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U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • iron • copper • zinc • oxidative stress • response surface • female rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and cancer
(Stevens et al. 1994
).
However, the link between dietary Fe and oxidative stress remains
controversial, and the role of other dietary factors that may
contribute to increases in Fe stores is not clearly elucidated. Some
investigators (Crosby 1978
) have warned that increased
Fe intake, which may be related to fortification of foods, may result
in an undesirable increase in Fe stores. Because of the substantial
interactions between Fe and other micronutrients, the intake of other
elements can also play a role in changes in Fe status and possibly
oxidative stress. Increased tissue Fe was observed in both zinc (Oteiza et al. 1995
) and copper deficiencies (Weisenberg et al. 1980
) in animal models. The oxidative damage observed in these
conditions appears to be sex-specific (most pronounced in males)
and is thought to be a consequence of increased free radical generation
secondary to tissue Fe accumulation and/or reductions in Zn- or
Cu-dependent antioxidant processes (Fields et al. 1992
, Lai et al. 1995
, Oteiza et al. 1995
).
The body's defense against reactive oxygen species includes
antioxidant nutrients (e.g., vitamin E) and the antioxidant
metalloenzymes glutathione peroxidase, superoxide dismutases (Cu/Zn
SOD,
Mn-SOD)5
, ceruloplasmin and catalase (Johnson et al. 1992
). The
micronutrients Fe, Zn and Cu are important components of these
endogenous antioxidant enzyme systems (with the exception of the
selenoenzyme glutathione peroxidase), and their interactions may
modulate changes not only in Fe status, but also in the oxidative
stress response.
The overall goal of this study was to examine the effects of the
interaction of Fe, Zn and Cu with emphasis at low-to-adequate, while
including extremes, of intakes for these nutrients. Whereas others have
included dietary levels as high as 30,000 µg/g diet when studying the
effects of Fe overload on oxidative stress (Dabbagh et al. 1994
), the current study employs dietary Fe concentrations up
to about 10 times the requirement (NRC 1985
) as a
realistic level that could be consumed, for instance, by people taking
Fe supplements. As most studies of oxidative stress have used male
rats, and as low-to-marginal Fe status and intakes are more relevant to
females, the current study used female rats to further examine the
effects of interactions among dietary Cu, Fe and Zn on both Fe status
and oxidative stress. Oxidative stress was evaluated by measuring the
activities of the related antioxidant metalloenzymes (Cu/Zn SOD,
Mn-SOD, catalase and ceruloplasmin) as well as heart and liver
lipid peroxidation and vitamin E concentrations in these animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seventy-two female Sprague-Dawley rats (35–40 g; Sasco Labs,
Omaha, NE) were housed in individually suspended stainless steel cages.
A 12-h light-dark cycle was used with lights on at 0600. With the
use of a three-factor central composite response surface design
(Myers and Montgomery 1995
), the animals were randomly
assigned to 15 groups of four rats each, except for the central group,
which had 16 animals (Table 1). The rats were fed modified AIN-93G basal diets (Reeves et al. 1993
), containing casein as the source of protein and corn
starch and sucrose as the sources of carbohydrates, for 6 wk. The diets
contained various amounts of Fe and Zn (7.0, 15.5, 45.8, 135.6, or 300
µg/g diet) added as the citrate and carbonate salts, respectively,
and Cu (0.5, 1.1, 3.2, 9.2, or 20 µg/g diet) added as the carbonate
salt (Table 1)
. The range of dietary concentrations was chosen to
provide minimal intakes without affecting body weight gain or mortality
and maximal intakes comparable to those practically achievable by
supplementation of human diets. All experimental procedures conformed
to the US National Institute of Health, Public Health Service, and
Animal Welfare Act guidelines for the ethical treatment of animals
(NRC 1985
).
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Aliquots of each diet were digested with concentrated nitric and 70%
perchloric acids by method (II)A of the Analytical Methods Committee
(1960)
. The trace element concentration of the digestates were
determined by inductively coupled argon plasma emission
spectrophotometry.
