© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effects of Lovastatin Therapy on Susceptibility of LDL to Oxidation During 
-Tocopherol Supplementation
From the Department of Internal Medicine, Kanta-Häme Central Hospital, Hämeenlinna (A.P.), and the Departments of Internal Medicine (K.M.) and of Clinical Chemistry (O.M., T.S.), Tampere University Hospital, Tampere, Finland.
Correspondence to Ari Palomäki, MD, Kanta-Häme Central Hospital, Department of Internal Medicine, FIN-13530 Hämeenlinna, Finland.
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Abstract |
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Abstract—A randomized, double-masked, crossover clinical trial was carried out to evaluate whether lovastatin therapy (60 mg daily) affects the initiation of oxidation of low density lipoprotein (LDL) in cardiac patients on
-tocopherol supplementation therapy (450 IU
daily). Twenty-eight men with verified coronary heart disease
and hypercholesterolemia received
-tocopherol with lovastatin or with dummy
tablets in random order. The two 6-week, active-treatment periods were
preceded by a washout period of at least 8 weeks. The oxidizability of
LDL was determined by 2 methods ex vivo. The depletion times for LDL
ubiquinol and LDL -tocopherol were determined in timed
samples taken during oxidation induced by
2,2-azobis(2,4-dimethylvaleronitrile). Copper-mediated oxidation of LDL
isolated by rapid density-gradient ultracentrifugation
was used to measure the lag time to the propagation phase of
conjugated-diene formation. -Tocopherol supplementation
led to a 1.9-fold concentration of reduced -tocopherol
in LDL (P<0.0001) and to a 2.0-fold longer depletion
time (P<0.0001) of -tocopherol compared
with determinations after the washout period. A 43% prolongation
(P<0.0001) was seen in the lag time of conjugated-diene
formation. Lovastatin decreased the depletion time of
reduced -tocopherol in metal ion–independent oxidation
by 44% and shortened the lag time of conjugated-diene formation in
metal ion–dependent oxidation by 7%. In conclusion,
-tocopherol supplementation significantly increased the
antioxidative capacity of LDL when measured ex vivo, which was
partially abolished by concomitant lovastatin therapy.
Key Words: -tocopherol • clinical trials • lipid oxidation • lovastatin • ubiquinol
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Introduction |
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High serum cholesterol concentrations and oxidation of LDL seem to play key roles in the pathogenesis of an atherosclerotic lesion. An effective means to treat hypercholesterolemic patients is through the use of statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA ) reductase inhibitors. They effectively decrease serum LDL cholesterol, increase HDL cholesterol levels slightly, and improve the prognosis of coronary heart disease (CHD) patients.1 2 The prominent antioxidant in LDL is
-tocopherol, because in every LDL particle there are
6 to 8 molecules of -tocopherol.3 4 5
-Tocopherol acts as a powerful antioxidant after the
very early stage of LDL oxidation and especially during intense
oxidative stress.6 7 8 However, under mild oxidation,
-tocopherol may act as a prooxidant, thus shifting the
peroxidative insult from the surface to the inner part of the LDL
particle.8 9 10 This event could be prevented by
ubiquinol.11
-Tocopherol supplementation may have a significant role
in the prevention of CHD. Retrospective data indicate an inverse
relation between plasma concentrations of vitamin E and the risk of
angina pectoris.12 According to 1 prospective study, the
serum -tocopherol concentration may affect cardiac risk
(ie, nonfatal myocardial infarction or cardiovascular
death) in patients with coronary disease.13
Similar results have been obtained in both men and
women,14 15 with a high intake of vitamin E being
correlated with a lowered risk of CHD. Although vitamin E
supplementation at pharmacological doses may reduce the rate of
nonfatal myocardial infarction, its effect on cardiac or all-cause
mortality is unclear.16
We found earlier in hypercholesterolemic CHD patients
not under any antioxidant treatment that the oxidation times of both
LDL ubiquinol and LDL -tocopherol were shortened
significantly during lovastatin therapy.17
This finding was associated with a shortened lag time to
conjugated-diene formation, suggesting a diminished resistance of LDL
particles to the early phase of oxidative stress. This oxidative
resistance, at least partially, may be restored with ubiquinone
supplementation.18 -Tocopherol
supplementation prolongs the lag time to conjugated-diene formation,
when LDL oxidation occurs at high concentrations of
oxidants.19 20 However, even during
-tocopherol treatment, a prominent change in diet can
affect the susceptibility of LDL to oxidation.21
In the present trial, we studied whether effective
lovastatin therapy modifies the oxidizability of LDL,
measured as the lag time to diene formation, in CHD patients who were
on -tocopherol supplementation. We also studied whether
lovastatin in this context affects the depletion times of
LDL -tocopherol or ubiquinol during metal
ion–independent LDL oxidation.
