Acute administration of vitamin E triggers preconditioning via K(ATP) channels and cyclic-GMP without inhibiting lipid peroxidation.
ABSTRACT Vitamin E (VitE) is considered an antioxidant agent. One or more brief periods of ischemia (isc), followed by short reperfusion (rep), increase the tolerance of the heart to a subsequent prolonged ischemia, a phenomenon known as ischemic preconditioning (PC). Mitochondrial KATP channels (mitoKATP), cyclic-GMP (cGMP), and free radicals are involved in the mechanism of PC, whereas some antioxidants abolish this benefit. The purpose of this study was to evaluate the effect of VitE on infarct size, PC, and the oxidative status in vivo. Male rabbits were divided into seven groups and were subjected to myocardial ischemia (isc) and reperfusion (rep) with the following interventions: (1) control (no intervention); (2) E150 (iv VitE at a dose of 150 mg/kg for 75 min, starting 40 min before index isc and lasting through 5 min of rep); (3) E300 (iv VitE 300 mg/kg as previously described); (4) PC (two cycles of 5 min isc and 10 min rep), (5) combined E150-PC; and (6) combined E300-PC. In the last two groups VitE was given 40 min before index ischemia. Blood samples were taken for malondialdehyde (MDA) and conjugated dienes (CDs) measurement. In a second series of experiments heart tissue samples were taken at the time of long ischemia for MDA and CD determination and for cGMP assay. In order to test whether combined treatment with VitE (as the E150 group) and the mitoKATP blocker 5-hydroxydecanoic acid (5-HD) changes the infarct size, an additional group was assessed in the first series of experiments. Tissue VitE concentration was evaluated in myocardium. VitE at both doses reduced the infarct size (19.7 +/- 2.8% for E150 and 18.8 +/- 4.9% for E300 vs 47.4 +/- 2.6% in control, P < 0.05) without attenuating the effect of PC (10.2 +/- 3.1% for E150-PC, 12.4 +/- 2.2% for E300-PC, vs 13.5 +/- 3.3% for PC). Combined VitE and 5-HD treatment abrogates this benefit (37.4 +/- 6.5%, P < 0.05 vs E150 and NS vs control). VitE increases intracellular cGMP and CDs levels (P < 0.05 vs control) to the same extent as PC (P < 0.05 vs control), with no effect on MDA (P = NS between all the groups). Peripheral markers of oxidative stress are increased during reperfusion in all groups (P < 0.05 vs baseline). Overall, VitE limits infarct size via mitoKATP and cGMP, while preserving the benefit of ischemic PC.
Article: Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.[show abstract] [hide abstract]
ABSTRACT: Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.AJP Heart and Circulatory Physiology 03/2003; 284(2):H727-34. · 3.71 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The phenomenon of ischemic preconditioning, in which a period of sublethal ischemia can profoundly protect the cell from infarction during a subsequent ischemic insult, has been responsible for an enormous amount of research over the last 15 years. Ischemic preconditioning is associated with two forms of protection: a classical form lasting approximately 2 h after the preconditioning ischemia followed a day later by a second window of protection lasting approximately 3 days. Both types of preconditioning share similarities in that the preconditioning ischemia provokes the release of several autacoids that trigger protection by occupying cell surface receptors. Receptor occupancy activates complex signaling cascades which during the lethal ischemia converge on one or more end-effectors to mediate the protection. The end-effectors so far have eluded identification, although a number have been proposed. A range of different pharmacological agents that activate the signaling cascades at the various levels can mimic ischemic preconditioning leading to the hope that specific therapeutic agents can be designed to exploit the profound protection seen with ischemic preconditioning. This review examines, in detail, the complex mechanisms associated with both forms of preconditioning as well as discusses the possibility to exploit this phenomenon in the clinical setting. As our understanding of the mechanisms associated with preconditioning are unravelled, we believe we can look forward to the development of new therapeutic agents with novel mechanisms of action that can supplement current treatment options for patients threatened with acute myocardial infarction.Physiological Reviews 11/2003; 83(4):1113-51. · 26.87 Impact Factor
Article: Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection.[show abstract] [hide abstract]
ABSTRACT: Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.AJP Heart and Circulatory Physiology 02/2004; 286(1):H468-76. · 3.71 Impact Factor
Acute administration of vitamin E triggers preconditioning via KATP
channels and cyclic-GMP without inhibiting lipid peroxidation
Ioanna Andreadoua,b,⁎, Efstathios K. Iliodromitisa, Konstantinos Tsovolasa,
Ioanna-Katerina Aggelic, Anastasia Zogaa, Catherine Gaitanakic, Ioannis A. Paraskevaidisa,
Sophia L. Markantonisd, Isidoros Beisc, Dimitrios Th. Kremastinosa
aSecond University Department of Cardiology, Medical School, Attikon General Hospital, University of Athens, Rimini 1, 124 62 Athens, Greece
bDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupolis, 155 71, Athens, Greece
cDepartment of Animal & Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, 157 84, Greece
dLaboratory of Biopharmaceutics and Pharmacokinetics, School of Pharmacy, University of Athens, Panepistimioupolis, 155 71, Athens, Greece
Received 20 March 2006; revised 7 June 2006; accepted 19 June 2006
Available online 4 July 2006
Vitamin E (VitE) is considered an antioxidant agent. One or more brief periods of ischemia (isc), followed by short reperfusion (rep), increase
the tolerance of the heart to a subsequent prolonged ischemia, a phenomenon known as ischemic preconditioning (PC). Mitochondrial KATP
channels (mitoKATP), cyclic-GMP (cGMP), and free radicals are involved in the mechanism of PC, whereas some antioxidants abolish this benefit.
