Prooxidant Effects of Ferrous Iron, Hemoglobin, and Ferritin in Oil
Emulsion and Cooked-Meat Homogenates Are Different
from Those in Raw-Meat Homogenates1
D. U. AHN,*,2 and S. M. KIM†
*Animal Science Department, Iowa State University, Ames, Iowa 50011, and †Food Science Department,
KyungSan University, KyungSan, Korea
ascorbate was present. Hemoglobin and ferritin had no
prooxidant effect in raw-meat homogenates. The status
of heme iron and the released iron from hemoglobin had
little effect on the prooxidant effect of hemoglobin in oil
emulsion and cooked meat homogenate systems. The
prooxidant effect of ferrous iron in oil emulsion and
cooked-meat homogenates disappeared in the presence
of superoxide (.O2–), H2O2, or xanthine oxidase systems.
In raw-meat homogenates, however, ferrous had strong
prooxidant effects even in the presence of .O2–, or H2O2.
The status of free iron was the most important factor in
the oxidation of oil emulsion and cooked-meat
homogenates but the impact in raw-meat homogenates
was small.
ABSTRACT Oil emulsion and raw and cooked tissue
homogenates were used to determine the mechanisms of
various iron forms on the catalysis of lipid peroxidation.
Flax oil (0.25 g) was blended with 160 mL maleate buffer
(0.1 M, pH 6.5) to prepare an oil emulsion. Raw or
cooked turkey leg meat was used to prepare meat
homogenates. Samples were prepared by adding iron
from each of the various sources, reactive oxygen
species, or enzyme (xanthine oxidase and superoxide
dismutase) systems into the oil emulsion or meat
homogenates.
In oil emulsion and cooked-meat homogenates,
ferrous iron and hemoglobin had strong prooxidant
effects, but ferritin became prooxidant only when
(Key words: hydrogen peroxide, superoxide, iron sources, oil emulsion, meat homogenates)
1998 Poultry Science 77:348–355
hydrogen peroxide (H2O2) to form hydroxyl radicals
(.OH). Ahn et al. (1993a), on the other hand, found that
iron chelated to iron binders or storage proteins had
very weak or no catalytic effect on lipid peroxidation
reaction.
Halliwell and Gutteridge (1986) showed that transferrin, an iron carrier protein that binds ferric iron tightly,
did not participate in .OH generation at physiological
concentrations. The work of Baldwin et al. (1984)
indicates that partly saturated transferrin protects cells
from damage by binding iron that might catalyze .OH
formation from superoxide (.O2–) and H2O2. Transferrin,
however, can release iron at low pH (below pH 5.6) and
thus can accelerate lipid peroxidation under those
conditions. Ferritin is regarded as a safe iron storage
protein, but has been shown to be involved in the
formation of .OH, if the iron ions are released from
ferritin by ascorbate, dithionite, or .O2– radicals (Carlin
INTRODUCTION
Organic and inorganic iron compounds are involved
in the catalysis of various stages of lipid peroxidation;
however, the catalytic effects of free ionic iron (both
ferric and ferrous irons), bound iron, and heme
pigments on lipid oxidation, and the mechanisms by
which the lipid peroxidation is catalyzed, are still
controversial. Kanner et al. (1988) reported that free ionic
iron is the major catalyst for lipid oxidation in meat
products. Johns et al. (1989), however, found that all
forms of inorganic iron have little prooxidant activity in
exhaustively washed muscle fibers and concluded that
heme pigments are more powerful catalysts of lipid
oxidation than inorganic iron compounds. The work of
Halliwell and Gutteridge (1990) indicates that all of the
simple iron complexes are capable of decomposing
Received for publication January 13, 1997.
Accepted for publication October 7, 1997.
1Journal paper Number J-16849 of the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa. Project Number 2794, and
supported by Hatch Act, NC-183 and State of Iowa funds.
2 To whom correspondence should be addressed: duahn
@iastate.edu
Abbreviation Key: BHA = butylated hydroxyanisol; DDW =
deionized distilled water; Hb = hemoglobin; MDA = malonaldehyde;
SOD = superoxide dismutase; TBA = 2-thiobarbituric acid; TBARS =
2-thiobarbituric acid reactive substances; TCA = trichloroacetic acid;
XOD = xanthine oxidase.
348
PROOXIDANT EFFECTS OF IRON SOURCES IN DIFFERENT TESTING SYSTEMS
and Djursater, 1984; Biemond et al., 1986; Decker and
Welch, 1990).
