe, Dettenheim-Liedolsheim,

Germany

R Greiner, Centre for Molecular Biology, Federal

Research Centre for Nutrition, Karlsruhe, Germany

Introduction

0001 The proper chemical designation for phytic acid

is myo-inositol(1,2,3,4,5,6)hexakisphosphoric acid.

Salts of this acid, designated as phytates, are found

in plants, animals and soil. Phytate has been con-

sidered as an antinutrient due to its inhibitory effect

on the bioavailability of essential dietary minerals.

During food processing and digestion, phytate can be

dephosphorylated to produce degradation products,

such as myo-inositol pentakis-, tetrakis-, tris-, bis-,

and monophosphates. Besides the adverse effects

of phytate and other highly phosphorylated myo-

inositol phosphates on mineral bioavailability, some

novel metabolic effects of phytate and some of its

degradation products have been recognized. Certain

myo-inositol phosphates have been suggested to

have positive effects on heart disease by controlling

hypercholesterolemia and atherosclerosis and to pre-

vent renal stone formation. The most extensively

studied positive aspect of myo-inositol phosphates is

their potential for reducing the risk of colon cancer.

Furthermore, much attention has been focused on

myo-inositol with fewer than six phosphate residues,

since some of these compounds have been shown to

play an important part as intracellular second mes-

sengers and some have shown important pharmaco-

logical effects, such as the prevention of diabetes

complications and antiinflammatory effects. The

position of the phosphate groups on the myo-

inositol ring is therefore of great significance for

their physiological function. Thus, it is important to

have reliable techniques available to determine quali-

tatively and quantitatively myo-inositol phosphates

not only by the number of phosphate groups, but

also by the position of the phosphate groups on the

myo-inositol ring. (See Plant Antinutritional Factors:

Characteristics.)

4546 PHYTIC ACID/Properties and Determination

Structure, Occurrence, and Biological

Significance

0002 Phytate is a meso compound and consequently pos-

sesses a plane of symmetry with either five equatorial

and one axial phosphate groups (5-eq/1-ax) or five

axial and one equatorial phosphate groups (5-ax/1-

eq: Figure 1). The carbon bearing the single axial or

equatorial phosphate group is numbered C2 and the

other ring carbons can be numbered C1–C6 from a

C1 atom either side of C2, proceeding around the ring

in a clockwise or counterclockwise fashion. Some less

phosphorylated myo-inositol derivatives are optically

active (Table 1). Their absolute configuration must be

clearly defined. According to convention, a counter-

clockwise numbering gives rise to myo-inositol phos-

phates with a d-prefix and a clockwise numbering to

myo-inositol phosphates with an l-prefix. The choice

of prefix is normally determined by giving preference

to that which results in the lowest numbering of

substituents (Figure 2). The predominant confor-

mation of the myo-inositol phosphates depends on

the specific myo-inositol phosphate, pH value, type

of cations present, and ionic strength. At pH values

above 9.5, phytate exists exclusively in the 5-ax/1-eq

conformation, whereas with myo-inositol pentakis-

phosphates a small amount of the 5-eq/1-ax con-

former is found in equilibrium with the predominant

5-ax/1-eq conformer. In myo-inositol phosphates

with fewer than five phosphate residues, the myo-

inositol ring appears to have a conformation in

which only the phosphate group at C2 is axially

oriented. Below pH 9.5 the 5-eq/1-ax conformer is

predominant with all myo-inositol phosphates. The

binding of some cations such as Cu

2þ

and Ca

2þ

proposed to occur mainly via phosphate groups at the

equatorial position of phytate; other cations such as

−2

OPO

2−

OPO

2−

OPO

2−

OPO

2−

OPO

2−

OPO

2−

6(4)

1(3)

4(6)

3(1)

5-eq/1-ax

5-ax/1-eq

6(4)

1(3)

4(6)

3(1)

fig0001 Figure 1 Possible chair conformations of phytate.