The purpose of this study was to evaluate the effect of VitE on infarct size, PC, and the oxidative status in vivo. Male rabbits were divided into
seven groups and were subjected to myocardial ischemia (isc) and reperfusion (rep) with the following interventions: (1) control (no intervention);
(2) E150(iv VitE at a dose of 150 mg/kg for 75 min, starting 40 min before index isc and lasting through 5 min of rep); (3) E300(iv VitE 300 mg/kg
as previously described); (4) PC (two cycles of 5 min isc and 10 min rep), (5) combined E150–PC; and (6) combined E300–PC. In the last two
groups VitE was given 40 min before index ischemia. Blood samples were taken for malondialdehyde (MDA) and conjugated dienes (CDs)
measurement. In a second series of experiments heart tissue samples were taken at the time of long ischemia for MDA and CD determination and
for cGMP assay. In order to test whether combined treatment with VitE (as the E150group) and the mitoKATPblocker 5-hydroxydecanoic acid (5-
HD) changes the infarct size, an additional group was assessed in the first series of experiments. Tissue VitE concentration was evaluated in
myocardium. VitE at both doses reduced the infarct size (19.7±2.8% for E150and 18.8±4.9% for E300vs 47.4±2.6% in control, P<0.05) without
attenuating the effect of PC (10.2±3.1% for E150–PC, 12.4±2.2% for E300–PC, vs 13.5±3.3% for PC). Combined VitE and 5-HD treatment
abrogates this benefit (37.4±6.5%, P<0.05 vs E150and NS vs control). VitE increases intracellular cGMP and CDs levels (P<0.05 vs control) to
the same extent as PC (P<0.05 vs control), with no effect on MDA (P=NS between all the groups). Peripheral markers of oxidative stress are
increased during reperfusion in all groups (P<0.05 vs baseline). Overall, VitE limits infarct size via mitoKATPand cGMP, while preserving the
benefit of ischemic PC.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Vitamin E; Ischemic preconditioning; Infarct size; Ischemia/reperfusion; Lipid peroxidation
Ischemic preconditioning is an endogenous protective
mechanism in which one or more brief periods of myocardial
ischemia and reperfusion render the heart resistant to a
subsequent potentially lethal ischemic insult . Although the
hard end point of myocardial protection is the limitation of
infarct size , the mechanism of preconditioning still remains
Free Radical Biology & Medicine 41 (2006) 1092–1099
Abbreviations: mitoKATP, mitochondrial KATP channels; cGMP, cyclic-
GMP;PC,preconditioning; ROS,reactiveoxygenspecies; NO,nitric oxide;I/R,
infarct to risk area; CDs, conjugated dienes; MDA, malondialdehyde; 5-HD, 5-
⁎Corresponding author. Department of Pharmaceutical Chemistry, School of
Pharmacy, University of Athens, Panepistimioupolis, Zografou, Athens 157 71,
Greece. Fax: +30 210 7274747.
E-mail address: email@example.com (I. Andreadou).
