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The effect of pharmacological doses of different antioxidants on oxidation parameters and atherogenesis in hyperlipidaemic rabbits
Atherosclerosis 154 (2001) 387– 398 www.elsevier.com/locate/atherosclerosis
The effect of pharmacological doses of different antioxidants on oxidation parameters and atherogenesis in hyperlipidaemic rabbits Sirus Djahansouzi, Jan Hinrich Braesen, Kai Koenig, Ulrike Beisiegel, Anatol Kontush * Biochemisches Labor, Pa6. 39, Medizinische Kern- und Poliklinik, Uni6ersita¨tskrankenhaus Eppendorf, Martinistraße 52, 20246 Hamburg, Germany Received 30 November 1999; received in revised form 31 March 2000; accepted 7 April 2000
Abstract The oxidation hypothesis of atherosclerosis implies that antioxidants are able to inhibit lipoprotein oxidation in the arterial wall and thereby retard atherogenesis. Since most of the animal studies performed have used very high doses of antioxidants, it is to date unknown whether antioxidants are effective antiatherosclerotic agents when given in pharmacological doses. Here we addressed this question using homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits as an animal model of atherosclerosis. The rabbits were divided into four groups, each consisting of ten animals. They received either a standard diet or a diet containing 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. After 12 months, the extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio in 14 cross sections equally distributed over the whole aorta. To evaluate the antioxidant effects of the diet, lipophilic and hydrophilic antioxidants, lipids, fatty acids and plasma oxidizability were measured after 0, 3 and 6 months of feeding. We found that supplementation with probucol significantly decreased aortic intima-to-media ratio compared to controls. The antiatherosclerotic action of probucol was accompanied by its beneficial action on plasma oxidizability and some plasma antioxidants. No decrease in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits, despite the fact that both antioxidants decreased plasma oxidizability and ubiquinone-10 increased the plasma levels of antioxidants. Taken together, these data suggest that pharmacological doses of probucol retard atherogenesis in WHHL rabbits by an antioxidant mechanism, while ubiquinone-10 and vitamin E at these dosages are ineffective in this highly hyperlipidaemic model. The measurement of some oxidation-related parameters in plasma, such as lipophilic antioxidants, polyunsaturated fatty acids and lipoprotein oxidizability, may be useful in assessing the risk of atherogenesis in humans. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Lipoprotein oxidation; Antioxidants; Watanabe heritable hyperlipidaemic rabbits; Probucol; Vitamin E; Ubiquinone10
1. Introduction The oxidation hypothesis of atherosclerosis implies that the oxidation of low density lipoprotein (LDL) in the arterial wall plays an important role in atherogenesis [1,2]. Since LDL contains a variety of antioxidants able to inhibit its oxidation, increasing the antioxidant content of LDL should be able to retard atherogenesis. The antioxidant content of LDL can be easily increased by dietary supplementation [3]. Accordingly, supple* Corresponding author. Tel.: + 49 40 428034449; fax: +49 40 428034592. E-mail address: [email protected] (A. Kontush).
mentation with different antioxidants, such as probucol [4–6], vitamin E [7–9] and butylated hydroxytoluene (BHT) [10], retards atherogenesis in animal models, such as Watanabe heritable hyperlipidaemic (WHHL) [4–7] or cholesterol-fed [8–10] rabbits. However, to obtain these effects, all of these antioxidants were given in amounts clearly exceeding those typically used in humans [11]. In most studies, the antioxidant content of the rabbit diet was approximately 0.5 –1% wt/wt, corresponding to about 10–20 g of daily intake in humans (assuming a daily dietary intake of 100 g and a weight of 3.5 kg for rabbits and a weight of 70 kg for humans).