Hirano and Olcott (1971) reported that heme with
iron in either the ferrous or ferric states was an effective
catalyst in lipid oxidation to unsaturated fatty acids.
Kaschnitz and Hatefi (1975) reported that only the ferric
forms of hemes were active catalysts of lipid oxidation
in muscle. Others (Sato and Hegarty, 1971; Love and
Pearson, 1974; Ahn et al., 1993b) found that heme
pigments have little influence on the development of offflavors or 2-thiobarbituric acid reactive substances
(TBARS) in meat. Hidalgo et al. (1990) suggested that
heme proteins may compete with other molecules for
oxidant radicals and, thus, serve as antioxidants. Kanner
and Harel (1985) showed that metmyoglobin alone had
no effect on membranal lipid peroxidation, but that
metmyoglobin activated by H2O2 had a significant
effect. The interaction of natural heme pigments with
H2O2 produces an active species that can initiate lipid
peroxidation (Kanner and Harel, 1985). The incubation
of heme pigments with a molar excess of H2O2 or
heating could release ionic iron from heme (Chen et al.,
1984; Gutteridge, 1986), which can catalyze lipid oxidation. Addition of NaCl into meat during processing
procedures significantly increases the release of free
ionic iron from heme pigments or other binding
macromolecules (Kanner et al., 1991; Ahn et al., 1993b).
According to Haber-Weiss reaction [4] (Halliwell and
Gutteridge, 1990), .O2– reduces ferric iron to the ferrous
form [1], and ferrous iron splits H2O2 formed by the
reaction [2] to produce .OH radicals, which catalyze
lipid oxidation.
.O2–
+ Fe3+ → Fe2+ + O2
[1]
.O2–
+ 2H+ → H2O2
[2]
H2O2 + Fe2+ → Fe3+ + OH– + .OH
.O2–
+ H2O2 → OH– + .OH (metal catalyst)
[3]
[4]
Miller et al. (1990), however, rejected the traditional
Fenton type reaction [3] (Halliwell and Gutteridge, 1990)
and suggested that an Fe(II):Fe(III) complex is the
catalyst of lipid oxidation. Other researchers (Kanner
and Harel, 1985; Halliwell and Gutteridge, 1990; Shen et
al., 1992) also suggested that the initiator of lipid
oxidation formed from the reactions [3] and [4] is not
.OH but ferryl or perferryl radicals, and the iron
involved could be free or bound and heme irons.
Various systems used to study the catalytic effect of iron
sources, and the controversial results from those studies
suggesting that iron sources may have different catalytic
effect in different study systems. Also, the mechanisms
3Fisher
4Sigma
Scientific, Pittsburgh, PA 15219-4785.
Chemical Co., St. Louis, MO 63178-9916.
349
by which lipid oxidation is initiated and propagated
could be affected by the study systems, iron sources, and
other conditions.
The objectives of this study were: 1) to determine the
mechanisms of various iron forms on the catalysis of
lipid peroxidation in oil emulsion, and raw and cooked
tissue homogenates; and 2) to find the effect of various
conditions on the release of iron from iron proteins and
its consequence on the catalysis of lipid peroxidation in
oil emulsions and raw and cooked tissue homogenates.
MATERIALS AND METHODS
Chemicals
Ascorbate was purchased from Fisher,3 and butylated
hydroxyanisol (BHA), superoxide dismutase (SOD), xanthine, xanthine oxidase (XOD), catalase, trichloroacetic
acid (TCA), 2-thiobarbituric acid (TBA), chelex-100 (50-100
mesh, sodium form), ferrozine (3-(2-pyridyl)-5,6-bis (4phenyl sulfonic acid)-1,2,4-triazine, and neocuproine were
obtained from Sigma.4 All chemicals used were reagent
grade.
Reagents
Ascorbate (1,000 ppm), SOD (100 U/mL), xanthine (40
mM), XOD (4.528 U/mL), desferrioxamine mesylate (20
mM), and potassium superoxide (KO2, 14 mM) were
prepared by dissolving the appropriate amount of each
chemical directly in deionized distilled water (DDW).
Fifty parts per million iron (0.895 mM) equivalent
solutions were prepared by dissolving the appropriate
amount of each protein in distilled water [hemoglobin
(Hb), 15.6 mg/mL; ferritin, 250 mg/mL]. The ionic iron
solutions were prepared by dissolving 178 mg FeCl2.4H2O
or 242 mg FeCl3.6H2O in 1 L of 0.1 N HCl to make 50 mg
Fe/mL (0.895 mM) solution. The TBA/TCA stock solution
was prepared by dissolving 15% TCA (wt/vol) and 20
mM TBA in DDW. Seventy-five milligrams of ferrozine
and 75 mg neocuproine were dissolved in 25 mL DDW to
make ferroin color reagent (Carter, 1971). The BHA (72
mg/mL) was dissolved in 97% ethanol.