The rats were killed by ether inhalation and cardiac puncture
subsequent to blood collection from the descending vena cava. Whole
blood samples, with EDTA as the anticoagulant, were analyzed for
hemoglobin by spectrophotometry (Drabkin and Austin 1935
), hematocrit with a Coulter Counter (Model S Plus 4,
Hialeah, FL), and Zn protoporphyrin by hematofluorometry (Environmental
Sciences Associates, 1983
). Serum samples were analyzed for
cholesterol, HDL, (Allain et al. 1974
) and
triacylglycerols (Bucolo and David 1973
) by using a
Cobas Fara automated analyzer (Hoffman La Roche Inc., Nutley, NJ).
Serum samples were also analyzed for Zn and Cu by atomic absorption
spectrophotometry (Perkin Elmer , Norwalk, CT), ferritin by ELISA
(Spectro Rat Ferritin Test Kit, Ramco Laboratories, Houston, TX) and
ceruloplasmin (EC 1.16.3.1) by colorimetric measurement of its oxidase
activity using o-dianisidine dihydrochloride as the
substrate (Schosinsky et al. 1974
). To assure
uniformity, the same section of the hearts and livers from each animal
were used for each analysis. A portion of the liver or heart samples
was immediately homogenized in the presence of ethanolic BHT (20
mmol/L), as an antioxidant, and thiobarbituric acid reactive substances
(TBARS) were determined within 2 h (Gutteridge and Quinlan 1983
).
For vitamin E and enzyme analyses, portions of the liver and heart were
immediately frozen in liquid nitrogen and stored at -80°C. Vitamin E
was extracted by a modification of a previously described method
(Lang et al. 1986
). Briefly, to prevent autoxidation,
weighed pieces of liver or heart tissue were homogenized in deionized
water in the presence of ethanolic BHT (100 g/L, w/v) and briefly mixed
after the addition of 0.1 mol/L SDS. The resulting homogenate was mixed
after the addition of ethyl alcohol; the mixture was then extracted
twice with HPLC-grade hexane. The pooled hexane layers were dried
under nitrogen on a heating block (40°C), and the samples were
reconstituted in 100% ethyl alcohol and analyzed by HPLC (Milne and Botnen 1986
). All vitamin E procedures were conducted under
dim light.
Superoxide dismutase (EC 1.15.1.1) was measured colorimetrically in
liver and heart tissue homogenates by using pyrogallol inhibition
(Marklund and Marklund 1974
). Liver catalase activity
(EC 1.11.1.6) was determined by titration of residual
H2O2 with permanganate (Cohen et al. 1970
). The medial lobe of the liver was perfused with saline,
and liver nonheme Fe was extracted by acid hydrolysis and protein
precipitation (Kaldor 1954
) and determined by atomic
absorption spectrophotometry.
Trace element analyses.
The liver, heart and bone samples were frozen for trace element
analyses. Unfortunately, these samples were lost in a major flood that
affected the entire city of Grand Forks, ND, in the spring of 1997.
Thus, no data on the effects of dietary treatment on the trace element
concentrations of these tissues are available. The effects of
interactions of dietary Fe, Cu and ZN on the concentrations of these
minerals in the tissues has been previously reported (Storey and Greger 1987
) and reviewed (Davis 1980
).
Statistical design and analysis.
The concentrations of Cu, Fe and Zn in the experimental diets (Table 1)
were based on a three-factor, central composite response surface
design (Myers and Montgomery 1995
). This statistical
approach can be used to evaluate the interaction between different
nutrients, as independent variables, at several concentrations while
minimizing the required number of observations and treatment groups.
Dietary treatments were assigned on a logarithmic scale to increase the
observations at low to adequate dietary intakes, while testing a broad
range of concentrations. There were 15 dietary treatment groups in this
study.