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 5 1 0 - 4
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Influence of pharmacological doses of antioxidants, i.e. those not exceeding 1 g daily in humans, has not yet been extensively studied in animal models. Supplementation of WHHL rabbits with 0.025% wt/wt (equivalent to about 500 mg daily in humans) of probucol or vitamin E for 6 months did not result in a statistically significant change in aortic atherosclerosis when compared with control animals [12]. A slightly higher dose of probucol (0.05% in a diet) also failed to reduce atherosclerosis in cholesterol-fed rabbits [13]. Supplementation of atherosclerotic patients with 500 mg probucol a day was also ineffective in retarding the development of femoral atherosclerosis [14]. Similarly, only a trend towards decreased atherosclerosis was found in cholesterol-fed rabbits supplemented with 10 mg vitamin E/kg body weight (equivalent to about 700 mg daily in humans) [15]. However, vitamin E at lower doses retarded aortic atherosclerosis in hypercholesterolaemic hamsters [16,17]. Differences in the antiatherosclerotic activity of vitamin E have been reported to be determined by plasma levels of cholesterol, with a lower activity at higher cholesterol levels [16]. The ability of vitamin E to affect atherogenesis has also been assessed in large-scale randomized trials in humans. In the CHAOS study, a reduced risk of cardiovascular death and non-fatal myocardial infarction was found when vitamin E was given at a dosage of 400 or 800 IU/day [18]. However, there was no beneficial effect on the rate of cardiovascular disease by supplementing vitamin E at a dose of 50 mg/day in the ATBC study [19]. In most prospective observational and nested case-control studies, a beneficial effect of high levels of dietary vitamin E intake on the risk of cardiovascular disease has been reported (see [20] for review). In contrast, ubiquinone-10, another potent lipophilic antioxidant, seemed to be effective in decreasing the rate of cardiovascular disease when given at a dose of 200 – 400 mg/day [21]. These data clearly indicate that the question of antiatherosclerotic efficiency of pharmacological, i.e. those appropriate to humans, doses of antioxidants remains equivocal. In the present study we investigated whether pharmacological doses of chemically different antioxidants influence atherogenesis under conditions of extreme hyperlipidaemia in homozygous WHHL rabbits. The rabbits were fed with a standard diet alone or a diet containing either 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily (equivalent to about 300 mg ubiquinone-10 and vitamin E and 1 g probucol in humans). After 12 months feeding, the extent of aortic atherosclerosis was assessed. To ensure efficient uptake of the antioxidants, their plasma concentrations were measured. Plasma levels of antioxidants and other oxidation-related parameters have been implicated as clinically important
markers for the development of atherosclerosis [22 –26]. To estimate the predictive value of these measurements and to evaluate antioxidant effects of the diet, lipophilic antioxidants, hydrophilic antioxidants, lipids, fatty acids and oxidizability in vitro were measured in plasma at the beginning of the study and after 3 and 6 months of supplementation.
2. Methods
2.1. Animals and diets Forty homozygous WHHL rabbits bred at the animal facilities of the University Hospital of Hamburg were divided into four groups of ten animals matched according to sex and litter at the age of 3 months. The control group was given an Altromin Standard Rabbit Diet 2110 (Altromin International, Lage, Germany) which contained 0.006% wt/wt vitamin E. The supplemented groups received the standard diet containing added antioxidants vitamin E, ubiquinone-10 or probucol. Antioxidants were added to the diet either by Altromin International (a-tocopherol and probucol) or as a solution in diethyl ether followed by rigorous mixing and overnight drying to remove the ether (ubiquinone10). The diets were given in a fashion such that the first portion of the daily food intake contained the body weight-adjusted dose of the supplement and the rest contained the standard chow (total 100 g). The ubiquinone-10-supplemented group received 4.3 mg/kg body weight ubiquinone-10 (HPH, Hamburg, Germany), the vitamin E-supplemented group 4.3 mg/kg body weight DL-a-tocopherol acetate (Sigma, Deisenhofen, Germany) and the probucol-supplemented group 15 mg/kg body weight probucol (Sigma, Deisenhofen, Germany) daily for 12 months (assuming an average rabbit body weight of 3.5 kg). The doses were chosen to approximately match pharmacological daily doses used in humans, i.e. 300 mg ubiquinone-10 and vitamin E and 1 g probucol. Dietary consumption and weight gain did not differ among the four groups of animals during the study period.