Sample Preparations
For the oil emulsion, 50 mL Tween-20 was dissolved in a
250-mL beaker containing 20 mL DDW and 8 mL 1 M
maleate buffer, pH 6.5, and then 0.25 mL oil (flax oil) was
added dropwise while stirring. Maleate buffer was chosen
in this study because it has relatively high buffering
capacity at pH 6.5 range, and has no iron chelating effect.
After 5 to 10 min of mixing, two to three pieces of KOH
(about 0.4 g) were added to improve saponification. After
mixing for 5 min, the volume of the oil emulsion (pH 10 to
11) was adjusted to 160 mL by slowly adding Chelex100-treated DDW. The pH of the diluted emulsion was
adjusted to pH 6.5 with 5 N HCl and then used
350
AHN AND KIM
immediately for the subsequent study. Addition of KOH
improved emulsification of oil but had no effect on the rate
of lipid oxidation. The flax oil used in this study was
composed of 5.42% C16:0, 2.69% C18:0, 12.08% C18:1n9,
16.65% C18:2n6, and 63.17% C18:3n3 fatty acids.
For meat homogenates, fresh hand-deboned turkey
thigh meat without skin was obtained from a local turkey
processor, ground twice in a Hobart meat grinder5
through 8- and 3-mm plates, vacuum packaged (200 g
each), and stored in a –20 C freezer until used. Cookedmeat homogenates were prepared after cooking the
ground meat (100 g in a plastic bag) at 85 C for 30 min in a
water bath to an internal temperature of 78 C. A
5-g meat sample was placed in a 50-mL test tube and
homogenized with 15 mL of DDW by using a Brinkman
Polytron6 for 15 s at speed 7 to 8.
Iron from each of the various sources (0.1 mL) and 0.5
mL oil emulsion or meat homogenate were added to
disposable test tubes (13 × 100 mm). The homogenates
were mixed and then 0.1 to 0.4 mL DDW and prooxidant
treatment were added. In ascorbate-containing
homogenates, 0.1 mL ascorbate solution was added
instead of DDW to give a total volume of 1 mL. Iron was
added to control samples without addition of enzyme
solution.
containing 1 mL sample mixture were vortexed and
incubated for 1 h in a 37 C water bath. The sample mixture
was added with 1 mL of 11.3% TCA and 50 mL of 5% BSA
solution to precipitate the oil emulsion. Subsequently, 0.8
mL of 10% ammonium acetate and 0.2 mL of ferroin color
reagent were added, mixed, and centrifuged at 3,000 × g
for 10 min. The absorbance of the supernatant was read at
562 nm against a blank (2 mL distilled water + 0.8 mL of
10% ammonium acetate + 0.2 mL ferroin color reagent)
after 5 min.
For the release of iron from iron proteins, a
4-mL oil emulsion prepared as previously described, was
mixed with an iron source (5 ppm iron equivalent in oil
emulsion, final concentration) and treatment (none,
ascorbate, KO2, H2O2, and XOD system) combinations.
Samples were filtered through a Centricon membrane
filter7 (cut-off size: 10,000 kDa), and the filtrate was used
to determine the amount of free iron released from iron
sources under the various treatment conditions. The
filtration was accomplished by centrifuging the Centricon at 3,000 × g for 120 min. The filtrate and filtration
residue (unfiltered) collected after the centrifugation were
also used to determine their effects on lipid oxidation in
oil and meat homogenates as described above as needed.
Statistical Analysis
Lipid Peroxidation
Lipid peroxidation was determined by the method of
Buege and Aust (1978). Test tubes containing 1 mL sample
mixture, prepared as described above, were incubated for
1 h in a 37 C water bath. Immediately after incubation, 50
mL 7.2% BHA and 2 mL TBA/TCA solution were added.
The mixture was vortexed and then incubated in a boiling
water bath for 15 min to develop color. After color
development, the samples were cooled in cold water for 10
min and then centrifuged for 15 min at 2,000 × g. The
absorbance of the resulting supernatant solution was
determined at 531 nm against a blank containing 1 mL DW
and 2 mL TBA/TCA solution. The TBARS numbers were
expressed as milligrams malonaldehyde (MDA) per liter
of incubating homogenates.