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Methods |
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Study Design
In this randomized, double-blind, crossover trial, the effects of lovastatin treatment were compared with those of placebo in patients receiving
-tocopherol. A 12-week-long period
of -tocopherol supplementation was preceded by a washout
period of at least 8 weeks. During -tocopherol treatment
(300 mg/d, or 450 IU daily), concomitant lovastatin (LT
period) or placebo resembling lovastatin (PT period) was
taken. In other words, 14 patients started with the LT
(lovastatin+-tocopherol) period and
continued, after 6 weeks, with PT (placebo+-tocopherol),
whereas the other half of the patients had the PT period first. The
clinical part of the study was carried out in the
cardiology outpatient clinic at Kanta-Häme
Central Hospital, Hämeenlinna, Finland. The study was approved by
the Ethics Committee of the hospital and the National Agency for
Medicines. Written, informed consent was received in advance from every
patient. Monitoring was carried out according to Good Clinical Trial
Practice,22 and the recommendations of the Declaration of
Helsinki were followed during the study protocol.
Inclusion criteria were checked during the prestudy visit. Except for
the study medication, the entire trial was performed without any
antioxidants, vitamins, or treatments affecting lipid
metabolism while all other medications were kept constant.
The study products were Esol (100 mg
RRR--tocopherol, Leiras) and Lovacol (20 mg
lovastatin, Orion, under license from MSD). The patients
took 1 pill of -tocopherol 3 times daily during both
6-week periods. One lovastatin tablet was taken daily
during the first week, 2 tablets per day during the second week, and 3
tablets daily during weeks 3 through 6. This dosage protocol and the
final daily lovastatin dose of 60 mg had proved to be well
tolerated and effective with a similar patient
group.18 The placebos were taken in identical
fashion. Compliance was checked by tablet counting, and diet was
assessed on each visit. Clinical evaluation of the safety of therapy
was completed by analyzing serum enzymes indicating the function of the
muscles, liver, and kidneys.
Subjects
The study was carried out with 28 well-informed and cooperative
men at an average age of 56±8 years (mean±SD; range, 40 to 69 years)
and a body mass index of 25.4±1.4 kg/m2. All of
them had definite CHD and primary hyperlipidemia. The
diagnosis of CHD was confirmed either by coronary angiography
(17 patients) or by verified previous heart infarction (11 patients) at
least 6 months before the study. The inclusion criteria for fasting
serum lipids were as follows: LDL cholesterol >4.0
mmol/L or total to HDL cholesterol ratio >5.5. Exclusion
criteria were concomitant steroid therapy; diabetes; alcoholism or
misuse of narcotics; overt
hypertriglyceridemia (>5 mmol/L);
liver, renal, or endocrine disease; malignant tumor; or chronic
terminal disease. All of the patients were on a
cholesterol- lowering diet. There were 2 current smokers
(>2 cigarettes per day). Twenty-seven of the patients were on
ß-blocking–agent therapy, 4 used angiotensin-converting
enzyme inhibitors, 11 were taking long-acting nitrates, and
7 used calcium channel– blocking agents.
