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This is to certify that the

thesis entitled

Bioavailability of Copper in Kidney and Garbanzo Beans

presented by

Yin-Chieh Li

has been accepted towards fulfillment
of the requirements for

Masters degree inHuman Nutrition

 

 

Major professor

Date W97

0-7639 MSU is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Michigan State
University

 

 

 

PLACE It RETURN BOX to remove thie checkout tram your record.

TO AVOID FINES return on or before date due.

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MSU Ie An Affirmative Action/Equal Opportunity Institution
WWJ

 

BIOAVAILABILITY OF COPPER IN KIDNEY AND GARBANZO BEANS

By

Yin-Chieh Li

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition
1994

ABSTRACT

BIOAVAILABILITY OF COPPER IN KIDNEY AND GARBANZO BEANS

By

Yin-Chieh Li

In the first of two experiments to evaluate effects of phytate in
legumes on copper and zinc bioavailability, rats were fed diets containing
2 pg/ g Cu supplied by garbanzo beans, kidney beans or copper carbonate.
The garbanzo bean and kidney bean diets contained 0.13 and 0.19% phytate,
respectively. Ceruloplasmin activity and serum zinc at the end of 3 wk were
significantly different among groups: control > garbanzo bean > kidney bean.
Serum copper was significantly higher in the control group. In the second
experiment, all rats were fed a copper deficient diet (0.12 pg Cu / g) for 4 wk
At the end of a 7 day repletion period (2 pg Cu / g), ceruloplasmin activity
and serum copper were significantly higher in rats fed the diets without
phytate (control and phytase-treated kidney bean diets) than in those
containing phytate (0.14% in kidney bean and control+phytate diets). In
both experiments, tissue concentrations of copper and zinc were similar in
all groups. The overall results indicate that copper is less bioavailable in
diets containing phytate. The apparent failure of phytate to inhibit zinc
bioavailability may have been related to the low phytate : zinc molar ratios

of the diets.

Tomyfamily

iii

ACKNOWLEDGMENTS

I wish to express my gratitude to my advisor, Dr. Wanda L
Chenoweth for her generous assistance and valuable advice throughout
the entire study. Sincere appreciation are extended to my committee
members, Dr. Maurice R Bennink and Dr. Dale R Romsos for being on
my committee and providing any guidance and help they could.

I would also like to thank Ritu Ummadi, Mei-Chuan Chen and
Yongsoo Chung for their friendship and support through the best and
worst of times. I want to thank each member of my family for their
understanding and support.

Last but not least, this thesis is dedicated to my husband, Kuo-Ding

for his patience, encouragement and love.

iv

TABLE OF CONTENTS

LIST OF TABLES eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee vii

LIST OF FIGURES eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

INTRODUCTION eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

REVIEW OF LITERATURE
1. Copper metabolism and functions eeeeeeeeeeeeeeeeeeeeeeeeeee
2. Dietary factors affecting copper utilization in
humans and animals 00.000.000.000.00000000000000000000.0000
a. Ascorbic acid-copperinteractions ooooououuouoouuuu
b. Carbohydrates-copper interactions ooooooouooouuouuou
C. Zinc-copper interactions eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

3. Bioavailability of trace elements uuuuuuuuuuuu«no
4, Legumes O000000.0000000000000000000000000CC...0.00.00.00.00
5. Phytate in legumes

Chemistry of phytic acid eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Occurrence eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Functions and uses of phytate eeeeeeeeeeeeeeeeeeeeeeeeeeee

Digestion and bioavailability of phytate in

humans and animals eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
e, Phytase eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

6. Phytate-minera] interactions eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
a. Interactions \Nith zinc eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
b. Interactions with copper eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

7. Summary 0.00.00.00.00.00000000000COOOOOOOOOOOOOOODOOOOOOOO

.o-np‘s»

MATERIALS AND METHODS
1. Experimental design
a_ General procedures eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
2. Experiment1

a, Animals andtreatmentseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
b, Diets eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

C, Bean preparatlon OOOOOOCCDOOOOOOOOOOOOOOOOO0.0.0.0000...

ix

31

31
32
34

3. Exp
a.
b.
c.

eriment 2
Animals and treatments OOOOOOOOOOOOOOOOOOOOOOO0.0.000...

Diets OOOOOOOOOOOOOOOOOOCOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Bean and dephytinized bean preparation oooouooouuoouo

4. Analytical methods

Protein content in diets ....0..........0..0.0...0.0.0.00.

. Tissue and diet copper and zinc determination o o o o o o o o o o o 0

Serum copper and zinc determination oonoouuuoouuu
Determination of serum ceruloplasmin activity 0 o o o o o o o o . o

. Determination of phytate phosphorus in beans 0 o o . . . o . o . . .

HPLC analysis of inositol phosphates in beans 0 o o o o o o n o . o o

5. Statistical methods .00......00.0.0...0...0....0.0..00...0.0.

a.

b

c.

d.

e

f.
RESULTS
1 . Exp

a.
b.

c.
d.
e.
2. Exp
a.

b.

C.

:‘h

eriment 1

Copper, zinc, iron, phytic acid and protein content of diets o 0
Body weight, weight gain, food intake, efficiency of food
utilization and total mineral intake 0 noon 0 o o u no u o o o o 0
Organ wet weights, dry weights and

0/0 body weight of organs ...00......0..0..00......0.00...0
Ceruloplasmin activity, serum concentrations of

copper and zinc ....00.000..00.0...000000.00.....00.0..0.
Tissue concentrations of copper and zinc o o c o o o o o o o o o o o o o o o
eriment 2

Copper, zinc, iron, phytic acid and protein content of diets o 0
Body weight, weight gain, food intake, and efficiency of food
utilization 0.000.00.000.00.00.0.0.0...000.000.000.000...
Organ wet weights, dry weights and .

% body weight of organs 00.00.0.........000.0......0.000.
Ceruloplasmin activity, serum copper and zinc

concentration 0.0.0.0...00......0.00....000......00....0.
Tissue concentrations of copper and zinc o n o o o o o o o u" o o o o
Inositol phosphate analyses using high pressure liquid

chromatography 0.....0.........0..00......0..0.........

DISCUSSION 0..0.0.0000...........0........0......00.000........

CONCLUSION 0.0.0.000000.0..........................0..00....00

FUTURE RESEARCH 000....000.................0.....0.00.0000...

LIST OF REFERENCES .000....00.0........................00.0....

vi

34
35
37

38
38
39
39
40
4‘1
42

43

43

45

49
49

49

55

57

57
61

6‘1
66
74
76
77

TAB LE

TAB LE

TABLE

TABLE

TABLE

TABLE

TAB LE

TABLE

TABLE

TABLE

TAB LE

TABLE

TABLE

10:

‘11:

12:

13:

: Compositions of diets, Experiment 1

: Compositions of diets, Experiment 2

: Body weights and food intake of rats, Experiment 1

: Organ wet weights and dry weights, Experiment 1

: Organ wet weight to body weight ratio, Experiment ‘1

LIST OF TABLE

OOOOOOOOOOOOOOOOO.

: Analyzed mineral and phytic acid content of

diets, Experiment 1

: Total mineral intake from diets, Experiment 1 o o o o . o o o o o

00....

: Ceruloplasmin activity, serum copper and zinc

concentrations, Experiment ‘1 0......000000.0...0.00.00

: Concentration and total content of copper in

tissues, Experiment 1

Concentration and total content of zinc in
tissueS, Experiment ‘1 ...0.....0.0.000.0.00..00..0.000
Analyzed mineral and phytate content of

diEtS, Experiment 2 ......0....0...........000.0000.00
Body weights and food intake of rats during

the repletion period, Experiment 2 ouuoououooouou

Organ wet weights and dry weights, Experiment 2

vii

33

36

44

46

47

48

50

51

52

53

54

56

58

TABLE 14 :

TABLE 15 :

TABLE 16 :

TABLE 17 :

TABLE 18 :

Organ wet weight to body weight ratio of
rats, Experiment 2 00.0.0.0...0.....00.00......00000.0

Ceruloplasmin activity at initial, 3d and 7d
of repletion period, Experiment 2 ooouooououuouou

Serum copper and zinc concentrations after
7 days of repletion, Experiment 2 ouooouuouoouuou

Concentration and total content of copper
in tissues, Experiment 2 .0.0......0....0.....000..00..

Concentration and total content of zinc
in tissues, Experiment 2 .00....00000....00....00000000

viii

59

60

62

63

64

LISl'OFFIGURI'S

FIGURE 1: Basic aspects of mammalian copper metabolism ”on" 5
FIGURE 2A; Structure ofphytic acid0.0.0......0...00.00000.....0.0 ‘15

FIGURE 2B: Phytic acid chelate at neutral pH uouuooouuuouon 15

ix

INTRODUCTION

Recognition of the biological importance of copper is of recent
origin. Copper is an essential component of metalloenzymes such as lysyl
oxidase, cytochrome c oxidase and superoxide dismutase. Activities of
these enzymes are decreased by copper deficiency. Although copper is
widely distributed in foodstuffs, its absorption and utilization may be
affected by various dietary factors.

Consumption of legumes is increasing as people become aware of
their potential nutritional benefits (Morrow, 1991). Legumes contain high
amounts of complex carbohydrate, dietary fiber, protein, certain vitamins
and minerals including copper and zinc. However, legumes also contain
antinutritional factors such as phytate, which accounts for up to 85% of
total phosphorus in many cereals and legumes. Concern about the
presence of phytate in cereals and legumes arises from the evidence that it
decreases the bioavailability of essential minerals such as zinc, calcium
and iron by forming insoluble complexes and rendering them unavailable
for intestinal absorption. This interference with intestinal absorption of
minerals may lead to mineral deficiencies in humans and animals.

It is known that zinc in beans is poorly available for animals and
humans. Phytate is one of factors that contributes to this low

bioavailability. The effect of phytate on copper bioavailability, however, is

1

2

still controversial. Based on animal studies, some researchers claimed
phytic acid had no effect on copper bioavailability, whereas others reported
that it enhanced copper bioavailability (Lo et al., 1984; Lee et al., 1988).
Although extensive work has been done on the effect of phytic acid on
zinc bioavailability, little is known about the bioavailability of copper in

various kind of beans which contain variable amounts of phytic acid.

REVIEWOF LITERATURE

qupermelamism all! fmcliuis

Although copper can be absorbed from all portions of the small
intestine as well as from stomach, the upper portion of the small intestine
is the site of maximal absorption of copper. The precise biochemical
mechanism for absorption of copper across the brush border surface of the
small intestine is not known. It is believed that copper absorption
involves mechanisms other than simple diffusion. Bronner and Yost
(1985) reported that at least two mechanisms were involved in copper
absorption. One was saturable suggesting an active transport process; the
other was unsaturable indicating passive diffusion. Like transport systems
for other nutrients, when dietary copper concentration is low, copper is
primarily absorbed via the active transport pathway, whereas passive
transport occurs when the dietary copper concentration is high.

In the blood stream, copper is distributed among three major
plasma constituents: the protein ceruloplasmin, albumin, and free amino
acids. Over 90% of the total plasma copper is tightly bound to
ceruloplasmin. Most of the remainder is less tightly bound to albumin,
and 1-2% of the nonceruloplasmin bound copper is loosely attached to
amino acids (DiSilvestro and Cousins, 1983). Albumin and amino acids

are the ligands that transport copper in the portal circulation following its

3

4

intestinal absorption and that deliver the metal to the liver. Once in the
liver, copper is incorporated into ceruloplasmin which is released into the
blood where it transports copper to extra hepatic tissue for synthesis of
cuproenzyrnes. Copper is excreted primarily via the gastrointestinal tract.
Bile contributes the major portion of endogenous fecal copper, especially
in humans. A small amount of copper is lost in the urine. Figure 1
summarizes the basic aspects of mammalian copper metabolism.

