© 1997 American Heart Association, Inc.
Articles |
Effect of Iron Overload and Iron Deficiency on Atherosclerosis in the Hypercholesterolemic Rabbit
From the Evans Memorial Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.
Correspondence to Balz Frei, PhD, Linus Pauling Institute, Oregon State University, 571 Weinger Hall, Corvallis, OR 97331. E-mail baiz.frei{at}orst.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract It has been suggested that iron plays an important role in the pathogenesis of atherosclerosis, primarily by acting as a catalyst for the atherogenic modification of LDL. Although some epidemiological data suggest that high stored iron levels are an independent risk factor for coronary artery disease and that iron has been detected in both early and advanced atherosclerotic lesions, the evidence is often contradictory and inconclusive. We used the New Zealand White rabbit to investigate the effects of iron overload (FeO) and iron deficiency (FeD) on atherosclerosis. Groups of 7 rabbits were either iron loaded by injections of iron dextran (FeO group), iron depleted by phlebotomy (FeD group), or given injections of saline (control group) for a total of 9 weeks. All rabbits were fed a chow diet containing 1% (wt/wt) cholesterol for the last 6 weeks of the study. Iron and antioxidant status and cholesterol levels were assayed in plasma before cholesterol feeding (week 3) and at the time that the rabbits were killed (week 9). In addition, the susceptibility of LDL to oxidation was measured and pathological examination of the aortic arch and thoracic aorta performed at the end of the study. FeD significantly decreased the levels of blood hemoglobin, serum iron, and transferrin saturation compared with controls. Conversely, FeO significantly increased transferrin Fe saturation. FeO but not FeD decreased plasma cholesterol levels compared with control animals both before (P<.05) and after (P=.055) cholesterol feeding. Neither FeO nor FeD had a significant effect on the levels of antioxidants and lipid peroxidation products in plasma and aortic tissue or on the susceptibility of LDL to ex-vivo oxidation. FeO significantly decreased aortic arch lesion formation by 56% compared with controls (P<.05), whereas FeD had no significant effect. These results indicate that in this animal model, FeO decreases rather than increases atherosclerosis, likely because iron dextran exerts a hypocholesterolemic effect. Our data do not support the hypotheses that elevation of Fe stores increases or that a reduction of Fe stores by phlebotomy decreases the risk of coronary artery disease.
Key Words: LDL • antioxidants • lipid peroxidation • free radicals • metal ions
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The development of atherosclerosis involves arterial inflammation and healing processes in a hyperlipidemic environment,1 where LDL oxidation is thought to be an important causal factor. The precise mechanism of LDL oxidation is unclear, but transition metal ions such as iron or copper2 3 may be involved by virtue of their ability to catalyze the production of highly toxic hydroxyl radicals and the chain reaction of lipid peroxidation. In vitro studies have demonstrated that iron or copper is required for LDL oxidation by endothelial cells,4 macrophages,5 and smooth muscle cells2 and that hemin, alone or in the presence of low concentrations of H2O2, can induce LDL oxidation.6 Iron may also promote smooth muscle proliferation,7 a key step in atherosclerotic lesion progression; may indirectly increase the susceptibility of LDL to oxidation by reducing the levels of antioxidants in plasma8 ; or may promote atherogenesis by affecting blood lipid levels.9 10
Iron within ferritin11 and hemosiderin12 13 has been detected in human fatty streaks and atherosclerotic plaques but not in the intervening normal intima. In subjects with increased iron stores due to hemochromatosis, hemosiderin iron is remarkably more prominent in aortic fatty streaks than in those of subjects without hemochromatosis.12 13 Bleomycin-detectable iron capable of stimulating lipid peroxidation has been detected in gruel samples from advanced atherosclerotic lesions.14 Although these observations suggest that iron is involved in the pathogenesis of atherosclerosis and that redox-active iron may be involved during the advanced stages of the disease, the mechanism of such involvement and the role of redox-active iron in the early stages remain to be elucidated.
Epidemiological and animal studies have provided conflicting evidence for the role of iron in atherosclerosis and coronary artery disease. For example, epidemiological data by Salonen et al3 indicate that high stored iron levels as measured by serum ferritin are an independent risk factor for acute myocardial infarction. However, Stampfer et al15 did not observe a correlation between serum ferritin levels and coronary risk in a study of US physicians with or without myocardial infarction, and similar results have been reported by Cooper and Liao.16 Surprisingly, some studies have suggested an inverse association between iron stores and mortality from cardiovascular disease,17 18 and a statistically significant inverse relation between iron overload resulting from hemochromatosis or multiorgan siderosis and the prevalence of coronary atherosclerosis has been reported.19 Animal studies of the role of iron in experimental atherosclerosis are limited and also have yielded conflicting results. While one study suggested that excessive iron loading in hypercholesterolemic rabbits was beneficial and significantly reduced lesion formation,20 another similar study suggested a detrimental role for iron loading in the same model.21
The data summarized above illustrate that the role of iron in atherosclerosis remains unresolved. Therefore, we investigated the effect of FeO on atherosclerosis in the hypercholesterolemic rabbit. Because a reduction in body iron stores by blood donation has been proposed as a possible prophylactic measure in coronary artery disease,22 we also investigated the effect of mild FeD by phlebotomy.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Sodium-heparin Vacutainers (for plasma preparation), Vacutainers without addition (for serum preparation), needles, and plastic syringes were purchased from Becton Dickinson. Acrodisc LC13 syringe filters were from Gelman Sciences, HPLC columns from Supelco, Chelex-100 resin from Bio-Rad Laboratories, and 15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid from Cayman Chemical Co. Organic solvents (HPLC grade) were purchased from Fisher Scientific. All other chemicals were obtained from Sigma Chemical Co. CuSO4 · 5H2O was prepared in Chelex-treated water adjusted to pH 3.0 and diluted from a 10 mmol/L stock solution just before use in experiments.