Blood Samples
Venous blood samples were drawn at 8 AM on all study
visits. The patients were advised to fast; not to take any medication,
coffee, or other beverages; and not to smoke for 12 hours before blood
sampling. Alcohol was prohibited for 36 hours before sampling. Blood
samples were taken in the sitting position after a rest of at least 15
minutes. Plasma was separated by centrifugation
immediately after cooling the samples on ice in the dark for 5 minutes.
After separation, the samples were kept frozen at -80°C until
analyzed.
Metal Ion–Dependent Oxidation of LDL
The copper-induced LDL oxidation method used in this trial has
been described earlier.18 In brief, LDL was isolated by
single-step ultracentrifugation for 30 minutes at
338 000 g (Beckman TL-100) with a TLV-100 rotor. A 1-mL
sample of desalted and in gel– filtered LDL solution was standardized
to 0.05 mg protein per mL with PBS. The sample was oxidized at 37°C
as described previously.23 The final concentration of
CuSO4 in the mixture was 1.67 µmol/L. UV
absorbance at 234 nm was monitored every minute for 300 minutes with
the use of a Perkin Elmer Lambda Bio 10 spectrometer. Lag time to the
start of the propagation phase of diene formation was defined as the
intersection of the tangents of the initial phase (first 5 minutes) and
maximal propagation.
Metal Ion–Independent Oxidation of LDL
The method of LDL oxidation induced by
2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) has been described
thoroughly in our previous article.17 In brief, LDL was
precipitated from EDTA-plasma with heparin (Noparin, Novo Nordisk) and
trisodium citrate (pH 5.0) in acid-washed Kimax glass
tubes.24 The lipid fraction of LDL was dissolved in
chloroform/methanol (1:1, vol/vol). Peroxyl radicals were produced by
AMVN (Polysciences Inc), with a final AMVN concentration of 2.1
mmol/L. Reduced -tocopherol and ubiquinol were
determined by high- performance liquid
chromatography and redox-sensitive
electrode.25 Samples of 100 µL were taken every 3
minutes from a test tube that had been placed in a
temperature-controlled incubator (37°C). Oxidation was stopped by
deep-freezing the samples in LN2, wherein they
were stored until HPLC analyses. The results were standardized
against LDL phosphate concentrations. LDL phosphate (Pi) concentration
was determined using a colorimetric method and an
inorganic ammonium molybdate reaction.
Determination of Lipids and Apolipoproteins
Total cholesterol, HDL cholesterol, and
triglycerides were analyzed immediately after
separation of the serum samples. LDL cholesterol
concentration was calculated with the Friedewald formula. The other
biochemical determinations of plasma samples were made after the entire
clinical phase of the study had been completed. Apo A-I and B were
measured by a nephelometric method (Behring) using highly specific
antisera.
Numerical Analyses
The rates of individual consumption of ubiquinol and reduced
-tocopherol during AMVN-induced oxidation were
calculated by linear regression analysis. The effects of the
lovastatin and placebo interventions during
-tocopherol treatment were compared by 2-way ANOVA with
equal replications (RANOVA) and with the factors treatment and
treatment order. Data for the 3 periods were included in the overall
analyses: initial washout (WO), lovastatin+
tocopherol (LT), and placebo+tocopherol (PT).
The treatment periods were further compared pairwise by t
test–based contrast analysis (user contrast, BMDP
Solo).26 A value of P
0.05 was regarded
as statistically significant. Mean±SEM are presented unless
stated otherwise.
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Results |
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During the trial, no severe adverse effects were reported or observed. Patient compliance was good, as measured by adherence to the protocol and the consumed- tablet counts, which were, on average, 96% for both lovastatin (or placebo resembling lovastatin) and
-tocopherol. No clinically
significant changes were observed in weight, diet, living habits, serum
enzymes, hematological parameters, or clinical status
(Table 1
).