Like other essential trace metals, the essentiality of copper is due to
its presence as part of the structure of essential enzymes. For example,
copper is required in collagen synthesis (lysyl oxidase), energy production
via oxidative phosphorylation (cytochrome c oxidase), and disposal of
peroxides in tissues (superoxide dismutase). Copper also plays a key role
in the metabolism of other elements such as iron which requires
ceruloplasmin (ferroxidase) to oxidize iron to ferric iron for attachment to
transferrin for transport and distribution. As interest in copper‘s role in
metabolism has grown so has interest in the factors which might affect its

absorption or utilization by the body.

Dietary faclms affecting comer uliizalim in lumans and animals

The absorption and utilization of copper in food may be influenced
by a large number of dietary factors such as ascorbic acid, fructose, sucrose
and possibly phytate. These dietary components can reduce copper
absorption. In addition, other trace minerals, if present in sufficiently
high concentrations, have an antagonistic effect on copper absorption and
the antagonistic effect of zinc on copper bioavailability is perhaps of

greatest practical significance (O'Dell, 1990).

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6

Ascorbic acid-copper interactions. A high dietary intake of ascorbic
acid is likely to decrease copper utilization in several species. Rats fed
copper deficient diets supplemented with 1-5% ascorbic acid had higher
mortality rates, increased cardiac and/or vascular abnormalities, lower
body weights, and lower hematocrit and hemoglobin levels than control
animals (Johnson, 1986; Johnson and Murphy, 1988). In a short term study
using orally administered radiolabeled copper to measure copper status,
Van Campen and Gross (1968) found that high level of ascorbic acid
decreased the absorption of copper from ligated intestinal segments, and
decreased the retention of copper in the liver and whole body of rats.

From these studies, it was proposed that ascorbic acid impairs copper
utilization through decreased copper absorption or through increased
turnover of copper. In human studies, Finley and Cerklewski (1983)
investigated the influence of ascorbic acid supplementation on the copper
status of young men. Results showed that ingestion of 1500 mg ascorbic
acid daily for 60 days significantly reduced serum ceruloplasmin activity by
26% and lowered serum copper concentrations. Serum copper was
significantly increased 20 days after the ascorbic acid supplements were
discontinued. This study confirmed that a high ascorbic acid intake is
antagonistic to the copper status of men supporting results found in

laboratory animals.

Carbohydrates-copper interactions. It has been reported that the
severity and rate of development of copper deficiency in rats are related to
the carbohydrate component of the diet (Fields et al., 1984; Fields et al.,

1986). These effects could be caused by a difference in bioavailability of

7

copper in the presence of different carbohydrate, or by differences in
excretion of copper when different carbohydrates are fed. Studies with
radiolabeled copper were conducted by Fields et a1. (1986) who investigated
the effect of different dietary carbohydrates (fructose and starch) on the
absorption, tissue distribution and excretion of copper with copper
deficient or copper supplemented rats. Forty eight and 96 hours after
intubation of 67Cu-labeled diets, there was a significantly higher retention
of 67Cu in the gastrointestinal tract accompanied by decreased whole body
retention and urinary excretion in copper deficient rats fed the fructose-
based diet compared to those fed a starch-based diet. No differences due to
dietary carbohydrates were found in copper supplemented rats. Another
study conducted by Johnson and Gratzek (1986) showed that male rats fed
copper deficient diets with 20% sucrose or fructose rather than starch or
glucose had lower growth rates, higher mortality rates, higher heart and
liver weights and were more anemic after 3-4 weeks feeding The
mortality rate of copper deficient rats fed fructose or sucrose was greater
than 60% after 7-9 weeks. In contrast, the mortality rate of copper deficient
rats fed starch or glucose generally ranged from 0-30% (Fields et al., 1984).
Results from these studies indicate that fructose may be the dietary
component which has an adverse effect on copper status. However, in rats
fed a marginal copper diet (2.5 ug/ g), Johnson et al. (1988) found no
significant differences in copper absorption due to different carbohydrates
in a single meal. These results showed that carbohydrate component of

the diets can affect copper bioavailability on a long term feeding.

8

Zinc-copper interactions. Zinc is an essential nutrient required for
numerous metalloenzymes, cell replication and differentiation,
reproduction, growth and many other essential functions (Hambidge et al.,
1986). Zinc and copper are two elements with very similar molecular
weight, and their electronic structures are quite close; therefore, they may
compete with each other for absorption. Several investigators have
reported inhibition of copper absorption and deposition of copper in the
tissues when the dietary or intraluminal Zn : Cu ratio was from 500 : 1 to
1000 : 1 (Van Campen and Scaife, 1967; Evans et al., 1974). O'Dell (1985)
reported that high zinc diets (120 or 240 ug/ g daily) significantly lowered
the activities of liver Cu, Zn-superoxide dismutase and heart cytochrome c
oxidase, even when the diet contained an adequate level of copper (6
ug/g).

Oestreicher and Cousins (1985) conducted a series of experiments to
test the influence that copper and zinc exert on each other's absorption by
using an isolated, vascularly perfused rat intestine technique. The dietary
copper and zinc concentrations were held at 6 mg/ kg and 30 mg / kg,
respectively, while the metal concentrations in the luminal perfusate were
changed (from 1 to 36 mg/ L and from 5 to 180 mg/ L for copper and zinc,
respectively). Results indicated that medium and high luminal zinc
concentrations (30 and 180 mg/ L, respectively) significantly decreased
copper absorption when the luminal copper concentration was low (1 mg
Cu/ L). The results of luminal copper concentration on zinc absorption
showed that medium and high copper concentrations (6 and 36 mg/ L,
respectively) in the lumen perfusate significantly decreased zinc

absorption when the luminal zinc concentration was also high (180 mg

9

Zn/ L). In contrast, there was no significant difference on zinc or copper
absorption when the luminal zinc and copper concentrations were 36 and
6 mg/ L respectively. Results of studies in humans were similar to those
obtained in animal studies. Fischer et al. (1984) reported that consumption
of 50 mg Zn/ day for 6 weeks by young men significantly decreased Cu, Zn-
superoxide dismutase activity in red blood cells. However, Turnland et al.
(1988) observed that relatively small changes in zinc intake did not appear
to affect copper absorption. For example, an increase in dietary zinc from
5.5 to 16.5 mg Zn/day, which is approximately the RDA for zinc (15

mg / day), did not increase fecal losses of copper or decrease the absorption
of copper in young men fed 1.3 mg Cu/day. The results obtained from
these studies indicate that a competition and/or inhibition of copper or
zinc uptake into intestinal cells occurs when the luminal concentration of
either metal is very high, whereas no copper-zinc antagonism occurs at
the absorptive level when the copper and zinc intakes are near

requirement level.

Bbavaialiityofh'amelanals

Bioavailability can be defined as the proportion of the total mineral
in a food, meal or diet that is utilized for normal body functions. It
involves various stages, each of which is affected by different dietary and
physiological factors (Fairweather—Tait, 1992). The amount of a mineral
that is available for absorption is dependent upon dietary composition,
gastrointestinal secretions, and luminal interactions. Several studies
indicated that the competitive interaction between minerals has an effect

on bioavailability of minerals and it can occur in the food during

 

10

harvesting processing or storage or in the lumen of the intestine during

the gastrointestinal phase of digestion. It also can occur as minerals bind

to the brush border of the intestinal cell or are incorporated into the
intestinal cell (Mertz, 1980; Sandstead, 1981). Clydesdale (1989) reported
that the interaction between minerals in the gastrointestinal tract was
directly related to their electronic structure, that is, those elements whose
physical and chemical properties are similar will act antagonistically to
each other biologically.

In foods, the mechanisms involved in mineral-mineral
interactions which have the potential to affect bioavailability have been
broadly defined into four categories (Clydesdale, 1988):

1. Mineral displacement: Displacement of a mineral from a complex
with another mineral to form a soluble (available) or insoluble
(unavailable) complex This category would include mineral reactions
with fiber or phytate. For instance, lignin, an inhibitor, provides a
good example of this since it readily binds most minerals, causing them
to precipitate and then become unavailable. However, the inhibition
of lignin on mineral was variable in several studies. This variability
may have occurred because in some studies only one mineral was
present and thus it would be inhibited, whereas in other studies other
minerals were also present which may compete for binding.

2. Polymineral-ligand complexes: The addition of a second or third
mineral to a soluble mineral-1i gand complex causing precipitation by
forming a polymineral-ligand complex This category includes the
phenomena which occur when calcium and zinc are present together

with phytate. Platt et al. (1987) analyzed the solubility of calcium in the

11

presence of phytate added with or without zinc. When the
phytate/calcium/zinc ratio was 3 : 6 : 1, 19% of the calcium and 50.2% of
the zinc remained soluble. Another study conducted by Graf and Eaton
(1984) indicated that calcium exhibited a bimodel effect on the solubility
of zinc For example, low concentrations of calcium increase the
solubility of zinc whereas high concentrations of calcium potentiate the
precipitation of zinc by phytate.

. Polymineral-polyligand: The addition of a mineral causing a mineral-
ligand complex to form and / or more than one mineral binding to
more than one substrate (ligand) and forming a polymineral-polyli-
gand complex This third category includes complexes such as an iron—
zinc—phytate-protein complex, the protein-phytate-zinc complex and
the zinc-phytate complex. When only one mineral and one ligand are
present in the system, precipitation may occur. This complex is usually
mineral-specific and ligand-specific so that this ligand will not form a
complex with all minerals. In addition, this complex may remain
soluble unless another mineral and [or another ligand combines with
this complex to form insoluble polymineral-polyligand complexes.
Platt et al. (1987) analyzed the interactions of iron, alone and in
combination with zinc, copper, with a phytate-rich, fiber-rich (PRFR)
fraction of wheat bran and a sodium phytate sample under
gastrointestinal pH conditions. Results showed that 31.2% of iron
remained soluble when iron was added alone to the PRFR fraction; the
solubility of protein and phytate was also decreased. This result
indicated that an insoluble iron-phytate-protein may have been

formed. In contrast, iron in the sodium phytate sample was completely

12

soluble. It is probable that the iron-phytate complex is soluble. When
both iron and zinc were added to PRFR samples, there was a large
decrease in iron solubility (8.2%) and the solubility of protein and
phytate. Zinc solubility was also very low (10.6%). This result
indicated that an insoluble zinc-iron-phytate-protein complex was
formed whereas zinc and iron remained soluble in the sodium phytate
sample. This demonstrated that phytate complexes existed when
protein is not present. In addition, when copper was added with iron
there was little change in iron solubility in either the PRFR or phytate
samples. However, the solubility of copper was lower in the PRFR
(65.9%) and higher in the sodium phytate (93.6%) sample. In this
situation, there are several factors that would affect solubility of
minerals such as proteins, fiber or phytate and the presence of other
minerals which may either increase or decrease solubility.

. Enzyme susceptibility of complexes: The formation of a polymineral-
ligand complex which changes the susceptibility of the mineral-ligand
bonds to cleavage by digestive enzymes. Digestive enzymes in the
gastrointestinal tract may increase the solubility of minerals by cleaving
mineral-ligand bonds. Although some mineral-ligand complexes are
susceptible to degradation by digestive enzymes, thus releasing the
mineral for absorption, it is possible that some complexes form which
are not susceptible to digestive enzymes. The total bioavailability of a
mineral depends on the proportion of mineral in complexes that can

be degraded by digestive enzymes.