The primary assumption of a response surface design is that the
measured response can be approximated over the region of interest, by a
polynomial. The polynomials used in this study included all linear,
quadratic and cross-product terms as potential predictors. Below is
an example of a full model, where y is the measured response;
b0–b9 are the estimated regression
coefficients; and Z, C and F are the natural logarithms of the dietary
concentrations of Zn, Cu and Fe:
 |
Step-wise regression was used to select the minimum set of
predictors that significantly (P < 0.05) maximized the
model R2. The selected models (Table 2) were used to predict the dietary
intakes of Fe, Cu and Zn at which the minimum and maximum response
occurred for each dependant variable. Semi-partial correlation
coefficients (Rp2) were determined for each
predictor in the selected models (Table 2)
. Semi-partial
correlation coefficients can be used to assess the relative
importance of each predictor in the selected model.
|
. Thus, in the
interpretation of the data, much more confidence can be placed on the
observations in the inner portion of the response surface compared to
its outer edges. To facilitate interpretation, the observed mean
responses for each treatment group were reported in a tabular format in
addition to depicting the predicted responses, by using the selected
models, in three-dimensional graphs. For these graphs, the two most
influential variables were arbitrarily chosen as x-y–axes,
with the third variable fixed at the value that maximized the response
of the dependent variable. To help with the interpretation of the
response surface, a two-dimensional projection is shown on the
floor of each graph. Values are means ± SEM. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dietary treatment did not affect final body weights. Mean initial and final body weights were 74 ± 1 g and 199 ± 2 g, respectively, with an average weight gain of 125 ± 2 g in 6 wk.
Fe status measurements.
There was a significant effect of dietary Fe, but not of dietary Cu or
Zn, on hemoglobin and hematocrit measurements. An increase in
hemoglobin and hematocrit was predicted up to dietary Fe intakes of 77
and 79 µg/g diet, respectively, beyond which there was a predicted
decline as a result of a strong quadratic effect of dietary Fe (Table 2)
. However, observed differences in hemoglobin and hematocrit were
minimal when dietary Fe concentrations met or exceeded 15 µg/g diet
(Table 3). Serum ferritin varied primarily as a function of dietary Fe,
which explained 31% of the variability in the data, with a small
contribution from the interaction between Cu and Zn (Table 2)
. Similar
to hemoglobin and hematocrit measurements, an increase in serum
ferritin concentrations was moderated by a quadratic effect of dietary
Fe. Serum ferritin concentrations were predicted to reach a maximum at
intakes of 14, 87 and 109 µg/g diet for Cu, Fe, and Zn, respectively
(Fig. 1
). Similarly, dietary Fe was the main determinant of liver nonheme
Fe, accounting for ~63% of the variability in the data, with small
contributions from dietary Zn and attenuating effects from the
interactions between Cu and Zn and that between Fe and Zn (Fig. 2
). By using the selected model (Table 2)
, liver nonheme Fe
concentration was calculated to be maximal at low dietary Cu (2 µg/g
diet), high dietary Fe (215 µg/g diet) and adequate dietary Zn
intakes (17 µg/g diet). The correlation between hepatic Fe stores, as
measured by liver nonheme Fe concentration, and serum ferritin was very
weak but significant (R2 = 0.16, P
= 0.0008). Dietary treatment did not significantly affect Zn
protoporphyrin (Table 3)
.
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Dietary Fe was the only significant predictor of variations in liver
TBARS and liver vitamin E concentration (Tables 2
and
Table 4
). However, the influence of dietary Fe on these variables was weak, as
indicated by the model R2 of 0.09 for TBARS and
0.06 for vitamin E. By using the selected regression models (Table 2)
,
the maximal liver TBARS concentration (139.4 nmol/g) was predicted to
be at the highest dietary Fe (300 µg/g diet) and the minimal
concentration (84.9 nmol/g) at the lowest (7 µg/g diet). Liver
vitamin E concentration, on the other hand, was predicted to be the
lowest (0.158 µmol/g) in the rats fed the most Fe and highest (0.198
µmol/g) in the rats fed the least Fe. In the heart, diet did not
affect organ weight, TBARS or vitamin E concentration (Table 4)
.