Animals and Diets
Twenty-eight male NZW rabbits (
3 kg body weight) were divided
into 4 groups of 7 animals each and given the following treatments
throughout the 9-week study: (1) Rabbits in the FeD group were iron
depleted by twice-weekly phlebotomy from a marginal ear vein (15 to 30
mL of blood per week). This method has been shown to produce mild
anemia without excessive iron deficiency.23 (2) Rabbits in
the FeO were given intramuscular injections of iron dextran (INFeD;
Schein Pharmaceutical, Inc) in doses of 25 mg iron (0.5 mL of a 50
mg/mL solution of iron dextran) every 3 days for the first 3
weeks of the study (total dose of 150 mg iron, or 50 mg/kg
body weight) and every 5 days for an additional 6 weeks (total dose by
the end of the study was 350 mg iron, or 120 mg/kg body
weight). A relatively moderate degree of FeO was chosen so as not to
confound the detrimental effects due to excessive FeO alone with the
effects of increased body iron stores on
atherosclerosis. (3) The control group of rabbits was
given intramuscular injections (0.5 mL) of sterile saline. (4) An
additional group of 7 rabbits was left untreated (the NC group).
All rabbits were fed standard Agway rabbit chow (Ralston Purina Co) for the first 3 weeks of the study. The control, FeO, and FeD groups were fed the standard chow diet containing 1% (wt/wt) cholesterol for an additional 6 weeks. Cholesterol-supplemented chow was prepared weekly by dissolving pure cholesterol in ether and spraying the appropriate amount over the standard chow. The NC group of rabbits continued to be fed the standard chow. All rabbits were allowed to consume the chow and water ad libitum. Blood was obtained from a marginal ear vein at 3, 6, and 9 weeks of the study. Plasma was prepared by centrifugation (1000g) for 15 minutes at 4°C and either used immediately or stored at -70°C (protected from light) for subsequent assays.
At the end of the study, fasted animals were killed with pentobarbital (120 mg/kg body weight) injected into a marginal ear vein. The rabbits were incised midventrally; blood was collected from the left ventricle into both heparinized and nonheparinized Vacutainers; and the liver, aortic arch, and thoracic aorta were excised. The aorta was subdivided so that same-sized sections from the same location of the aorta of each animal were used for the respective assays. Tissue and plasma or serum samples were either used immediately or frozen at -70°C until the time of analysis (within 6 weeks). All animal studies were approved by the Boston University Medical Centre Institutional Animal Care and Use Committee.
Iron Status
Serum iron, TIBC, and transferrin iron saturation were measured
spectrophotometrically in nonhemolyzed serum samples as
described.24 Colorimetric determination of
hemoglobin concentration in whole blood was performed with Sigma kit
procedure No. 525 based on the method of Drabkin and
Austin.25 Total liver and thoracic aorta iron were
measured by atomic absorption spectroscopy as previously described by
us8 and the results expressed in micrograms of iron per
gram of wet tissue.
Markers of Lipid Peroxidation
Fresh plasma, liver, and thoracic aorta samples were
analyzed for acylated F2-isoprostanes, a marker for
in vivo lipid peroxidation.8 26 Lipids were extracted by a
modified Folch procedure and base hydrolyzed.27 After
purification and derivatization, the resulting free
F2-isoprostanes were measured by gas
chromatography/negative-ion chemical ionization mass
spectrometry as described.27 Neutral lipid hydroperoxides
in n-hexane extracts of fresh plasma samples were measured
by HPLC with isoluminol chemiluminescence detection as
described.28 29
Antioxidant Status
Ascorbic acid was measured in fresh plasma extracts by HPLC with
electrochemical detection.30 
-Tocopherol in
plasma was extracted into n-hexane and stored at -20°C
until assayed by HPLC with electrochemical detection.31
Because levels of circulating -tocopherol are influenced
by increases in serum lipid levels,32 plasma
-tocopherol levels were expressed as the ratio of
-tocopherol (in micromoles per liter) to total
cholesterol plus triglycerides (in millimoles
per liter) as described.33
For analysis of -tocopherol levels in the liver
and thoracic aorta, the samples were cleaned of extraneous tissue and
homogenized in 1.0 mL of ice-cold 10 mmol/L
PBS, pH 7.4, containing 5.0 mmol/L of the metal chelator
DTPA to prevent ex vivo lipid peroxidation. A sample of tissue
homogenate was subjected to base hydrolysis by addition of
1.0 mL of 10N NaOH and 1% SDS for analysis of tissue protein
contents. The remaining tissue homogenate was then
extracted with 1.0 mL of methanol and 5.0 mL n-hexane, and
the lipid-soluble antioxidants in the hexane extract were measured as
described.31
Plasma Lipids
Plasma total cholesterol and
triglycerides were quantified enzymatically as previously
described.34 35 HDL cholesterol was measured
after phosphotungstic acid–MgCl2 precipitation of
apoB-containing lipoprotein fractions.36 The difference
between total cholesterol and HDL cholesterol
was calculated as the combined cholesterol in the VLDL and
LDL fractions.