13

Leguns

Legume production and consumption are increasing as more
people become aware of legumes' nutritional benefits. Morrow (1991)
reported that the 1990 per capita consumption levels of dry beans was 7.2
lb, an increase of 30.9 % over the previous year. Legumes are high in
complex carbohydrates, protein, certain vitamins (thiamin and niacin),
minerals (phosphorus, potassium, calcium, zinc, copper and magnesium),
and dietary fiber, especially water-soluble fiber. The high content of water
soluble fiber is particularly effective in lowering cholesterol in the blood,
while water insoluble fiber provides bulk, promoting the movement of
food through the intestines at a faster rate. In addition, beans are a good
alternative to red meat. Since beans offer more protein per dollar than
other foods, they become an important economical source of protein in
the diet of many developed and developing countries (Morrow, 1991).
However, legumes also contain antinutritional factors such as trypsin
inhibitors, lectins, protease inhibitors, polyphenols or phytates. Since
legumes contain a considerable amount of phytate, concern has been
expressed that their content of phytate may have an adverse effect on

mineral metabolism, particularly on trace elements (Reddy et al., 1982).

mummies-m

Chemistry of phytic acid. Phytic acid was discovered by Hartig in
1855 and through extensive studies by many researchers, it is accepted that
phytic acid is myoinositol 1, 2, 3, 4, 5, 6-hexaphosphate with an empirical
formula of C6H18024P6 (Reddy et al., 1982; Gibson and Ullah, 1990). The

14

name of phytic acid has been used interchangeably in the literature with
the term phytin which more correctly refers to the mixed Ca-Mg-K salts of
the acid in plants. A partially dissociated Anderson-based structure for
phytic acid might occur at neutral pH. At neutral pH, the phosphate
groups have negatively charged oxygen atoms so that cations can chelate
between two phosphate group or within a phosphate group (Reddy et al.,
1982). Figure 2 is the structure of phytic acid and phytic acid chelate at

neutral pH.

Occurrence. Phytic acid widely occurs in plant seeds and/ or grains
(O'Dell, 1979), root and tubers (McCance and Widdowson, 1935), and fruits
and vegetables (Larbi and M'Barek, 1985). Phytic acid occurs primarily as a
salt of mono— and divalent cations in discrete regions of cereal grains and
legumes. It rapidly accumulates in seeds and [or grains during ripening
period, accompanied by other storage substance such as starch and lipids
(Nahapetian and Bassiri, 1975). The accumulation site of phytate in cereals
such as wheat, rice, barley and rye is near the outside of the seed coat,
whereas in legumes, phytate is distributed throughout the entire protein
complex of the seed (O'Dell et al., 1972; Reddy et al., 1982). Phytate
represents most of the phosphorus in cereals and legumes. Values for the
concentration of phytate in various foods have been summarized by
Reddy et al. (1982). In general, the amount of phytate varies from 0.14%
(rice, polished short grain) to 1.16% (barley) in cereals and from 0.28%
(garbanzo bean) to 2.06% (red kidney bean) in legumes.

15

o
eoL_.rIo H
AW
0 o
mlhloi H H claim
m m

 

 

 

(A)
O

 

 

eOIon H
O

O O
H __ __
DIWIOI H H IOIWI

\O 09

a

C

/

0'.

H
m

 

OH

Figure 2. (A) Structure of Phytic acid,

(B) Phytic acid chelate at neutral pH

16

Functions and uses of phytfl Several physiological roles have been
suggested for phytic acid in plants. These include its role as a storage of
phosphate, an energy source, a source of myoinositol and an initiator of
dormancy (Gibson and Ullah, 1990). Besides having a role in the
physiological function, phytate may have an effect on the production of
aflatoxin. Gupta and Venkatasubramanian (1975) reported that phytate in
soybean seeds prevented aflatoxin production by making zinc, which is
necessary for aflatoxin production, unavailable to the mold. In addition,
Graf et al. (1987) suggested that phytate may serve as a natural antioxidant
in seeds during dormancy based on its ability to bind iron to form an iron
chelate thus inhibiting iron-catalyzed hydroxyl radical formation and
suppressing lipid peroxidation.

Sands et al. (1986) summarized the food and medical applications of
phytic acid and suggested several additional uses for phytic acid. Because
of the chelating and antioxidant properties of phytic acid, it has been
proposed for use as an additive to preserve a variety of foods. A number
of medical applications have been suggested for phytic acid based on its
interaction properties, and its antineoplastic action has become the topic of
intense investigation at present. Phytic acid has been shown to have
antineoplastic action in colon carcinogenesis. Graf and Eaton (1985)
reported that inositol hexaphosphate consumption is considered to be a
major etiologic factor for the approximate 50% reduction in the incidence
of large intestinal cancer in Finland compared to Denmark Since Finnish
people consume 20-40% more phytate than do Danes, it was suggested that
diets high in phytate suppress colonic carcinogenesis and other

inflammatory bowel diseases by inhibiting intracolonic hydroxyl radical

 

 

17

generation via the chelation of iron. Moreover, Shamsuddin and his co—
workers have done a series of experiments to investigate the
antineoplastic action of phytic acid (Ullah and Shamsuddin, 1990;
Sakamoto et al., 1993; Vucenik and Shamsuddin, 1994; Shamsuddin et al.,
1988 and 1992). They demonstrated that phytate exerted chemopreventive
and chemotherapeutic effects in rodent colon and mammary
carcinogenesis models. However, Thompson (1988) indicated that the use
of antinutrients such as phytic acid as therapeutic agents needs to be
carefully considered because of the adverse effects associated with their

high intakes.

Digestion and bioavailability of phytate in humans and animals. In
mature cereal grains, legumes, and oil seeds, the major portion of the total
phosphorus is present in the form of phytic acid, and the availability of
phosphorus when present in the form of phytate depends on the species,
the age of animal, and the level of phytase activity in the intestinal tracts
of the species. Due to the rumen microorganisms, ruminants were able to
utilize most of the dietary phytate. Nelson et al. (1976) studied the
hydrolysis of natural phytate phosphorus from soybean meal, sorghum
grains, and corn meal in the intestinal tract of calves and steers. They
found that the initial phytate hydrolysis occurred in the rumen and was
complete before the feed reached the other parts of the digestive system.

In general, biological availability of dietary phytate phosphorus to
ruminants is 80% or greater (Reddy et al., 1982). However, the biological
availability of phytate phosphorus to nonruminants is poor compared to

ruminants. Reddy et al. (1982) reported that the biological values of

 

 

18

phytate phosphorus for pigs were from 25% to 40%. Lei (1992) reported
that phytate phosphorus from barley and corn was about 17% and 8%
digestible, respectively, for growing pigs when phytate was the major
source of phosphorus in the diets.

It has been reported that rats can utilize phytate phosphorus. The

 

average of phytate phosphorus that can be utilized by rats is 27% to 41%
(Reddy et al., 1982). A study found that rats adapt to low phosphorus diets
by an increase in phytate digestion (Moore and Veum, 1983). The
adaptation may result from increased synthesis of phytase or alkaline
phosphatase by intestinal microorganisms when there is a low level of
phosphorus in the diet. Although the number of experiments to
determine the bioavailability of phosphorus from legumes phytate in
humans have been limited, Sandberg et al. (1982, 1986, and 1987) reported
that 40% to 60% of the phytate phosphorus from wheat bran was available
to man. They also indicated that the hydrolysis of phytate by human
phytase or alkaline phosphatase does occur in the stomach and small

intestine, and is not due to microbial phytase activity.

Phflase. Phytase, myoinositol hexaphosphate phosphohydrolase, is
an enzyme which is widely distributed in plants, animals, and fungi.
Phytase is capable of hydrolyzing myoinositol hexaphosphate to inorganic
orthophosphate and a series of lower phosphoric esters of myoinositol or
to free myoinositol (Reddy et al., 1982). Maiti et al. (1979) reported that
degradation of phytate by phytase occurs in a stepwise manner starting
with dephosphorylation from position 6 followed by removal of

phosphorus from positions 5 and 4, 1 and 3, or 1 and 4, with the phosphate

19

at position 2 being stable. Recently, Gibson and Ullah (1990) suggested that
there are two major types of phytase. One is 3-phytase (E. C. 3. 1. 3. 8)
which initiates the removal of phosphate groups attached to position 1 or
3 of myoinositol. The other one is 6-phytase (E. C. 3. 1. 3. 26) which first
frees the phosphate at the 6 position. Phytase characterized from
microorganisms and the fungi belong to 3-phytase, while the 6-phytase is
found in seeds of higher plants. In general, both types of activity are
referred to as phytase.

The pH and temperature optima for phytase from certain cereals
and legumes are different. According to Reddy et al. (1982), the optimal
pH for phytase activity appears to be in the range 4-7.5. The optimum
temperature for phytase activity varies from source to source but appears
to be in a high temperature range (45-60 °C). The unit of phytase activity
described in this thesis is defined as the amount of enzyme that liberates
1 umol of inorganic phosphorus from sodium phytate per minute at pH
5.15 and 55 °C.

It has been shown that the bioavailability of trace elements can be
improved by the presence of supplemental phytase in diets which contain
a high amount of phytate; this will be discussed in the following section

on phytate-mineral interactions.

H l I - I - l I'
Under certain condition, phytate may form insoluble complexes
with di- and trivalent cations at neutral pH potentially rendering these

minerals unavailable for intestinal absorption (Graf and Eaton, 1984). The

insolubility of metal-phytate complexes may have adverse nutritional

 

20

implications since it has been claimed to interfere with the bioavailability
of calcium, zinc, and iron in humans (Morris, 1986). The formation of
these complexes is pH dependent. Oberleas (1973) reported that although
the decreasing order of metal complexion is Cu++ > ZnH > C0“ > Mn++ >
Fe+++ > Ca++ at pH 7.4, maximum precipitation of zinc-phytate or phytate-
zinc-calcium occurs at pH 6.0 which is the approximate pH of the
duodenum. Nutritionally, mineral-phytate binding at pH 6.0 is more
important since the duodenum is the place where maximum absorption

of divalent metal ions takes place.

Interactions with zinc. Phytic acid is thought to be the primary
inhibitory factor in soybean products that results in reduced zinc
bioavailability. A reduction in phytate is reported to improved the
bioavailability of soybean zinc (Ellis and Morris, 1981; Lonnerdal et al.,
1988; Zhou et al., 1992). A negative effect of phytate on zinc bioavailability
was first shown by O'Dell and Savage (1960) who reported that the phytic
acid associated with plant proteins was responsible for decreased
availability of zinc in diets prepared from natural plant proteins. They
found that animals fed animal protein-based diets containing the same
level of zinc grew normally without any deficiency symptoms compared
to those fed on plant protein-based diets. To verify this, they conducted a
second experiment in which phytic acid from soybean was added to a
casein-based diet and the grth response of chicks fed this diet was
compared to those fed soybean proteins as the only source of protein.
Results showed that phytic acid decreased the bioavailability of zinc with

the casein-based diet and produced symptoms similar to those observed in

21

animals fed soybean protein diets containing a comparable level of
phytate. Therefore, they concluded that the zinc requirement was
increased in the presence of soybeans in animal diets.

Momcilovic et al. (1976) evaluated the biological availability of zinc
in bovine milk and soy protein-based infant formulas using total femur
zinc of growing rats. A zinc deficient diet containing egg white protein
was supplemented with graded levels of zinc (0, 3, 6, 9, 12 ug/ g) from zinc
sulfate, bovine milk, or soy protein-based infant formulas. By using a
slope-ratio bioassay model, the relative biological availability of zinc in
bovine milk-based formula and soy-based formula was 0.86 and 0.67,
respectively compared to zinc sulfate (zinc sulfate = 1). It was evident
from these results that the relative biological availability of zinc in soy-
based formula was about 20% less than that in the milk-based formula. In
a similar study, Forbes and Parker (1977) measured zinc bioavailability
from full fat soy flour using a slope-ratio assay procedure in male
weanling rats. These researchers reported that when the log of total femur
zinc content was used as a criterion of response, zinc from the flour was
utilized 34% as efficiently as was zinc from zinc carbonate. In a second
experiment, however, when zinc carbonate was added to a whole fat soy
flour diet, the zinc utilization was 94% as efficient as when zinc carbonate
was added to an egg white diet. Results indicated that zinc bioavailability
in full fat soy flour was lower than zinc carbonate and it was increased by
adding zinc carbonate.