0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
obscure with several unknown facts with respect to the
intracellular signaling pathways triggered . The opening of
mitochondrial KATPchannels, with subsequent generation of
reactive oxygen species (ROS), is considered to be a pivotal
step in the mechanism of preconditioning . It is of interest
that NO, guanylate cyclase, cGMP, and PKG are significant
mediators, which result in the opening of mitoKATPchannels
. Infusion of free radical scavengers, superoxide dismutase
and N-2-mercaptopropionyl glycine, prevents the protection
from preconditioning in rabbits  and rats . Ascorbic acid
which is known to scavenge oxygen-derived free radicals has
also been shown to abolish the benefit of ischemic precondi-
tioning in pigs . However, we have previously shown that the
administration of melatonin and N-acetylcysteine to rabbits
does not prevent the protection from ischemic preconditioning
via limiting the infarct size but it does protect from oxidative
damage during ischemia and reperfusion .
Vitamin E belongs to the most powerful group of lipid-
soluble chain-breaking antioxidants that prevent lipid peroxida-
tion and disruption of membrane integrity . Epidemiologic
evidence supports an inverse relation between vitamin E intake
and risk of coronary artery disease . However, this general
statement is much debated today, since previous epidemiologic
findings were not supported in a recent large, multicenter trial of
patients with increased risk of coronary artery disease .
Furthermore, the findings of a recent large trial do not support
vitamin E supplementation for cardiovascular disease or cancer
prevention among healthy woman .
In the present study we sought to determine the effect of
vitamin E as an antioxidant agent on (a) the beneficial effect of
ischemic preconditioning by reducing myocardial infarct size,
(b) lipid peroxidation and oxidative stress during ischemia
reperfusion, (c) the effect of vitamin E on tissue cGMP levels as
well as the effect of combined vitamin E and PC on cGMP
Materials and methods
New Zealand white male rabbits weighing between. 2.3 and
3.1 kg were used. All animals received proper care in
compliance with the Principles of Laboratory Animal Care,
published by the National Society for Medical Research, and the
Guide for the Care and Use of Laboratory Animals, prepared by
the Academy of Sciences and published by the National
Institutes of Health (Institute of Laboratory Animal Resources
Commission on Life Sciences, 1996).
All animals were anesthetized by slowly injecting pento-
barbital (30 mg/kg) into an ear vein. They were then subjected
to tracheal incision and intubation for mechanical ventilation
with a respirator for small animals (MD Industries, Mobile,
AL). The ventilator was properly adjusted at a rate of
approximately 35 respirations/min, in order to maintain blood
gases and pH within the normal range. Two polyethylene
catheters were inserted; one was positioned in the carotid artery
for continuous blood pressure monitoring, and the other in the
jugular vein for fluid infusion (1 ml of normal saline containing
1000 IU heparin/100 ml was administered every 30 min), drug
administration, and additional anesthesia when necessary. A
bipolar chest lead was used for continuous electrocardiographic
recording. Blood pressure and heart rate were continuously
monitored. The chest was opened via a left thoracotomy in the
fourth intercostal space and, after pericardiotomy, the beating
heart was exposed. A 3-0 silk thread was passed through the
myocardium around a prominent branch of the left coronary
artery and by pulling the ends of the suture through a small
segment of a soft tube induced regional ischemia; the tube was
then firmly attached against the artery with a clamp. The
successful induction of ischemia was verified by visual
inspection (cyanosis) and by ST elevation on the electrocardio-
gram. Reperfusion was achieved by unclamping the tube .
Forty six rabbits were randomly divided into seven groups;
all animals were subjected to the following interventions prior
to 30 min regional ischemia of the heart and 3 h reperfusion.
Control group (CTL) (n=10); no intervention.
E150 group (n=6): Vitamin E (DL-all-rac-α-tocopherol,
Sigma Aldrich Chemical Co., St. Louis, MO) was administered
intravenously as 10 ml of an oil-in-water emulsion containing
DL-all-rac-α-tocopherol with a few drops of Tween 80 in
normal saline at a total dose of 150 mg kg−1for 75 min, starting
40 min before index ischemia and lasting up to 5 min of
reperfusion. Vitamin E has been previously administered
intravenously as an oil-in-water emulsion to humans .