Fatty Streak Quantification
The extent of atherosclerotic lesion development in the aortic
arch and thoracic aorta was determined by methods previously
described.37 Aortic segments were pinned at in situ
length, fixed for en face preparation with 4% phosphate-buffered
paraformaldehyde (pH 7.0) at room temperature for 1
hour, and then stored at 4°C overnight. For each en face preparation,
the segments were cleaned of adventitia and rinsed in 60% isopropanol,
and the inner aortic surface was stained for 25 minutes with a
saturated solution of oil red O in 60% isopropanol. The aorta was
counterstained with Gill's type hematoxylin (1:20 dilution) for 1
minute. The area of oil red O staining (lesion area) was quantified
with a Bioscan Optimas 4.02 image analysis (Aldus Corp), and
the degree of lesion formation was expressed as a percentage of the
total area examined (20 mm2).
LDL Oxidation
At the time when the rabbits were killed, LDL was prepared from
fresh plasma by the method of Chung and colleagues38 by
using single vertical-spin discontinuous density gradient
ultracentrifugation as previously described by
us.39 Standard incubation of LDL for susceptibility to
oxidation was performed at a concentration of 0.1 mg LDL protein per
milliliter in the presence of 1.25 µmol/L
CuCl2 at 37°C. Lipid peroxidation was determined by assay
of sample absorbance at 234 nm (diene conjugation) at 10-minute
intervals with a Hitachi U-2000 spectrophotometer equipped with a
thermostatic six-cell holder. LDL susceptibility to lipid peroxidation
was quantified by the duration of the lag phase before the propagation
phase of diene conjugation as described by Esterbauer and
colleagues40 as well as by the rate of lipid peroxidation
during the initiation and propagation phases.
Statistics
Unless otherwise specified, all results are expressed as
mean±SD. For comparisons between the different groups, unpaired
Student's t test was used. Pearson's correlations were
used to determine relationships between covariates. Statistical
significance was accepted if the null hypothesis was rejected at the
P<.05 level.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Iron, Cholesterol, and Antioxidant Levels in NC Rabbits
Groups of 7 rabbits were either given intramuscular injections of iron dextran (FeO group) or saline (control group) or were phlebotomized (FeD group) for 3 weeks. In the FeD group, significantly decreased serum iron levels (P<.01), transferrin iron saturation (P<.01), and hemoglobin levels (P<.01) were observed compared with the control group (Fig 1
). Iron dextran injections (a total of
150 mg of iron per animal) in the FeO group did not significantly
affect serum iron or hemoglobin levels but significantly
(P<.05) decreased TIBC and increased (P<.05)
transferrin saturation (Fig 1) values compared with those in control
animals. FeO animals also exhibited a 23% decrease in plasma
cholesterol levels compared with control animals
(P<.05), whereas FeD did not affect plasma
cholesterol levels (Table 1).
As also shown in Table 1, neither FeO nor FeD had an effect on plasma
levels of either the water-soluble antioxidants (ascorbic acid and uric
acid) or the lipid-soluble antioxidant -tocopherol. In
addition, no plasma lipid hydroperoxides were detected (<5
nmol/L) in any of the groups (data not shown).
|
), control; (
), FeO;
(
), FeD. *P<.05, 
P<.01 vs
control.
|
Lipoproteins, Iron, and Antioxidant Status in
Hypercholesterolemic Rabbits
Cholesterol feeding (1% wt/wt) for 6 weeks led to a
significant increase in plasma total and VLDL plus LDL
cholesterol levels in all treatment groups (control, FeO,
and FeD) compared with NC animals (Table 2). This effect was less marked in the
FeO group, and the difference between the FeO and the control group was
of marginal statistical significance (P=.055). There was no
difference in the plasma levels of HDL cholesterol between
the groups (Table 2). Triglyceride levels were higher in
the cholesterol-fed rabbits than in the NC animals but not
different between the treatment groups (control, FeO, and FeD; Table 2).
|
The iron status of the treatment groups was assessed after 3 weeks of
cholesterol feeding (week 6 of the study) and at the end of
the study (week 9; Fig 1
). At week 6, levels of serum iron and blood
hemoglobin were significantly lower in the FeD group than in control
rabbits (P<.05 and P<.01, respectively).
Furthermore, significantly decreased TIBC (P<.05) and
increased transferrin saturation (P<.01) values were
observed in the FeO rabbits compared with control animals (Fig 1). At
week 9, the differences in transferrin saturation between the groups
were similar to those observed at week 6 of the study (Fig 1C).
Interestingly, there was a decrease in serum iron levels as a result of
cholesterol feeding in the control group
(P=.077, week 3 versus week 9). As a result of this
decrease, serum iron levels at week 9 were no longer different between
FeD and control animals but were now significantly (P<.01)
different between the FeO and control animals (Fig 1A). Furthermore, a
significant decrease in the TIBC level (P<.05) was observed
in control rabbits from the start of cholesterol feeding to
the time when the rabbits were killed. Because high concentrations of
plasma lipids interfere with the hemoglobin assay, hemoglobin levels
could not be determined at week 9.
