Effects of Meat and Selected Food Components on the
Valence of Nonheme Iron during In Vitro Digestion
MARIA KAPSOKEFALOU
ABSTRACT
Our objective was to determine whether meat and other dietary factors
have the capacity to reduce nonheme ferric iron to the ferrous form
during digestion. Beef, selected organic acids, selected purified pro-
teins, red blood cells, whole blood, or blood plasma was mixed with
FeCIJ and subjected to simulated gastrointestinal digestion. Ferrozine
was used to monitor the formation of dialyzable Fe(H). Ascorbic acid,
glutathione and cysteine produced large increasesin Fe(H) while meat
(raw and cooked), hemoglobin and red blood cells yielded smaller
increases. Casein, plasma, bovine serum albumin and egg albumin
did not affect Fe(I1) formation. Our Results suggest dietary factors
which enhance iron absorption in vivo promote Fe(W) reduction in
the intestinal lumen.
INTRODUCTION
IT IS WELL ESTABLISHED that certain dietary components
can markedly enhanceor inhibit nonheme iron absorption. For
example absorption of dietary nonheme iron was enhancedby
meat and ascorbic acid but inhibited by tea and wheat bran
(Hallberg, 1981). Layrisse et al. (1968) were the first to report
that meat (beef, pork, and lamb), poultry, and fish promoted
nonheme iron absorption in humans, an observation later con-
firmed by numerousinvestigators(Martinez-Torreset al., 1971;
Cook and Monsen, 1976; Hallberg et al:, 1978; Layrisse et
al., 1984; Hurrell et al., 1988). The magnitude of the enhance-
ment varied but was usually in the range of two to threefold.
Several hypotheseshave been advancedto explain the mech-
anism for the enhancing effect of meat. One is that meat diges-
tion products chelate iron in the intestinal lumen thereby
preventing hydrolysis and precipitation (Berner and Miller,
1985). This hypothesis is supported by studies which showed
that iron solubility increased when meat was digested in vitro
(Kane and Miller, 1984; Slatkavitz and Clydesdale, 1988).
Presumably, the chelating ligands are amino acids or peptides.
Using human subjects, Martinez-Tones and Lay&se (1970)
showed that 1OOgof fish, and a mixture of free amino acids
of similar composition to that in fish, produced similar en-
hancement of nonheme iron absorption. Layrisse et al. (1984)
showed that glutathione and cysteine, but not other amino acids,
enhancednonheme iron absorption by humans, suggesting the
effect may be a function of amino acid composition. However,
the enhancing effect of meat was not found in all foods that
contained significant amounts of high quality protein. Milk,
cheese (Lay&se et al., 1974), casein (Hurrell et al., 1989),
egg, egg albumin (Cook and Monsen, 1976), and bovine serum
albumin (Hurrell et al., 1988), did not significantly increase
nonheme iron absorption by human subjects.
Another hypothesis is that the mechanism for enhancement
of nonhem iron absorption may involve the reduction of Fe(II1)
to Fe(I1) during digestion. Fe(I1) is more soluble and less likely
to hydrolyze under conditions present in the gastrointestinal
lumen (May et al., 1978; Forth and Schafer, 1987). Moreover,
Fe(I1) complexes are less stable than Fe(II1) complexes and
The authors are with the Institute of Food Science, New York
State College of Agriculture & Life Sciences, Stocking Hall, Cor-
nell Univ., Ithaca, NY 14853.
352-JOURNAL OF FOOD SCIENCE-Volume 56, No. 2, 1991
and DENNIS D. MILLER
therefore reduction of Fe(II1) associated within soluble food
components may promote its release into solution (Reddy et
al., 1986). Evidence from human studies (Bothwell et al.,
1979; Forth and, Shafer 1987; Dietzfelbinger, 1987) suggested
