1 The Evans Memorial Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston 02118; 2 The Core Laboratory, Beth Israel Hospital and Deaconess Medical Center, Boston, Massachusetts 02115; 3 The Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331; and 4 Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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Ascorbate is a strong antioxidant;
however, it can also act as a prooxidant in vitro by reducing
transition metals. To investigate the in vivo relevance of this
prooxidant activity, we performed a study using guinea pigs fed high or
low ascorbate doses with or without prior loading with iron dextran.
Iron-loaded animals gained less weight and exhibited increased plasma
-N-acetyl-D-glucosaminidase activity, a
marker of tissue lysosomal membrane damage, compared with control
animals. The iron-loaded animals fed the low ascorbate dose had
decreased plasma
-tocopherol levels and increased plasma levels of
triglycerides and F2-isoprostanes, specific and sensitive markers of in vivo lipid peroxidation. In contrast, the two groups of
animals fed the high ascorbate dose had significantly lower hepatic
F2-isoprostane levels than the groups fed the low ascorbate dose, irrespective of iron load. These data indicate that 1)
ascorbate acts as an antioxidant toward lipids in vivo, even
in the presence of iron overload; 2) iron loading per se
does not cause oxidative lipid damage but is associated with growth
retardation and tissue damage, both of which are not affected by
vitamin C; and 3) the combination of iron loading with a low
ascorbate status causes additional pathophysiological changes, in
particular, increased plasma triglycerides.
antioxidant; ascorbate; F2-isoprostanes; guinea pigs; lipid peroxidation
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INTRODUCTION |
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OXIDATIVE STRESS,
defined as an imbalance in prooxidant vs. antioxidant species in favor
of the former, results in oxidative damage to biological macromolecules
(13, 26). Increased oxidative stress has been implicated
in iron overload conditions, such as homozygous hemochromatosis and
treatment of -thalassemia (24, 43), and is thought to
be due to iron-catalyzed generation of hydroxyl and alkoxyl radicals
through Fenton chemistry (reactions 1 and 2; Ref.
19). Ascorbate (vitamin C) is a strong antioxidant capable
of scavenging a wide variety of reactive oxygen and nitrogen species
(19). Ascorbate is the most effective water-soluble antioxidant in human plasma against lipid peroxidation induced by
aqueous peroxyl radicals, activated neutrophils, or the gas-phase of
cigarette smoke (15-17). However, under certain in
vitro conditions, ascorbate can act as a prooxidant by reducing
transition metal ions (reaction 3), thereby driving the
Fenton reaction (19).
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(1) |
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(2) |
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(3) |
Contrary to its putative prooxidant role in the presence of redox-active transition metal ions, we have found that ascorbate not only effectively protects low-density lipoprotein from metal ion-dependent oxidation (35, 36) but also acts as an antioxidant toward lipids in iron-overloaded human plasma in vitro (3). Furthermore, ascorbate did not increase lipid peroxidation in 3T3 fibroblasts incubated with iron (10), and the addition of ascorbate to the diet of guinea pigs supplemented with iron inhibited, rather than promoted, lipid peroxidation in an ex vivo microsomal system (9). Given this mounting evidence for an antioxidant role of ascorbate in the presence of excess iron in vitro and ex vivo, the question arises whether ascorbate acts as a prooxidant or an antioxidant in vivo under conditions of iron overload.
Therefore, we conducted a study in a well-characterized guinea pig
model of iron overload (1, 37) by use of a two-by-two factorial design of iron loading and ascorbate feeding. The guinea pig
is a physiologically relevant model to study the effects of dietary
ascorbate manipulation, because it is one of the few animals that, like
humans, lack a functional enzyme, L-gulono--lactone oxidase, required for de novo biosynthesis of ascorbate. We assessed oxidative damage by measuring F2-isoprostanes, which are
specific and sensitive markers of oxidative lipid damage
(29).
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METHODS |
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Iron loading.
