Nutritional Bioavailability of Iron - American Chemical Society

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Downloaded by CALIFORNIA INST OF TECHNOLOGY on February 6, 2017 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch008

Phytate, Wheat Bran, and Bioavailability of Dietary Iron EUGENE R. MORRIS and REX ELLIS U.S. Department of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Beltsville, MD 20705

Monoferric phytate is the major fraction of iron in wheat bran, and is a highly bioavailable form of dietary iron in contrast to insoluble di- or tetraferric phytate. Monoferric phytate equilibrates with the miscible nonheme iron pool of a meal in extrinsic label iron absorption tests. Whole wheat bran depressed absorption by humans of nonheme iron in a meal. Dephytinized wheat bran also inhibited nonheme iron absorption by humans and the inhibition could not be clearly attributed to either the insoluble or soluble fractions of the dephytinized bran. Adult men who consumed 36 g of wheat bran per day had positive iron balances. Iron balance was not increased when dephytinized bran was consumed. The form of ferric phytate must be known to properly explain the effect of phytic acid on iron absorption. The overall meal composition must be considered to evaluate the effect of wheat bran on iron nutrition of humans. Based on USDA estimates of per capita consumption of wheat flour, one-third of the adult woman's Recommended Dietary Allowance (RDA) for iron could be obtained i f we consumed whole wheat products (1). The iron i n wheat, however, i s thought to be poorly bioavailable to humans, primarily attributable to the effect of phytate. B r i t i s h investigators found that the iron balance of individuals was lower when they ate largely whole meal bread than when they ate bread made with white flour (2). When the test bread made with white flour contained either sodium or f e r r i c phytate, postprandial serum iron rise was depressed (£)• They theorized that the phytate present i n the brown bread formed an insoluble iron salt and rendered the iron unabsorbable. That theory was supported by the work of Moore et al. (4) at Washington University, who tested the response of anemic individuals administered therapuetic doses of f e r r i c This chapter not subject to U.S. copyright. Published 1982 American Chemical Society. Kies; Nutritional Bioavailability of Iron ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

122

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION


phytate and concluded the iron was poorly absorbed. Hussain et a l . (5) found that iron deficiency e l i c i t e d considerably less enhancement i n absorption of wheat iron than of iron from either f e r r i t i n or iron salts. They concluded that the mucosal regulation was less effective i n this instance because wheat iron was a low bioavailability form of iron. About a decade ago we began studies to identify the chemical nature of iron i n wheat as possibly a f i r s t step i n devising a means to improve the assimilation of the iron i n wheat-based foods. In this communication we w i l l discuss the evidence for our belief that monoferric phytate i s the endogenous form of iron i n bran, some physical-chemical characteristics of monoferric phytate, and studies i n animals and humans of the bioavailability of iron i n wheat bran and monoferric phytate. Monoferric Phytate-Isolation, Identification and Properties Isolation and identification.

The i n i t i a l isolation of mono-

f e r r i c phytate was from aqueous 1.0-1.2 M ammonium acetate or

sodium chloride extracts of wheat bran (6»). Approximately 70% of the iron i n bran can be extracted i n this manner. The extraction of iron i s reduced by about 50% i f the molarity of the salt solution i s reduced to 0.5. Practically no iron can be extracted by water from untreated wheat bran (7). Ammonium sulfate solution, 1M, i s also an e f f i c i e n t extraction medium for the iron i n wheat bran. Phytic acid i s i n o s i t o l hexaphosphate. Analysis of the iron containing product isolated from the wheat bran extracts for i n o s i t o l , phosphorus and iron gave molar ratios of 0.97:6.26:1, respectively, corresponding to a theoretical ratio of 1:6:1 for monoferric phytate. The isolated product exhibited a characteri s t i c absorption spectrum i n the 200- to 320-nm range (Figure 1) that corresponded to the spectrum for synthetic monoferric phytate. The Mbssbauer spectra (Figure 2) of the iron i n wheat bran and of synthetic monoferric phytate also correspond (8). Solubility characteristics of monoferric phytate. Although a solution of high ionic strength i s required to extract monof e r r i c phytate from wheat bran, free monoferric phytate, whether extracted from bran or synthesized, i s readily water soluble. We have not tried to determine i t s maximum solubility i n water, but found i t insoluble i n 50$ ethanol:water solution. This later fact enabled i t s precipitation when prepared from f e r r i c chloride and sodium phytate (6, 9). The product produced by the synthetic method developed by E l l i s , described i n detail by Lipschitz et a l . (9), i s a finely divided pale yellow solid.

Kies; Nutritional Bioavailability of Iron ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


8.

MORRSI AND ELLIS

123 Effects of Phytate and Wheat Bran

Figure 1. UV absorption spectra of monoferric phytate in 1.2 M ammonium acetate. Solid line, sodium phytate; short dashed lines, monoferric phytate purified from bran; longer dashed lines, synthetic monoferric phytate. (Reprinted, with permission, from Ref. 6. Copyright 1976, American Institute of Nutrition.)