that Fe(I1) was more readily absorbed than Fe(III), although
conflicting evidence existed (Forth and Schafer, 1987). Reddy
et al. (1988)proposed that the enhancedmentof iron bioavail-
ability is a function of the concentration of Fe(I1) in the prox-
imal small intestine. Wollenberg and Remmel (1987) concluded
that liminal iron reduction is a prerequisite for uptake by mu-
cosal cells. This hypothesis is supported by data from Layrisse
et al. (1984), who demonstrated that cysteine and glutathione
enhanced nonheme iron absorption by humans. Cysteine and
glutathione are capable of reducing Fe(II1) to Fe(I1) at low pH
(Hamed et al., 1983). Studies involving the effect of ascorbic
acid and also support this hypothesis. Ascorbic acid is a re-
ducing agent and a potent enhancer of iron absorption by hu-
mans.
The objective of our study was to determine whether meat
and other dietary components reduced Fe(II1) to Fe(I1) under
simulated gastrointestinal conditions.
MATERIALS & METHODS
Materials
Distilled, deionized water was used throughout the experiment. All
glasswarewas washed with detergent, rinsed with water, soaked over-
night in 1N HCl, rinsed again, and dried.
Nonheme ferric iron. A FeCl, solution, 1000 ppm in Fe (Certified
Atomic Absorption Standard(Fisher SO-I-124)), was used as the source
of Fe(II1) in all treatments.
Pepsin. 4.Og porcine pepsin preparation (Sigma P7000) was sus-
pended in 0.01N HCl and diluted to 100 mL with O.lN HCl.
Pancreatinlbile mixture. OSg porcine pancreatin (Sigma P-1750)
and 3.Ogbile extract (Sigma B-8631) were suspendedin O.OlN NaHCOB
and diluted to 250 mL with 0.1 N NaHC03.
PIPES buffer. 0.15N. PIPES ( piperazine-N,N’ -bis[2-ethane-sul-
fonic acid]) disodium salt (Sigma‘?3?68) was dissolved in water and
adiusted to PH 6.1 or 6.3 using concentratedHCl. The DH 6.1 PIPES
solution was used for the coniol, ascorbic acid, cysteihe, and gluta-
thione treatments. The pH 6.3 PIPES solution was used for the re-
maining treatments, to compensatefor their higher buffering capacity.
HEPES buffer. 0.3N. pH 9.9. (N-2-hydroxyethyl-piperazine-N’-
2-ethanesulfonic acid) sodium salt (Sigma H7006).
Protein precipitant solution (reducing). 1OOgtrichloroacetic acid
and 50 g hydroxylamine monohydrochloride were dissolved in water,
100 ml concentrated HCl were added and the volume was diluted to
1L with water.
Protein precipitant solution (nonreducing). The same as the pro-
tein precipitant solution (reducing) except that hydroxylamine monoh-
vdrochloride was omitted.
Ferrozine chromogen solution (5 mg/mL). Ferrozine (3-(2-pyri-
dvll-5.6-bis(4-nhenvl-sulfonic acid)-l-2.4-triazinel. disodium salt.
(Sigma P97k2j wasdisolved in watkr.’ . ”
Dialysis tubing. Sepectrapore”I dialysis tubing with a molecular
weight cut-off of 6000-8000 (Fisher 08-670C) was cut into 20 cm
lengths and soaked in water for at least 1 hr prior to use.
Preparation of treatments
Ascorbic acid. 88 mg ascorbic acid (Sigma A-0278) were mixed
with 0.05 mmol FeCl,. The volume was diluted to 100 mL with O.OlN
HCl, yielding a solution 0.5 mM in iron and 5 mM in ascorbic acid.
 Cysteine and glutathione. 78.8 mg L-cysteine hydrochloride hy-
drate (Sigma C-7880) or 153.6 mg glutathione (Sigma G-4251) were
mixed with 0.05 mmol Fe&. Volumes were diluted to 100 mL with
O.OlN HCl, yielding solutions 0.5 m M in iron and 5 m M in cysteine
or glutathione. Amounts of cysteine and glutathione were chosen to
yield sample thoil concentrations similar to those present in the raw
beef treatment. Attempts to measure thiol content of meat using the
Ellman reagent were unsuccessful so the values for meat reported by
Layrisse et al. (1984) were used.