tbl0001 Table 1 myo-inositol phosphate isomers

No. of

phosphate

residues

No. of

isomers

No. of

enantiomeric

pairs

myo-inositolphosphate isomers

610 I(1,2,3,4,5,6)P

562

-I(1,2,3,4,5)P

-I(1,2,4,5,6)P

, I(1,2,3,4,6)P

, I(1,3,4,5,6)P

4156 D-I(1,2,3,4)P

/L-I(1,2,3,4)P

, D-I(1,2,4,5)P

/L-I(1,2,4,5)P

-I(1,2,5,6)P

/L-I(1,2,5,6)P

D-I(1,2,4,6)P

/L-I(1,2,5,6)P

-I(1,3,4,5)P

/L-I(1,3,4,5)P

-I(1,4,5,6)P

, I(1,2,3,5)P

I(1,3,4,6)P

, I(2,4,5,6)P

3208 D-I(1,2,4)P

/L-I(1,2,4)P

, D-I(1,2,5)P

/L-I(1,2,5)P

-I(1,2,6)P

/L-I(1,2,6)P

-I(1,3,4)P

/L-I(1,3,4)P

D-I(1,3,6)P

/L-I(1,3,6)P

-I(1,4,5)P

/L-I(1,4,5)P

, D-I(1,4,6)P

-I(1,4,6)P

-I(2,4,5)P

/L-I(2,4,5)P

I(1,2,3)P

, I(1,3,5)P

, I(2,4,6)P

, I(4,5,6)P

2156 D-I(1,2)P

/L-I(1,2)P

-I(1,4)P

, D-I(1,5)P

/L-I(1,5)P

, D-I(1,6)P

-I(1,6)P

-I(2,4)P

-I(4,5)P

/L-I(4,5)P

, I(1,3)P

,I(2,5)P

, I(4,6)P

162

-I(1)PI

-I(1)P,

-I(4)P/L-I(4)P, I(2)P, I(5)P

Myo-inositol phosphate isomers found in nature are indicated in italics.

PHYTIC ACID/Properties and Determination 4547

2þ

,Mn

2þ

, and the alkali metal ions seem to have

preference for phosphate groups at the axial position.

A single-crystal X-ray analysis of the sodium salt

showed the 5-ax/1-eq conformer. In solution, the ino-

sitol ring also occurs over a wide pH range in a chair

conformation in which five phosphate residues are

arranged axially and only the phosphate at C2 is

equatorially-oriented. Raman data indicate that

alkali metal ions preferentially bind to, and thus sta-

bilize, the 5-ax/1-eq phytate conformer in the order

* Na

>Cs

. In contrast, the Raman spectrum

of solid Ca

-phytate is characterized by the 1-ax/5-eq

conformer.

Plants

0003 Phytate is ubiquitous among plant seeds and/or

grains, comprising 0.5–5% (w/w). It is primarily pre-

sent as a salt of the mono- and divalent cations K

2þ

, and Ca

2þ

and accumulates in the seeds during

the ripening period. In dormant seeds phytate repre-

sents 60–90% of the total phosphate. Only a very

small part of the myo-inositol phosphates exists

as myo-inositol penta- and tetrakisphosphate of

unknown isomeric state. The function of these high

phytate concentrations in plant seeds is unclear. It has

been suggested that phytate may serve as a store of

phosphate, of cations, of the cell wall glucuronate

precursor, of high-energy phosphoryl groups and, by

chelating free iron, as a potent natural antioxidant. In

amoeba, two diphospho-myo-inositol pentakisphos-

phate isomers and one bis(diphospho)-myo-inositol

tetrakisphosphate isomer are present in concentra-

tions exceeding that of phytate and thus may in fact

represent a compact store of high-energy phosphate.

0004 Until now only little is known of the pathway of

phytate synthesis in either the plant or animal king-

dom. A study in the slime mould Dictyostelium dis-

coideum established that phytate synthesis from myo-

inositol proceeds via Ins(3)P, Ins(3,6)P

, Ins(3,4,6)P

Ins(1,3,4,6)P

, and Ins(1,3,4,5,6)P

. Early studies

of phytate synthesis in plants led to the proposal

that phytate synthesis from Ins(3)P was mediated by

phosphoinositol kinase(s) via a series of undefined

myo-inositol phosphates. Recently the first descrip-

tion of the synthetic sequence to phytate in the plant

kingdom was given. From the identities of myo-ino-

sitol phosphates found in duckweed (Spirodela poly-

rhiza L.), at a development stage associated with

massive accumulation of phytate, it was concluded,

that synthesis of phytate from myo-inositol proceeds

according to the sequence d-I(3)P, d-I(3,4)P

, d-

I(3,4,6)P

, d-I(3,4,5,6)P

, I(1,3,4,5,6)P

. An un-

answered question that relates to the pathway of phy-

tate synthesis in plants concerns the source of d-I(3)P.