Fifty-two female albino guinea pigs (Charles River Scientific,
Wilmington, MA), ~21 days old and weighing between 200 and 250 g, were housed in groups of three animals in stainless steel cages and
given a commercial diet containing 1 mg of ascorbate/g of chow (Purina
Guinea Pig Chow #5025, St. Louis, MO) and water ad libitum. The guinea
pigs were weighed twice weekly throughout the study. Twenty-six guinea
pigs were iron loaded over the course of 4 wk by intraperitoneal
injections of iron (Fe3+) dextran (Sigma, St. Louis, MO) on
alternate days (83 mg
Fe · kg1 · injection
1) to
achieve a total iron load of 1.5 g/kg body wt (37). The remaining 26 guinea pigs were injected on alternate days with the
equivalent volume of dextran (Sigma). At the end of 4 wk and an
additional 1 wk of recovery, six guinea pigs from each group were
killed, and their tissues were collected for iron analysis.
Ascorbic acid manipulation. The diet of the remaining 40 guinea pigs was switched to a casein-based test diet (Purina Mills #5710C-6, Richmond, IN) containing no detectable ascorbate. To achieve chronic hypovitaminosis C without symptoms of scurvy, one-half of the animals (10 iron-loaded, 10 controls) were given a maintenance dose of 1.0 mg ascorbate (Sigma) every other day by gastric gavage, while the other 20 guinea pigs (10 iron-loaded, 10 controls) were given 100 mg ascorbate every other day. This regimen was continued for 4 wk, after which the guinea pigs were killed and the tissues collected for analysis. Ascorbate was withheld 2 days before death, and animals were killed after an overnight fast. Two guinea pigs in each of the iron-loaded groups (given either the high or low ascorbate dose) died before completion of the study. Necropsies showed severe inflammation, especially of the small bowel, and deposits of hemosiderin in several tissues, most markedly in the liver, as well as browning of the peritoneum.
Sample collection.
The guinea pigs were anesthetized by intramuscular injection of 50 mg/kg xylazine and 30 mg/kg ketamine (Butler, Westfield, MA). Blood was
collected by cardiac puncture and aliquotted into either
anticoagulant-free tubes (for serum), heparinized tubes, or
EDTA-containing tubes, the latter for F2-isoprostane
analysis. The blood was centrifuged (1,250 g, 10 min), and
the plasma samples were snap frozen in liquid nitrogen and stored at
70°C until analyzed, with two exceptions. Plasma samples used for
ascorbate analysis were mixed with an equal volume of ice-cold 5%
(wt/vol) metaphosphoric acid containing 1 mM of the metal chelator
diethylenetriaminepentaacetic acid and centrifuged, and the supernatant
was stored at
70°C. Plasma samples used for
-tocopherol analysis
were extracted with an equal volume of HPLC-grade methanol and 10 vol
of HPLC-grade hexane and centrifuged, and the hexane phase was stored
at
20°C. After blood collection, the animals were killed by
injection of 60 mg pentobarbital sodium into the heart, and the heart,
liver, spleen, and adrenals were harvested. The tissues were rinsed
with 10 mM phosphate-buffered saline and blotted dry and cut into
pieces, which were either snap frozen in liquid nitrogen and stored at
70°C (for F2-isoprostane analysis), placed in a Kimax
glass tube pretreated with hydrochloric acid, and stored at
20°C
(for iron analysis), or homogenized in ice-cold phosphate-buffered
saline and further processed for ascorbate or
-tocopherol analysis.
Measurements.
Ascorbate and -tocopherol were analyzed by reversed phase HPLC with
electrochemical detection as described (15, 40). Total
cholesterol, triglycerides, and serum iron levels were determined spectrophotometrically using Sigma kits #352, #339, and #565, respectively. Total iron levels in liver, heart, and spleen were measured by atomic absorption spectroscopy. Protein levels were determined spectrophotometrically using the Lowry assay (Sigma procedure P5656) or the Bradford assay (Bio-Rad, Hercules, CA). The
determination of
-N-acetyl-D-glucosaminidase
activity was adapted from a method by Skrha et al. (38).
Briefly, 100 µl of plasma were added to 1.25 ml of sodium citrate
buffer (100 mM, pH 4.4), which was then incubated for 5 min at 37°C
with 0.25 ml of 10 mM
p-nitro-phenyl-
-N-acetyl-D-glucosaminide
(Sigma). The reaction was terminated by adding 1.5 ml sodium carbonate (200 mM, pH 10.4), and the absorbance was measured at 405 nm. For
analysis of free and acylated F2-isoprostanes, lipids in
plasma or liver homogenates were extracted by a modified Folch
procedure and base hydrolyzed (27). The resulting free
F2-isoprostanes were measured after purification and
derivatization by capillary gas chromatography-negative ion chemical
ionization mass spectrometry (GC-MS) as described (28).
