Antiatherosclerotic effect of probucol in WHHL rabbits: are there plasma
parameters to evaluate this effect?
B. Finckh, A. Niendorf , M. Rath, and U. Beisiegel
EUR J CLIN PHARMACOL (1991) 40 [SUPPL 1]: S 77 -
Probucol is well known to reduce cholesterol levels in blood and to
induce regression of xanthomata in patients with familial hypercholesterolemia
. The biochemical mechanisms responsible for the antiatherosclerotic
effects of probucol are of special interest. In addition to its cholesterol-lowering
effect, probucol acts as an antioxidant. As such, it is able to prevent
the oxidative modification of LDL in vitro . Moreover, probucol has
been reported to enhance the HDL-mediated cholesterol efflux from human
skin fibroblasts .
WHHL rabbits are characterized by a inherited LDL receptor defect. They
have cholesterol levels about 20 times those of normal rabbits and develop
severe arteriosclerosis at an early age with a diet of normal rabbit
chow [4, 5].
Materials and methods
Female and male WHHL rabbits were used at different ages. In all, 22
animals were used in four experiments, and in experiments 1-3 only animals
from the same litter were used in any one experiment. All rabbits were
fed normal rabbit chow. The probucol (1% w/w) was mixed in this chow.
Total cholesterol and total lipids were determined by standard colorimetric
methods (Diagnostica, Boehringer Mannheim, FRG; Merck, Darmstadt FRG).
Probucol levels and the concentration of the physiological antioxidants
(alpha and gamma tocopherol) were measured by HPLC [6, 7]. Thiobarbituric
acid-reactive substances (TBARS) were determined in plasma as a parameter
of lipid peroxidation . The plasma parameters were obtained before
treatment and after the indicated treatment period.
In addition to the biochemical measurements the plaque area was analysed
macroscopically and microscopically in the rabbit aortas to check the
antiatherosclerotic effect and correlate it to the biochemical parameters.
Macroscopically the plaque area was analysed with the aid of computer-assisted
planimetry. For the histological examination the tissue was formalin-fixed
and paraffin-embedded according to standard procedures. Special attention
was paid to representative cross sections through the entire aortic
wall. Therefore, 30 sections of each vessel were embedded in three
paraffin blocks, and then cut and stained.
Four different experiments were performed with WHHL rabbits to determine
the effect of probucol. Notes of age of animals at beginning and end
of the experiments, length of feeding period, and sex of animals are
given in Table 1. The percent plaque area measured in the aortas of the
rabbits at the end of the experiment is given in Table 2. A macroscopical
picture of the aorta of an untreated animal compared to a sibling treated
with probucol (expt.2) is given in Fig.1. In each of experiments 2-4
one animal was sacrified at the beginning of the experiment and its plaque
area was measured as pre-treatment control. In Fig. 2 an overview of
the middle part of the aorta o a probucol-treated animal and a non-treated
control is shown in low-power magnification of a histological section.
It is evident that the non-treated animal has a far greater lesioned
area than the probucol-treated rabbit. Histological examination reveals
that (1) the major type of lesions in the control animal are complicated
atheroma (i.e. lesions including necrosis and calcification) in contrast
to more fibrous and generally less elevated lesions in the control rabbit;
(2) the non-treated animal has more foam cells in its atheromas than
the probucol-treated animal.
The mean cholesterol levels of all animals at the beginning of and during
the experiment is given in Fig. 3. A 20-30% decrease in total cholesterol
could be observed after 6, 12 months of treatment. The TBARS, probucol
and tocopherol values in plasma are given for the treated rabbits and
for the untreated controls from all experiments in Fig. 4. The probucol
levels vary between 3.2 and 9.3 mg/mg total lipids. The TBARS are decreased
in the treated animals. This might be an indication of a lower degree
of lipid peroxidation in the blood or the arterial wall of these animals.
A slight increase could be seen in the alpha tocopherol and the measurable
gamma tocopherol that had appeared in the treated animals.
In the first experiment animals were only 1 month old when we started
the feeding experiment. We therefore did not kill an animal as a pre-treatment
control, since at this age we did not expect any visible plaque development.
After the 6 months of the experiment only around 5% plaque area was
measured in the aorta of the untreated animals. This means that this
litter was less prone to the development of atherosclerotic plaques
than the other animals seen in this study (see expt.3). The probucol-treated
rabbits showed an even smaller percent, age plaque area (1-2%) after
the 6 month period. The mean cholesterol values were 686 mg/dl in the
controls and 569 mg/dl in the probucol-treated animals. Only very slight
differences were observed in TBARS, but the treated animals with the
lowest TBARS (0.07 nmol/mg total lipid) also had the smallest plaque
area, with 0.9%.