A study conducted by Welch and House (1982) demonstrated that
both endogenous phytate in plant proteins and added phytate from

sodium phytate impaired zinc absorption. In the first experiment, rats

22

were given either immature soybean seeds (0.6% phytic acid) or mature
soybean seeds (1.7% phytic acid) which were intrinsically labeled with
65Zn. A single meal of the intrinsically labeled soybean supplement was
supplied after a 16-hour fast. The results showed that approximately 89%
of the 55Zn was absorbed from the immature and 60% from mature seeds.
In the second experiment, sodium phytate (1.7% phytic acid) was added to
a basal egg white diet to simulate the phytate level in the diet containing
mature soybean seeds. The rats were fed a zinc deficient diet for five days
before the 16 hours fast and administration of a single meal. 65Zn as zinc
sulfate was added extrinsically in the case of the basal diet and as intrinsic
label in the case of the soybean seeds. Results showed that the phytate
supplement decreased the absorption of 65Zn from both sources. From
these experiments, it is clear that both endogenous and added phytate
impair zinc absorption.

From these studies, it is clear that phytate has a negative effect on
zinc bioavailability. A question arises with regard to whether the factor or
factors responsible for the low zinc bioavailability in cereals and legumes
can be either removed or overcome. Lonnerdal et al. (1988) evaluated the
effect of phytate removal from soybean formula on zinc absorption using
suckling rat pups and infant rhesus monkeys as animal models. The
absorption of zinc from human milk, whey-predominant formula (60 : 40
whey : casein), casein-predominant formula (18 : 82 whey : casein), soy
formula and dephytinized soy formula was determined by whole body
counting. In infant rhesus monkeys, zinc absorption was 65% from
human milk, 60% from whey-predominant formula, 46% from casein—

predominant formula and only 27% from conventional soy formula.

23

However, the zinc absorption from dephytinized soy formula was
increased to 45%. Similar results were obtained in suckling rat pups. Zinc
absorption was 60% from human milk, 52% from whey-predominant
formula, 53% from casein-predominant formula compared to 16% from
conventional soy formula and 47% from dephytinized soy formula.
Results suggested that the low bioavailability of zinc from soy formula was
attributed to its phytate content and it could be overcome by the removal
of phytate. These results were confirmed by Zhou et al. (1992) who
investigated zinc bioavailability in rats fed either egg white-based or
soybean isolate-based diets. Two commercial soybean isolates containing
either normal (2.20%) or reduced levels (0.47%) of phytic acid were used.
Results showed that zinc bioavailability from soybean isolate—based diets
containing normal levels of phytic acid was significantly reduced
compared to the egg white-based diets; furthermore, the reduced phytic
acid soybean isolate-based diets resulted in a significant increase in zinc
bioavailability compared to the soybean isolate-based diets containing
normal phytic acid level. These results coupled with other reports
indicate that phytic acid is the primary inhibitory factor in soybean
products that results in reduced zinc bioavailability, and that phytate
reduction in soybean protein increases zinc bioavailability.

In human studies, Prasad (1976) reported that patients who were
characterized by dwarfism and hypogonadism showed an improvement in
growth and sexual maturity in response to zinc supplementation of a
hospital diet which consisted mainly of phytate-rich cereals and beans.
Analysis of foods consumed by humans suffering from zinc deficiency

symptoms revealed that the amounts of zinc from foods were adequate

24

according to published nutrient requirement literature. Later it was
realized that the presence of phytate in plant products was an important
factor in the reduction of zinc absorption. Several studies on the effect of
phytate on zinc deficiency in humans were reported from developing
countries since people in these countries consume diets primary
consisting of mixtures of cereals and legumes (Reinhold, 1971 and 1973;
Reinhold et al., 1981). The average daily phytate intake may be higher
when compared to developed countries, where the diets contain primary
animal proteins. Thus, zinc deficiency in humans in Middle Eastern
countries was in part caused by the high phytate contents of breads that are
the staple food of the poor.

Several researchers evaluated zinc absorption in humans from
composite meals containing varied levels of phytate (Turnlund, et al.,
1984; Sandstrom, et al., 1980; Morris and Ellis, 1985). Most of these studies
concluded that high levels of phytate in the meals decrease zinc
absorption. Turnlund et al. (1984) studied the effects of oz-cellulose and
phytate on zinc absorption in young men. A semipurified liquid formula
diet supplying 15 mg of zinc daily was given, and zinc absorption was
measured by monitoring fecal excretion of a stable isotope of zinc Results
showed that average zinc absorption was 34% for the basal diet, but
dropped to 17.5% when 2.34 g of phytate as sodium phytate was added to
basal semipurified liquid formula. They concluded that phytate inhibits
zinc absorption and high levels of dietary phytate could result in zinc
deficiency in humans. However, from this study, it is not known whether
or not the effect would be the same if the same level of phytate were

consumed in a natural food diet. Morris et al. (1988) investigated mineral

25

absorption of adult men consuming whole or dephytinized wheat bran
which contained 2 or 0.2 g phytic acid, respectively. Results showed that
apparent absorption tended to be lower when whole bran was consumed.
Sandstrom et al. (1985) observed that retention of a radioactive tracer of
zinc in humans was inversely related to the amount of phytate in meals
that were based on cereal grains.

The molar ratio of phytate to zinc in phytate-rich food has been
suggested as a predicator of the availability of zinc This concept was first
proposed by Oberleas and Prasad (1976). Later, this hypothesis was
supported and demonstrated by Davies and Olpin (1979) and Morris and
Ellis (1980). The latter investigators used semipurified diets to test the
bioavailability of dietary zinc at different phytate : zinc molar ratios in rats.
Growth and femur zinc were used as parameters to evaluate zinc
bioavailability. They observed that grth was not adversely affected
when the dietary phytate : zinc molar ratio was about 12 or less and the
dietary zinc was near the minimum requirement (12 ppm). When the
phytate : zinc molar ratio was greater than 12, grth was not depressed if
the dietary zinc concentration was 2.5 or 5 times the minimal requirement
for growth. However, the accumulation of zinc in femurs was depressed.
In agreement with the findings of Morris and Ellis, Lo et al. (1981) found
that, in general, ratios of approximately 12 and above reduced zinc
bioavailability. Lo and co-workers determined the zinc bioavailability in
isolated soy protein by giving an oral dose of 65Zn and measuring the liver
uptake and disappearance from the gastrointestinal tract of rats. Results
showed that when rats were fed a low zinc diet (5 ug Zn /mg) for 2 weeks

before the absorption trial , the isolated soy protein source had an negative

 

26

effect on zinc absorption. In a four hour period, 65Zn absorption was
12.7% when administered with soybean protein compared to 30.4% when
given with egg white. The liver uptake was 4.2% for soybean protein diet
compared to 10.1% for egg white. The phytatezzinc ratio in soybean
protein was 12.5 and zinc absorption was 40% of that in the egg white
protein which contained no phytate. In contrast, when rats were fed
adequate dietary zinc prior to 65Zn administration, the percentage
absorption and liver uptake were the same for both proteins. From these
experiments, it was demonstrated that phytate had a marked effect on zinc
absorption when the zinc status of the animal was low and the ratio of
phytate : zinc was high but had little effect when the phytate : zinc ratio

was 9 or less and the zinc status was adequate.

In addition to phytate : zinc molar ratios, Morris and Ellis (1980) also
showed the importance of the calcium content of the diet to the phytate :
zinc molar ratio on zinc bioavailability. Growth of rats was not affected by
phytate : zinc molar ratios of 12 or less if the level of dietary calcium was
0.75% but was depressed if the level of calcium was 1.75%. In contrast,
when the dietary zinc concentration was 2.5 or 5 times higher than the
minimal requirement, grth was not depressed at the dietary calcium
levels of 0.75% and 1.75%. These findings were confirmed by Graf and
Eaton (1984), who suggested that calcium ions potentiated zinc ion

precipitation only at high phytate : zinc ratios.

Interactions with copper. Although the effect of phytate on the
absorption and availability of copper has been studied, compared to zinc

the effect of phytate on copper bioavailability was less pronounced and the

27

results were often conflicting. Johnson, P. E. et al. (1988) evaluated copper
bioavailability in sunflower seeds, peanuts, cooked garbanzo beans, cooked
shrimp and cooked beef compared to copper sulfate using an extrinsic 67Cu
label. Rats were fed a diet containing 2.5 ppm copper for 2 weeks before
administration of a test meal. The amount of copper absorbed from
sunflower seeds, peanuts, cooked garbanzo beans, cooked shrimp, and
cooked beef was 46%, 41 %, 30%, 50%, and 46%, respectively. Copper
absorption from the control diet which contained copper sulfate was 46%.
These results suggested that copper bioavailability in cooked garbanzo
beans tended to be lower compared to other foods in a single meal. In an
earlier study, Davies and Nightingale (1975) found that the addition of 1%
phytate as sodium phytate to an egg white diet significantly decreased
copper absorption. The whole body retention of copper was also
significantly reduced by 57% in rats when they received a diet containing a
phytate to copper molar ratio of 40.

In contrast, the results of Lee et al. (1988) suggested that phytic acid
increased copper bioavailability in rats by its ability to bind other dietary
components, such as zinc, that compete with copper at the site of intestinal
absorption. In their study, depletion and repletion feeding techniques
were used to evaluate the biologically available copper. Liver copper, liver
Cu, Zn-superoxide dismutase activity, serum copper and serum
ceruloplasmin were used as physiological indicators to determine the
bioavailability of copper in the diet. In the repletion period, rats were fed
diets containing either 1.4, 3.0, 5.2, or 10.5 Jig Cu / g as copper carbonate and
0, 0.4, or 0.8% phytic acid as sodium phytate at each copper level. The

results of this study indicated that phytic acid added to the diets containing

 

28

5.2 ug/ g copper significantly enhanced copper utilization during the 3d
repletion period, whereas there were no differences among the other
levels of copper. This enhanced utilization was evident for both amounts
of dietary phytate tested and for all copper indices examined.

In an experiment conducted by Lo et al. (1984), the bioavailability of
copper in isolated soybean protein was evaluated with growing rats using
a depletion and repletion feeding approach. After a 21 day copper
depletion period, rats were fed diets containing different levels of copper
either from copper carbonate or isolated soybean protein for one week
Serum and liver copper contents were used as indicators to determine the
bioavailability of copper. Results suggested that copper was available
equally from isolated soybean protein and copper carbonate.

Turnlund et al. (1985) studied the effects of phytate on copper
absorption in young men. The young men were given a liquid formula
diet with and without 2.35g of phytic acid as sodium phytate. The average
copper absorption was 35% (25.3%—45.3%) from the basal diet and 31.4%
(22%-44.6%) from the diet containing added phytic acid. They concluded
that high levels of phytate do not affect copper absorption. Morris and
Ellis (1985) similarly concluded that intake of 0.5, 1.7 or 2.9 g of phytic acid

daily had no effect on apparent absorption of copper in adult men.

 

29
Summary

There is considerable potential for increased utilization of cereals
and legume-based foods in developed countries, and a high consumption
of these foods already exists in developing countries. These plant
products, however, contain considerable quantities of phytic acid which
may affect mineral bioavailability. I

Phytic acid, myoinositol hexaphosphate, is a storage form of
phosphorus in plants and is common constituent in cereals and legumes.
Its molecule is highly charged with six phosphate groups extending from
the central inositol ring structure and therefore is an excellent chelator of
cations. At neutral pH, phosphate groups of phytate have either one or
two negatively charged oxygen atoms, and it is apparent that various
cations could strongly chelate between two phosphate groups or weakly
within a phosphate group.

Numerous studies have suggested that phytate in plant foods
reduces bioavailability of essential trace elements. The negative effect of
phytate on zinc bioavailability is clear and occurs in both human and
animal studies. The degree of its effect on zinc bioavailability depends on
the phytate : zinc molar ratio and zinc nutritional status of the animal.
Whether the phytate is added to the diet as sodium phytate or as
endogenous phytate seems to make little difference in its effect on zinc
absorption.

Compared to zinc, the effect of phytate on copper utilization has not
been studied extensively and results often are conflicting. Results from
experiments reported in the literature indicate that further studies are

need to clarify the effect of phytic acid on copper bioavailability. In

 

30

addition, most of the available information related to the effect of food
phytate on mineral utilization were from studies on soybean or soybean
products. The data on soybean may not be completely applicable to other
types of beans. The effect of phytate in other legumes on mineral

utilization should be also investigated.