As shown in Table 3,
cholesterol feeding also led to a significant
(P<.05) reduction in lipid-standardized plasma
-tocopherol levels but had no effect on plasma levels of
ascorbic acid or F2-isoprostanes, a marker of lipid
peroxidation. Neither FeO nor FeD significantly affected plasma levels
of F2-isoprostanes compared with the
cholesterol-fed control group (Table 3).
|
As expected, liver iron levels significantly (P<.01)
increased in the iron dextran–treated group and decreased in the
phlebotomy group compared with the control group. Similar but
nonsignificant trends were observed for aortic iron levels (Fig 2A). As shown in Fig 2B, aortic
-tocopherol levels did not differ between any of the
groups. However, liver -tocopherol levels were higher in
the control group than in the NC group (P<.05), an
observation that probably reflects increased hepatic
cholesterol levels as a result of cholesterol
feeding.20 41 Within the treatment groups, hepatic levels
of -tocopherol were significantly (P<.01)
higher in the FeO than the control rabbits (Fig 2B).
F2-isoprostane levels in the thoracic aorta did not differ
significantly among any of the groups. However, hepatic
F2-isoprostane levels in the control group were
significantly (P<.05) lower than in the NC group (Fig 2C).
|
Aortic Atherosclerotic Lesion Formation
Lesions were not observed in the NC rabbits but were present
in both the aortic arch, and, to a lesser degree, in the thoracic aorta
of the cholesterol-fed animals (Fig 3). There was a significant
(P<.05) 56% decrease in aortic arch lesion coverage in the
FeO group compared with the control group. In contrast, FeD led to a
32% increase in aortic arch lesions, but this difference was not
statistically significant (P=.19). Thoracic aorta lesion
coverage was lower in the FeO group than in the control rabbits
(P=.09). Aortic arch but not thoracic aorta lesions were
significantly (P=.01) and positively correlated with plasma
cholesterol levels (r=.54).
|
Ex Vivo LDL Oxidation
The susceptibility of LDL to oxidation was determined ex vivo by
using LDL concentrations standardized to protein (0.1 mg LDL protein
per milliliter). No significant differences in either the lag phase or
the Ri of lipid peroxidation were observed between any of
the groups. However, cholesterol feeding led to a dramatic
increase in the Rp of lipid peroxidation
(P<.001, control versus NC group; Table 4). Because the animals were fed
cholesterol and the cholesterol contents of LDL
may vary between animals, we measured the cholesterol
content in isolated LDL. As expected, the LDL from control animals
contained significantly (P<.001) higher levels of
cholesterol per milligram of LDL protein than the LDL from
NC rabbits (Table 4). We also found that the LDL
cholesterol content was positively correlated with total
plasma cholesterol (r=.52, P=.028)
and Rp (r=.62, P=.006) and negatively
correlated with the lag phase (r=-.54; P=.021).
These data are in agreement with previous observations of a significant
negative correlation between the lag phase and LDL
cholesterol content in humans.42 To adjust for
cholesterol content, the lag phase, Ri, and
Rp were expressed per micromole of LDL
cholesterol (Table 4). This analysis showed that
the LDL from control rabbits was significantly more susceptible to ex
vivo oxidation than the LDL from NC animals, as indicated by a
decreased lag phase (P<.05) and an increased Rp
(P<.001). However, FeO or FeD did not affect the
susceptibility of LDL to ex vivo oxidation compared with controls
(Table 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The present study shows that FeO does not increase oxidative stress or atherosclerotic lesion formation in hypercholesterolemic rabbits. To the contrary, FeO decreased atherosclerosis, an effect possibly related to the hypocholesterolemic effect of iron loading with iron dextran. Our study also shows for the first time that FeD by phlebotomy does not ameliorate the development of atherosclerosis in hypercholesterolemic rabbits.
The degrees of FeO and FeD used in the present study were
relatively moderate. Intramuscular iron dextran injections led to an
4.5-fold increase in body iron stores, as measured by total liver
iron levels, compared with the corresponding values in saline-injected
control animals. In contrast, in patients with symptomatic
hemochromatosis, there is a 16- to 30-fold increase in liver iron
levels compared with that in normal individuals.43
Phlebotomy treatment of the animals led to an 30% decrease in body
iron stores, as measured by liver iron levels. Such mild FeD is
comparable to the difference between healthy adult men and
premenopausal women44 and would consequently provide a
physiologically relevant and realistic
prophylactic approach in human studies.
Because iron has been proposed to affect
atherosclerosis by increasing oxidative stress and LDL
oxidation, we assessed the oxidative stress status of the animals.
Neither FeO nor FeD influenced the plasma levels of antioxidants
(ascorbic acid, uric acid, and -tocopherol) or lipid
peroxidation products (lipid hydroperoxides and
F2-isoprostanes). Moreover, F2-isoprostane
levels in the thoracic aorta did not differ between any of the groups.
It must be noted, however, that the thoracic aorta exhibited only mild
atherosclerosis (<10% lesion coverage), and we did
not compare lesion versus nonlesion areas. Therefore, the measured
F2-isoprostane levels do not represent those within
the lesions themselves. The observation that FeO did not increase
oxidative stress in the rabbits may be explained by the facts that
injected iron dextran is mainly confined to the Kupffer cells in the
liver, and only at higher doses is parenchymal iron deposition
observed.45 Studies have shown that oxidative stress
occurs in cases of parenchymal iron loading but not when iron
deposition is confined to the Kupffer cells.46 For
example, parenteral administration of iron dextran at doses >800
mg/kg body weight has been reported to lead to parenchymal iron
deposition and increased lipid peroxidation.47 In
contrast, in the present study, the animals were loaded with only
120 mg iron/kg body weight.