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The interaction between dietary Cu and Zn significantly affected serum
ceruloplasmin, with direct influences from the interactions between Zn
and Fe and Cu and Fe moderating influences from the quadratic effect of
dietary Cu, and linear effects of dietary Zn and dietary Fe (Tables 2
and Table 4
, Fig. 3
). By using the model (Table 2)
, ceruloplasmin activity was
predicted to be minimal at very low dietary Cu intakes, with Fe and Zn
intakes of 62 µg each/g diet, , and maximal at moderately high
dietary intakes of Cu, Fe and Zn (9, 144, 134 µg/g diet,
respectively). Observed differences in ceruloplasmin activity, however,
were minor at dietary Cu intakes of >1.1 µg/g diet (Table 4)
.
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and 4
,Fig. 4
). The interaction between dietary Cu and Fe significantly affected
liver Mn-SOD, with moderating effects from the quadratic effect of
Fe and the interaction between Cu and Zn. Liver Cu/Zn SOD activity was
mainly determined by the linear effect of dietary Cu and was tapered by
the quadratic effect of dietary Cu (Table 2)
. The maximum activity for
both liver total SOD and Cu/Zn SOD was predicted to occur at moderately
high Cu (8 µg/g diet), low Fe (14 µg/g diet) and adequate Zn
intakes (14 µg/g diet). The maximal liver Mn-SOD was predicted to
occur at high intakes of Cu (13 µg/g diet) and Fe (81 µg/g diet)
and adequate intake of Zn (16 µg/g diet). Liver total and Cu/Zn SOD
activities were predicted to be minimal at very low Cu intake, adequate
Fe and moderately high Zn intakes (~0.5, 47 and 45 µg/g diet for
Cu, Fe, and Zn, respectively). Minimal liver Mn-SOD activity was
predicted at low dietary Cu, and moderately high dietary Fe and Zn
(0.8, 155 and 41 for Cu, Fe and Zn, respectively). Dietary treatment
did not significantly affect liver catalase activity (Table 4)
.
|
. Similar dietary
effects were also observed with heart Cu/Zn SOD. Maximal heart total
and Cu/Zn SOD activities were predicted to result when dietary Cu
intakes were moderate (6 µg/g diet), Fe was low (13 µg/g diet) and
Zn was adequate (13 µg/g diet). The activities of these enzymes were
predicted to be minimal in the heart when dietary Cu was very low (0.5
µg/g diet) and dietary Fe and Zn were at moderate amounts (56 µg/g
diet for both Fe and Zn). There were no effects of dietary treatment on
heart Mn-SOD activity (data not shown). Serum lipids.
The quadratic effect of dietary Fe predicted a small, but significant, proportion of the variability in serum triglycerides; dietary treatment did not affect serum total or HDL cholesterol (data not shown).
Serum Cu and Zn.
Serum Cu was primarily predicted by the interaction between dietary Cu
and Fe, with small contributions from the interactions between dietary
Fe and Zn and that between Cu and Zn (Table 3)
. The increase in serum
Cu was significantly attenuated by the quadratic effects of dietary Cu,
with small contributions from the quadratic effect of dietary Fe and
that of dietary Zn. The highest serum Cu was calculated to occur at
moderately high Cu intakes of ~11 µg/g diet and high Fe and Zn
intakes of 123 µg each/g diet. Serum Cu was expected to be at minimum
when dietary Cu was very low and Fe and Zn were moderate (0.5, 73 and
63 µg/g diet, respectively). There was a significant correlation
between serum Cu and ceruloplasmin (R2 = 0.90,
P = 0.0001).