The objectives of this study were:
1. to evaluate the bioavailability of copper in kidney beans and garbanzo
beans by comparing the utilization of copper from copper carbonate,
kidney bean or garbanzo bean.

2. to determine the effect of phytate in beans on copper utilization.

 

MATERIAIS AND METHODS

EXPERIMENTAL DESIGN

General gocedures. Weanling male Sprague-Dawley rats (Harlan,
Indianapolis, IN) were housed individually in stainless steel wire cages in
a temperature (21-23 °C) and humidity (68-70%) controlled room with 12—
hour cycles of light and dark (light cycle, 7 am-7 pm). Deionized water was
available ad libitum using water bottles with plastic caps and stainless steel
drinking tubes. Food intakes and body weights were recorded twice a
week Experimental protocol and procedures were approved by the All
University Committee on Animal Use and Care at Michigan State

University.

Expelinent‘l

Animals and treatments. The purpose of this experiment was to
evaluate the utilization of copper in kidney beans and garbanzo beans.
After an adjustment period of 2 days, 24 weanling male Sprague-Dawley
rats were randomly allocated to the following three diet treatment groups:
1) control, 2) kidney bean and 3) garbanzo bean. Beans were selected for

the experiment based on literature values for their phytate content:

31

32

garbanzo bean, 0.65%; kidney bean, 2.06% (Reddy et al., 1982). Rats were
fed the respective diets for 3 weeks. After an overnight fast at the end of
the feeding period, rats were anesthetized with methoxyflurane and blood
collected by open cardiac puncture using 5 ml syringes rinsed with 1000
units/ml sodium heparin solution. Blood samples were centrifuged
(Centrific Centrifuge, Fisher Scientific, Itasca, IL) at 200 x g for 20 min.
Serum was removed immediately and stored at -20 °C for later
determination of ceruloplasmin activity and serum copper and zinc.
Liver and kidneys were removed, washed with cold saline, blotted,
weighed and frozen on dry ice. Tissues were stored at -20 °C and freeze-
dried (Unitrap II, Virtis Co., Gardiner, NY) for later analysis. On the first
day of the experiment, four additional rats were killed and blood and

tissues obtained for baseline analyses.

fleLs, Table 1 shows the compositions of diets used in this
experiment. The diets were formulated according to the general
recommendations of the American Institute of Nutrition (Reeve et al.,
1993) with exception of the vitamin mix and the mineral mix Diets
provided adequate and approximately equal amounts of total energy,
protein and fat. Protein was supplied by egg white or a combination of egg
white and beans. The amount of beans used in garbanzo bean and kidney
bean diets was seleted to provide approximately 2 mg Cu [kg which is
lower than the AIN-93G recommendation (6 mg / kg). The same amount
of copper was provided by copper carbonate in the control diet. The
purpose of using a lower copper level was to maximize the chance of

showing differences in bioavailability. A copper-, zinc— and iron- free

 

Compositions of diets, Experiment 1

33

TABLE 1

 

 

Ingredient Control Kidney bean Garbanzo bean
(g/100g)

I<idney bean1 — 30.7 —
Garbanzo bean2 — — 32.7
Egg whites 20.0 11.5 12.7
Cornstarch 39.8 19.6 15.8
Dyetrose4 13.2 13.2 13.2
Sumose 10.0 10.0 10.0
S Qlka-Floc 5.0 3.1 3.7
(B W 200 cellulose)

Choline bitartrate 0.14 0.14 0.14
L— cystine 0.3 0.3 0.3
A IN-76 vitamin mix4 1.0 1.0 1.0
Mineral mix5 3.5 3.5 3.5
‘Fe, Zn, Cu free)

Soy oil 7.0 7.0 7.0
(m g/ 100g)

Zinc carbonate 2.30 0.62 0.28
Ferric citrate 21.10 11.20 10.30
Clapric carbonate 0.38 — —

 

f
iPigeon Co—op Co., Pigeon, MI
2Manchester Co., Shawnee Mission, KS
3Teklad Test Diet Co., Madison, WI
4Dyets Inc, Bethlehem, PA
5Supplies (g/ kg mineral mix): CaHPO4, 500; NaCl, 74; K3C6H507.HzO, 220;

K2504, 52; MgO, 24; MnCOg, 3.5; K103, 0.01; NazSe03.5H20, 0.01 ;
CrK(SO4)2.12H20, 0.5; sucrose, 125.93.

 

34

mineral mix was prepared to provide the recommended amount of
minerals for rats. The amount of zinc carbonate, ferric citrate and cellulose
added to the diets was adjusted for amounts of zinc, iron and fiber
provided by the beans so that the total amount of these minerals and
insoluble fiber in the diets would be equal. The copper, zinc and iron
content of experimental diets was analyzed by atomic absorption

spectrophotometry before feeding.

Bean preparation. Kidney beans (Pigeon Co-op Co., Pigeon, MI) and
garbanzo beans (Manchester Co., Shawnee Mission, KS) were cooked in
deionized water for 4 hours at a ratio of 1 : 2 and 1 : 3 (beans : water),
respectively. The cooked beans were dried in a forced air convectional
drying oven (Proctor & Schwartz Inc., Philadelphia, PA) at 60°C for 48
110- Irrs. The dried beans were ground to fine powder using a micro-mill
(Chemical Rubber Co., Cleveland, OH) and analyzed for protein, copper,

Zinc, iron before incorporation into the diets.
W2

Animals and treatments. The purpose of the second experiment
was to clarify results obtained in experiment 1 using a different
Qwerimental approach. After a 2 day adjustment period, four rats were
l,illed on day 1 of the feeding study to obtain baseline values. The
remaining 32 rats were fed a copper deficient basal diet (0.12 mg Cu/ kg )
for 4 weeks. Rats were trained to meal feed by restricting food intake (9:00-

5:00 pm). The purpose of the meal feeding was to avoid differences in

 

 

35

food intake that had occurred in experiment 1. During the fourth week,
food intakes were recorded daily to obtain the average daily food intake.
At the end of 4 weeks, blood samples were taken from tail arteries to
determine serum ceruloplasmin. Rats were divided into 4 groups (n=8) of
equal mean weight and fed the respective experimental diets for 7 days.
The amount of diet fed (15 g) was based on the average daily food intake
during the fourth week of the basal copper deficient diet. The dietary
treatments were: 1) control (basal copper deficient diet with the addition of
copper at 2 mg /kg diet as copper carbonate), 2) control diet with the
addition of sodium phytate (Sigma Chemical, St. Louis, M0) at 1.53 g / kg
diet, 3) kidney bean diet and 4) dephytinized kidney bean diet. The copper
content of the bean diets was 2 mg/kg provided by the beans. At the end of
the four weeks of copper depletion, 1 ml blood was taken from tail artery
into a 1 ml syringe rinsed with sodium heparin for ceruloplasmin activity
datermination. At the third day of copper repletion period, blood samples
Were taken from the tail artery again. After seven days of copper repletion,
rats were anesthetized and killed by open cardiac puncture. Blood and

tissues were collected as previously described in experiment 1.

1213;; Composition of the basal copper deficient diet and four test
diets is listed in Table 2. Diets were prepared in the same manner as in
Qweriment 1 and formulated to provide adequate and equal amounts of
,otal energy, protein and fat. Protein sources in the experimental diets
were supplied by egg white or a combination of egg white and kidney
beans. The protein content of the diets was analyzed using standard

Kjeldahl nitrogen analysis (AOAC, 1984). In this experiment, a AIN-93G

 

36

TABLE 2

Compositions of diets, Experiment 21

 

Basal Control Control Kidney bean

 

Dephytinized
Ingredient +phytate kidney bean
(g/100g)
Kidney bean — — — 30.0 30.0
Egg white 20.0 20.0 20.0 11.6 11.6
Cornstarch 39.8 39.8 39.7 20.2 20.2
Dyetrose 13.2 13.2 13.2 13.2 13.2
Sucrose 10.0 10.0 10.0 10.0 1 0.0
Solka-Floc 5.0 5.0 5.0 3.0 3.0
(BW 200 cellulose)
Choline bitartrate 0.14 0.14 0.14 0.14 0.14
L-cystine 0.3 0.3 0.3 0.3 0.3
AIN-76 vitamin mix 1.0 1.0 1.0 1.0 1.0
AIN-93 mineral mix2 3.5 3.5 3.5 3.5 3.5
(Fe, Zn, Cu free)
Soy oil 7.0 7.0 7.0 7.0 7.0
Sodium phytate3 — — 0.1 —- ——
(mg/ 100g)
Zinc carbonate 2.30 2.30 2.30 0.60 0.60
Ferric citrate 21.00 21.00 21.00 11.00 1 1.00
Cupric carbonate — 0.32 0.32 —— —

 

1The sources of ingredients in this experiment were the same as in
experiment 1 with the exception of mineral mixture, sodium phytate
and phytase.

2Dyets Inc, Bethlehem, PA

3Sigma Chem. Co., St. Louis, MO

37

mineral mix without copper, zinc and iron was purchased from Dyets
(Bethlehem, PA). Zinc carbonate, ferric citrate and copper carbonate were
premixed with sucrose and then incorporated into the test diets and basal
copper deficient diet as indicated in Table 2. Concentrations of copper, zinc
and iron in experimental diets were analyzed by atomic absorption

spectrophotometry.

Mam] Dephytinized bean preparation. Kidney beans were
obtained from the same source as in experiment 1. Beans were mixed
with deionized water in a ratio of 1 : 1.2 (beans : deionized water) and
soaked for 2 hours. Beans were then cooked by a conventional home
cooking procedure for 2 hours and dried in an air oven at 100°C for 48
hours. The dried beans were ground to a fine powder using a coffee mill
(Robert Krups, Denver, CO).

Dephytinzed beans were prepared according to procedures described
by Sandberg and Svanberg (1991) modified as follows: 200 g of ground
kidney bean and 2 g wheat phytase (Sigma Chem. Co., St. Louis, M0) were
suspended in 300 ml of deionized water. The pH was adjusted to 4.5 using
1N hydrochloric acid and then gradually increased to 5.15, the optimal pH
for phytase in wheat. The suspension was incubated at 55°C in a water
bath with mild mechanical agitation. The suspension was removed after
17 hours, dried in an oven for 24 hours and ground using a coffee mill
(Robert Krups, Denver, CO).

Beans were analyzed for protein, copper, zinc, iron before they were

mixed with other ingredients in the preparation of the complete diet.

38

Inositol phosphates (Ip3 to Ip6) were also determined using ion exchange

chromatography and high pressure liquid chromatography techniques.
Alnlylimlmefllods

Protein content in diets. Protein content of the diets was
determined by the standard Kjeldahl nitrogen analysis using a
modification of the AOAC (1984) method. Concentrated sulfuric acid (5
ml) and one catalyst table (potassium sulfate and selenium; Tecator Co.,
England) were added to each of the digestion tubes containing preweighed
samples. The samples were digested using gradually increased
temperatures until digestion was complete. The protein content was

calculated on a dry weight basis using a nitrogen conversion factor of 6.25.

fissue and diet copper and zinc determination. Diets and tissues
were digested using a modified wet-ash procedure (Clegg et al., 1981).
Duplicate samples of diets, freeze-dried livers, kidneys and tibias were
weighed and put into 50 ml Erlenmeyer flasks. Following the addition of
10 ml of concentrated nitric acid, flasks were heated at low temperature for
30 min to oxidize the more reactive organic matrix without an overly
vigorous reaction. The temperature was raised after the initial chemical
reaction subsided. For some samples additional nitric acid was needed.
When the nitric acid digest had been reduced to near dryness, hydrogen
peroxide (30%) was then added to oxidize the remaining particles. After
evaporation of hydrogen peroxide, a white ash was obtained. The flask

contents were cooled and diluted with 0.1N HCl (Cu: 10 ml, Zn: 20 ml).

 

39

Samples were analyzed for copper, zinc and iron by atomic absorption
spectrophotometry (Perkin-Elmer model 2380, Norwalk, CT). Mineral
standards in appropriate concentrations were prepared from stock
standard solutions (J. T. Baker Chem. Co., Phillipsburg, NJ) and analyzed
by atomic absorption spectrophotometry.