Nonheme Iron Determinations
The ferrozine method of Carter (1971), modified for the
use in meat samples (Ahn et al., 1993c), was used to
analyze reduced iron and total iron when needed. To
determine the content of ferrous iron in raw-meat
homogenates, 0.5 mL meat homogenate, 0.1 to 0.4 mL
treatment combinations (0.1 mL for each component) and
0.1 to 0.4 mL DDW were added to a disposable test tube
(13 × 100 mm) to give a total volume of 1 mL. The test tubes
5Model 84185, Hobart Manufacturing Corp., Troy, OH 44859.
6Type PT 10/35, Brinkman Instruments Inc., Westbury, NY
11590-0207.
7Amicon Inc., Beverly, MA 01915.
The experiment was designed primarily to determine
the catalysis of lipid peroxidation in oil emulsion, and raw
and cooked tissue homogenates under various reactive
oxygen species. The data (four replications) for the meat
homogenate and oil emulsion treatments were analyzed
separately by SAS software (SAS Institute, 1986).
Analyses of variance were conducted to test treatment
effects within a meat homogenate or oil emulsion system.
The treatments compared were control, H2O2, KO2, KO2 +
H2O2, XOD, SOD, and SOD + XOD. These treatments
were compared with the presence of Fe2+, Fe3+ +
ascorbate, and no added iron; Hb, Hb + ascorbate, and no
added Hb; and ferritin, ferritin + ascorbate, and no added
ferritin. The Student-Newman-Keuls multiple range test
was used to compare differences among mean values.
Mean values and SEM are reported, and replications were
used as the error terms for the calculations.
RESULTS AND DISCUSSION
Ferrous Iron and Oxidation
There were clear differences in the way ferrous iron
catalyzed lipid oxidation in meat homogenates and oil
emulsions (Table 1). In oil emulsions, ferrous iron had a
strong prooxidant effect; however, the strong prooxidant
effect of ferrous iron disappeared in the presence of .O2–,
H2O2, or the XOD system (XOD + xanthine). The TBARS
value of oil emulsion with XOD system was greater than
those of .O2– and H2O2 but less than those of control, SOD,
and SOD + XOD systems. This result contradicts the
351
PROOXIDANT EFFECTS OF IRON SOURCES IN DIFFERENT TESTING SYSTEMS
TABLE 1. Effect of ferrous iron1 on the TBARS values of oil emulsion, and raw-meat and cooked-meat homogenates
with added reactive oxygen species, SOD, or XOD systems2
Oil emulsion
Raw-meat homogenate
Treatment
None
Fe2+1
Fe2+ +
asc.
Control
H2O2
KO2
KO2 + H2O2
XOD system
SOD system
SOD + XOD systems
SEM
0.02b
0.01b
0.01b
0.05b
0.03b
0.16a
0.17a
0.00
2.82a
0.19d
0.29d
0.16d
0.51c
2.85a
1.20b
0.02
3.15a
1.30d
2.11c
1.16d
1.44d
2.82b
1.98c
0.03
a–eMeans
Fe2+
None
(mg MDA/L reaction
0.26ab
1.19bc
0.19bc
1.39a
0.29ab
1.27b
0.18bc
1.44a
0.15c
0.23d
0.36a
1.44a
0.11c
0.18d
0.01
0.01
Cooked-meat homogenate
Fe2+ +
asc.
None
Fe2+
Fe2+ +
asc.
mixture)
0.77c
1.18b
1.25b
2.01a
1.16b
0.67c
0.55d
0.01
0.89a
0.87a
0.83a
0.82a
0.58b
0.91a
0.63b
0.01
1.70b
0.69e
0.93d
0.63e
0.76e
1.95a
1.17c
0.01
3.94b
2.71d
3.51c
2.77d
2.70d
5.00a
4.15b
0.02
within a column with no common superscript differ significantly (P < 0.05). n = 4.
1There was 5 ppm iron (89.5 mM); XOD system: 0.145 U XOD plus 2 mM xanthine; SOD system: 500 U catalase plus 50 U SOD/mL; KO , 100 ppm (1.4
2
mM); ascorbate, 100 ppm (0.57 mM); H2O2, 2 mM (final concentrations).