In agreement with the findings of one group20 but not with those of another,21 we found that parenteral iron dextran administration led to a decrease in plasma cholesterol levels in NZW rabbits. This hypocholesterolemic effect of iron loading with iron dextran is unlikely related to iron-mediated liver damage, since liver iron levels in the present study were only moderately increased (about 4.5-fold; see above). In contrast, >20-fold increases in liver iron levels are required to cause liver damage.43 48 Some49 50 but not all51 52 studies have demonstrated that large doses of dextran infusions lower plasma cholesterol levels. Although significantly lower levels of dextran (as a component of iron dextran) were used in the present study, dextran may be partially responsible for the observed hypocholesterolemic effects in the iron dextran–treated group.
The present study showing that moderate FeO (0.35 g iron per
rabbit) decreases atherosclerosis in the NZW
hypercholesterolemic rabbit is in agreement with a
previous study by Nadkarni et al,20 in which a protective
effect of massive loading with iron dextran (up to 7.5 g iron per
rabbit) against atherosclerosis was observed.
These20 and our results, however, conflict with those of
Araujo and coworkers,21 who, using the same
cholesterol-fed rabbit model of
atherosclerosis, reported that iron loading with iron
dextran (1.5 g iron per rabbit) augmented the formation of
atherosclerotic lesions. Obviously, these discrepant results cannot be
explained by differences in the degree of FeO. A more likely
explanation may come from the fact that, in the study by Araujo et
al,21 plasma cholesterol levels (600
mg/dL) were dramatically lower than in our study (1200
mg/dL) and the study by Nadkarni et al (1700
mg/dL).20 It appears, therefore, that the effect of
iron loading on atherosclerosis depends on the degree
of hypercholesterolemia and that in the setting
of severe hypercholesterolemia, the
cholesterol-lowering effect of iron dextran has a more
profound effect on lesion formation than under moderate
hypercholesterolemic conditions. On a similar note,
Parker and coworkers53 hypothesized that the relative
contribution of oxidative stress to atherosclerosis
depends on the degree of hypercholesterolemia
and found that "excessive"
hypercholesterolemia can outweigh the
therapeutic effectiveness of antioxidants. It must be noted, however,
that excessive hypercholesterolemia may explain
the lack of a detrimental effect of FeO in our study, but it seems less
conceivable as an explanation for the observed antiatherosclerotic
effects of FeO.
Our study also demonstrated that FeD by phlebotomy does not ameliorate the development of atherosclerosis in the hypercholesterolemic rabbit. Bari and Rahman54 found that iron deficiency anemia did not offer any protection against atherosclerosis in chickens. In fact, a slight potentiating effect was observed. Furthermore, in a study of 30 necropsied cases of pernicious anemia, no significant difference was found in the amount of atherosclerosis compared with that in 60 control cases matched by age, sex, and race.55
At first glance, the results of the present study appear somewhat
surprising in view of the plethora of recent studies implicating iron
in LDL oxidation2 4 5 6 and
atherosclerosis.3 11 12 13 14 21 However, on a
closer look, one finds about an equal number of studies suggesting a
detrimental role for iron3 11 12 13 14 21 as there are
suggesting a beneficial role17 18 19 20 or none at
all15 16 Furthermore, epidemiological and clinical studies
on iron status and the risk of coronary artery disease use
indirect methods (serum ferritin, iron, TIBC, or transferrin
saturation) as a measure of iron stores. These methods, though
convenient, lack sensitivity and/or specificity and hence are not
always accurate indicators of body iron stores.44 For
example, increased serum ferritin levels can indicate either increased
iron stores or the presence of an acute-phase response, cancer, or
liver disease. Similarly, a decrease in serum iron levels or hematocrit
may indicate the presence of ACD or a true iron deficiency anemia. The
ACD is well characterized in chronic inflammatory conditions, such as
rheumatoid arthritis.56 Atherosclerosis is
increasingly thought of as a chronic inflammatory
disease,57 and hence, disturbances in iron
metabolism characteristic of ACD or the presence of an
acute-phase response are likely to occur and confound the observations
of epidemiological studies. For example, serum iron levels were lowest
and liver iron levels highest in patients with severe versus mild
calcific arteriosclerosis.58
Furthermore, plasma C-reactive protein was reported to significantly
increase with increasing severity of
atherosclerosis,59 and patients with
chronic arterial disease had significantly increased levels
of plasma haptoglobin and 1-acid glycoprotein (both
acute-phase proteins) and a decreased plasma transferrin concentration
compared with healthy control subjects.60 Such changes are
characteristic of ACD. Similarly, experimental
hypercholesterolemia is associated with a
decrease in hematocrit,21 61 serum iron levels, and TIBC
(the present study) and an increase in plasma ferritin
levels,21 thus suggesting the presence of chronic
inflammation and an acute-phase response. Therefore, in
atherosclerosis, serum ferritin may serve as a marker
of the extent of the disease rather than as a measure of body iron
stores. Also, the presence of iron (within ferritin and
hemosiderin) in the fatty streak and atherosclerotic
plaque may simply be due to the withholding of iron by
macrophages that usually occurs under inflammatory
conditions.