Statistical analysis. For comparisons involving three or more groups, the Kruskal-Wallis nonparametric ANOVA test was used to determine whether the medians of each group were different. If they were different, the Dunn multiple comparison test was performed to determine which pairs were different. For comparisons involving only two groups, the Wallace nonparametric two-tailed t-test was performed. For comparisons of groups with two levels of independent variables, the two-way ANOVA test was performed with the Dunn multiple comparison test included to determine which pairs were different. The level of statistical significance was set at P < 0.05 for all tests.
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RESULTS |
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Iron loading of the guinea pigs was accomplished by intraperitoneal injections of iron dextran over the course of 4 wk to achieve a final load of 1.5 g iron/kg body wt. Control animals were injected with equal volumes of a dextran solution. After a 1-wk recovery period, ascorbate was fed to the guinea pigs by gastric gavage for another 4 wk, either at a high dose (50 mg/day) or a low, maintenance dose (0.5 mg/day) causing chronic hypovitaminosis without scurvy (18). Thus the 9-wk study protocol produced four groups of guinea pigs: high ascorbate/control (n = 10); low ascorbate/control (n = 10); high ascorbate/iron-loaded (n = 8); and low ascorbate/iron-loaded (n = 8).
At the end of the study period, the animals in the iron-loaded groups
weighed significantly less than the animals in the control groups
(Table 1), indicating that iron loading,
but not ascorbate dose, adversely affected the animals' growth rate.
Growth retardation has been observed previously in guinea pigs
(37) and humans suffering from iron overload (14,
44). Plasma
-N-acetyl-D-glucosaminidase activity, a
marker of tissue lysosomal membrane damage (1), was about
fourfold higher in the iron-loaded animals compared with the control
animals, irrespective of ascorbate dose (Table 1). This result is in
agreement with earlier findings of severe tissue damage in iron-loaded
guinea pigs (1, 37).
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Plasma total cholesterol levels were ~20% lower (P < 0.05) in the iron-loaded compared with the control animals (Table
1), in agreement with previous observations in iron-loaded rabbits (12). In contrast, plasma triglyceride levels were more
than doubled in the iron-loaded animals fed the low dose of ascorbate compared with the other three groups (Table 1). Interestingly, in the
same group of animals, lipid-standardized plasma -tocopherol levels
were decreased significantly (Table 1), suggesting the presence of
increased oxidative stress. However, hepatic
-tocopherol levels were
not affected by either ascorbate feeding or iron loading (Table 1).
The iron-loaded guinea pigs had ~30-fold elevated hepatic iron levels
compared with control animals (Fig.
1A). Significant increases in
iron levels by ~10- and 20-fold were also observed, respectively, in
heart and spleen, which had the highest iron levels of the tissues
examined (not shown). Interestingly, there was no effect of ascorbate
dose on hepatic iron levels (Fig. 1A). No significant
differences were observed in serum iron levels between control and
iron-loaded animals fed the high or low dose of ascorbate (Fig.
1B).
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Hepatic levels of ascorbate were significantly higher in the animals
fed the high compared with the low dose of ascorbate (Fig.
1C, Table 2). Significant
ascorbate accumulation was also observed in spleen, heart, and adrenals
(Table 2). The highest tissue ascorbate levels were found in adrenals,
followed by spleen, liver, and heart, in agreement with previous
studies (21). Interestingly, iron load did not have a
significant effect on ascorbate levels in the liver (Fig.
1C) or any of the other tissues examined (Table 2). Plasma
ascorbate concentrations also were significantly higher in the animals
fed the high compared with the low dose of ascorbate but were not
affected by iron status (Fig. 1D).