Hemoglobin, casein, egg albumin, and bovine serum albumin.
4g of bovine hemoglobin (Sigma H-2500), or purified casein powder
(Sigma C-5890), or chicken egg albumin (ovalbumin) (Sigma A-5503),
or bovine serum albumin (Sigma A-4503), were mixed with 0.05
mmol FeCI,. The pH was lowered to 2 with 1N HCI and the volume
was diluted to 100 mL with O.OlN HCI.
Blood. Heparinized whole blood was obtained fresh from a steer
at time of slaughter. The hemoglobin content of the blood was deter-
mined using Sigma diagnostic kit No. 525. The amount of blood used
(29.6 mL) contained 4 g hemoglobin. Blood was mixed with 0.05
mmol FeCI,, diluted with O.OlN HCI, acidified to a final pH of 2,
and diluted to 100 ml with O.OlN HCl.
Red blood cells. Red blood cells were isolated from heparinized
fresh blood by centrifugation at 3000 x g for 20 min. The blood
plasma was decanted and collected. The hemoglobin content of the
blood and the red blood cells was determined using Sigma diagnostic
kit No. 525. The amount of red blood cells used (15.6 mL) contained
4g hemoglobin. Red blood cells were mixed with 0.05 mmol FeCl,,
diluted with 0.01 N HCI, acidified to a final pH of 2, and diluted to
100 mL with O.OlN HCI.
Blood plasma. Blood plasma was isolated from blood as described
above. The amount used (14.0 mL) was equvalent to the amount
present in a volume of whole blood that contained 4g hemoglobin.
The blood plasma was mixed with 0.05 mmol FeCI,, diluted with
O.OlN HCI, acidified to a final pH of 2, and diluted to 100 ml with
O.OlN HCI.
Meat. Top round steak, purchased at a local supermarket, was
trimmed of visible fat before use. Three meat treatments were pre-
pared. Raw meat: An amount containing 4g protein (based on protein
content of 22.03 g/lOOgmeat, Agriculture Handbook 8-13) was minced,
mixed with 0.05 mmol Fe& and water and homogenized with a
Kinematica’a mixer. The pH of the homogenatewas adjusted to 2 with
1N NC1 and the volume was made to 100 ml was O.OlN HCL. Broiled
meat: Pieces (3.5 x 3.5 x 5 cm3) were broiled to a gas broiler 10
minutes on each side. Piecesof the broiled meat containing 4 g protein
were treated as described for the raw meat treatment. Microwaved
meat: Pieces(3.5 x 3.5 x 5 ems) were cooked 30 min in a microwave
oven equipped with a cooking probe. The oven was programmed to
cook to a maximum internal temperature of 170°C. The cooked meat
was treated as described for broiled meat.
METHODS
In vitro digestion. A modification of an in vitro digestion method
developed by Miller et al. (1981) was used to assess relative iron
bioavailability. The modified method is described below and in Fig.
.1.
Ten mL aliquots of each of the treatments and their respective
blanks were transferred to vials, in triplicate, and mixed with 10 ml
O.OlN HCI. Pepsin suspension (1 mL) was added to each vial. The
mixture was incubated at 37°C in a shaking water bath for two hours.
At the end of the pepsin incubation, a dialysis bag containing 20 mL
PIPES buffer was placed in each vial. The samples were incubated
30 min. Five mL of the pancreatin-bile mixture were added to each
vial and the incubation continued another 2 hr. At the end of the
pancrcatin-bile incubation the dialysis bags were removed and rinsed
by dipping in water. Bag contents were transferred to tared beakers
and weighed. The experiment was conducted on a timed schedule so
that all samples were incubated for the same period of time. The pH
of each dialysate and retentatewas measuredat the end of the in vitro
incubation
Iron assay. Iron concentrations in dialysates (Fe(B) and total) and
retentates (Fe(I1) only) were measured using a modification of the