E300 group (n=5): Vitamin E was administered intrave-
nously, as described before, at a total dose of 300 mg kg−1for
75 min, starting 40 min before index ischemia and lasting up to
5 min of reperfusion. The 300 mg kg−1dosage of vitamin E has
been administered intravenously to pigs in an earlier protocol
Preconditioning group (PC, n=7): Two cycles of precondi-
tioning, each including 5 min of regional ischemia and 10 min
Combined E150–PC group (n=6): Vitamin E at a total dose
of 150 mg kg−1was administered as in group E150, and all
animals were subjected to two cycles of preconditioning.
Combined E300–PC group (n=6): Vitamin E at a total dose
of 300 mg kg−1was administered as in group E300, and all
animals were subjected to two cycles of preconditioning.
In a second series of experiments 24 additional rabbits (four
in each group) were subjected to the above interventions up to
20 min of sustained ischemia and tissue samples were taken
from the ischemic and nonischemic regions for analysis of
tissue lipid peroxidation products (malondialdehyde (MDA)
and conjugated dienes (CDs)) and determination of cGMP
In order to investigate the role of mitoKATPchannels on
infarct size, a final group (n=6) was treated as the E150group
but with the addition of the mitoKATPblocker 5-hydroxydeca-
noic acid (5-HD). 5-HD (Sigma Aldrich Chemical Co.) was
dissolved in normal saline (10 mg kg−1) and was given as a
1093I. Andreadou et al. / Free Radical Biology & Medicine 41 (2006) 1092–1099
bolus dose of 5 mg kg−1into the jugular vein 40 min before
sustained ischemia as previously described .
Blood samples were taken at different time points (baseline,
before sustained ischemia, and at 20 min of reperfusion), and
plasma was obtained for MDA and CD measurement as lipid
Also, in order to determine the tissue concentrations of
vitamin E, 10 additional rabbits were treated with vitamin E 150
or 300 mg kg−1(n=5 in each group) as previously described
until 20 min of sustained ischemia while 5 rabbits were used as
controls. In total 90 rabbits were used to complete this study.
The experimental protocol is shown in Fig. 1.
Risk area and infarct size
At the end of the experiment, hearts were removed, mounted
on a perfusion apparatus, and perfused for 2 min retrogradely
via the aorta with normal saline (20 ml/min, 50 mm Hg, room
temperature). When all residual blood had been removed from
the arteries, the coronary ligature was retightened to the same
extent as before and 5 ml of green fluorescent microspheres (2–
9 μm diameter; Duke Scientific Corp., Palo Alto, CA,
suspended in saline) were slowly infused over 5 min for the
delineation of the normally perfused tissue from the risk zone.
Hearts were then frozen for 24 h at –20°C and sliced into 3-mm-
thick sections from the apex to the base. The slices were then
incubated in 1% triphenyl tetrazolium chloride solution (TTC)
for 20 min at 37°C and the infarcted area was defined as the
negatively stained region. The heart slices were then immersed
in 10% formaldehyde solution for 24 h to delineate the infarcted
(tetrazolium chloride negative) areas more clearly. To clarify the
borders between the risk zone and the normal area, slices were
examined under ultraviolet light (wavelength 366 nm).
Infarcted, risk, and normal areas were traced onto an acetate
sheet, photographically enlarged, and quantified by planimetry
(Scion Image Program, Epson Perfection 1200S scanner, Adobe
Photoshop 6.0). The areas of infarction and myocardial tissue at
risk for infarction were automatically transformed to volumes
by multiplying by the slice thickness (3 mm). Infarct and risk
area volumes were expressed in cubic centimeters and the
percentage of infarct to risk area (I/R) was calculated as
previously described .
Measurement of malondialdehyde
MDA plasma concentrations at different time points (under
basal conditions, before sustained ischemia, and at 20 min of
reperfusion) were determined spectrophotometrically and
expressed as micromolar using a commercial kit (Oxford
Biomedical Research Colorimetric Assay for lipid peroxidation)
with some modifications [8,18]. The method we used is based
on the reaction of the chromogenic reagent N-methyl-2-
phenylindole with MDA. One molecule of free MDA combines
with two molecules of the chromogenic agent to produce a
stable chromophore with a maximal absorbance at 586 nm.
Briefly, 0.65 ml of 10.3 mM N-methyl-2-phenylindole in
acetonitrile was added to 0.2 ml of plasma. After vortexing for
3–4s and adding 0.15 ml of HCl 37%, samples were mixed well
and incubated at 45°C for 60 min. The samples were then
cooled on ice and centrifuged at 3500g for 20 min and the
absorbance at 586 nm was measured. A standard curve of an
accurately prepared standard MDA solution (from 2 to 20 μmol/
ml) was also run for quantitation. Measurements for each group
were performed in triplicate.