The accuracy of the ashing procedure and the atomic absorption
spectrophotometry was checked by analyzing N IST bovine liver-1577b
(National Institute of Standards and Technology, Gaithersburg MD).

Serum copper and zinc determination. Serum samples were
diluted and analyzed for copper and zinc by atomic absorption
spectrophotometry. Serum was diluted with an equal volume of
deionized water for copper analysis and diluted 1 : 5 with deionized water
for zinc analysis. Copper and zinc standards were prepared by diluting the
stock standard solution with 10% (v/v) or 5% (v/v) glycerol, respectively.
A 10% or 5% glycerol solution was used in order to maintain similar
viscosity in samples and standards. Absorbance of samples was recorded

and compared to standard solutions treated in a similar manner.

Determination of serum ceruloplasmin activity. Ceruloplasmin
activity of serum samples was determined by the colorimetric procedure of
Schosinsky et al. (1974). This method is based on the oxidase activity of
ceruloplasmin on diamine groups using o-dianisidine dihydrochloride as
substrate (Sigma Chem. Co., St. Louis, MO). This reagent is converted to a

yellowish-brown product in the presence of ceruloplasmin and oxygen at

 

40

pH 5. Acidification stops the enzymatic reaction, and a stable purplish-red

solution is formed that absorbs maximally at 540 nm.

Determination of phflate phosphorus in beafl A modified
method of ion-exchange procedure (Graf and Dintzis, 1982) and
colorimetric measurement (Fiske and Subbarow, 1925) was used for
determination of phytic acid. Dried ground bean samples (0.5g) were
extracted in 20 ml 0.5M hydrochloric acid using vigorous mechanical
agitation for 2 hours at 20 °C. The mixture was centrifuged at 1800 rpm for
30 min (International Centrifuge, Model UV) and supernatant decanted,
frozen overnight and filtered through a 0.45 micrometer membrane filter
(Gelman Sciences, Ann Arbor, MI) under pressure. The filtrate was
diluted with 10 ml distilled deionized water and passed through an ion
exchange column containing 0.65 ml resin (AG 1-X8, 200-400 mesh) at 0.4
ml /min follow by 10 ml of 0.025 M HCl. Inositol phytates were removed
and collected from the column with ten 1 ml portion of 2M HCl. The final
eluent was then digested with 5 ml nitric acid and 5 ml hydrogen peroxide
until white ash was obtained. The ash was dissolved in 10 ml deionized
water, and a 0.5 ml aliquot was then taken for phosphorus determination
(Fiske-Subbarow, 1925). The final sample was allowed to stand for 15 min
for color formation; absorbance was read at 640 nm (Spectronic 21, Bausch
& Lomb). A standard curve was prepared by diluting a stock standard
solution (100 pg P / ml) made from potassium acid phosphate. A factor of

3.55 was used to convert phytate phosphorus into total phytate content.

41

HPLC analysis of inositol phosphates in hem The purpose of
determining the inositol phosphates in kidney beans and dephytinized
kidney beans was to quantify the inositol hexaphosphates in the kidney
beans and dephytinized kidney beans and evaluate the extent of
degradation of phytic acid in dephytinized kidney beans used in
experiment 2. Phytate content in kidney bean and dephytinized kidney
bean was determined by ion exchange chromatography and high pressure
liquid chromatography (HPLC) techniques using modifications of methods
described by Graf and Dintzis (1982) and Sandberg and Ahderinne (1986).
Samples were prepared using the same ion exchange procedure as
described previously for phytate phosphorus determination. The final
eluent was evaporated to dryness and diluted with 1 ml of HPLC water. A
100 umole / ml stock sodium phytate solution was made by dissolving
sodium phytate (Sigma, St. Louis, MO) in HPLC water. Standard solutions
contained 1, 2, 5, 10 and 20 umol / ml of phytate. The mobile phase
consisted of 0.05M formic acid : methanol (46 : 54) to which was added 1.5
ml/100 ml of tetrabutyl ammonium hydroxide. The pH was adjusted to
4.3 by addition of 9M sulfuric acid. The mobile phase was filtered through
a millipore filter (0.45 pm) under vacuum and degassed by sonication for
20 min. A reverse phase Supelcosil LC-18 column (25 cm x 4.6 mm)
(Supelco, A ROHM and HAAS Co., Bellofonte, PA) 5 micron particle size
was equilibrated with the mobile phase for one hour each day prior to
beginning the sample analysis. Samples were analyzed using a Model M45
Waters HPLC pump (Waters Associates, MA), Rheodyne injector (Model
7010, Rheodyne Inc. CA) with a 20 ill loop at a flow rate of 2 ml / min.

Inositol phosphates were detected using a differential refractometer

42

(Model R401, Waters Associates, MA). Retention times and peak areas
were measured with an analog to digital board and Peak Simple II
integration software (SR1 Instruments, CA). The linearity of phytate
concentration versus peak area was determined using 20 ul injections of
standard solutions (1-20 umol/ ml) and a standard curve was obtained by
calculating the regression equation for peak area vs standard solutions.
Phytate content of the samples were computed from sample peak areas

and the regression equation.

Statistical methods

All data were analyzed by one-way analysis of variance. If the
difference was significant at P< 0.05, the Bonferroni/ Dunn test was used
for comparisons among groups. Tests were conducted with statistical

software (Statview version 4.01, Abacus Concepts, Inc, 1992-1993).

 

RESULTS

lbtper'lntmti

Copper, zinc, iron, phytic acid and protein content of diets. The

analyzed amounts of copper, zinc, iron were similar in all diets (Table 3).

 

The accuracy of analytical procedures was verified by analysis of NIST
bovine liver. The analyzed copper, zinc and iron concentrations in bovine
liver were within the range of certified values. The two kind of beans
were selected to represent beans with low and high contents of phytate.
Literature values for the phytate content of garbanzo and kidney beans are
0.65% and 2.06%, respectively (Reddy et al., 1982). However, the analyzed
values for the beans used in this experiment were low (0.4% and 0.6% for
garbanzo bean and kidney bean, respectively) and also similar to each
other. Concentrations of phytate in the diets containing garbanzo bean
and kidney bean (Table 1) were 0.13% and 0.19%, respectively (Table 5).
The analyzed protein concentrations of kidney beans and garbanzo beans
were 22.1% and 17.8%, respectively. The amount of protein in the two
bean diets was then adjusted with egg white to be the same as in the

control diet .

Body weight, weight gain. food intake. efficiency of food utilization

and totaflnineral intake. The mean final body weight of rats in the
43

44

TABLE 3
Analyzed mineral and phytic acid content of diets, Experiment 1

 

 

Group Phytic acid Copper Zinc Iron
(mg/ g) (03/ 3)

Control — 2.53 12.64 37.40

Garbanzo bean 1.3 2.37 11.93 36.25

Kidney bean 1.9 2.15 12.00 39.00

 

45

control group (208220 g) was significantly lower than that of the garbanzo
bean (23315.6 g) and kidney bean (23614.8 g) groups (Table 4). There was
no significant difference in the final body weight of the garbanzo bean and
kidney bean groups. Rats fed the control diet gained significantly less
weight than rats fed the garbanzo bean and kidney bean diets. The lower
total weight gain of rats in the control group, which was 80 % of that in the
garbanzo bean group and 78% of that in the kidney bean group, may be
related in part to differences in food intake, since their food intake was
significantly less than that in the bean groups. However, decreased
efficiency of food utilization was also observed in rats fed the control diet
compared to the garbanzo bean or kidney bean diets.

The total dietary intakes of copper, zinc and iron are shown in
Table 5. Because of differences in food intake, the total copper and zinc
intakes were significantly (P< 0.05) lower in rats fed the control diet than

in those fed the garbanzo bean or kidney bean diets.

_O_rgan wet weights. dry weights and % body weight of organ_s. Rats
fed the garbanzo bean and kidney bean diets had significantly higher liver
and kidney weights than rats fed the control diet (Table 6). However, there
were no significant differences in the mean wet weights of the livers and
kidneys between rats fed garbanzo bean and kidney bean diets. The same
trend was observed in the mean liver and kidney dry weight values for
rats fed the three diets. These results could be related to differences in
body weights. Body weights of rats in garbanzo bean and kidney bean diets
were also significantly higher than those fed the control diet. Therefore,

when the liver and kidney weights were expressed per 100 g body weight,

46

TABLE 4
Body weights and food intake of rats, Experiment 11

 

 

Group Initial body Final body Weight Food efifggrcicy
weight weight2 gain2 intake2 ratiozi3
(g) (g) (g) (g/ d) (%)
Control 94:2 20812.08 114:1.38 13.21028 36.610.5a
Kidney bean 92:3 233:5.6b 141 :43b 14.7:03b 41.611.0b
Garbanzo bean 91 :3 236:4.8b 145:4.8b 14.6:03b 416:0.9b

 

1Values are means 2 SEM (n=7 in control group and n=8 in kidney bean
and garbanzo groups).

2Values in the same column with different superscript letters are
significantly different (p<0.05) by Bonferroni/ Dunn test.

3Food efficiency ratio (%) = weight gain / total food intake x 100.

47

TABLE 5
Total mineral intake from diets, Experiment 11

 

 

Total Total Total
Group food intake copper intakezt3 zinc intakezi4
(3) (ug) (113)
Control 312168 7861158 39401 718
Garbanzo bean 33918b 882119ID 44061 93b
Kidney bean 34917b 858121b 42861103b

 

1Values are means 1 SEM (n=7 in control group and n=8 in kidney bean
and garbanzo groups).

2Values in the same column with different superscript letters are
significantly different (p < 0.05) by Bonferroni/Dunn test.

3Total copper intake = copper content in diet (ug/ g as fed) x total food
intake (g).

4Total zinc intake = zinc content in diet (ug/ g as fed) x total food intake (g).

48

TABLE 6
Organ wet weights and dry weights, Experiment 11

 

 

 

 

Wet weight2 Dry weight2
Group Liver Kidney Liver Kidney
(g) (g)
Control 9.11038 1,610.08 2.810.18 0,410.08
Garbanzo bean 10.1103b 1.810.0b 3.110.1b 0.410.0b
Kidney bean 10.1103b 1,810.1b 3.1101b 0410.0b

 

1Values are means 1 SEM (n=7 in control group and n=8 in kidney bean
and garbanzo groups).

2Values in the same column with different superscript letters are
significantly different (p<0.05) by Bonferroni/ Dunn test.

49

there were no significant differences between groups (Table 7).

Ceruloplasmin activity. serum concentrations of copper and zinc.
Serum ceruloplasmin activity was significantly different among groups :
control > garbanzo bean > kidney bean (Table 8). A similar pattern in
serum copper was observed although the difference between the garbanzo
bean and kidney bean groups were not statistically significant. Serum zinc
concentrations followed the same trend as ceruloplasmin activity with
statistically significant differences between groups and rats fed the control

diet having the highest concentrations (Table 8).

Tissue concentrJations of copper and zinc. The concentration of
copper in livers and kidneys of rats were unaffected by dietary treatments
(Table 9). The total kidney copper in rats fed garbanzo bean or kidney bean
diets was significantly higher than that in rats fed the control diet. Total
liver copper also was higher in the bean-fed rats although the differences
were not statistically significant. The dietary treatments did not
significantly affect zinc concentration and total zinc content of kidney.
The total liver zinc content is significantly higher in rats fed bean diets
although liver zinc concentration were not significant in all groups (Table

10).

W2

Copper, zinc, iron, phytic acid and protein content of diets. Table 11

lists the mineral and phytate content of the depletion and repletion diets.

50

TABLE 7
Organ wet weight to body weight ratio, Experiment 11

 

 

 

Ratio (%)
Group Liver Kidney
Control 4.4101 0.8100
Garbanzo bean 4.3100 0.8100
Kidney bean 4.3101 0.8100

 

1Organ ratio (%) = (organ wet weight / body weight) x 100.