method proposed by Reddy et al. (1986). Briefly, for total iron mea-
surement, reducing protein precipitant solution (1 mL) was added to
2 mL aliquots of each dialysate. For Fe(B) measurement, non-reduc-
ing protein precipitant solution (1 mL) was added to 2 mL aliquots of
each dialysate and retentate. Samples were held overnight at room
temperature. Subsequently they were centrifuged in a bench-top cen-
Volume 56, No. 2, 7991-JOURNAL
trifuge at 2575 x g for 10 min. Aliquots of the supematants (1 mL
in duplicate) were transferred to separate tubes. Ferrozine solution
(0.25 mL) and HEPES buffer (2.0 ml) were added to each tube.
Absorbances(at 562 nm) were measuredimmediately after chromogen
addition for the Fe(I1) determination or 1 hr after addition for the total
iron determination. Sample iron concentrations were calculated from
the absorbancereadings using a regression equation derived from data
generated from standards.
Preoaration of control and blanks. FeCl-- (0.50 \ mM) in O.OlN
HCI was used as control.
Since the iron content of the ascorbic acid, cysteine, and glutathione
treatments was negligible, blanks for the Fe(B) and total iron mea-
surement in these treatments was O.OlN HCI.
The nonheme iron content of the protein-containing samples (hemo-
globin, whole blood, red blood cells, blood plasma, casein, bovine
serum albumin, egg albumin, raw meat, broiled meat, and micro-
waved meat) was variable. Therefore each of these samples had an
individual blank for Fe(B) and total iron measurements.These blanks
were preparedby the same procedure described for sample preparation
except Fe& was not added, i.e. blanks contained the protein source
but not the added iron.
Calculations. Dialyzable ferrous iron (D-Fe(II)), dialyzable total
iron (D-(Fe(B) +Fe(III))), nondialyzable ferrous iron (nonD-Fe(II)),
and total ferrous iron (D-Fe(B) + nonD-Fe(B)) were expressed as
percentagesof the total added Fe(II1) in each vial. We assumed that
dialyzable iron had equilibrated across the dialysis membrane by the
time the dialysis bag was removed at the end of the digestion.
D-Fe(I1):
‘[Fe(II)], (p&ml) x Ztotal volume (mL) x IO0
Fe(II1) in original sample (pg)
‘ferrous iron concentration in the dialysate.
*dialysate volume + retentate volume.
D-(Fe(I1) + Fe(III)):
3[Fe(II) + Fe(III)]u (pg/mL) x total volume (mL) x 1oo
Fe(II1) in original sample (pg)
?otal iron concentration in the dialysate.
D-Fe(B) + nonD-Fe(B):
[Fe(II)]u (pg/mL) x 4dialysatevolume (mL)
+
‘[Fe(II)]s (bg/mL) x ’ retentate volume (mL) x loo
Fe(II1) in original sample (kg)
%veight of the dialysate assuming a density of 1 g/mL.
Sferrousiron concentration in the retentate.
“total volume minus volume of dialysate.
nonD-Fe(B):
([Fe(B)], - [Fe(II)]u) (pg/mL) x retentate volume (mL) x loo
Fe(II1) in original sample (kg)
Blank values for D-Fe(II), D-Fe(I1)+ Fe(III)), D-Fe(I1)+ nonD-Fe(II),
and nonD-Fe(I1) were subtracted from samples values.
Statistics
Data were analyzed by Tukey’s Q test. Treatments were compared
with the control using the paired t-test (Snedecor and Cohran, 1980).
A probability level 0.05 was used for determining statistical signifi-
cance.
RESULTS & DISCUSSION
Values for D-Fe(H) fell into three distinct groupings (Fig. 2).
Plasma, casein, egg albumin, and bovine serum albumin did
not increase D-Fe(U) over the control. Meat and hemoglobin
caused a 6-fold increase in D-Fe(H) while ascorbic acid, cys-
teine, and glutathione yielded a 1Zfold increased in D-Fe(I1)
over the control.
Cysteine and glutathione treatmentsyielded significantly more
D-Fe(I1) than the meat treatments. If the effect of meat were
Of FOOD SCIENCE-353
 IRON VALENCE DURING DIGESTION. . .