2TBARS = thiobarbituric acid reactive substances; SOD = superoxide dismutase; XOD = xanthine oxidase; MDA = malondialdehyde; H O =
2 2
hydrogen peroxide; KO2 = potassium superoxide, asc. = ascorbate.
widely accepted superoxide-driven Fenton reaction, in
which .OH is produced from .O2– and iron to initiate lipid
peroxidation. This unexpected result, however, was
caused by the oxidation of ferrous iron to ferric form by
the .O2– (KO2) or H2O2 added in oil emulsion. Ahn and
Kim (1997, unpublished data) observed that both .O2– and
H2O2 oxidized ferrous iron to ferric form rapidly and the
ferric form of iron had no reactivity with both .O2– and
H2O2. There are no reducing agents that can reduce ferric
iron to ferrous form present in the oil emulsion. Therefore,
the production of .OH in oil emulsion with .O2– (KO2) or
H2O2 lasted only for a short time, which limited the
prooxidant effect of ferrous iron.
The TBARS value of the oil emulsion with SOD system
(SOD + catalase) was similar to that of the control,
suggesting that the SOD system had no effect on the
prooxidant effect of ferrous iron in oil emulsion. However,
the addition of the SOD system in the presence of the XOD
system increased the TBARS values of the oil emulsion.
The prooxidant effect of catalase contributed to the high
TBARS values of the oil emulsion with SOD system (data
not shown), and the dismutation of .O2– (SOD) and
degradation of H2O2 (catalase) should be responsible for
the higher TBARS value in oil emulsion with SOD + XOD
system than that with XOD system (Table 1).
The addition of ascorbate in the oil emulsion containing
ferrous iron significantly increased the TBARS values of
the oil emulsion under all reactive oxygen species
treatments, except the SOD system, which had little effect
on the prooxidant effect of ferrous iron in oil emulsion
with or without ascorbate. The added ascorbate reduced
ferric iron to the ferrous form and maintained the high
prooxidant effect of iron even in .O2–, H2O2, and XOD
system treatments. The cycling of the valence of iron
(ferrous iron ↔ ferric iron) in oil emulsion with ascorbate
and XOD system produced .OH continuously and the
oxidation of lipids continued while ascorbate was available.
The prooxidant effects of ferrous iron in raw-meat
homogenates were totally different from those in oil
emulsion under various reactive oxygen conditions, and
the influences of ascorbate, .O2–, or H2O2 were also
dramatically different from those in oil emulsion. In rawmeat homogenates, only the samples containing the XOD
system nullified the prooxidant effect of ferrous iron via
the continuous production of .O2– and H2O2. The XOD
system reduced the TBARS values of raw-meat
homogenates more than that of the .O2–, H2O2, H2O2 plus
.O2– or the SOD systems. The .O2– and H2O2 produced by
the XOD system should have maintained the iron in ferric
form.
When ascorbate plus ferrous iron was added, the
TBARS values of raw meat homogenates with H2O2, .O2–,
.O2– + H2O2 or the XOD system were significantly higher
than that of the control; however, the TBARS values of raw
meat homogenates with the SOD system maintained the
same or less than that of the control. The addition of
ascorbate in raw-meat homogenates decreased the prooxidant effect of ferrous iron in the control, H2O2, and SOD
systems, but increased that effect in .O2– + H2O2, and XOD
systems. The large increase of TBARS values in raw meat
homogenates with the XOD system can be explained by
the continuous production of .O2– and H2O2 and the
regeneration of ferrous iron by the added ascorbate. The
decrease in TBARS values in meat homogenates with SOD
system should be caused by the removal of .O2– and H2O2
by SOD and catalase.
The prooxidant effects of ferrous iron (with and
without ascorbate) in cooked-meat homogenates with
various reactive oxygen species conditions were similar to
those of oil emulsion. The .O2–-generating systems (XOD
system and KO2) and their products (H2O2 and .O2–)
reduced the TBARS values of cooked-meat homogenates
by converting the active form of iron (ferrous) to an
inactive form (ferric).
The effect of ascorbate on the oxidation of oil emulsion,
and raw and cooked meat homogenates were different.
352
AHN AND KIM
TABLE 2. Effect of hemoglobin (Hb)1 on the TBARS values of oil emulsion, and raw-meat and cooked-meat homogenates
with added reactive oxygen species, SOD, or XOD systems2
Oil emulsion
Raw-meat homogenate
None
Hb1
Hb + asc.
None
Hb + asc.
None
Hb1
Hb + asc.