Two enzyme activities that are capable of synthesizing

d-I(3)P have been identified. These are myo-inositol

phosphate synthase (EC 5.5.1.4), which converts glu-

cose-6-phosphate, the ultimate source of myo-inositol

in plants, to d-I(3)P, and myo-inositol kinase (EC

2.7.1.64), which converts myo-inositol to d-I(3)P.

The spatial and temporal distribution and the relative

contribution of these two enzymes to phytate synthe-

sis via d-I(3)P are unclear.

0005During germination, phytate is rapidly hydrolyzed

in a stepwise manner by phytate-specific phospho-

hydrolases (phytases, EC 3.1.3.8, EC 3.1.3.26) or a

concerted action of phytases and other phosphatases

to supply the nutritional needs of the plant without an

accumulation of less phosphorylated myo-inositol

intermediates. Neither the isomer structure of these

intermediates nor the final product of phytate degra-

dation is known to date. From in vitro investigations

on the stereospecificity of phytate hydrolysis by puri-

fied phytases from cereals it was established that these

enzymes dephosphorylate phytate in a stereospecific

way by sequential removal of phosphate groups via

d-I(1,2,3,5,6)P

, d-I(1,2,5,6)P

, d-I(1,2,6)P

, and d-

I(2,6)P

to finally I(2)P. Moreover, the phytases from

bacteria and fungi investigated for phytate degra-

dation release five of the six phosphate groups, and

the end product was identified as I(2)P. Thus, the

−2

OPO

2−

OPO

2−

6(4)

1(3)

4(6)

3(1)

D-I (1,4,5)P

(L-I (3,5,6)P

)

(

D-I (3,5,6)P

)

L-I (1,4,5)P

6(4)

1(3)

4(6)

3(1)

fig0002 Figure 2 Absolute configuration of D- and L-myo-inositol(1,4,5)trisphosphate.

4548 PHYTIC ACID/Properties and Determination

phosphate at C2 seems to be particularly resistant to

enzymatic cleavage. (See Enzymes: Functions and

Characteristics.)

Soil

0006 In the soil, phytate as well as less phosphorylated

myo-inositols are found. Their biological sources are

unknown.

Animals

0007 In animal tissue, a considerable number of myo-inosi-

tol phosphates containing one to six phosphate resi-

dues have been found. In most tissues stimulation of

the phosphatidylinositol pathway (Figure 3) causes

release of d-myo-inositol(1,4,5)trisphosphate, which

is subsequently metabolized to a wide range of myo-

inositol phosphate isomers. For d-I(1,3,4)P

, meta-

bolism is complex, involving both dephosphorylation

via d-I(1,4)P

to d-I(4)P and phosphorylation to d-I

(1,3,4,5)P

, this latter compound being eventually de-

graded to d-I(1)P or d-I(3)P. The full significance of

this complex metabolism is not clear, but there is now

evidence that certain products of the phosphoinositide

metabolism play second messenger roles in most cells.

d-I(1,4,5)P

and d-I(1,3,4,5)P

bind to specific recep-

tors and regulate Ca

2þ

release from or movement

between intracellular Ca

2þ

stores. d-I(1,3,4,5)P

also the starting point for metabolic pathways gener-

ating other myo-inositol tetrakisphosphate isomers as

well as higher phosphorylated myo-inositols. There

are no known functions for these higher phosphoryl-

ated myo-inositols; these metabolites comprise the

bulk of myo-inositol phosphate content in mamma-

lian cells, but evidence for their association with cell

signaling was recently suggested.

0008d-myo-inositol(1,3,4,5,6)pentakisphosphate was

also found in the erythrocytes of birds, turtles, and

frogs. The functional importance of this compound as

a key regulator of oxygen affinity becomes evident

with the discovery that erythrocytes of adult birds

contain virtually no 2,3-bisphosphogylcerate, the

potent allosteric regulator of hemoglobin in mamma-

lian erythrocytes.

Chemical Properties

0009Numerous studies have been made on the protona-

tion constants of phytate, but the results are often

conflicting. This could be due to the fact that the

protonation constants of phytate to a large extent

are dependent on the ionic strength of the medium.