Conjugated dienes evaluation
Peroxidation of polyunsaturated fatty acids leads to the
formation of a conjugated diene system, with a characteristic UV
absorption maximum of 234 nm. Briefly, plasma (100 μl) was
mixed with 1 ml water, and 3 ml chloroform:methanol (2:1 v/v),
and was vortexed vigorously for 2 min. Samples were centrifuged
Fig. 1. Diagrammatic presentation of the experimental protocol used to evaluate the effect of acute administration of vitamin E and preconditioning on infarct size.
1094I. Andreadou et al. / Free Radical Biology & Medicine 41 (2006) 1092–1099
for 5 min at 2000g; the lower organic layer was removed and
dried under nitrogen. The dry residue was solubilized in 1 ml
hexane and the absorbance was measured at 234 nm, with
hexane used as a blank. All measurements were performed in
duplicate [19,20]. The amount of plasma CDs was expressed as
Determination of MDA and CDs in tissue preparation
Samples of the ischemic and nonischemic areas were
obtained at 20 min of sustained ischemia for each group,
frozen, and kept at −70°C until assay. On the day of analysis,
tissue samples were washed in ice-cold NaCl 0.9% (w/v),
blotted on absorbent paper, and weighed. Each sample was then
minced in a small volume of ice-cold 20 mM Tris-HCl buffer,
pH 7.4, and homogenized, in a ratio 1:10 (w/v), using a Teflon
pestle. After centrifugation at 3000g for 10 min at 4°C [20,21],
the clear homogenate supernatant was used for the biochemical
assays as described above. The results for MDAwere expressed
as micromolar per milligram protein. For the determination of
CD the results were expressed as nanomole hydroperoxide per
milligram protein using εmax=25,200 M−1cm−1. Protein
concentrations were determined using the BioRad Bradford
Heart powders were homogenized with 5 ml/g absolute
ethanol and extracted on ice for 10 min. The samples were
centrifuged (10,000 g, 5 min, 4°C) and supernatants were used
for cGMP determination by radioimmunoassay using the cGMP
[3H] assay Biotrak system (TRK 500) according to the
manufacturer’s protocol (Amersham Biosciences, Buckingham-
shire, HP7 9NA, England). cGMP concentration was normal-
ized to tissue weight.
Vitamin E determination
Myocardial levels of vitamin E were determined with high
performance liquid chromatography (HPLC) using the method
of Good et al.  for tissue sample preparation and the method
of Talwar et al.  for HPLC analysis at a wavelength of
295 nm for concentrations between 1 and 90 μg/ml. Within- and
between-run precision was calculated to be <10%, while the
limit of detection and limit of quantification were found to be
0.553 and 1.675 μg/ml, respectively.
Data analysis and statistics
All results are presented as mean±standard error (SE).
Comparisons of absolute values of variables from the groups
were analyzed using one-way analysis of variance model
(ANOVA) with Bonferroni correction and with Duncan post
hoc analysis. A calculated P value of less than 0.05 was
considered to be statistically significant.
A one-factor repeated measures ANOVA model was used to
compare each variable separately during the treatment period
(baseline until 20 min of reperfusion). All tests were two-sided
with a 95% significance level. Statistical analysis was carried
out using the statistical package SPSS ver 10.00 (Statistical
Package for the Social Sciences).
The infarct-to-risk zone ratio, which is the most reliable
index of protection [4,7,8], was 47.4±2.6% in the control
group as shown in Fig. 2. Administration of vitamin E at a
dosage of either 150 mg kg−1or 300 mg/kg/BW reduced the
infarct size, to 19.7±2.8 and 18.8±4.9%, respectively
(P<0.01 vs control). The PC group had a significantly smaller
infarct size 13.5±3.3% (P<0.01 vs control). Administration of
vitamin E did not affect the reduction in myocardial infarct
size obtained in the PC group: the mean infarct sizes in the
E150–PC group and the E300–PC group were 10.2±3.1, and
12.4±2.2%, respectively (P<0.01 vs control), (Fig. 2).
Combined treatment with vitamin E and 5-HD abrogated the
benefit obtained by VitE treatment (37.4±6.5%, P<0.05 vs
E150and NS vs control values).