ITreatment preparation I
40

(Pepsin incubation (2 h @ 37"C)( g 30

I I
=
z 20
A
Dialysis bag addition
(incubation for 30 min @ 37°C)
IO

0
Pancreatin-bile incubation (2 h @ 37 "C) C AA Cys GTH Hb Bd RBC PI Gas EA BSA R Br MW

Fig. 2. -Ferrous dialyzable iron (D-Fe(I)) formed during in vitro

digestion. Percentages of Fe(lll) in samples at start of incuba-
tion. Each value is mean 2 standard deviation for three obser-
vations. * = not significantly different from the control.
Dialysis bag removal Abbreviations: C-control, AA-ascorbic acid, Cys-cysteine, GTH-
(Contents transferred to beakers glutathione, Hb-hemoglobin, Bd-whole blood RBC-red blood
and weighed) cells, PI-blood plasma, Cas-casein, EA-egg albumin, BSA-bovine
serum albumin, R-raw meat, Br-broiled meat, MN-microwave
cooked meat.

Protein precipitant solution addition to SO

aliquots from dialysate and retentate,

/ sample held ;"' 12 hours. 1 40

30

lCentri';"""onl
20

10

l~~~~'d:Er~;~~~~~n~
Fig. l.-Schematic representation of the in vitro experimental
procedure.
due to thiol concentration alone, we would have expected them
to be the same since concentrations for cysteineand glutathione
were based on meat thiol concentration. Cell membranes or
lipids present in meat may have bound iron and inhibited its
dialysis and/or reduction.
Our data showed that D-Fe(II) was a reasonable predictor
for iron bioavailability. Components known to enhance non-
heme iron absorption by humans e.g. ascorbic acid (Lynch and
Cook, 1980), cysteine (Layrisse et al., 1984), glutathione
(Layrisse et al., 1984), and meat (Layrisse et al., 1968; Mar-
tinez-Torres and Layrisse 1971), produced an increase in D-
Fe(II) over the control. Components that did not promote non-
heme iron absorption, e.g. casein (Berner and Miller, 1985),
bovine serum albumin (Hurrell et al., 1988), and egg albumin
(Hurrell et al., 1988), were ineffective at reducing Fe(II1).
Ascorbic acid also reduced more Fe(U) than meat. Monsen
and Balintfy (1982) proposed that 1 mg ascorbic acid was
equivalent to 1.3g of raw meat in iron absorption enhancing
properties. We used a higher ratio of ascorbic acid to meat (1
mg ascorbic acid to 0.21g meat). Since the iron absorption
enhancing properties of ascorbic acid have been shown to be
dose-dependent,it was not surprising that we observed higher
D-Fe(I1) with the ascorbic acid treatment. The capacity of mus-
cle tissue to reduce Fe(II1) is biologically plausible since Fe(I1)
is required in cellular metabolism. Egyed and Saltman, (1984)
0
C AA CyS GTH Hb Bd RBC PI Cas EA BSA R BP MW
Fig. 3- Total dialyzable iron (D-(FellI) + Fe(lll))) formed during
in vitro digestion. Percentages of Fe(lll) in samples at start of
incubation. Each value is mean ? standard deviation for three
observations. * = not significantly different from the control.
For abbreviations see Fig. 2.
showed that the redox environment within both reticulocytes
and mature red cells permitted the generation and mobilization
of Fe(H) in an oxygenated environment.
Treatment effects were also observed for D-(Fe(I1) + Fe(II1))
(Fig. 3). The pattern, however was less clear-cut and effects
were smaller. There was considerable variability among the
meat treatments, the average increase over the control being
about twofold. Ascorbic acid, cysteine, and glutathione in-
creased D-(Fe(II)+Fe(III)) bout the same amount as meat.
The most striking difference between D-Fe(I1) and D-
(Fe(I1) +Fe(III)) was with the bovine serum albumin treat-
ment. It had no effect on Fe(I1) formation but a large effect
on D-(Fe(I1)+ Fe(II1)) indicating that digestion products of BSA
did not reduce Fe(II1) but formed relatively stable complexes
with Fe(II1) that are apparentlysoluble and low molecular weight.
More than 80% of the iron was present as soluble Fe(I1) in the
hemoglobin and ascorbic acid treatments (Fig. 4). NonD-Fe(U)
was unexpectedly high for those treatmentssuggesting that iron
either polymerized or (in the case of hemoglobin) was bound
to high molecular weight species. Although we did not see any
effect of cooking on D-Fe(I1) formation, a significant cooking