124


4

1

4

h

±

VELOCITY (mm/sec) Figure 2. Mossbauer spectra. Top: solid monoferric phytate, 212 mg at 80 K. Center: wheat seeds, 1.54 g at room temperature. Bottom: dissected wheat bran, 9.5 mg at 80 K. All materials enriched in Fe. (Reprinted from Ref. 8. Copyright 1980, American Chemical Society.) 57



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MORRSI AND ELLIS


Recovery of monoferric phytate subjected to g e l - f i l t r a t i o n chromatography over a pH range of 1.5 to 10.5 i s shown i n Figure 3. In 0.05 M buffer recovery was 90$ or greater at pH 6.5 to 10.5, but only about 70$ at pH 4 to 6, and very low at pH less than 3* Recovery was reduced by at least 50$ when 0.5 M sodium chloride was included i n the buffer. Further experiments showed that monoferric phytate undergoes a transformation and forms an insoluble product i n either 0.1 M HC1 or high chloride concentration. In the transformation iron precipitates as insoluble d i f e r r i c phytate (10). However, 1.3 moles excess of phytate anion prevents this transformation, and the monoferric phytate remains soluble. The presence of excess phytate i n the extract made possible the i n i t i a l extraction i n 1 M sodium chloride of monoferric phytate from wheat bran. The transformation does not occur i n acetic acid or ammonium acetate solution. Endogenous form of iron i n wheat bran. Because monoferric phytate formed readily i n mixtures of sodium phytate and f e r r i c ion, we wondered whether the monoferric phytate i n extracts of bran represented an a r t i f a c t of the extraction procedure rather than represented the chemical form of iron that i s endogenous i n wheat bran. Two lines of information support our b e l i e f that iron exists as monoferric phytate i n wheat bran. F i r s t , when Mossbauer spectra were compared between seeds and bran of wheat grown on hydroponic media enriched in 57Fe and synthetic iron phytates (8), the spectra of the seeds and dissected bran (Figure 2) are very similar to the spectrum of solid monoferric phytate. The spectral parameters, quadrupole s p l i t t i n g and isomer shift (Table I ) , of wheat seeds and bran are identical with those of solid monoferric phytate; a l l are within the range of those found with high-spin f e r r i c compounds. Table I.

Mdssbauer Spectral Parameters of Wheat Seed, Wheat Bran, and Monoferric Phytate (8) a

Sample

Quadrupole

splitting

Isomer shift

Wheat seeds

0.55

0.76

Wheat bran

0.56

0.77

Monoferric phytate

0.55

0.77

a

Values are i n millimeters per second relative to sodium nitroprusside and were measured at 80 K. Reprinted from Ref. 8.

Copyright 1980, American

Chemical


Society.


126


Figure 3. Effect of pH on recovery of monoferric phytate subjected to gel-filtration chromatography. Monoferric phytate samples (40 mg) were chromatographed on a 2 X 50 cm Biogel P-4 column equilibrated with respective buffer.


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MORRSI AND ELLIS



The parameters indicate that the same iron configuration exists in the wheat seeds and bran and i n the monoferric solid. The Mdssbauer measurements revealed differences between d i f e r r i c and monoferric phytate (8). The second independent line of evidence was derived i n the preparation of the low-phytate wheat bran used for zinc bioavaila b i l i t y studies (11). The endogenous phytase of bran was i n a c t i vated by autoclaving the dry bran. The autoclaved bran was then extracted with 0.1 M acetate buffer, pH 4.5, at 37°. About 90$ of the phytate was extracted by this procedure; phytic acid concentrations i n the whole bran and the low-phytate bran were 3^42 and 0.36$, respectively. Only a small amount of iron was present i n the 0.1 M acetate extract. The iron i n the low phytate product, however, could be extracted as from whole bran by 1.2M ammonium acetate. Gel f i l t r a t i o n chromatography of the 1.2 M ammonium acetate extracts of whole and low-phytate bran showed almost equal amounts of monoferric phytate, although total phytate' was much less i n the extract of the low phytate bran. Thus, even though the bulk of the phytate was extracted by the 0.1 M acetate solution, the iron was not extracted and remained bound i n the low-phytate residue. The higher ionic strength solution was required to extract the iron and monoferric phytate was identified as the iron species i n the extract. There was a small excess of phytate i n the 1.2 M acetate extract of the low phytate residue, but because of the d i f f e r e n t i a l extractability, we view the evidence to indicate that monoferric phytate represents the major endogenous fraction of iron i n wheat bran. Bioavailability of Ferric Phytates Animal studies. In the i n i t i a l studies (6), a partially purified monoferric phytate prepared from extracts of wheat bran and two synthetic preparations were bioassayed using rats. The hemoglobin depletion-repletion method was used and the relative biological value (RBV) was computed by slope ratio. Compared to the response to ferrous ammonium sulfate as 100, the RBV for the monoferric phytate prepared from wheat bran was 99, and for the two synthetic preparations 101 and 97. The 95$ confidence interval for the preparation from wheat bran was 87-111. The iron of monoferric phytate i s highly bioavailable to rats. E l l i s and Morris (10) tested d i - and tetraferric phytates as dietary iron sources for rats. We harvested d i f e r r i c phytate from the precipitate that formed when monoferric phytate was suspended i n dilute hydrochloric acid and we made tetraferric phytate by adding excess f e r r i c ion to a dilute acid solution of sodium phytate and harvesting the resultant precipitate. After a 3-week feeding period, the hemoglobin responses to monoferric phytate and ferrous ammonium sulfate were about equal (Table I I ) .


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NUTRT IO INAL BO IAVAL IABL IT IY OF R ION Table I I . Comparison of Ferric Phytates as Dietary Iron Source for Rats Iron Source

Hemoglobin'

1

g/dl 4.0 + 0.5

Basal Fe(NH4) (S04) -6H 0

10.3 + 1.2

FePhytate

10.6 + l . l

2


a

2

b

2

b

Fe Phytate

5.4 + 1.1°

Fei^Phytate

7.4 + 0.5

2

d

1

Added iron for a l l diets except basal was 18 pg/g. Diets were fed for 3 weeks. Basal diet contained 6 yg/g of iron.

2

Mean + S.E.M. Values with different superscripts are significantly different (P

Nutritional Bioavailability of Iron - American Chemical Society

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