51

TABLE 8
Ceruloplasmin activity, serum copper and
zinc concentration, Experiment 11

 

 

Ceruloplasmin Serum copper Serum zinc
Group activity2 concentration2 concentration2
(U/L) (rig/d1) (ug/ d1)
Control 107.712.78 88.314.98 188017.28
Garbanzo bean 94.412.9b 76.311.7b 155.714.4b
Kidney bean 84.6120C 72.412.6b 140.1131c

 

1Values are means 1 SEM (n=7 in control group and n=8 in kidney bean
and garbanzo bean groups).

2Values in the same column with different superscript letters are
significantly different (p < 0.05) by Bonferroni/Dunn test.

52

TABLE 9

Concentration and total content of copper in tissues, Experiment 11

 

Tissue total copper2

 

 

Group Liver Kidney Liver Kidney
(118/ g dry weight) (118)

Control 11.1103 15.8105 31.1114 6010.1a

Garbanzo bean 10810.5 15.8103 33911.9 6.8102b

Kidney bean 10810.6 16.2104 33.8121 6.9102b

 

1Values are means 1 SEM (n=7 in control group and n=8 in kidney bean

and garbanzo groups).

2Values in the same column with different superscript letters are
significantly different (p < 0.05) by Bonferroni/Dunn test.

53

TABLE 10
Concentration and total content of zinc in tissues, Experiment 11

 

Tissue total zinc

 

 

Group Liver Kidney Liver Kidney
(118/ g dry weight) (118)

Control 37011.6 24.2116 10301493 9.3107

Garbanzo bean 38.4114 23.3108 120.5149b 9.0104

Kidney bean 40912.1 24.3109 127.2168b 10310.4

 

1Values are means 1 SEM (n=7 in control group and n=8 in kidney bean
and garbanzo groups).

54

TABLE 11

Analyzed mineral and phytate content of diets, Experiment 2

 

 

Diet Phytic acid Copper Zinc Iron
(mg/ g) (118/ g)
Depletion diet — 0.12 11.42 43.44

Repfeb'on diets

Control — 1.94 12.86 35.61
Control+ 1.4 2.36 13.02 33.04
phytate‘l

Kidney bean2 1.4 2.20 12.41 33.89
Dephytinized3 ND 2.10 13.01 35.43
kidney bean

 

11.53g of sodium phytate (Sigma, St. Louis, MO) was added to the control
diet to make the phytate content equal to the amount of phytate in the
kidney bean diet.

2The phytate content in this diet was calculated as total phosphorus and
then converted it to total phytate.

3The phytate content in dephytinized kidney beans was not detectable
(ND) using HPLC analysis.

55

The amounts of copper, zinc and iron were similar in all repletion diets
although the copper content was somewhat lower in the control diet. The
analyzed concentration of phytate in kidney beans was 0.5%. HPLC
analysis showed no phytic acid and little evidence of lower inositol
phosphates in the dephytinized kidney beans. The amount of sodium
phytate added to the control+phytate diet was based on the amount of
phytate in the kidney bean diet (0.14%). The kidney beans used in this
experiment were from the same source as in the first experiment;

therefore, the protein content of the beans was assumed to be the same.

Body weim. weigl_rt_gain, food intake and efficiency of food
utilization. There were no significant differences in the mean initial and
final body weights among groups in the repletion period (Table 12),
although the final body weights in the control and control+phytate groups
tended to lower than those in the kidney bean and dephytinized kidney
bean groups. The mean body weights at the beginning of the depletion
period also were not significantly different. The total weight gain and
efficiency of food utilization in rats fed both the kidney bean and
dephytinized kidney bean diets were significantly higher than those fed
the control and control+phytate diets. These results were not due to
differences in food intake since food intake in the four groups was
controlled. The food intake of rats in each group was limited to the

average food intake of all rats during the last week of the depletion period

(15 g/day).

56

TABLE 12
Body weights and food intake of rats during the repletion period,
Experiment 2‘1

 

 

Food
Group Initial body Final body Weight Food efficiency
weight2 weight2 gain3 intake ratio3:4

(g) (g) (g) (g/ d) (%)
Control 237110 26219 25139 15 27.8130a
Control+ 2371 7 26416 2711a 15 30311.6a
phytate
Kidney bean 2371 8 27616 3912b 15 43.1128b
Dephytinized 2371 9 27219 3612b 15 40612.4b
kidney bean

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

2Initial body weights represent weight at the end of the depletion period,
and the final body weights were measured at the end of the repletion
period.

3Values in the same column with different superscript letters are
significantly different (p<0.05) by Bonferroni/ Dunn test.

4Food efficiency ratio (%) = weight gain / total food intake x 100.

57

Orgawet weights. dry weights and % body weight of orgafl The
mean wet weights and dry weights of the livers, kidneys and tibias in rats
fed the control, control+phytate, kidney bean and dephytinized kidney
bean diets were not significantly different (Table 13 and 14). Similarly,
when the liver, kidney and tibia weights were expressed per 100 g body

weight, the differences also were not statistically significant.

Ceruloplasmin activity, serum copper and zinc concentration.
During wk 3 of the copper depletion period, rats were randomly chosen to
test serum ceruloplasmin activity. The range of ceruloplasmin activity
was from 3.1 U / L to 51.3 U / L (n=9). The ceruloplasmin activity values
obtained in baseline rats ranged from 62.2 U / L to 103.4 U/ L Although the
values of ceruloplasmin activity in the copper depleted rats were low
compared to the values of baseline rats, individual variation was high.
Consequently, the depletion period was extended one more week before
the repletion period was started. After four weeks of copper depletion, the
serum ceruloplasmin activity was determined in all rats, and rats were
divided into four groups having similar mean ceruloplasmin values
ranging from 3.3 to 6.4 U / L (Table 15). After consuming the repletion diets
providing approximately 2 pg Cu / g for 3 days, rats showed an increase in
ceruloplasmin activity irrespective of copper source in the diet. Rats fed
either the control or dephytinized kidney bean diets had significantly
higher ceruloplasmin activity than rats fed the control+phytate or kidney
bean diets. The serum ceruloplasmin activity was not increased further by
an additional four days of feeding the marginal copper diets; thus, at 7 days

differences between groups were unchanged. Results in this study

TABLE 13
Organ wet weights and dry weights, Experiment 21

 

 

 

Wet weight Dry weight
Group Liver Kidney Tibia Liver Kidney Tibia
(g) (g)

Control 11.1106 2.2101 0.8100 3.4102 0.5100 0410.0
Control+ 10.9104 2.1101 0710.1 3.3102 0510.0 0.4100
phytate

Kidney bean 11.1105 2.2101 0810.1 3.4102 0510.0 0.4100
Dephytinized 11.0105 2.3101 0.8100 3.3103 0.5100 0.4100

kidney bean

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

59

TABLE 14
Organ wet weight to body weight ratio of rats, Experiment 21

 

 

 

Ratio (%)
Group Liver Kidney Tibia
Control 4.2101 0.8100 0.3100
Control+ 4.1 10.2 0.8100 0310.0
phytate
Kidney bean 4010.2 0.8100 0310.0
Dephytinized 4.0102 0.8100 0310.0
kidney bean

 

1Organ ratio (%) = (organ wet weight / body weight) x 100.

60

TABLE 15
Ceruloplasmin activity at initial, 3d and 7d of repletion period,
Experiment 21

 

 

 

Repletion3
Initial2
Group 3d 7d
U/ L
Control 6,412.6 113.4121 a 113.3126
Control+ 3.4126 91.313.3b 95.2140b
phytate
Kidney bean 3.3119 82.413.9b 83.215.0b
Dephytinized 6411.3 1 1 4012.33 11 5911.93
kidney bean

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

2Values represent the ceruloplasmin activity at the end of the copper
depletion period.

3Values in the same column with different superscript letters are
significantly different (p<0.05) by Bonferroni/ Dunn test.

61

demonstrated that 3 days of copper repletion at 2 pg Cu / g of intake is
sufficient to replete serum ceruloplasmin. Changes in serum copper
values were consistent with those in serum ceruloplasmin activity (Table
16). When rats were fed the control or dephytinized kidney bean diets,
serum copper concentrations were significantly higher than in rats fed the
control+phytate or kidney bean diets. There were no significant

differences in serum zinc among the four groups.

Tissue concentrations of copper and zinc. The concentrations and

 

total contents of copper and zinc in livers, kidneys and tibias are given in
Tables 17 and 18. The concentrations and total contents of copper and zinc
in livers, kidneys, tibias were not significantly different among groups.
However, the copper concentrations and total liver copper in rats fed
control and dephytinized kidney bean diets tended to be higher as
compared to rats fed the control+phytate and the kidney bean diets. The
same pattern was observed in zinc concentration and total zinc content of

livers and tibias.

Inositol phosphate analyses using high pressure liquid
chromatography. There was a strong linear relationship between

 

integrated values of refractive index detector response and amount of
inositol hexaphosphates injected (Y=231.50X—18222, r=0.99). Peak areas of
kidney bean and dephytinized kidney beans extracts were measured and
the concentrations of inositol hexaphosphates in the kidney beans and

dephytinized kidney beans were calculated by the equation obtained from

the standard calibration curve. Results showed that there was 0.5% of

 

62

 

 

TABLE 16
Serum copper and zinc concentrations after 7 days of repletion,
Experiment 21
Group Serum copper2 Serum zinc
ug/dl

Control 106.6133a 154.0156
Control+ 87.014.5b 158.1153
phytate
Kidney bean 84314.6b 152.2187
Dephytinized 110.5125a 150.9135
kidney bean

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

2Values in the same column with different superscript letters are
significantly different (p<0.05) by Bonferroni/ Dunn test.

63

TABLE 17
Concentration and total content of copper in tissues, Experiment 21

 

Tissue total copper

 

 

Group Liver Kidney Tibia Liver Kidney Tibia
(113/ g dry weight) (rig)
Control 8.5103 9.7104 1.3102 29.1112 5.1103 0.6101

Control+ 8.3102 8.8103 1.7105 27211.3 4.6102 0.6102
phytate

Kidney bean 8.3103 9.4105 1.2105 27.7116 5.1103 0.5103

Dephytinized 9.1106 10.3103 1.4103 29.4121 5.6102 0.7102
kidney bean

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

64

TABLE 18

Concentration and total content of zinc in tissues, Experiment 21

 

Tissue total zinc

 

Group Liver Kidney Tibia Liver Kidney
(rig/g dry weight) (118)

Tibia

 

Control 30.4109 26.8108 1803172 1001174 14.1105

Control+ 27.2125 26211.7 178.2199 89.9163 13.7108
phytate

Kidneybean25.811.2 26.3108 175.8186 86416.7 14.2103

Dephytinized28.612.0 27.6115 183.0130 94218.5 14.8105
kidney bean

78.5123
69.5120

70316.5
78.4142

 

1Values are expressed as means1SEM (n=7 in dephytinized kidney bean
group and n=8 in control, control+phytate and kidney bean groups).

65

phytic acid in kidney beans and phytic acid in dephytinized kidney beans

was undetectable.

DISCUSSION

In experiment 1, when food intakes were not controlled, rats fed the
kidney bean and garbanzo bean diets tended to ingest more food than the
control group. The factor(s) responsible for this apparent preference for
the bean diets are not clear but may have included differences in
palatability or digestibility of the diets. This phenomena was also observed
by Zhou et al. (1992) who reported that soybean protein-fed rats consumed
greater amounts of diet than egg white fed rats. In experiment 1, higher
total weight gains in the bean groups were also observed and it was
thought that this difference was related to higher food intakes. In
experiment 2, however, total weight gains were also significantly higher in
the kidney bean and dephytinized kidney bean groups compared to the
control and control+phytate groups. In this experiment food intakes were
controlled, and theoretically, the total weight gains should be the same in
all groups. Although diets were planned to be as similar as possible, small
compositional differences in the diets may have contributed in part to the
differences in weight gain.