Phytic acid contains six strong acid groups which are

completely dissociated in solution (pK

1.1–3.2),

three weak acid protons (pK

5.2–8.0), and three

very weak acid protons (pK

9.2–12). There unusually

high pK

values for the second protonization step

seem to be due to intramolecular hydrogen bonding

between the syn-axial phosphate residues at C1 and

C3 as well as C4 and C6. These pK

values imply that

phytic acid will be strongly negatively charged over a

wide pH range and have immense potential for bind-

ing positively charged species, such as cations or pro-

teins. Free phytic acid is an unstable compound and

decomposes to yield lower myo-inositol phosphates

and orthophosphate. It is generally isolated as a

sodium or calcium salt. In its free form, phytic acid

is a light-yellow to light-brown syrupy liquid, soluble

in polar solvents (water, methanol, ethanol, 2-propa-

nol, acetone tetrahydrofuran, dimethyl sulfoxide,

dimethyl formamide), but insoluble in nonpolar

solvents (benzene, toluene, hexane, chloroform). In

G-protein

Intracellular

Inositol

Receptor

PIP

membrane Extracellular

D-I(1,4,5)P

-I(1,3,4,5)P

-I(1,3,4)P

-I(1,3,4,6)P

-I(1,3,4,5,6)P

I(1,2,3,4,5,6)P

-I(1,4,)P

-I(3,4)P

I(1,3)P

-I(4)P

D-I(3)P

D-I(1)P

fig0003 Figure 3 The phosphatidylinositol pathway. PIP

, phosphatidylinositol 4,5-bisphosphate.

PHYTIC ACID/Properties and Determination 4549

contrast to free phytic acid, their salts are very stable

compounds. The phosphate groups can be removed

hydrolytically by enzymes or acid/heat to yield a large

number of homologs and positional isomers ranging

from myo-inositol mono- to pentakisphosphates

(Table 1). In spite of the considerable number of

isomers identified in vivo, they still represent only a

small percentage of the number possible in theory.

Above pH 5, there is almost no decomposition of

phytate at 100



C within 10 h. The rate of acid hy-

drolysis is low – as in other orthophosphoric esters it

reaches a maximum at pH 4, e.g., 27% of phytate is

cleaved after 6 h at 100



C, and even the use of strong

acids leads to an only moderate increase in the rate

of hydrolysis. In 5 mol l

1

HCl 47% of phytate is

cleaved after 6 h at 100



C. Final products of acid

hydrolysis are myo-inositol and myo-inositol(2)mo-

nophosphate. As with enzymatic cleavage, the axial

phosphate at C2 seems to be particularly resistant to

hydrolysis. Complete decomposition of phytate was

achieved with 3 mol l

1

at 165



C for 4 h.

Under acidic conditions and higher temperatures in

particular, monophosphorylated cis-diol groups of

the inositols, in competition with hydrolysis, exhibit

the phenomenon of phosphate migration. Thus, for

example, d-myo-inositol(1)phosphate can yield a mix-

ture of d-myo-inositol(1)phosphate, myo-inositol(2)-

phosphate and l-myo-inositol(1)phosphate. For this

migration, an intermediate formation of cyclic phos-

phodiesters is essential which is only sterically favored

in cis-diol groups.

Phytate–Cation Interaction

0010 Phytate forms complexes with numerous divalent and

trivalent cations. The stability and solubility of the

cation–phytate complexes depend on the specific

cation, pH value, phytate-to-cation molar ratio, and

the presence of other compounds in the solution.

Phytate has six reactive phosphates and meets the

criterion of a chelating agent. In fact, a cation can

complex not only within one phosphate group or

between two or more phosphate groups of one phy-

tate, but also between two or more phytate molecules

(Figure 4).

0011Studying the solubility and relative stability of vari-

ous phytate–metal complexes by potentiometric titra-

tion, the following order of stability at pH 7.4 was

found: Cu

2þ

>Zn

2þ

>Ni

2þ

>Co

2þ

>Mn

2þ

>Fe

3þ

2þ

. Most phytates tend to be more soluble at lower

than at higher pH values. The pH value below which

the solubilities increase is about 5.5–6.0 for calcium,

7.2–8.0 for magnesium, and 4.3–4.5 for zinc phytate.