Control
H2O2
KO2
KO2 + H2O2
XOD system
SOD system
SOD + XOD systems
SEM
0.02b
0.01b
0.01b
0.05b
0.03b
0.16a
0.17a
0.00
3.15b
2.79c
3.65a
2.07e
2.37d
3.54a
2.40d
0.02
3.24a
2.66b
3.08a
2.78b
2.17c
3.28a
2.14c
0.03
(mg MDA/L reaction mixture)
0.26ab
0.07b
0.14
0.19bc
0.08b
0.14
0.29a
0.09b
0.14
0.18bc
0.09b
0.13
0.15c
0.20a
0.14
0.36a
0.08b
0.12
0.11c
0.10b
0.13
0.01
0.00
0.00
0.89a
0.87a
0.83a
0.82a
0.58b
0.91a
0.63b
0.01
1.09c
1.72a
1.78a
1.69a
0.71e
1.27b
0.87d
0.01
1.95a
2.02a
2.13a
1.89a
1.19b
1.87a
1.36b
0.02
a–eMeans
Hb1
Cooked-meat homogenate
Treatment
within a column with no common superscript differ significantly (P < 0.05). n = 4.
1Hb: 5 ppm iron (89.5 mM) equivalent (1.56 mg/mL, final). XOD system: 0.145 U XOD plus xanthine (2 mM)/mL; SOD system: 500 U catalase plus 50
U SOD/mL; KO2. 100 ppm (1.4 mM); ascorbate, 100 ppm (0.57 mM); H2O2, 2 mM (final concentrations).
2TBARS = thiobarbituric acid reactive substances; SOD = superoxide dismutase; XOD = xanthine oxidase; MDA = malondialdehyde; H O =
2 2
hydrogen peroxide; KO2 = potassium superoxide; asc = ascorbate.
Addition of ascorbate increased the prooxidant effect of
ferrous iron in oil emulsion and cooked-meat
homogenates, especially with superoxide-generating systems and reactive oxygen species. However, the effect of
added ascorbate reduced the TBARS values of raw meat
homogenates in control, H2O2, and SOD system, but
increased in the KO2 plus H2O2, XOD system and SOD +
XOD systems. This result indicated that raw meat
homogenate has strong reducing power, which was
different from that of the added ascorbate. The reducing
power in raw meat homogenates could maintain most of
the added iron to raw meat homogenates in the ferrous
form even under .O2–, H2O2, or both. A relatively low
ascorbate content in meat homogenates (approximately 6
ppm) suggests that only a small part of the reducing
power of the raw meat homogenate could originate from
ascorbate. Ghiselli et al. (1995) also reported that there are
many antioxidant compounds present in plasma, and
ascorbate plus thiol groups are only about 25% of the total
antioxidant capability.
Oxygen plays a more critical role in the oxidation of
lipids in cooked-meat than it does in the oxidation of
lipids in raw meat (Ahn et al., 1993d). Therefore, the
involvement of certain heat-labile substances, possibly
reducing enzymes or organelles (e.g., mitochondria) that
can maintain reducing conditions by consuming oxygen
in raw-meat homogenates, could be responsible for the
differences in the prooxidant mechanisms of iron between
oil emulsion/cooked-meat homogenates and raw-meat
homogenates.
Hb and Oxidation
The mechanism and the prooxidant effect of Hb in lipid
peroxidation of oil emulsions are quite different from
those of meat homogenates, especially raw-meat
homogenates. As shown in Table 2, Hb had very strong
prooxidant effects in oil emulsions regardless of the
presence or absence of ascorbate and reactive oxygen
species. As far as the prooxidant effect of Hb in oil
emulsion was concerned, heme iron did not appear to
have an effect; however, the presence of H2O2 or XOD
system reduced the prooxidant effect of Hb in oil
emulsions. In raw meat homogenates, Hb was not a
prooxidant under all ascorbate and reactive oxygen
species conditions. In cooked-meat homogenates,
however, Hb had some catalytic effects on lipid oxidation.
The catalytic effects of Hb in cooked-meat homogenates
was increased when .O2–, H2O2, or .O2– + H2O2 were
present.
The TBARS values of cooked-meat homogenates with
ascorbate were higher than those without ascorbate,
indicating that free ionic iron was also involved in the
oxidation of cooked-meat homogenates. The reason for
the dramatic difference in the prooxidant effects of
hemoglobin in oil emulsions, and raw and cooked-meat
homogenates is not clear; however, substances other than
reducing agents, such as ascorbate, are involved.
Johns et al. (1989) found a powerful prooxidant effect of
heme pigments in exhaustively washed muscle fibers.