Although our study is not conclusive in terms of the role of excess iron on experimental atherosclerosis (as FeO also led to a decrease in plasma cholesterol levels), it demonstrates that iron depletion by phlebotomy does not ameliorate lesion formation. Therefore, these data do not support the hypothesis that higher levels of iron stores increase the risk of coronary artery disease or the suggestion that reducing iron stores by phlebotomy may be beneficial. In view of the limited number of experimental studies on the role of iron in atherosclerosis, it appears that further studies are needed to clarify the relationship, if any, between iron status and coronary artery disease.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
Financial support for this work was provided by National Institutes of Health grants HL49954 and HL56170 (to B.F.). J.F.K. Jr is the recipient of a clinical investigator development award from the National Institutes of Health (HL03195). We thank Jason D. Morrow and L. Jackson Roberts II, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn, for the analysis of F2-isoprostanes by gas chromatography–mass spectrometry. We also thank John Zeind, Core Laboratory, Beth Israel Hospital, Boston, Mass, for measuring tissue iron by atomic absorption spectroscopy.
Received March 24, 1997; accepted May 14, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Nerem RM. The pathogenesis of atherosclerosis: an overview. Clin Cardiol. 1991;14(suppl):I-1-I-16.
2. Heinecke JW, Rosen H, Chait A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells. J Clin Invest. 1984;74:1890-1894.
3.
Salonen JT, Nyyssönen K, Korpela H, Tuomilehto
J, Seppänen R, Salonen R. High stored iron levels are
associated with excess risk of myocardial infarction in Eastern Finnish
men. Circulation. 1992;86:803-811.
4.
Martin A, Frei B. Both intracellular and
extracellular vitamin C inhibit atherogenic modification of LDL by
human vascular endothelial cells.
Arterioscler Thromb Vasc Biol. 1997;17:1583-1590.
5. Leake DS, Rankin SM. The oxidative modification of low-density lipoproteins by macrophages. Biochem J. 1990;270:741-748.[Medline] [Order article via Infotrieve]
6.
Balla G, Jacob HS, Eaton JW, Belcher JD, Vercellotti
GM. Hemin: a possible physiological mediator
of low density lipoprotein oxidation and endothelial
injury. Arterioscler Thromb. 1991;11:1700-1711.
7.
Porreca E, Ucchino S, Di Febbo C, Di Bartolomeo N,
Angelucci D, Napolitano AM, Mezzetti A, Cuccurullo F.
Antiproliferative effect of desferrioxamine on vascular smooth muscle
cells in vitro and in vivo. Arterioscler Thromb. 1994;14:299-304.
8. Dabbagh AJ, Mannion T, Lynch SM, Frei B. The effect of iron overload on rat plasma and liver oxidant status in vivo. Biochem J. 1994;300:799-803.
9.
Berg LN, Bonna KH, Nordoy A. Serum ferritin,
sex hormones, and cardiovascular risk factors in
healthy women. Arterioscler Thromb. 1994;14:857-861.
10. Feussner G, Ziegler R. Type III hyperlipoproteinemia in a patient with idiopathic hemochromatosis. Hum Genet. 1991;86:326-328.[Medline] [Order article via Infotrieve]
11. Pang JH, Jiang MJ, Chen YL, Wang FW, Wang DL, Chu SH, Chau LY. Increased ferritin gene expression in atherosclerotic lesions. J Clin Invest. 1996;97:2204-2212.[Medline] [Order article via Infotrieve]
12. Jankus A, Ardlie NG, Edwards RG, Schwartz CJ. Marked aortic hemosiderin deposits in a patient with hemochromatosis. Arch Path. 1967;84:418-421.[Medline] [Order article via Infotrieve]
13. Schwartz CJ, Ardlie NG, Carter RF, Paterson JC. Gross aortic sudanophilia and hemosiderin deposition: a study on infants, children and young adults. Arch Pathol. 1967;83:325-332.[Medline] [Order article via Infotrieve]
14. Smith C, Mitchinson MJ, Aruoma OI, Halliwell B. Stimulation of lipid peroxidation and hydroxyl radical generation by the contents of human atherosclerotic lesions. Biochem J. 1992;286:901-905.
15. Stampfer MJ, Grodstein F, Rosenberg I, Willett W, Hennekens C. A prospective study of plasma ferritin and risk of myocardial infarction in US physicians. Circulation. 1993;87:688-693.
16. Cooper RS, Liao Y. Iron stores and coronary heart disease: negative findings in the NHANES I epidemiologic follow-up study. Circulation. 1993;87:686-694.
17.
Sempos CT, Looker AC, Gillum RF, Makuc DM. Body
iron stores and the risk of coronary heart disease.
N Engl J Med. 1994;330:1119-1124.
18. Reunanen A, Takkunen H, Knekt P, Seppanen R, Aromaa A. Body iron stores, dietary iron intake and coronary heart disease mortality. J Intern Med. 1995;238:223-230.[Medline] [Order article via Infotrieve]
19.
Miller M, Hutchins GM. Hemochromatosis, multi
organ hemosiderosis, and coronary artery disease.
JAMA. 1994;272:231-233.
20. Nadkarni BB, Solomon RD. Effect of iron dextran on atherogenesis in cholesterol and corn oil fed rabbits. Ind J Med Res. 1966;54:157-164.[Medline] [Order article via Infotrieve]
21.
Araujo JA, Romano EL, Brito BE, Parthe V, Romano M,
Bracho M, Montaño RF, Cardier J. Iron overload augments
the development of atherosclerotic lesions in rabbits.
Arterioscler Thromb Vasc Biol. 1995;15:1172-1180.