Six 2month-old animals were used in the second experiment. No plaques
were detected in the pre-treatment control. The untreated animals developed
36% and 37% plaque area in the aortas over the 12 months of the experiment
(see Fig. 1). In comparison, the three probucol-treated animals developed
only 7-21% plaque area. The cholesterol at the end of treatment was 715
m/dl in the controls and 515 mg/dl in the probucol-treated animals. The
difference in TBARS between the treated animals and the controls was
rather small (0.086 versus 0.1 nmol/mg total lipids). In this experiment
probucol was able to slow down the early plaque development in rabbits
age 2-14 months.
In the third experiment we started the treatment at the age of 5 months.
The pre-treatment control sacrified at this age had 55% plaque area in
its aorta. After the 6 months of feeding, in controls the aortic plaque
area had increased to 62% and 80%, while in the treated siblings we measured
only 42% and 20% plaque area, i.e. smaller areas than in the pre-treatment
control. This indicates that regression of early plaques may be possible
in young animals after probucol treatment. The values for lipid peroxidation
in plasma, the TBARS; were 0.25 nmol/mg total lipid in controls versus
0.1 nmol/mg total lipid in the probucol-treated animals. The mean cholesterol
values were 788 mg/dl in the controls versus 438 mg/dl in the treated
animals. Both biochemical parameters show significant differences, which
might be responsible for the regression of the plaque area seen in this
The question as to whether probucol can also have an effect in very
old rabbits was addressed in the fourth experiment. The 15-month-old
female animals were not from the same litter but had the same parents.
The three pre-treatment controls already had an average of 83% plaque
area in the aortas. The probucol treatment did not have any effect on
plaque development (94% versus 95%). The cholesterol was 631 mg/dl in
the controls and 706 mg/dl in the treatment group. This experiment shows
that in older animals probucol is not effective in cholesterol lowering
and that existing severe plaques are also not affected by the treatment.
In this study we measured plasma lipids, antioxidants and lipid peroxidation
parameters and compared these biochemical values with the plaque development
in the aortic intima of rabbits. We used animals from the same litter
in each of experiments 1-3 to ensure the same genetic background in the
animals we wanted to compare. One interesting observation on comparison
of the litters was that even though they were derived from the same colony
they differed in the age of onset of plaque development. The 5-month-old
rabbits in experiment 3 already had 55% plaque area, while the 7-month-old
control animals in experiment 1 had only 1-8% plaque area; and the 14-month-old
female in experiment 2 had 37% plaque area while the females in experiment
4, which were only 1 month older, already had around 88% plaque area.
Littermates, in contrast, were more similar in the amount of plaque development
at the same age. We have no explanation for this observation as yet,
but we consider it makes it even more important to compare only animals
from the same litter.
Probucol was able to reduce the cholesterol level significantly in younger
animals, but no effect was observed in the 15-month-old female animals.
The TBARS were lower in the treated animals, but the difference were
not significant and we are not yet sure whether we can detect measurable
differences for this parameter in human plasma of patients treated with
The increase in alpha tocopherol and the appearance of gamma tocopherol
in the plasma of the treated animals confirm earlier observations that
treatment with probucol preserves the physiological antioxidant tocopherol.
When the plaque area measured in the aortas of WHHL rabbits after treatment
is compared with that in untreated controls it becomes obvious that probucol
slows down the progression of atherosclerosis in WHHL rabbits. The histological
investigation allows a more detailed analysis of the plaque composition.
The most striking histological finding in the probucol-treated animals
is a reduction in foam cells in the early period of life. Thus, inhibition
of LDL oxidation (as performed here in an in vivo experiment) prevents
the overloading of macrophages with cholesterol. This, together with
stimulation of the reverse cholesterol transport, might be the basis
of the antiatherogenic potential of probucol.
These data confirm observations published by Kita et al.  and Carew
et al. . In these author studies, however, the prevention of progression
was not correlated to biochemical parameters for lipid peroxidation.
With our study we want to contribute to the explanation of the mechanism
by which probucol prevents plaque development. If this were understood
it might enable us to find diagnostic parameters allowing a better estimation
of the individual risk of early coronary heart disease in individual
patients. Moreover, it might allow us to measure the efficiency of the
treatment with antioxidants, what is not possible at present
In four experiments with 22 WHHL rabbits we demonstrated that probucol
treatment decreased the progression of atherosclerotic plaques by a combination
of cholesterol lowering and antioxidative effect. In one litter we could
even show a reduction of plaque area by the treatment in comparison with
a sibling sacrified at the beginning of the experiment. This indicates
that regression might be possible in WHHL, rabbits. Our study demonstrates
that differences in biochemical parameters for lipid peroxidation such
as TBARS can be measured in probucol-treated animals versus controls and
might later be used to check the efficiency of the drug treatment. In
addition, the increased tocopherol level in the plasma of the animals
might indicate the positive drug response. The possibility has to be evaluated
whether increased levels of tocopherol can be used as a measure of antiatherogenic
effects in patients.
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