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Lipids and Lipoproteins
LT treatment had significant effects on all lipid and lipoprotein
parameters measured. The only parameters on
which -tocopherol alone (ie, the PT phase) seemed to
have an influence were HDL cholesterol and apo A-I. When LT
was compared with the initial WO period, HDL cholesterol
increased by 15.9% and apo A-I by 5.9%, whereas with PT, the
elevations were more modest: 10.2% and 3.4%, respectively.
When treatments including -tocopherol therapy were
compared, lovastatin highly significantly decreased both
serum total and LDL cholesterol, by 32.4% and 41.1%,
respectively. Furthermore, a slight but significant increase in serum
HDL cholesterol (5.2%) was measured during LT, as well as
a decrease in triglycerides by 28.3%. Apo B decreased
significantly by 31.1%, but apo A-I remained at the same level as
during PT. The LDL to HDL ratio decreased by 44.1%, and the LDL
cholesterol to apo B ratio dropped by 13.2%. The absolute
means and probability values are presented in Table 1
.
LDL Antioxidant Consumption During Metal Ion– Independent
Oxidation
The effects of different therapy on the kinetic
parameters of LDL antioxidants during oxidation induced by
AMVN are compiled in Table 2.
Tocopherol supplementation led to a significant increase in
the reduced -tocopherol content in LDL, by 81.4%. The
consumption rate of -tocopherol was decreased
nonsignificantly by 15.6%, and the prolongation of depletion time was
100.9% (P<0.0001). LT did not change LDL
-tocopherol levels when compared with the values after
PT. However, the rate of -tocopherol consumption was
hastened and the depletion time shortened significantly, by 44.5% and
44.1%, respectively. It is noteworthy that the depletion time during
LT was shortened to almost the starting level (WO, Table 2) due
to the significant acceleration (P=0.0003) of LDL
-tocopherol consumption. A
representative example of LDL
-tocopherol consumption is presented in Figure 1.
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, WO; 
, LT; and 
, PT.
The LDL ubiquinol level did not change during PT, but surprisingly,
there was a tendency for faster ubiquinol consumption (35.7%,
P=0.057). The total consumption time of ubiquinol was
shortened significantly, by 14.3%, compared with WO. During LT, the
LDL ubiquinol level decreased by 18.7% from values during PT. The rate
of LDL ubiquinol consumption was not further changed by LT, and the 9%
shortening of the depletion time of ubiquinol was not statistically
significant (P=0.084). A representative
example of LDL ubiquinol consumption during AMVN-induced oxidation is
presented in Figure 2.
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Conjugated-Diene Formation in LDL During Metal Ion–Dependent
Oxidation
During copper-mediated oxidation, -tocopherol
therapy increased the lag time by 43.3% and decreased the maximal
oxidation rate by 15.8% (Table 3). The
maximal protein-normalized diene formation had a significant but small
tendency to decrease when compared with WO.
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During LT, the lag time decreased by 7.1% when compared with PT. The
small decrease, 5.2%, in the oxidation rate during LT did not reach
statistical significance (P=0.096) when compared with PT.
Lovastatin did not significantly alter the maximal,
protein-normalized conjugated-diene level. A
representative example of conjugated-diene formation
during copper-induced oxidation of LDL is presented in Figure 3.
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Ubiquinol concentration in LDL was related to the changes in LDL lag time. When the study group was evenly divided into 2 groups according to ubiquinol level during PT, the lag time diminished during LT, from 87.9 to 78.6 minutes in the lower-ubiquinol subgroup and from 93.6 to 90.0 minutes in the higher-ubiquinol subgroup. These changes did not differ significantly (P=0.17) from each other. When the same subdivision was made according to the LDL ubiquinol level during LT, the changes in lag times were significantly different (P=0.03): from 95.0 to 82.5 minutes in the lower- ubiquinol subgroup and from 86.5 to 86.1 minute in the higher-ubiquinol subgroup.