In contrast, ferric phytate is insoluble at pH values in

the 1–3.5 range at equimolar Fe

3þ

-to-phytate molar

ratios. Solubility increases above pH 4, reaching 50%

at pH 10. When Fe

3þ

-to-phytate molar ratio is in-

creased to 3.5:1, there is increased solubility below

pH 2, reaching a maximum of 90% at pH 1.5 and

lower solubility at pH values above pH 4. By forming

a complex with Fe

3þ

that lacks iron-coordinated

water and thus is unable to catalyze the formation

of hydroxyl radicals in the Fenton reaction, phytate is

a good antioxidant.

0012Another important fact is the synergistic effect of

secondary cations, among which Ca

2þ

has been most

prominently mentioned. Two cations may, when pre-

sent simultaneously, act together to increase the quan-

tity of phytate precipitation. For example, Ca

2þ

enhanced the incorporation or adsorption of Zn

2þ

into phytate by formation of a Ca-Zn phytate. The

effect of Ca

2þ

on the amount of Zn

2þ

coprecipitated

with phytate is dependent on Zn

2þ

-to-phytate molar

ratios. For high Zn

2þ

-to-phytate molar ratios, Ca

2þ

displaces Zn

2þ

from phytate-binding sites and in-

creases its solubility. The amount of free Zn

2þ

directly proportional to the Ca

2þ

concentration. For

low Zn

2þ

-to-phytate molar ratios, Ca

2þ

potentiates

the precipitation of Zn

2þ

as phytate. The higher the

2þ

level, the more extensive the precipitation of the

−2O

−2

POO

−

OPO

2−

OPO

2−

fig0004 Figure 4 Phytate–cation interaction. DG, diacylglycerol.

4550 PHYTIC ACID/Properties and Determination

ions. Mg

2þ

also has been shown in vitro to potentiate

the precipitation of Zn

2þ

in the presence of phytate;

however, Mg

2þ

has been found to exert a less pro-

nounced effect on Zn

2þ

solubility than Ca

2þ

0013 The knowledge about the interaction of the lower

myo-inositol phosphates with different cations is

limited. Recent studies have shown that myo-inositol

pentakis-, tetrakis-, and trisphosphates have a lower

capacity to bind cations (Ca

2þ

,Cu

2þ

,Zn

2þ

,Fe

2þ

3þ

) at pH values in the 5–7 range. The capacity to

bind cation was found to be a function of the number

of phosphate groups on the molecule. The cation-

myo-inositol phosphate complexes seem to become

more soluble as the number of phosphate groups

decreases. There is also some evidence for weaker

complexes when phosphate groups are removed from

phytate. Furthermore, the binding affinity of cations

to myo-inositol phosphates has been shown to be

affected by the orientation of the phosphate groups.

Phytate–Protein Interaction

0014 Phytate interactions with proteins are pH-dependent.

Phytate is known to form complexes with proteins at

both acidic and alkaline pH (Figure 5). At pH values

below the isoelectric point of the protein, the anionic

phosphate groups of phytate bind strongly to the

cationic groups of the protein to form insoluble com-

plexes that dissolve only below pH 3.5. The a-NH

terminal group, the e-NH

of lysine, the imidazole

group of histidine, and guanidyl group of arginine

have been implicated as protein-binding sites for phy-

tate at low pH values. These low-pH protein–phytate

complexes are disrupted by the competitive action of

multivalent cations.

0015 Above the isoelectric point of the protein, both

protein and phytate have a negative charge, but in

the presence of multivalent cations soluble protein–

cation–phytate complexes occur. The major protein-

binding site for the ternary complex appears to be the

unprotonated imidazole group of histidine. The ion-

ized carboxyl group of the protein are also suggested

sites. These complexes may be disrupted by high ionic

strength, high pH (> 10), and high concentrations of

the chelating agents.

0016Protein–phytate complexation may effect changes

in protein structure that can decrease enzymatic activ-

ity, solubility, and vulnerability to attack by proteo-

lytic enzymes. Phytate has been shown to reduce the

activity of lipase, a-amylase, pepsin, trypsin, and chy-

motrypsin in vitro. The inhibitory effect increases with

the number of phosphate groups per myo-inositol

molecule and the myo-inositol phosphate concentra-

tion. (See Protein: Interactions and Reactions Involved

in Food Processing.)