Serum Zn was mainly determined by the quadratic effect of dietary Zn,
with some moderation from the linear effect of dietary Zn and the
interaction between dietary Cu and Fe (Table 3
, Fig. 5
). The maximum serum Zn was expected to occur at moderately low
dietary Cu, moderate dietary Fe and high Zn intakes (3, 43 and 293
µg/g diet, respectively). Minimum serum Zn was calculated to occur at
moderately high dietary Cu and Fe and moderate dietary Zn intakes (15,
113 and 29 µg/g diet, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, Fe status, as indicated by hemoglobin, hematocrit, serum
ferritin and liver nonheme Fe concentrations, was mainly determined by
dietary Fe. Based on the response surface model, serum ferritin would
increase as dietary Fe increased up to moderately high Fe
intakes, and when dietary Fe intake exceeded 87 µg/g, no further
increases were predicted. It is not clear whether the response of serum
ferritin to increases in dietary Fe intake is truly parabolic, or
whether it follows a broken-line model (Robbins et al. 1979
)(Table 3)
. Regardless of the model, these data indicate
the limited sensitivity and responsiveness of serum ferritin to dietary
Fe intake, and point not only to the moderating effects of very high
dietary Fe on serum ferritin, but also to the interactions among
dietary Cu and Zn, which accounted for 7% of the variability in the
serum ferritin concentrations. The relative lack of correlation between
serum ferritin and liver nonheme Fe (R2 = 0.16)
indicates that, in rats, serum ferritin is a poor indicator of Fe
stores.
The interaction between Fe and other micronutrients and the resulting
effect on Fe status have been observed in both human (Yadrick et al. 1989
, Yokoi et al. 1994
) and animal studies.
For example, in rats fed excess Zn, the amount of hepatic ferritin was
about one-third that found in control animals, and the authors
concluded that the turnover rate of ferritin was faster in Zn toxicity
(Coleman and Matrone 1969
). In another study examining
the effects of severe Cu deficiency on Fe stores in male rats, serum
ferritin concentrations were markedly increased (from 402 to 807
µg/L) compared to the control animals (Klevay 1994
).
Similarly, in the present study, serum ferritin concentrations were
elevated relative to the overall mean (403 µg/L) in the group fed the
lowest dietary Cu and moderate amounts of Fe and Zn (0.5, 45, and 45
µg/g diet, respectively, Table 3
). However, the concentrations of
this protein were also elevated at the highest intake of Cu and at
moderate intakes of Fe and Zn (20, 45, 45 µg/g diet, respectively,
Table 3
). These results indicate that, in addition to Fe, other dietary
trace elements, such as Zn and Cu, also affect serum ferritin
concentrations. In practical terms, because elevated serum ferritin
concentrations have been implicated in the etiology of chronic
diseases, it should be noted that extremes of Cu intakes (very low or
very high) resulted in high serum ferritin values in this study.
Despite the low intakes of Cu in this study, Cu-deficiency anemia
(microcytic hypochromic) was not observed in this study. One possible
explanation is that, as reported by others, female rats are more
resistant to the development of Cu-deficiency anemia (Fields et al. 1992
), and also, for the groups fed the lowest dietary
Cu, dietary Fe was not a limiting nutrient and was provided at marginal
to high amounts. These observations are consistent with the findings of
other investigators (Cohen et al. 1985
) who, using
female rats, reported anemia only in the animals fed low dietary Cu
(0.7 µg/g) together with low dietary Fe (<25 µg/g).
Previous studies have demonstrated that dietary Fe overload, in male
rats fed as much as 25,000–30,000 µg carbonyl Fe/g diet, increases
total hepatic Fe (17.8 and 24 µmol/g of wet tissue, respectively)
(Dabbagh et al. 1994
, Wu et al. 1990
). In
the present study, however, liver nonheme Fe concentration was
predicted to be maximal (13.5 µmol/g) at moderately high Fe (215
µg/g diet), low Cu and adequate Zn intakes. This increase in liver Fe
with low Cu intake was reported previously in female (Cohen et al. 1985
) and male rats (Klevay 1994
,
Saari 1992
) and indicates that Fe metabolism
(mobilization and storage) in the liver of the rat is altered in Cu
deficiency. As ceruloplasmin is thought to be essential for the
oxidative incorporation of Fe into transferrin and ferritin
(Thiel 1987
), a low Cu status may result in a decreased
liver Fe mobilization and subsequently lead to increased hepatic Fe
deposits and anemia (Cohen et al. 1985
). This connection
between ceruloplasmin and Fe metabolism is also observed in individuals
suffering from aceruloplasminemia who exhibit hemosiderosis
characterized by low serum Fe, high serum ferritin and tissue Fe
accumulation (Mukhopadhyay et al. 1998
).