Mean blood pressure and heart rate were not affected by
administration of 150 mg/kg BW or 300 mg/kg/BW vitamin E
and /or PC.
Measurement of circulating MDA
MDA production at baseline, before sustained ischemia, and
at 20 min of reperfusion as an index of lipid peroxidation is
shown in Table 1. There were no significant differences
between the different study groups compared at each time
point. However, there were significant increases in MDA
concentration at 20 min of reperfusion when compared to
Fig. 2. The effect of various interventions on infarct size (expressed as a
percentage of risk zone) in rabbit hearts following 30 min of ischemia and
180 min of reperfusion. CTL, control group; E150, group treated with vitamin E
150 mg/kg/BW; E300, group treated with vitamin E 300 mg/kg/BW; PC,
preconditioning with 2 cycles of 5 min ischemia–10 min reperfusion; E150-PC,
group treated with vitamin E 150 mg/kg/BW and subjected to preconditioning;
E300-PC, group treated with vitamin E 300 mg/kg/BW and subjected to
preconditioning; E150+5-HD, group treated with vitamin E150 mg/kg/BW and
5-HD 10 mg/kg/BW *P<0.01 versus control group and E150+5-HD group.
1095 I. Andreadou et al. / Free Radical Biology & Medicine 41 (2006) 1092–1099
baseline values in all study groups (*P<0.05 vs baseline values)
including those treated with vitamin E.
Table 2 shows plasma CD concentrations at baseline, at the
time before sustained ischemia, and at 20 min of reperfusion.
There were significant increases in CD concentrations at
20 min of reperfusion when compared to baseline values in
the CTL and PC groups. Both doses of vitamin E resulted in
an increase in CDs at 20 min of reperfusion (P<0.05 vs
The ratio of cardiac MDA levels (μM/mg protein) in
ischemic/nonischemic areas is shown in Fig. 3. Increased
MDA levels were noted in all areas at risk in all study groups.
Vitamin E and PC exhibited a tendency to increase MDA
concentrations during ischemia but not to a statistically
Fig. 4 shows CD concentrations expressed as nanomole
hydroperoxide per milligram protein obtained in the different
study groups at 20 min of sustained ischemia. In the CTL group
a significant decrease in the ratio of CD concentrations in
ischemic and nonischemic areas was obtained when compared
to the PC group and to the groups treated with different doses of
vitamin E (P<0.05 vs CTL group).
Fig. 5 shows the ratio of cGMP levels in ischemic/
nonischemic areas. PC and vitamin E-treated groups or
combined PC with vitamin E was found to enhance the
cGMP levels in the ischemic myocardium after 20 min of
sustained ischemia (*P<0.05 vs CTL group).
Concentration of vitamin E in myocardium
The myocardial concentration of vitamin E in the control
group was 0.150±0.06 nmol/mg tissue. Acute intravenous
treatment with 150 mg kg−1vitamin E increased the myocardial
vitamin E concentration to 11.46±1.7 nmol/mg tissue, whereas
treatment with 300 mg kg−1increased the vitamin E
concentration to 21.6±5.8 nmol/mg tissue at 20 min of
The present study demonstrates that the acute administration
of vitamin E reduces infarct size and maintains the beneficial
effect of ischemic preconditioning without altering plasma
MDA or cardiac CDs during ischemia and reperfusion. Vitamin
E increases the levels of tissue cGMP in the ischemic
Plasma-conjugated dienes (ΔABS/ml) assessed under basal conditions, at the
time before sustained ischemia, and at 20 min of reperfusion
GroupBaseline Before ischemia 20 min rep
Data are means±SE.
aP<0.05 compared to E150, E300, PC, and E150–PC groups at 20 min of
⁎P<0.05 compared to baseline values.
Fig. 3. The ratio of cardiac malondialdehyde (MDA) levels (μM/mg protein)
determined in the ischemic and nonischemic areas.
Fig. 4. The ratio of cardiac-conjugated dienes (nmol hydroperoxide/mg protein)
determined in the ischemic and nonischemic areas. * P<0.05 versus CTL
Plasma malondialdehyde (MDA) assessed under basal conditions, at the time
before sustained ischemia, and at 20 min of reperfusion
GroupBaseline Before ischemia20 min rep
Data are means±SE.
⁎P<0.05 compared to baseline values.
1096I. Andreadou et al. / Free Radical Biology & Medicine 41 (2006) 1092–1099