Application

0017Phytate has found industrial application, including

uses in the food industry (Table 2). The focus of

research on phytates includes occurrence and func-

tions in plant seeds, nutritional significance, preserva-

tive applications in food technology, and potential

medical and industrial uses. (See Preservatives: Food

Uses.)

Determination

0018The measurement of myo-inositol phosphates in any

material requires an initial extraction. The reagents

most commonly used to extract myo-inositol phos-

phates from foodstuff and biological samples include

3% trichloroacetic acid and 2.4% hydrochloric

acid. Since myo-inositol phosphates do not have a

POO

−

OPO

2−

OPO

2−

OPO

2−

OPO

2−

−2

−CH

−protein

NH−protein

pH<5

5<pH<10

C−CH

−protein

−

fig0005 Figure 5 Phytate–protein interaction.

PHYTIC ACID/Properties and Determination 4551

characteristic absorption spectrum, nor can they be

identified using specific colorimetric reagents, the

determination of these compounds has remained a

persistent problem.

Qualitative Separation Methods

0019 Qualitative separation and detection of myo-inositol

phosphates have been developed in the 1950s and

1960s. Paper chromatography has been shown to be

useful for separating myo-inositol phosphates by the

number of phosphate groups. Myo-inositol mono- to

hexakisphosphate could also be resolved relatively

rapidly by electrophoresis. Thin-layer chromato-

graphy, even if successfully applicable, has not been

widely adopted for the separation of myo-inositol

phosphates.

Quantitative Separation Methods

0020 Precipitative methods Quantitative methods for

determining phytate often employ the addition of a

controlled amount of Fe

3þ

to an acidic sample extract

to precipitate the phytate. Phytate is subsequently

estimated either by determining the phosphate, inosi-

tol, or iron content of the precipitate (direct method),

or by measuring the excess iron in the supernatant

(indirect method). The indirect methods are generally

more convenient and reproducible, because the stoi-

chiometric ratio of phosphate to iron in Fe

3þ

-myo-

inositol phosphate precipitates is affected by several

variables, including the way in which the precipitate

is washed. These methods are not specific for phytate

due to coprecipitation of less phosphorylated myo-

inositols and should therefore be limited to the analy-

sis of material which contains negligible amounts of

these myo-inositol phosphates.

0021 Nonprecipitative methods Nonprecipitative methods

for myo-inositol phosphate determination include

P-Fourier transform nuclear magnetic resonance

(

P-FT NMR) spectroscopy, near-infrared reflect-

ance spectroscopy, low-pressure anion-exchange

chromatography, several high-performance liquid

chromatographic (HPLC) separation systems, and

capillary electrophoresis. The main limitation of

P-FT NMR and near-infrared spectroscopy is that

these methods are specific for phytate only when

the sample contains negligible amounts of less phos-

phorylated myo-inositols. Additionally, sophisticated

instruments which are not available in most labora-

tories are required.

0022Low-pressure anion-exchange chromatography

Low-pressure anion-exchange chromatography is

widely used in the determination of myo-inositol

phosphates. The method currently accepted by the

Association of Official Analytical Chemists (AOAC)

for measuring phytate in foods and feeds is based

on a step gradient (0.7 mol l

1

NaCl) anion-exchange

method (Dowex AG1-X8). Unfortunately, the anion-

exchange resin also retains less phosphorylated myo-

inositols. The method should therefore be limited to

the analysis of material with negligible amounts of

these myo-inositol phosphates. Myo-inositol mono-to

hexakisphosphate and even some positional isomers

could be resolved using anion-exchange chromato-

graphy with a linear eluting gradient of hydrochloric

acid or a stepwise elution with either hydrochloric

acid or ammonium formate/formic acid solutions

of increasing concentrations. Unfortunately, these

methods require long elution times (up to 24 h) and

a large number of eluate fractions must be hydrolyzed

for quantitation as phosphate or inositol, since these

systems preclude the use of refractive index and con-

ductivity detection methods. Methods designed by

those studying calcium metabolism are dependent

on the use of radiolabeled myo-inositol phosphates

to facilitate detection and quantitation, but it is not

feasible to label existing myo-inositol phosphates in

dietary constituents.

tbl0002 Table 2 Application of phytate

Action Application

Metal chelation Prevention of color and quality changes in processed agricultural (chestnut, bean sprouts, pickles,

asparagus, etc.) and fishery (tuna, clams, shrimps, crabs, etc.) products

Removing metal ions from wine

Rust-proofing and dissolving-out prevention inside cans

Prevention of oxidation in oil/water emulsion-type food such as cream, dressings, butter, chesses, soups

Additive for etching solution for offset printing

Anticorrosion agent for paints, antifreezes, and metal surfaces (steel, tin, aluminum, iron)

Stabilizer for perfumes and cosmetics

Antioxidant for industrial oils and greases

pH control Prevention of quality changes by controlling pH value

Fermentation promoter Improvement of product yield and quality by promoting the growth of microorganisms such as lactic acid

bacteria and yeasts (fermented food, antibiotics, methanol, etc.)