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To determine the extent of oxidative lipid damage, levels of
F2-isoprostanes were measured in liver and plasma using
GC-MS. As shown in Fig. 2A,
hepatic F2-isoprostane levels were significantly elevated
in the animals fed the low ascorbate dose (control 7.6 ± 2.8, and
iron-loaded 9.4 ± 2.6 ng/g) compared with the high ascorbate dose
(control 5.9 ± 2.2, and iron-loaded 4.9 ± 2.6 ng/g; P < 0.05). Interestingly, comparison of the liver
F2-isoprostane levels in iron-loaded animals with control
animals revealed no significant differences. Plasma
F2-isoprostane levels were significantly decreased and
increased, respectively, in iron-loaded compared with control animals
fed the high or low ascorbate dose (Fig. 2B), indicating
that ascorbate feeding lowers oxidative lipid damage in the plasma of
iron-loaded animals.
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In Fig. 3, the liver
F2-isoprostane levels of all 36 animals in the study are
plotted against liver iron (Fig. 3A) and ascorbate levels
(Fig. 3B). In contrast to iron levels
(r2 = 0.003, P = 0.73),
ascorbate levels were significantly (r2 = 0.32, P = 0.003) inversely correlated with
F2-isoprostane levels. These data indicate that ascorbate
protects against oxidative lipid damage in the liver, irrespective of
iron status, and that iron loading itself does not cause oxidative
lipid damage.
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DISCUSSION |
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This study represents the first demonstration that ascorbate
exhibits antioxidant activity, rather than prooxidant activity, in vivo
toward lipids in the presence of iron overload. Our results show the
following. 1) Iron loading per se does not cause oxidative lipid damage but nevertheless is associated with growth retardation, tissue damage, and hypocholesterolemia. This systemic dysfunction with
iron overload is not affected by vitamin C. 2) Iron loading combined with a low ascorbate status results in decreased
lipid-standardized plasma -tocopherol levels and substantially
increased plasma triglyceride levels. Vitamin C deficiency in guinea
pigs has been previously shown to be associated with increased plasma
triglycerides (4), and this may be exacerbated by iron
loading. 3) Ascorbate protects against oxidative lipid
damage in vivo, independent of iron status. This finding is best
illustrated in Fig. 3, which shows that tissue ascorbate levels, in
contrast to iron levels, are significantly inversely correlated with
F2-isoprostanes.
Oxidative damage in the present study was assessed by measuring
esterified and free F2-isoprostanes in, respectively, liver and plasma. F2-isoprostanes are stable, specific, and
sensitive markers of oxidative lipid damage that are found in almost
every tissue and biological fluid (29). Esterified
F2-isoprostanes are formed in vivo on
arachidonyl-containing lipids by a free radical-catalyzed mechanism
that is independent of cyclooxygenase; these esterified
F2-isoprostanes are released into plasma following hydrolysis by phospholipases (29). Elevated levels of
F2-isoprostanes have been detected under conditions of
increased oxidative stress (13, 29), and administration of
antioxidants, in particular ascorbate and -tocopherol, has been
shown to reduce their levels (26, 29).
The finding that iron load is not correlated with
F2-isoprostane levels in guinea pigs (Fig. 3A)
raises the interesting question of whether iron per se causes tissue
damage (as indicated by increased plasma activity of
-N-acetyl-D-glucosaminidase activity) through oxidative mechanisms. Dabbagh et al. (12), in a previous
study carried out in iron-loaded rabbits, also showed no increase in F2-isoprostanes in plasma and liver, while in a third
animal study (11), slightly increased hepatic, but not
plasma, F2-isoprostanes were observed in iron-loaded
rats. Several other studies investigating the effects of iron
overload in rats found increased markers of lipid oxidation, i.e.,
thiobarbituric acid-reactive substances and conjugated dienes (2,
5, 25). It should be noted, however, that rats synthesize
vitamin C and as such may not be relevant models for iron overload in
humans. Most importantly, unlike F2-isoprostanes,
thiobarbituric acid-reactive substances and conjugated dienes are
nonspecific markers of oxidative lipid damage, particularly in complex
biological samples (13, 19). Finally, some of the
inconsistent results may be explained by the fact that some studies
have employed intraperitoneal iron dextran injection (this study and
Ref. 12) and others iron carbonyl feeding (2, 5, 11,
25); the former regimen leads to the accumulation of iron
primarily in macrophages and Kupffer cells (22, 37), while
the latter delivers iron to parenchymal cells (31).