2.2. Blood sampling Fasting blood samples were obtained from the lateral ear vein at the start of the study and after 3 and 6 months of supplementation. Blood was taken into heparin- or ethylenediaminetetraacetic acid (EDTA)-containing tubes (15 IU heparin or 1.6 mg EDTA/ml blood; Sarstedt, Nu¨mbrecht, Germany). Plasma was obtained by centrifugation of the blood at 4°C for 10 min and stored at −80°C under nitrogen until assayed.
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2.3. Measurement of plasma parameters
2.4. Extent of aortic atherosclerosis
Plasma total cholesterol and triglycerides were determined by commercially available enzymatic kits (Boehringer-Mannheim, Mannheim, Germany). Lipophilic antioxidants a-tocopherol, g-tocopherol, a-carotene, b-carotene and ubiquinol-10 (in its reduced and oxidized form) were quantified in EDTA plasma samples by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection as described elsewhere [27,28]. Ubiquinol-9, ubiquinone-7 and d-tocopherol were used as internal standards. Plasma concentrations of lipophilic antioxidants were normalized to plasma lipids and expressed as pmol/(mg plasma cholesterol+triglycerides). Probucol was measured in EDTA plasma using HPLC with UV detection at 242 nm after extraction with diethyl ether as described elsewhere [29]. Total sulfhydryl (SH)-groups and bilirubin were measured as plasma hydrophilic antioxidants. SH-groups were determined spectrophotometrically at 412 nm after their reaction with dithionitrobenzene [30]. Bilirubin was measured at 530 nm after its reaction with ethyl anthranilate in dimethyl sulfoxide [31]. Fatty acid composition of plasma was characterized by capillary gas chromatography as described elsewhere [32]. Tricosanoic acid was used as an internal standard and t-butyl hydroxytoluene as an antioxidant. The total amount of fatty acids was calculated as the sum of polyunsaturated fatty acids (PUFAs, consisting of linoleic, linolenic, g-linolenic, eicosadienoic, eicosatrienoic, arachidonic, eicosapentaenoic, docosadienoic, docosatetraenoic, docosapentaenoic and docosahexaenoic acids), monounsaturated fatty acids (MUFAs, consisting of palmitoleic, oleic, vaccenic, eicosaenoic and docosaenoic acids) and saturated fatty acids (SFAs, consisting of myristic, palmitic, stearic, arachidic and behenic acids). Susceptibility of plasma lipoproteins to oxidation was characterized as in vitro oxidizability of heparin plasma [33]. To measure the oxidizablity, plasma samples (20 ml) were diluted 150-fold with phosphatebuffered saline (PBS), pH 7.4, containing 0.16 M NaCl. PBS was treated with Chelex 100 ion-exchange resin (Bio-Rad, Munich, Germany) to remove transition metal ions. PBS-diluted plasma was oxidized by CuSO4 (50 mM) or soybean lipoxygenase (25 U/ml). The oxidation was performed in a spectrophotometrical cuvette at 37°C and the change in sample absorbance at 234 nm was monitored. The absorbance was measured every 5 min over a period of 20 h. The level of conjugated dienes in plasma samples has been shown to correlate with other indices of lipid peroxidation and to be indicative of the oxidizability of plasma lipoproteins [33 – 35].