4552 PHYTIC ACID/Properties and Determination

0023 High-performance liquid chromatoraphy More re-

cently, HPLC techniques have been introduced into

myo-inositol phosphate determination. Purification

of crude acid extracts of biological samples is usually

required prior to injection on to the analytical HPLC

system. The techniques used for detection and quan-

titation of the myo-inositol phosphates is heavily de-

pendent on the system employed for their separation.

The myo-inositol phosphates may be separated using

anion-exchange, reverse-phase, micellar and ion

chromatography, and detected/quantified by a variety

of techniques, including refractive index, conducti-

vity, indirect photometry, online postcolumn spectro-

photometric detection, and offline phosphate or

inositol assay. Among these, ion-pair reverse-phase

and anion-exchange chromatography are largely

used. (See Chromatography: High-performance

Liquid Chromatography.)

0024 Ion-pair reverse-phase chromatography Ion-pair re-

verse-phase chromatography with refractive index

detection has been successfully applied to analysis of

myo-inositol phosphates. The retention of myo-inosi-

tol phosphates on reverse-phase packings is markedly

increased through the use of ion-pair reagents, allo-

wing the simultaneous separation of myo-inositol

tris- to hexakisphosphates, but neither myo-inosi-

tol mono- or bisphosphates nor the individual pos-

itional isomers are resolved (Figure 6). However,

sample extracts must be passed through anion-ex-

change resin to remove orthophosphate and concen-

trate the myo-inositol phosphates. Acidic column

eluent is then evaporated to dryness to remove hydro-

chloric acid and reconstituted in water prior to injec-

tion on to a silica-based C18 reverse-phase HPLC

column. The mobile phase consisted of formic acid/

methanol and tetrabutylammonium hydroxide. The

affinity of myo-inositol phosphates for the stationary

phase increases with the increasing number of phos-

phate groups on the inositol ring and with increasing

pH. (See Chromatography: High-performance Liquid

Chromatography.)

0025 Anion-exchange chromatography To date, pub-

lished procedures involving anion-exchange HPLC

fall into two categories: isocratic and gradient ion-

chromatographic techniques. The capability of resol-

ving the different myo-inositol phosphates depends on

the stationary phase used and the chromatography

conditions. Myo-inositol mono- to hexakisphosphates

have been successfully resolved by isocratic elution

from low-capacity weak anion-exchange columns.

These single eluent systems are compatible with re-

fractive index, indirect photometric, thermospray

mass spectrometric, and conductivity detection.

However, in the case of conductivity detection, sensi-

tivity is low unless counterions in the eluent are

continously removed using a suppressor column or

membrane suppressor system.

0026In the last few years a number of isomer-specific

ion-exchange chromatography methods with gradi-

ent elution for separation and quantitation of myo-

inositol phosphates in the picomolar range have been

developed. Eluents with high ionic strength, such as

formate, acetate, citrate, phosphate, nitrate, sulfate,

sodium chloride, or hydrochloric acid, have been

used. The most commonly used detection method

with gradient elution is online postcolumn derivatiza-

tion or complexation reactions followed by spectro-

photometric detection. Three approaches have been

employed in the postcolumn detection and quantita-

tion of myo-inositol phosphates. The first is based on

the direct reaction of myo-inositol phosphates with a

13.4−

26.7−

8.607

18.23

28.423

fig0006Figure 6 Chromatographic profile of a myo-inositol phosphate

standard by high-performance liquid chromatography (HPLC)

ion-pair chromatography on Ultrasep ES 100 RP18 (2 

250 mm). The column was run at 45



C and 0.2 ml min

1

of an

eluent consisting of formic acid:methanol:water: tetrabutylammo-

nium hydroxide (TBAH: 44:56:5:1.5 v/v), pH 4.25. Myo-inositol

phosphates were detected by refractive index a standard.