22. Sullivan JL. Blood donation may be good for the donor: iron, heart disease and donor recruitment. Vox Sang. 1991;61:161-164.[Medline] [Order article via Infotrieve]
23. Likhite V, Rodvien R, Crosby WH Jr. Depressed phagocytic function exhibited by polymorphonuclear leucocytes from chronically iron deficient rabbits. Br J Haematol. 1976;34:251-255.[Medline] [Order article via Infotrieve]
24. Persijn JP, van der Slik W, Riethorst A. Determination of serum iron and latent iron-binding capacity (LIBC). Clin Chim Acta. 1971;35:91-98.[Medline] [Order article via Infotrieve]
25.
Drabkin DL, Austin JH. Spectrophotometric
studies, II: preparation from washed blood cells; nitric oxide,
hemoglobin and sulfhemoglobin. J Biol Chem. 1935;112:51-65.
26. Morrow JD, Awad JA, Kato T, Takahashi K, Badr KF, Roberts LJ II, Burk RF. Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity: an animal model of lipid peroxidation. J Clin Invest. 1992;90:2502-2507.
27. Morrow JD, Harris TM, Roberts LJ. Non cyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal Biochem. 1990;184:1-10.[Medline] [Order article via Infotrieve]
28. Frei B, Yamamoto Y, Niclas D, Ames BN. Evaluation of an isoluminol chemiluminescence assay for the detection of hydroperoxides in human blood plasma. Anal Biochem. 1988;175:120-130.[Medline] [Order article via Infotrieve]
29. Yamamoto Y, Frei B, Ames BN. Assay of lipid hydroperoxides using high-performance liquid chromatography with isoluminol chemiluminescence detection. Methods Enzymol. 1990;186:371-380.[Medline] [Order article via Infotrieve]
30.
Frei B, England L, Ames BN. Ascorbate is an
outstanding antioxidant in human blood plasma. Proc Natl
Acad Sci U S A. 1989;86:6377-6381.
31.
Stocker R, Bowry VW, Frei B. Ubiquinol-10
protects human low density lipoprotein more efficiently against lipid
peroxidation than does -tocopherol. Proc
Natl Acad Sci U S A. 1991;88:1646-1650.
32.
Bieri JG, Poukka R, Thorp S. Factors affecting
the exchange of tocopherol between red blood cells and
plasma. Am J Clin Nutr. 1977;30:686-690.
33. Thurnham DI, Davies JA, Crump BJ, Situnayake RD, Davies M. The use of different lipids to express serum tocopherol: lipid ratios for the measurement of vitamin E status. Ann Clin Biochem. 1986;23:514-520.
34. Allain CC, Poon LS, Chan GS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470-475.[Abstract]
35. Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973;19:476-482.[Abstract]
36.
Assmann G, Schriewer H, Schmitz G, Hagele E.
Quantification of high-density-lipoprotein cholesterol by
precipitation with phosphotungstic acid/MgCl2.
Clin Chem. 1983;29:2026-2030.
37. Foxall TL, Shwaery GT, Stucchi AF, Nicolosi RJ, Wong SS. Dose-related effects of doxazosin on plasma lipids and aortic fatty streak formation in the hypercholesterolemic hamster model. Am J Pathol. 1992;140:1357-1363.[Abstract]
38. Chung BH, Segrest JP, Ray MJ, Brunzell JD, Hokanson JE, Krauss RM, Beaudrie K, Cone JT. Single vertical spin density gradient ultracentrifugation. Methods Enzymol. 1984;128:181-209.
39. Dabbagh AJ, Frei B. Human suction blister interstitial fluid prevents metal ion-dependent oxidation of low density lipoprotein by macrophages and in cell-free systems. J Clin Invest. 1995;96:1958-1966.
40. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989;6:67-75.[Medline] [Order article via Infotrieve]
41. Brattsand R. Actions of vitamins A and E and some nicotinic acid derivatives on plasma lipids and on lipid infiltration of aorta in cholesterol-fed rabbits. Atherosclerosis. 1975;22:47-61.[Medline] [Order article via Infotrieve]
42. Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation. J Lipid Res. 1993;34:2135-2145.[Abstract]
43. Tavill AS, Bipin KS, Bacon BR. Iron and the liver: genetic hemochromatosis and other hepatic iron overload disorders. Prog Liver Dis. 1990;9:281-305.[Medline] [Order article via Infotrieve]
44. Brittenham GM, Danish EH, Harris JW. Assessment of bone marrow and body iron stores: old techniques and new technologies. Semin Hematol. 1981;18:194-221.[Medline] [Order article via Infotrieve]
45. Martin LE, Bates CM, Beresford CR, Donaldson JD, McDonald F, Dunlop D, Sheard P, London E, Twigg G. The pharmacology of an iron-dextran intramuscular haematinic. Br J Pharmacol Chemother. 1955;10:375-382.[Medline] [Order article via Infotrieve]
46. Fletcher LM, Roberts FD, Irving MG, Powell LW, Halladay JW. Effects of iron loading on free radical scavenging enzymes and lipid peroxidation in rat liver. Gastroenterology. 1989;97:1011-1018.[Medline] [Order article via Infotrieve]
47. Schwartz KA, Fisher J, Adams ET. Morphologic investigations of the guinea pig model of iron overload. Tox Pathol. 1993;21:311-320.