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Discussion |
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The oxidation of LDL is a gradual reaction, wherein antioxidants are consumed according to different priorities.19 27 28 Besides
-tocopherol,
other known antioxidants like ubiquinol occur in very low
concentrations in LDL particles.3 19 Ubiquinol acts as an
effective antioxidant at the very beginning of the peroxidative
process.27 28 29 30 31 32 Its level may be reduced by high-dose
treatment with an HMG-CoA reductase
inhibitor.17 33 34 35 36 In the present study,
the LDL ubiquinol level decreased during the LT period. The lag time to
conjugated- diene formation in ex vivo, isolated LDL was prolonged by
-tocopherol treatment (450 IU/d), regardless of
concomitant treatment with lovastatin or placebo. In in
vitro studies on copper-induced oxidation of LDL, in which the
Cu2+ to LDL particle ratio is at least 3,
-tocopherol acts as an antioxidant.8 Our
finding accords with earlier studies, in which doses of not less than
400 IU/d have lengthened the lag time.7 37 The lag time
during LT was statistically significantly shorter than that during PT.
The results of the AMVN experiments are in accordance with those of
metal ion–dependent oxidation: LT accelerated the consumption of
reduced -tocopherol during AMVN-induced oxidation. In vitro studies using coincubation of LDL and statins have suggested that lovastatin, simvastatin, and fluvastatin themselves might have some direct antioxidative influence on LDL.38 39 40 Mevalonate is the precursor of many nonsteroidal derivatives, such as geranyl and farnesyl pyrophosphates. They are lipid moieties of prenylated proteins, which may regulate superoxide generation in neutrophils and macrophages.41 Other molecules that are affected by inhibition of HMG-CoA reductase and that may participate in the defense against oxidative stress are Rho-related proteins, like rac 1 and rac 2.41 Simvastatin has decreased the formation of superoxide in human monocytes,42 and lovastatin has inhibited the oxidizability of LDL by rabbit leukocytes.40 Finally, both vitamin E and lovastatin therapy have diminished the number of atherosclerotic lesions in rabbits.40 43 In contrast, lovastatin has also been found to potentiate superoxide-radical generation in the atherosclerotic vascular wall in rabbits.44 However, it is not conceivable that the shortening of lag time is due to the direct prooxidative effect of lovastatin.45
Lag time, an accepted marker of LDL susceptibility to oxidation, is associated with endothelial function and the severity of coronary atherosclerosis in cross-sectional settings.46 47 However, no controlled, prospective clinical trials have been carried out to compare the different measures of LDL antioxidative capacity as surrogates for CHD events. The amount and formation rate of conjugated dienes reflect later phases of LDL oxidation than does the lag time or antioxidant depletion time of AMVN experiments. Effective statin therapy has been reported to slow the oxidation rate and to decrease the maximal production of conjugated dienes.17 38 40 48 49 50 51 52 These phenomena may be secondary to a greater decrease in the lipid moiety than that of the proteins in LDL.17 49 53 Later phases of LDL oxidation can also be studied by using excessive, long-lasting oxidation with other measures for LDL peroxidation, like the determination of thiobarbituric acid–reactive substances and trinitrobenzenesulfonic acid,38 or LDL malondialdehyde, peroxides, and the total amount of conjugated dienes.54 This kind of long-lasting oxidation may also lead not only to peroxidation of the lipids in the LDL particle but also to the degradation of its protein, like the inner part of apo B.55 Therefore, the effect of vigorous lovastatin therapy may be simultaneously indifferent or favorable when judged according to oxidation rate or maximal diene formation and unfavorable when based on lag time or antioxidant depletion.