Peaks (8.607) IP

; (12) IP

; (18.23) IP

; (28.423) IP

PHYTIC ACID/Properties and Determination 4553

reagent to form a fluorescent complex or one which

has an absorbance in the ultraviolet or visible part of

the spectrum. For example, the eluate from the

column was mixed, online, with 0.1% Fe(NO

)

2% HClO

to form ultraviolet-absorbing phytate–

3þ

–ClO

complexes. The use of postcolumn deri-

vatization through ligand-exchange reaction between

the iron(III)-sulfosalicylate complex and eluted myo-

inositol phosphates has been described as an alterna-

tive. Furthermore, a complexometric technique based

on competition between myo-inositol phosphates and

the cation-specific reporter dye 4-[2-pyridylazo]resor-

cinol for the transition metal yttrium has been de-

scribed. The second approach is based on the online

enzymatic hydrolysis of myo-inositol phosphates

which is then mixed with a molybdate solution in

the reaction coil. The colored phosphomolybdate

complex may be quantified spectrophotometrically.

Finally, myo-inositol phosphates may be quantified

by online thermospray mass spectrometric techniques.

0027 A remaining problem, however, is to separate

isomers from the whole spectrum of myo-inositol

phosphates in the same run. Separation is generally

performed on HPLC columns with gradient elution

in two combined systems. Myo-inositol mono- to

trisphosphates have been acidic gradient-eluted, post-

column-derivatized, and ultraviolet-detected and

myo-inositol bis- to hexakisphosphates have been

alkali gradient-eluted and detected using chemically

suppressed conductivity detection. The sensitivity of

the analysis of myo-inositol mono- and bisphosphates

was improved 10–100 times by using sodium acetate

gradient elution in a sodium hydroxide environment

and pulsed amperometric detection.

0028 Capillary electrophoresis Recently capillary electro-

phoresis has been applied to the determination of

myo-inositol phosphates. Capillary electrophoresis is

attractive, since only a few nanoliters of sample are

used in each analysis, there is the potential for con-

current separation of mono- to hexakisphosphate

species in the same analysis, and run times are usually

short due to the intrinsically high efficiency of the

technique. Indirect ultraviolet detection was used to

allow the detection of the nonchromophoric myo-

inositol phosphates. Thus, no derivatization of the

compounds is needed. Separations of all six myo-

inositol phosphate groups in deionized water has

been achieved in about 13 min. The position isomers

myo-inositol(1)phosphate and myo-inositol(2)pho-

sphate are easily separated in a phthalate electrolyte

system, demonstrating the potential for separating

myo-inositol phosphate isomers. However, further

work is required on the development of capillary

electrophoretic methods for the separation and

quantiation of the different myo-inositol phosphate

isomers.

0029All in all, efficient analytical systems for separation

and quantitation of myo-inositol phosphate isomers

are available. One problem in developing methods to

determine myo-inositol phosphate isomers is their

availability as reference compounds. They may be

produced by chemical or enzymatic hydrolysis of

phytate. Then identification of these isomers is

needed. This requires sophisticated methods. The

earliest developed technique is chemical analysis by

oxidation with periodate, reduction, dephosphoryla-

tion, and subsequent identification of the polyols

found. Further information can be obtained from

the above-mentioned cis phosphate migration. In

the past few years, high-resolution nuclear magnetic

resonance (NMR) spectroscopy has evolved as a

much simpler technique in the identification of

the isomeric nature of myo-inositol phosphates.

Thirty-nine of the 63 theoretically possible myo-ino-

sitol phosphate isomers can be identified by NMR.

Only for the 24 enantiomeric pairs among these

isomers (Table 1) absolute configurations are

indistinguishable from NMR, but these enantiomers

are also not separated on the achiral columns in

use. Separation techniques using chiral columns

have to be developed to resolve the enantiomers.

The absolute configuration of such enantiomers may

be determined using high-affinity binding proteins or

enzymatic assays.

See also: Chromatography: High-performance Liquid

Chromatography; Electrophoresis: General Principles;

Enzymes: Functions and Characteristics; Phosphorus:

Properties and Determination; Physiology; Plant

Antinutritional Factors: Characteristics; Preservatives:

Food Uses