48. Bacon BR, Brittenham GM, Tavill AS, McLaren CE, Park CH, Recknagel RO. Hepatic lipid peroxidation in vivo in rats with chronic dietary iron overload is dependent on hepatic iron concentration. Trans Assoc Am Phys. 1983;96:146-154.[Medline] [Order article via Infotrieve]
49. Lafrance L, Leblanc J. Effects of compound 48/80 chlorpromazine, dextran, and reserpine in rabbits fed a 1% cholesterol diet. Can J Physiol Pharmacol. 1967;45:215-223.[Medline] [Order article via Infotrieve]
50. Flotte CT, Buxton RW. Reduction of serum cholesterol by dextran. Circulation. 1965;28:721. Abstract.
51. Yu-Chen L. Further study of the effect of dextran on experimental hyperlipemia in rabbits. Curr Ther Res.. 1970;12:242-246.
52.
Brahmankar DM, Conner WE. Effect of high
molecular weight dextran on experimental
hypercholesterolemia. Circ
Res. 1967;21:817-821.
53.
Parker RA, Sabrah T, Cap M, Gill BT. Relation of
vascular oxidative stress, -tocopherol, and
hypercholesterolemia to early
atherosclerosis in hamsters. Arterioscler
Thromb Vasc Biol. 1995;15:349-358.
54. Bari MA, Rahman MA. Effect of iron deficiency anemia on the development of atherosclerosis in chicks. Atherosclerosis. 1975;21:401-408.[Medline] [Order article via Infotrieve]
55. Mitchell IA, Mitchell SC. `The protective effect' of pernicious anemia on the development of atherosclerosis: a comparative study. Am J Med Sci. 1964;247:36-41.[Medline] [Order article via Infotrieve]
56.
Vreugdenhil G, Baltus CAM, Van Eijk HG, Swaak
AJG. Anaemia of chronic disease: diagnostic
significance of erythrocyte and serological parameters in
iron deficient rheumatoid arthritis patients. Br J
Rheumatol. 1990;29:105-110.
57. Munro JM, Cotran RS. Biology of disease—the pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest. 1988;58:249-261.[Medline] [Order article via Infotrieve]
58. Gerami S, Payan HM, Easley GW, Zimmermam B. Arteriosclerosis and iron metabolism. J Surg Res. 1970;10:105-110.[Medline] [Order article via Infotrieve]
59. Heinrich J, Schulte H, Schönfeld R, Köhler E, Assmann G. Association of variables of coagulation, fibrinolysis and acute-phase with atherosclerosis in coronary and peripheral arteries and those arteries supplying the brain. Thromb Haemost.. 1995;73:374-379.[Medline] [Order article via Infotrieve]
60.
Snyder S, Coodley EL, Durham BC, Pennock RS.
Serum glycoproteins in coronary disease.
Circulation. 1977;56:359-362.
61. Maxwell PW, Wiggans RG III, Mao R. Anemia and hypercholesterolemia in cholesterol-fed rabbits. J Lab Clin Med. 1970;75:893-902.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  N. Lamharzi, C. B. Renard, F. Kramer, S. Pennathur, J. W. Heinecke, A. Chait, and K. E. Bornfeldt Hyperlipidemia in Concert With Hyperglycemia Stimulates the Proliferation of Macrophages in Atherosclerotic Lesions: Potential Role of Glucose-Oxidized LDL Diabetes, December 1, 2004; 53(12): 3217 - 3225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ramakrishna, T. W Rooke, and L. T Cooper Iron and peripheral arterial disease: revisiting the iron hypothesis in a different light Vascular Medicine, August 1, 2003; 8(3): 203 - 210. [Abstract] [PDF]
|
|
|
|
 |
|
 S. M. Day, D. Duquaine, L. V. Mundada, R. G. Menon, B. V. Khan, S. Rajagopalan, and W. P. Fay Chronic Iron Administration Increases Vascular Oxidative Stress and Accelerates Arterial Thrombosis Circulation, May 27, 2003; 107(20): 2601 - 2606. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. L. Turbino-Ribeiro, M. E. Silva, D. A. Chianca Jr, H. de Paula, L. M. Cardoso, E. Colombari, and M. L. Pedrosa Iron Overload in Hypercholesterolemic Rats Affects Iron Homeostasis and Serum Lipids but Not Blood Pressure J. Nutr., January 1, 2003; 133(1): 15 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Choi, S. K. Kim, and S. H. Pai Changes in Serum Lipid Concentrations during Iron Depletion and after Iron Supplementation Ann. Clin. Lab. Sci., April 1, 2001; 31(2): 151 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Chen, J. Suh, A. C. Carr, J. D. Morrow, J. Zeind, and B. Frei Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload Am J Physiol Endocrinol Metab, December 1, 2000; 279(6): E1406 - E1412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. HEINECKE Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall FASEB J, July 1, 1999; 13(10): 1113 - 1120. [Abstract] [Full Text]
|
|
|
|
|
|
D. Pratico, M. Pasin, O. P. Barry, A. Ghiselli, G. Sabatino, L. Iuliano, G. A. FitzGerald, and F. Violi Iron-Dependent Human Platelet Activation and Hydroxyl Radical Formation : Involvement of Protein Kinase C Circulation, June 22, 1999; 99(24): 3118 - 3124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xing, J. Baffic, and C. P. Sparrow LDL oxidation by activated monocytes: characterization of the oxidized LDL and requirement for transition metal ions J. Lipid Res., November 1, 1998; 39(11): 2201 - 2208. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||