Their samples had only membrane components (mainly
phospholipids) and myofibrillar proteins and connective
tissues due to washing off all the water-soluble components were rinsed away, whereas the oil emulsion used in
our study contained only lipids. Because myofibrillar
proteins-only have no known effect on the oxidation of
lipids, the conditions of washed muscle fibers are basically
the same as those of the oil-only emulsion system. The
reactions of iron sources in exhaustively washed muscle
fibers, thus, became very similar to those of oil emulsion in
this study. These results (Johns et al., 1989 and Table 2)
indicated that the most of the components that prevent
heme pigments from being prooxidant would be watersoluble, and heat-labile cytoplasmic substances. Because
of those unknown substances the rate of lipid oxidation in
raw-meat with Hb is very slow, and the oxidation
mechanisms of raw-meat are different from those of
cooked-meat and oil emulsion systems. However, the
PROOXIDANT EFFECTS OF IRON SOURCES IN DIFFERENT TESTING SYSTEMS
353
TABLE 3. Effect of ferritin on the TBARS values of oil emulsions and raw-meat homogenates
with added reactive oxygen species, SOD, or XOD systems1,2
Oil-emulsion
Treatment
None
Ferritin2
Control
H2O2
KO2
KO2 + H2O2
XOD system
SOD system
SOD + XOD systems
SEM
0.02b
0.01b
0.01b
0.05b
0.03b
0.16a
0.17a
0.00
0.05c
0.04c
0.05c
0.06c
0.05c
1.26a
0.65b
0.01
Raw-meat homogenate
Ferritin
+ asc.
None
Ferritin
(mg MDA/L reaction mixture)
2.14b
0.26
0.11b
0.08e
0.19
0.19a
0.08e
0.29
0.22a
0.10e
0.18
0.11b
0.75d
0.15
0.15ab
3.24a
0.36
0.09b
1.67c
0.11
0.09b
0.02
0.01
0.00
Ferritin
+ asc.
0.10
0.14
0.16
0.20
0.13
0.12
0.11
0.00
a–eMeans
within a column with no common superscript differ significantly (P < 0.05). n = 4.
homogenate was not tested.
2TBARS = thiobarbituric acid reactive substances; SOD = superoxide dismutase; XOD = xanthine oxidase;
MDA = malondialdehyde; H2O2 = hydrogen peroxide; KO2 = potassium superoxide, asc = ascorbate.
3Ferritin: 5 ppm iron (89.5 mM) equivalent (25 mg/mL, final); XOD system: 0.145 U XOD plus xanthine (2 mM)/
mL; SOD system: 500 U catalase plus 50 U SOD/mL; KO2, 100 ppm (1.4 mM); ascorbate, 100 ppm (0.57 mM);
H2O2, 2 mM (final concentrations).
1Cooked-meat
SOD, and SOD + XOD systems. The presence of .O2–,
H2O2, .O2– + H2O2, or XOD in the oil emulsion
significantly reduced the TBARS values of the samples
containing both ascorbate and ferritin, indicating that not
only release of ferritin iron but also changes in the status of
free iron are important factors in the prooxidant capability
of ferritin. The ferritin had no prooxidant effect, and the
presence of ascorbate or reactive oxygen species and
XOD/SOD systems also had no influence on the catalytic
effect of ferritin in raw-meat homogenates (Table 3).
mechanisms of these heat-labile cytoplasmic components
can not be explained at this point.
Ferritin and Oxidation
Ferritin had no prooxidant effect in the oil emulsion
with control, .O2–, H2O2, .O2– + H2O2, or XOD but had
significant prooxidant effects when SOD was present
(Table 3). The high TBARS values in oil emulsion samples
with ferritin, however, were not caused by ferritin but by
catalase, an enzyme in the SOD system. Obviously,
catalase also accounts for part of the MDA produced in the
SOD system for both ferrous iron and Hb treatments
(Tables 1 and 2). Catalase-stimulated MDA formation was
also observed in phospholipid liposomes (Thomas et al.,
1985).
When ascorbate was added to the ferritin samples, it
increased the TBARS values of oil emulsion with control,
Amount and Status of Free Iron
Tables 2 and 4 support the view that Hb itself (not iron
released from Hb) is a strong prooxidant in oil emulsions,
and the status of heme iron and the release of free iron
from Hb by .O2–, H2O2, and the XOD system were less
important on the prooxidant effect of Hb in oil emulsions.