Table 4 compiles the
characteristics of earlier studies, wherein the effects of statin
treatment on lag time were measured during copper-induced oxidation ex
vivo. Two groups studied oxidation of the non-HDL fraction, including
VLDL, whereas all of the others used the LDL fraction. In 5 of 7
studies, lag time did not change significantly.48 49 50 51 52 Our
earlier study was a randomized, placebo-controlled, double- blind trial
with a crossover protocol, in which 60 mg daily of
lovastatin shortened the lag time by 7%.17
However, in an open, uncontrolled setting with 10 subjects,
fluvastatin at 40 mg/d was reported to be associated with a
lengthening of lag time by 2.5 times.39
Fluvastatin has also been studied in a randomized,
double-blind, parallel, placebo-controlled trial design, wherein the
daily dose of 80 mg did not change the lag time
significantly.48
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As in most of the earlier studies,17 48 49 50 51 52 oxidation tests were performed on frozen samples after the entire clinical phase had been completed. If some oxidation or conformational changes did occur in LDL, then lovastatin made LDL more vulnerable to minimal oxidation than placebo, because the conditions were the same for all samples and the treatment periods were balanced. Also, with this design, the possible interassay variability does not affect the results.
If cholesterol-lowering therapy is effective enough,
the nonbeneficial effect of lovastatin on the
by-products of cholesterol synthesis (eg, ubiquinol)
may become important.18 In the present study, serum
LDL cholesterol was decreased to levels below those in most
of the other statin trials in which antioxidative defense mechanisms
were studied (Table 4). LDL was isolated by a rapid procedure to
minimize oxidative stress before the analysis. The effect of
statin treatment on the antioxidative defense against the very initial
phase of LDL oxidation (eg, ubiquinol) may disappear if LDL is isolated
more slowly.
The statins may differ in their effect on LDL susceptibility to
oxidation. Treatment with pravastatin but not with
simvastatin has reduced the malondialdehyde content of the
vessel wall, at the same time enhancing
endothelium-dependent relaxation in
cholesterol-fed rabbits.56 The only
comparative study in humans consisted of 12 patients in the
simvastatin group and 11 patients in the
pravastatin group.49 However, the statistical
power of that study was not great enough even to show the different
efficacy of these 2 statins in lowering LDL cholesterol
(Table 4).49
The 450-IU daily dose of vitamin E slightly but significantly
elevated both apo A-I and HDL cholesterol levels. The
association between -tocopherol and HDL is
controversial.57 58 59 60 61 62 In a study with 24 volunteers, HDL
remained stable during dl--tocopherol therapy
of 800 IU (727.3 mg) daily.63 64 The initial HDL
level of patients in our study was 21% to 32% lower than in some of
the studies showing no change.7 37 63 64
-Tocopherol might be elementary in the synthesis of apo
A-I and HDL but may not have any elevating effect on them if the level
of HDL is already sufficient in the cholesterol
circulation. HDL is a vehicle for peroxidized lipids, and it may
protect LDL from peroxidation.65 66
To further elucidate the clinical relevance of statin treatment
on LDL antioxidative capacity, we need more randomized, controlled,
double-blind studies to compare statins with each other. It might also
be worthwhile to study different patient groups to evaluate whether
some of them have LDL that is more vulnerable to oxidation during
statin therapy than others. This trial supports the view that the
antioxidative protection of LDL by -tocopherol against
medium/high radical flux is incomplete. Despite the high-dose vitamin E
supplementation, effective lovastatin treatment (60 mg
daily) may affect the initial antioxidative mechanisms in the LDL
particle.
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Acknowledgments |
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This work has been supported by grants from the Hilkka and Väinö Kiltti Foundation, Hämeenlinna, Tampere University Hospital, and the Finnish Academy of Sciences (to A.P. and K.M.). We also thank Leiras Oy, Turku, Finland, for technical support in masking the study and monitoring the trial. The
-tocopherol pills were a generous gift from Leiras
Oy. We appreciate the professional technical aid of Kaarina
Siltanen, MSc, Nina Lehtinen, Elina Selkälä, Erja
Konttinen, and Irma Valtonen. We also thank Heli
Määttä and Marja-Leena Lampen for coordinating
help. Received June 22, 1998; accepted October 23, 1998.
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