Apparently, one important mechanism through which reactive oxygen
species damage cells is via peroxidation of membrane lipids. Liver
TBARS concentrations were widely used as an indicator of this process
(Cederbaum 1992
). Vitamin E, a fat soluble vitamin
present in most biological membranes, is one of the major defenses
against lipid peroxidation and readily reacts with free radicals to
terminate chain reactions (Kontush et al. 1996
). In the
present study, dietary Fe was the main predictor of increases in liver
TBARS (Tables 2and 4)
and of decreases in liver vitamin E; however,
these associations were weak, as indicated by the small
R2 for these models. Others have also reported
hepatic Fe accumulation in Cu-deficient female rats without an
increase in free radical generation, as measured by electron spin
resonance (Fields et al. 1992
).
In this study, the lowest concentration of liver vitamin E was observed
in the group with lowest dietary Zn intake (7 µg/g) (Table 4)
, which
may reflect a lower intestinal absorption of vitamin E (Kim et al. 1998
). Previous studies, using much higher amounts of
dietary Fe and male rats, have found stronger correlations between Fe
intake and lipid peroxidation. In a study of Fe overload that compared
200 and 30,000 µg/g dietary Fe as carbonyl in male rats, other
workers (Dabbagh et al. 1994
) reported substantial
hepatic lipid peroxidation, as measured by F2-
isoprostanes, and depletion of endogenous antioxidants, including
vitamin E. Similarly, in a study of Fe overload in young and old male
rats fed 25,000 µg/g of a highly bioavailable carbonyl Fe, urinary
TBARS were markedly elevated compared to the control animals. It was
concluded that Fe overload increased in vivo lipid peroxidation
(Wu et al. 1990
). The discrepancy in the magnitude of
lipid peroxidation and changes in vitamin E concentration between the
present study and those reported by others may be caused by the vast
differences in the amount and form of dietary Fe used in the
formulation of the experimental diets and also the use of female versus
male animals. It was previously shown that female rats, also used in
this study, are more resistant to oxidative stress (Fields et al. 1992
, Weglicki et al. 1969
) and have higher
hepatic vitamin E concentrations compared to male rats (Haw-Wen et al. 1992
). The diets in the current study provided Fe in the
form of ferric citrate that exceeded, by up to 10-fold, the
requirements for normal hemoglobin (Siimes et al. 1980
).
These results suggest that high dietary Fe, exceeding a range commonly
consumed by humans, has minimal influence on indices of oxidative
stress in female rats.
Reports of oxidative damage or increased susceptibility to oxidative
stress during Cu deficiency suggest that Cu could, at certain dietary
intakes, be characterized as an antioxidant nutrient (Johnson et al. 1992
). As mentioned earlier, Cu is a component of the
antioxidant metalloenzyme ceruloplasmin, which in addition to its
ferroxidase activity (Sullivan 1992a
), binds ~90% of
plasma Cu (Frieden 1986
). This close association between
serum Cu and ceruloplasmin was evident because of the high
correlation coefficient (R2 = 0.90) between these
two variables in this study. Cu is also a component of Cu/Zn SOD, which
regulates the intercellular concentration of superoxide anion by
converting it to hydrogen peroxide. A reduction in Cu/Zn SOD activity
may cause oxidative damage to membranes and other cellular structures
(Balevska et al. 1981
, Paynter 1980
).