Increased levels of lipid and protein oxidation products together with
decreased levels of ascorbate and -tocopherol have been observed in
the serum of hemochromatosis and
-thalassemia patients (24,
43). These findings were attributed to the iron overload
conditions; however, they do not demonstrate a prooxidant role of
ascorbate. In the present study, high levels of ascorbate suppressed,
rather than promoted, the formation of F2-isoprostanes, while low levels of ascorbate were associated with increased oxidative stress, as indicated not only by increased F2-isoprostane
levels but also decreased plasma
-tocopherol levels. In addition,
our earlier work showed that F2-isoprostane and protein
carbonyl levels were not significantly different in plasma of preterm
infants containing nonprotein bound, bleomycin-detectable iron
compared with plasma of preterm infants devoid of
bleomycin-detectable iron, despite the presence of high levels of
ascorbate (3). Finally, a recent report (42)
in which healthy volunteers were supplemented with both iron (14 mg/day) and ascorbate (60 or 260 mg/day) for 12 wk showed a modest
reduction of ex vivo low-density lipoprotein oxidizability and
beneficial effects on platelet function; no evidence for a prooxidant
effect of vitamin C and iron cosupplementation was observed.
It is possible that changes in oxidative damage to lipids differ from oxidative damage to other biological macromolecules. For example, oxidative damage may depend on iron binding sites present on proteins or DNA but not lipids (20). A study (34) investigating the effects of ascorbate and iron cosupplementation on 13 different oxidative DNA damage products in human leukocytes found that, although some oxidative products increased and others decreased, total base damage increased after 6 wk of supplementation and returned to baseline after 12 wk. Similarly, supplementation with ascorbate alone was found to increase 8-oxoadenine levels in human leukocytes, while also decreasing 8-oxoguanine levels (32). However, the findings from both of these studies (32, 34) are likely confounded by artifactual ex vivo DNA oxidation during GC-MS analysis, and other problems with these studies have been identified (23, 33).
In addition to addressing oxidative lipid damage, the present study
also provides some information on the possible interactions between
ascorbate and iron metabolism. The data indicate that in guinea pigs
iron overload does not affect tissue and plasma ascorbate levels and,
vice versa, ascorbate feeding does not affect serum and liver total
iron levels, as reported previously (7, 39). Similar
results were also observed recently in ascorbate-requiring osteogenic
disorder Shionogi rats injected intraperitoneally with 0.5 g iron
dextran/kg body wt and fed 150 or 900 parts per million ascorbate (N. Gorman and P. Sinclair, personal communication). In these rats, iron
loading only slightly lowered hepatic ascorbate levels and ascorbate
dose did not affect hepatic nonheme iron levels. In contrast, iron
overload in humans is associated with significantly decreased
leukocyte, platelet, and plasma ascorbate levels, suggesting that high
iron levels enhance the rate of ascorbate catabolism (24, 41,
43). Furthermore, vitamin C administration to patients with
-thalassemia major enhances the removal of iron by the chelator
deferoxamine and markedly increases transferrin saturation and serum
iron and ferritin levels (8, 30). These data suggest that ascorbate,
most likely through its reducing capacity, can mobilize excess tissue
iron in humans. The discrepancy between our and other researchers'
data in experimental animals may be explained by the fact that iron
dextran is deposited primarily in Kupffer cells (37),
while in humans excess iron is localized mainly to parenchymal cells
(31).
In summary, the results from the present in vivo study, in agreement with previous in vitro and ex vivo studies (3, 9, 10, 36, 42), demonstrate that ascorbate does not promote oxidative lipid damage but instead acts as an antioxidant toward lipids in vivo, even in the presence of iron overload. Further studies are needed to evaluate the effects of dietary and supplemental vitamin C on iron redistribution and oxidative damage in humans suffering from iron overload before the data of the present study can be applied clinically.
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ACKNOWLEDGEMENTS |
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We thank Alya Dabbagh for help in study design and tissue collection.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-49954 and HL-56170 to B. Frei, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48831 and National Cancer Institute Grant CA-77839 to J. D. Morrow. J. D. Morrow is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research.
Address for reprint requests and other correspondence: B. Frei, Linus Pauling Institute, Oregon State Univ., 571 Weniger Hall, Corvallis, OR 97331-6512 (E-mail: balz.frei{at}orst.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 May 2000; accepted in final form 24 August 2000.
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