354-JOURNAL OF FOOD SCIENCE-Volume 56, No. 2, 1991

 , D-Fe(W) + nonD-Fdll)
I dry1 groups were blocked with N-ethylmaleimide.
q nonD-Fd II)
Fig. 4- Total [D-Fe(h) -I- nonD-Fe(h)] and nondialyzable ferrous
iron (nonO- Fe(I)) formed during in vitro digestion. Difference
between the two bars represents dialyzable ferrous iron ID-Fefll)),
shown in Fig.2 Percentages of ferric iron present in samples at
start of incubation, Each value is mean ? standard deviation
for three observations, All treatments significantly different from
control for both values. For abbreviations see Fig. 2.
effect on D-Fe(B) + nonD-Fe(E)formation was observed(Fig.
4).
Role of thiol groups
The high levels of D-Fe(B) formed in the cysteine and glu-
tathione treatments suggestedthat thiol groups could reduce
Fe(II1) under conditions present in the gut. Thus it is likely
that thiol groups play a role in the “meat effect” through
Fe(II1) reduction. It was shown in humans that cysteine en-
hancediron absorptionbut basic, aromatic, or aliphatic amino
acids did not (Martinez-Torres and Layrisse, 1970).
If thiol groups are responsiblefor Fe(II1) reduction it might
be expectedthat cooking would reduce Fe(B) formation since
thiol oxidation may occur. Taylor et al. (1986) showed that
although reducedcysteine enhancediron absorption, oxidized
cysteine had no effect. When cysteine was mixed with food
before final cooking, it did not enhanceiron absorption. How-
ever, glutathion remainedactive even when addedbefore cook-
ing, producing an enhancingeffect on iron absorption similar
to that producedwhen addedafter cooking. Therefore, cysteine
residues in intact proteins or in peptides may not be readily
oxidized by cooking (Taylor et al., 1986).
Protein digestibility may also be a factor. The more com-
pletely digested the protein was, the more thiol groups would
be exposed and available for reducing iron. Politz and CIy-
desdale(1988) showed that iron solubility increasedwith time
in an in vitro digestion of chicken muscle. However Hurrell
et al. (1989) showed that predigestion of casein increaseddi-
alyzable iron but did not have any effect on iron absorptionby
humans. Productsof the proteolytic digestion could act as iron
ligands (Slatkavitz and Clydesdale, 1988) thereby enhancing
iron solubilization and promoting (or inhibiting) its dialysis.
Role of the heme group
High levels of Fe(B) were formed in the hemoglobin treat-
ment (Fig. 2 and 4) suggestingthat the heme group may par-
ticipate in Fe(II1) reduction. Eguchi and Saltman (1984, 1987)
showed that hemoglobin, in both aerobic and anaerobic sys-
tems reducednonhemeferric iron. Deoxymyoglobin, an Fe(B)
myoglobin derivative, predominatesat the low pOZconditions
found in meat (Livingston and Brown, 1981). Oxidation of
deoxymyoglobin to metmyoglobin, may be coupled to the re-
duction of nonhem Fe(III) under conditions in the gastrointes-
tinal tract. Thiol groups in hemoglobin could have also been
involved, but Eguchi and Saltman (1987) observedthat Fe(II1)
Volume 56, No. 2, 1991-JOURNAL
reduction was only slightly inhibited when hemoglobin sulfhy-
Compared to the hemoglobin treatment, blood treatments
were unexpectedly low in D-Fe(I1) formation. A possible ex-
planation is that ceruloplasmin, a ferroxidase present in blood
plasma (Jansson, 1990), could oxidize Fe(B). However, this
seems unlikely since ceruloplasmin would probably be inac-
tivated by digestive enzymes.
Dialyzable ferrous iron as an indicator of iron
bioavailability
Comparing the results of in vivo and in vitro studies, Miller
and Berner (1989) concluded that dialyzability following in
vitro digestion is a reliable predictor of iron bioavailability. In
general, our data supportedtheir conclusions. However, in the
case of bovine serum albumin, D-(Fe(I1) + Fe(II1)) measure-
ments predicted a greater enhancementof iron bioavailbility
than has been reported in studies with human subjects (Kane
and Miller, 1984; Hurrell et al., 1988) Our data showed that
D-Fe(I1) is a better predictor of the effect of bovine serum
albumin on iron bioavailability, i.e. bovine serum albumin did
not increase D-Fe(I1) nor does it enhanceiron absorption by
human subjects. The effect of the other treatments on iron
bioavailability was predicted by either D-Fe(H) or D-
(Fe(B) + Fe(III)).
To summarize; the hypothesisthat meat exerts its enhancing
effect through Fe(II1) reduction is supported by our data: an
increase in D-Fe(I1) in the meat treatments over the Fe(II1)
control was observedin our in vitro systemwhile egg albumin,
bovine serum albumin, and casein did not have any effect on
D-Fe(B) formation. Cysteine and glutathione were capable of
reducing Fe(II1). Under the conditions of our experiments,
published data for iron absorptioncorrespondedbetter with D-
Fe(B) than with D-(Fe(B) + Fe(III)). Therefore D-Fe(I1) may
be a better indicator of iron bioavailability than D-
(Fe(I1)+ Fe(II1)).
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