Several parameters were used to evaluate copper and zinc status in
this study. Results in experiment 1 showed that ceruloplasmin activity
was significantly different among groups (control > garbanzo bean >

kidney bean), and serum copper was significantly lower in the garbanzo

66

 

67

bean and kidney bean groups compared to the control group. The
concentrations of copper in liver, kidney and tibia were not significantly
different, although the liver copper concentrations were somewhat lower
in the garbanzo and kidney bean groups. These results suggest that the
availability of copper in garbanzo and kidney beans is lower than that of
copper carbonate. A feeding trial of more than 3 weeks may be needed to
show significant differences in tissue content in response to small
differences in bioavailability.

The results in experiment 2 using a different experimental approach
generally support findings in the first experiment. Serum copper and
serum ceruloplasmin activity in the kidney bean and control+phytate
groups were significantly lower than in the control and dephytinized
kidney bean groups. The concentrations of liver, kidney and tibia copper
were not significantly different among groups, although the kidney
concentrations tended to be lower in the kidney bean and control+phytate
groups compared to control and dephytinized kidney bean groups. Results
indicated that whether the phytate is added to the diet as sodium phytate
or as endogenous phytate seems to make little difference in its effect on
copper bioavailability and the low copper bioavailability in diets
containing phytate can be overcome by the removal of phytate. It is
speculated that the one week of repletion is not enough to show the
difference in tissue copper. The overall results from experiments 1 and 2
indicated that copper was less bioavailable in the diets that contained
phytate than in those that did not contain phytate.

These data are in agreement with the observations of Johnson et al.,

(1988) and Davies and Nightingale (1975). Davies et al. (1975) found that

68

dietary phytate significantly reduced the average daily accumulation and
whole body retention of copper. In their study, the amount of sodium
phytate fed (1%) was higher than the level of phytate in the present
experiment (0.13-0.19%). Although the phytate content of their diets was
higher, the copper level also was higher (25 pg Cu / g diet), approximatly 12
times higher than the copper level used in this study (2 1.1g Cu/ g diet).
Johnson et al. (1988) used a single test meal which contained 20 ug copper
extrinsically labeled with 67Cu to evaluate the bioavailability of copper in
various foods. Weanling rats were fed diets containing 2.5 ppm copper
and 20 ppm zinc for 16 days before the test meals were given. Results
showed that only 65% of copper in garbanzo beans was absorbed compared
to copper sulfate.

Other research in rats and humans has shown no effecf of phytate
on copper bioavailability or, in one study (Lee et al., 1988), an enhancing
effect. These conflicting results may be related to differences in
experimental conditions. Lo et al (1984) did not observe any difference in
copper bioavailability between copper carbonate and isolated soy protein
using a slope-ratio assay. In their study, rats were fed a copper deficient
diet (0.53 ltg Cu / g diet) for 3 weeks followed by 7 days of repletion diets
containing 0.5, 1.0, 1.5 or 2.0 ug Cu/g diet from copper carbonate or various
types of soy protein isolate. The phytate content in the diets ranged from
0.05% to 0.22% and was similar to that used in the present experiment.
Serum and liver copper also were used as indicators to evaluate copper
bioavailability in their study. They did not report the serum concentration
of copper at the end of the depletion period, however, and it may be that

rats were less responsive to the experimental diets because they were not

69

as depleted as those in the present study which were depleted for a total of
4 weeks. At the end of 3 weeks of copper depletion, ceruloplasmin activity
was still high in some rats in experiment 2 although the average activity
was decreased. At the end of 4 weeks depletion ceruloplasmin activity in
all rats was markedly decreased. Other characteristics of the diets also may
have affected the results, such as the amount of zinc used in the two
studies. Diets contained 50 pg Zn / g diet in their study which was higher
than the amount used in the present study (12 pg/ g). It is possible that in
the presence of a high level of zinc in the diet, phytate is more likely to
form a phytate-zinc complex leaving less available to complex with
copper.

Conflicting results were reported by Lee and co—workers (1988) who
concluded that copper bioavailability was enhanced by the presence of
phytate. In their study, rats were fed copper deficient diets (<1 pg Cu/ g
diet) for 4 weeks and then provided required amounts of copper (5 pg
Cu/ g diet) for 3 days. The level of copper in their study was higher than
the level used in this study (2 pg Cu / g diet). The phytate content of diets
(0.4 and 0.8%) were also higher than the content of phyate (0.12-0.18%) in
the present study. Differences in total copper intake may influence the
outcome. In their study, diets contained 20 pg Zn/ g which was also higher
than the amount used in present study (12 pg / g). It is possible that phytate
is more likely to form a complex with zinc allowing more available copper
to be absorbed; thus an enhancing effect occurred because copper level was
higher.

Two groups of investigators did not observe any negative effect of

phytate on copper absorption in humans (Morris and Ellis, 1985; Turnlund

 

70

et al., 1985).Relative to body weight the amount of phytate fed in these
experiments was less than that used in animal experiments. In addition,
the effect of phytate may be different in humans than in rats. Differences
between species have been demonstrated for dietary factors affecting non—
heme iron absorption. Reddy and Cook (1991) reported that whereas meat
enhanced and tea inhibited iron absorption in humans, minimal effects of
these substanecs were observed in rats.

In experiment 2, ceruloplasmin activity increased rapidly within 3
days and no further increases were observed at day 7. This result indicates
that ceruloplasmin activity is very sensitive to copper status. Although
the lower ceruloplasmin activity and serum copper in rats fed diets
containing phytate were statistically significant, they may have limited
biological significance. In experiment 1, the values of ceruloplasmin
activity in baseline rats ranged from 80 to 126.3 U / L and were 82.2 to 103.4
U / L in experiment 2. The values in the baseline rats were considered as
normal values because rats had been fed copper adequate diets. In both
experiments, final ceruloplasmin values for all groups were within the
range of baseline values although ceruloplasmin activity in the rats fed
diets containing phytate were lower.

Although an inhibitory effect of phytate on zinc bioavailability is
well established (O'Dell and Savage, 1960; Lonnerdal et al., 1988; Zhou et
al., 1992), there was limited evidence of an effect on zinc in the
experimental conditions used in this study. In experiment 1, serum zinc
concentrations were significantly higher in the control group than the
garbanzo bean and kidney bean groups. Although there were no

significant differences in liver and kidney zinc concentrations between

 

71

groups, the liver zinc concentrations tended to be lower in the garbanzo
bean and kidney bean groups. A longer period of feeding may have been
needed to show significant differences in tissues. The higher total liver
and kidney zinc contents in garbanzo bean and kidney bean groups might
be explained by the higher total zinc intake in these groups.

In experiment 2, there were no significant differences in serum,
liver, kidney, and tibia zinc concentrations. Since diets containing phytate
were only fed for one week there may not have been sufficient time for
the phytate to show an inhibitory effect. Another explanation could be a
decreased zinc requirement in older rats. It is known that zinc is required
for growth; therefore, it is more likely to see the effect of dietary treatment
in growing rats than in older rats. In experiment 2, rats were
approximately 7 weeks old before the experimental diets were initiated,
and rats of that age are considered as adult. It is assumed that zinc
requirement is lower for adult rats, therefore, the effect of dietary
treatment would be less and a longer period of feeding is necessary in
order to show differences.

Perhaps the most likely explanation for the weak evidence of an
effect of phytate on zinc bioavailability is the low phytate : zinc ratio in the
diets. It has been reported that the phytic acid : zinc molar ratio in the diet
can be used as a predictor for the effect of phytic acid on zinc bioavailability
(Oberleas and Prased, 1976; Morris and Ellis, 1980). In experiment 1, the
phytic acid : zinc molar ratios were 10.7 and 15.5 for the garbanzo bean and
kidney bean diets, respectively. In experiment 2, the phytic acid : zinc
molar ratios were 10.5 and 11.0 in the control+phytate and kidney bean

diets, respectively. Values greater than 12 have been reported to reduce

 

72

accumulation of zinc in femurs but not depress growth (Davies and Olpin,
1979; Morris and Ellis, 1980). Moreover, Zhou et al. (1992) reported that
the total tibia zinc gain tended to be lower when the phytate : zinc molar
ratio was between about 10 to 16 but the differences were not statistically
significant. They concluded that zinc utilization is not inhibited when the
ratio ranges from 10 to 16. Results in the present study confirm such a
conclusion. Lo et al. (1981) reported that there was no effect on zinc
bioavailability when the ratio was less~ than 9 and zinc status was adequate.

Work performed by Graf and Eaton (1984) demonstrated the
importance of dietary calcium on the effect of the phytate : zinc molar ratio
on zinc bioavailability. They indicated that a low calcium content in the
diet (0.75%) had no effect on zinc bioavailability when the phytate : zinc
molar ratio was 12 or less, whereas when the calcium content of the diet
was increased to 1.75%, growth of rats was significantly depressed. In the
present study, calcium concentrations were low and similar in all diets:
0.50, 0.51 and 0.52% in control, kidney bean and garbanzo bean diets,
respectively in experiment 1 and 0.50, 0.50, 0.51 and 0.51% in control,
control+phytate, kidney bean and dephytinized kidney bean diets in
experiment 2, respectively.

The lower phytate contents of beans used in these experiments
compared to literature values might be explained by food processing. It
has been reported that phytate can be hydrolyzed during various types of
food processing including cooking, soaking and fermentation (Chang,
1977; Reddy et al., 1982; Sandberg 1991). During such processes hydrolysis
occurs and various inositol phosphates are formed. Sandberg (1991)

reported that soaking of wheat bran resulted in hydrolysis of 95% of the

 

73

phytate within one hour and complete degradation within two hours and
concluded that the naturally occurring phytase in cereals are activated by
soaking under optimal conditions (pH 4.5-5.5, 60°C). Chang (1977) also
reported that incubation of presoaked beans in water at 60 °C for 10 hours
lowers their phytate content by 90%. Therefore, it is speculated that part of
the phytate in the beans used for the diets in this experiment was
hydrolyzed to lower inositol phosphates during cooking and were not

detected in the analysis for phytic acid.

CONCLUSION

The overall results of this study indicate that copper bioavailability
is lower in diets containing phytate than those without phytate. In
experiment 2, when phytate was added to the control diet, bioavailability
of copper was lower compared to the copper carbonate in the control diet.
In addition, results also indicate that when phytate was removed from
kidney beans, copper bioavailability increased and was similar to that of
copper carbonate. Therefore, it is concluded that whether the source of
phytate is sodium phytate or garbanzo or kidney beans seems to make
little difference, and the adverse effect of phytate on copper bioavailability
can be overcome by removal of phytate. Although serum copper and
ceruloplasmin activity were depressed, it is unclear if these changes have
any biological significance. The values of ceruloplasmin activity in the
present study were within the normal range. In other words, rats were not
copper deficient; however, the diet was fed for a relatively short period of
time. Therefore, a longer period of feeding would be needed to evaluate
long term effects of phytate on tissues.

Bioavailability of zinc in this study was not inhibited by phytate.
Although the serum zinc was significantly lower in the two bean groups
in experiment 1, no differences were found in liver, kidney and tibia zinc

concentrations. The phytate : zinc molar ratio used in present study
74

75
ranged from 10 to 16 and the ratio may have been too low to show
difference in tissues. It is concluded that zinc bioavailability is not

inhibited when the phytate : zinc ratio is less than 16.

 

FUTURE RISEARCH

It is well documented that expression of the phytate : zinc content of
diets on a molar basis is a satisfactory means of predicting dietary zinc
bioavailability. Results of numerous studies have demonstrated that an
increase in the phytate : zinc molar ratio will decrease zinc bioavailability.
In addition, some investigators have reported that the calcium content of
diet to phytate : zinc molar ratio is also a good indicator to predict the zinc
bioavailability since zinc bioavailability can be further decreased by high
dietary calcium to form a calcium, zinc and phytate complex However,
few studies have been done to evaluate the phytate molar relationship
with respect to copper. Using phytate : copper molar ratio to evaluate
copper bioavailability can be further studied. In addition, according to the
literature the copper is likely to interact with other minerals such as zinc
and iron; therefore, the interactions among phytate, calcium, zinc, iron
and copper expressed as molar ratio to evaluate the bioavailability of

copper should be further investigated.

76

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