Conversely, Cu is also a potent catalyst of the Fenton reaction
(Sokol et al. 1996
) and, like Fe, at high concentrations
may act as a pro-oxidant nutrient. Consistent with the findings
from other studies, which have reported depressed Cu/Zn SOD activities
in the organs of animals fed Cu-deficient diets (Prohaska 1990
, Prohaska and Wells 1974
), in the present
study, the liver and heart Cu/Zn SOD activities were predicted to be
minimal when dietary Cu was low. On the other hand, the activities of
these enzymes were predicted to be maximal when Cu intake was moderate.
The latter finding, combined with the observations on ceruloplasmin
activity, is consistent with the notion that moderate intakes of Cu
optimize the antioxidant capability of an organism and that high
intakes of Cu are not desirable in this regard.
Catalase, another important antioxidant metalloenzyme, acts
sequentially to SOD in the conversion of hydrogen peroxide to water
(Cohen et al. 1970
). In earlier investigations with male
rats, liver catalase activity was unaffected by dietary Fe
(Cusack and Brown 1965
) or was unchanged
(Gallagher et al. 1956
, Prohaska and Lukasewycz 1990
) or reduced in Cu deficiency (Chen et al. 1994
, Lai et al. 1995
). In this study, liver
catalase activity was not affected by dietary treatment. This may
reflect the resistance of female rats to oxidative stress or it may
suggest that the measurement of this enzyme may not be as sensitive an
indicator of oxidative stress as measurements of liver vitamin E and
TBARS.
Prior research has indicated that antioxidant enzyme activities are
greater in the liver than in the heart (Lai et al. 1995
,
Prohaska 1991
). In the present study, the mean total SOD
activity of the liver was twice that found in the heart. The lower
antioxidant defense system in the heart was implicated in its increased
susceptibility to oxidative damage, especially in Cu deficiency
(Chen et al. 1994
). In both organs, Cu/Zn SOD activity
constituted about two-thirds of the total SOD activity (Table 4)
.
The reciprocal relationship between Zn and Cu, reported by others
(Prasad et al. 1978
) was evident in the present study in
that serum Zn concentration was predicted to be the highest at
lower Cu intakes and lowest at the higher Cu intakes.
Zinc is thought by some to be a component of the antioxidant defense
system, which may exert its protective effect either directly by
competing with the pro-oxidant metals (i.e. Cu and Fe) for binding
sites (Bray and Bettger 1990
), or indirectly as a
structural component of antioxidant metalloenzymes, such as Cu/Zn SOD.
In the present study, the activities of heart or liver Cu/Zn SOD were
affected minimally by dietary Zn intake. A similar observation was
reported for rats where changes in Cu/Zn SOD activity under different
nutritional stresses were influenced by the tissue concentrations of Cu
but not Zn (Taylor et al. 1988
).
In conclusion, in this study Fe status was primarily determined by dietary Fe and was minimally influenced by dietary Cu or Zn. Feeding female rats a wide range of dietary Fe up to 10 times the estimated requirement, did not induce overt oxidative stress, as measured by the response of several components of the antioxidant defense system. This indicates that moderately high intakes of Fe, when combined with intakes of Cu and Zn as represented in this study, do not pose a major risk in increasing oxidative stress in female rats. The data also indicate that adequate to moderate intakes of Cu are beneficial both in preventing Fe overload and in optimizing the antioxidant defense capabilities in the female rat.
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ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Mention of a trademark or proprietary product
does not constitute a guarantee or warranty of the product by the US
Department of Agriculture and does not imply its approval to the
exclusion of other products that may also be suitable.
3 The US Department of Agriculture, Agricultural
Research Service, Northern Plains Area, is an equal
opportunity/affirmative action employer and all agency services are
available without discrimination.
4 To whom reprint requests and correspondence
should be addressed
5 Abbreviations used: SOD, superoxide dismutase;
TBARS, thiobarbituric acid reactive substances.
Manuscript received November 2, 1998. Initial review completed December 9, 1998. Revision accepted March 26, 1999.
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3. Balevska P. S., Russanov E. M., Kassabova T. A. Studies on lipid peroxidation in rat liver by copper deficiency. J. Biochem. 1981;13:489-493
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