TABLE 4. Effect of ascorbate, superoxide, H2O2, and the XOD system on the release
of iron from iron proteins in oil emulsion1,2
Hemoglobin
Treatment
Total Fe
Control
Ascorbate
KO2
H2O2
XOD + xanthine
SEM
0.30c
0.57b
0.28c
0.54b
1.22a
0.01
a–cMeans
Ferritin
Fe2+
Total Fe
(mg Fe/mL reaction mixture)
0.05b
0.30c
0.46a
0.51b
0.05b
0.23c
0.02b
0.54b
0.06b
0.64a
0.00
0.01
FE2+
0.04b
0.41a
0.01b
0.00b
0.01b
0.00b
within a column with no common superscript differ significantly (P < 0.05). n = 4.
ppm Fe equivalent (89.5 mM) for iron proteins (Hb 15.6 mg/mL, ferritin 25 mg/mL); Oil emulsion was
filtered through a membrane filter (cut-off size: 10 kd) by centrifugation and the filtrate was used for iron analysis.
XOD, 0.145 U/mL; KO2, 2 mM; ascorbate, 2 mM; xanthine, 2 mM, H2O2, 2 mM (final conc.) were used.
2H O = hydrogen peroxide; XOD = xanthine oxidase; KO = potassium superoxide.
2 2
2
15
354
AHN AND KIM
TABLE 5. Effect of H2O2, XOD, and SOD systems on the
content of ferrous iron1 in raw-meat homogenates2
Treatment
Fe2+
None
H2O2
SOD + catalase
XOD + xanthine
SEM
3.33c
3.23c
3.60b
5.61a
0.02
Fe2+ =
ascorbate
Fe2+
+ KO2
SEM
(ppm Fe in reaction mixture)
5.16c
2.66c
0.04
5.29c
2.80c
0.03
5.76b
3.27b
0.04
7.21a
4.64a
0.03
0.05
0.03
a–cMeans
within a column with no common superscript differ
significantly (P < 0.05). n = 4.
15 ppm ferrous iron (89.5 mM); XOD, 0.29 U/mL; catalase, 1,000 U/
mL; SOD, 200 U/mL; H2O2, 2 mM (final concentrations) were used.
2H O = hydrogen peroxide; XOD = xanthine oxidase; SOD =
2 2
superoxide dismutase; KO2 = potassium superoxide.
Ascorbate, H2O2, and XOD system mobilized more iron
from ferritin and Hb than those of control and KO2
treatments, only ascorbate kept the released iron in the
ferrous state and catalyzed the oxidation of oil emulsions.
Less than 10% of iron from the iron proteins was released
under the tested conditions, except for the XOD system,
and almost all of the free iron in oil emulsions without
reducing agents was in the inactive (ferric) form.
Table 5 shows that SOD and XOD systems had
significant effects on the content of analyzable ferrous iron
in meat homogenates. The content of ferrous iron in meat
homogenates with the XOD system was approximately 2
ppm greater than those of control, H2O2, and SOD
systems. The addition of ascorbate significantly increased
but .O2– reduced the amount of ferrous iron in meat
homogenates. Although the amount of ferrous iron and
TBARS values correlated well in the oil emulsion and
cooked-meat homogenates (Table 1), the amounts of
ferrous iron in Table 5 did not agree well with the TBARS
values of raw-meat homogenates in Table 1. This lack of
agreement indicates that the prooxidant effects and
mechanisms of iron, ferritin, and Hb in raw-meat
homogenates are different from those in oil emulsions and
cooked-meat homogenates. The results shown in this
study underscore a few important points: 1) ferrous iron
and hemoglobin had strong prooxidant effects in oil
emulsions, 2) the status of heme iron and the released iron
from Hb had minor effects on the catalytic effect of Hb in
oil emulsion, 3) Hb had no catalytic effect on the oxidation
of raw-meat homogenates under any circumstances, and
4) the reaction of Hb with H2O2 did not produce active Hb
that can catalyze lipid oxidation in raw-meat
homogenates.
We conclude that the prooxidant effects and the
mechanisms of ionic iron, ferritin, and Hb in raw-meat
homogenates are different from those in oil emulsion and
cooked-meat homogenates, and the differences were
caused by heat-labile components such as reducing
enzymes related to the balance of redox potentials in rawmeat homogenates. The presence of ascorbate, H2O2, and
XOD system can increase the release of iron from iron
proteins (Hb and ferritin). The status of free iron was more
important than the amount of free iron on the oxidation in
oil emulsion. Ferrous iron was the most important
prooxidant among all iron sources, and heme pigments
and ferritin had no catalytic effect on the oxidation of rawmeat homogenates during storage.
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