At the end of the 12-month study period, the rabbits were anesthesized through the lateral ear vein by a thiobarbituric acid derivate. This was followed by an intracardial exsanguination and overdose of the same derivate. Subsequently, a complete autopsy was carried out with special attention paid to quick dissection of the aorta from the left subclavian artery to the bifurcation and then the whole system of large and middle sized arteries, followed by other organ sampling. The aortas were cleaned of excess adventitial tissue, rinsed with saline buffer and quickly divided into 14 segments equally distributed over the aorta, each about 1 cm in length. The segments were alternatively asseverated in neutral buffered 4% formaldehyde for 12 h or flash frozen and kept in saline buffer for biochemical analyses. The seven formalin fixed aortic segments were routinely dehydrated and paraffinized and the endings were brought to the bottom of the resulting paraffin blocks after transverse bisection in an upright position, resulting in 14 cross sections equally distributed over the whole aorta. Serial sections were performed and stained histochemically according to routine methods and Masson-Goldner-ElasticaKossa combined stain as described elsewhere [36], as well as immunohistochemically with the monoclonal mouse antibodies HHF35 (Enzo Diagnostics, New York, USA) concentrated as delivered for smooth muscle cells, and RAM11 (Dako, Hamburg, Germany) diluted 1:50 for macrophages using the ABC-DAB peroxidase technique (Vector Laboratories, Burlingame, USA). Morphometrical analyses were carried out with computer-assisted measurements using a Zeiss unit supported by Kontron software KS400 (Zeiss/Kontron, Eching, Germany). The thickness of the intima and media were measured perpendicular to the internal elastic lamina at 0.5 mm intervals on all 14 cross sections by the same operator in a blinded fashion. The extent of atherosclerosis was assessed as the intima thickness, media thickness as well as the ratio of intima to media thickness (intimato-media ratio) for each of the 14 cross-sections and presented as means for all cross sections of each animal.
2.5. Statistical analysis Differences between groups fed with different diets were analyzed by Mann –Whitney U-test. Differences between different time-points within the same group were analyzed by Wilcoxon’s matched pairs test. Pearson correlation coefficients were calculated to evaluate relationships between variables. All results are presented as means9 SD.
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3. Results
3.1. Plasma parameters
3.1.1. Lipids As expected, WHHL rabbits had already developed severe hyperlipidaemia at the beginning of the study, i.e. at the age of 3 months. At this time-point, plasma total cholesterol and triglyceride levels reached 7579 71 and 260964 mg/dl, respectively (averaged for all 40 animals). Plasma cholesterol levels further increased significantly in all study groups after 3 months of supplementation and remained elevated during the following 3 months (Table 1). Plasma triglycerides increased significantly only in the vitamin E- and probucol-fed rabbits after 6 months of supplementation. Low significance of the differences in plasma lipid levels was partly due to their high variability within the same feeding group, as indicated by high S.D. values.
There was no significant difference in plasma cholesterol levels between the feeding groups at any of the three time-points studied, indicating that no cholesterol-lowering effect of probucol or vitamin E was observed. In contrast, plasma triglycerides were significantly lower in the ubiquinone-10-supplemented animals than in the controls after 3 months feeding.
3.1.2. Antioxidants a-Tocopherol, a major lipophilic antioxidant in rabbit plasma, increased during the first 3 months of the study in all feeding groups (Fig. 1). The increase in plasma a-tocopherol was not associated with the increase in plasma lipids, since the effect was seen when a-tocopherol concentration was normalized to the lipids. After both 3 and 6 months of supplementation, a-tocopherol levels remained significantly elevated in all groups, except ubiquinone-10-fed rabbits who had significantly less a-tocopherol in their plasma compared with the controls, after 6 months. As expected, the
Table 1 Increase in total cholesterol and triglyceride levels in plasma of WHHL rabbits fed with antioxidants for 3 and 6 monthsa Group
Control Ubiquinone-10 Vitamin E Probucol
Increase in total cholesterol (% of the initial level)
Increase in triglycerides (% of the initial level)
0–3 months
0–6 months
0–3 months
0–6 months
15.69 10.5* 15.89 10.1* 12.999.7* 7.498.9*
3.6 9 26.5 8.0 9 14.1 3.1 915.4 1.89 14.0
17.8 942.6 −13.2919.6§ 13.9 9 38.4 24.1 941.4
35.0 958.1 7.1 954.6 31.7 9 35.7* 42.7 950.5*
a The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. Each feeding group consisted of ten animals. * PB0.05 versus corresponding value of plasma cholesterol before feeding. § PB0.05 versus control group.
Fig. 1. a-Tocopherol in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. a-Tocopherol was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *PB0.05, **PB 0.01 versus corresponding value before feeding; §PB 0.05 versus control group.
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Fig. 2. b-Carotene in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. b-Carotene was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *P B0.05, **PB 0.01 versus corresponding value before feeding; §P B0.05, §§PB 0.01 versus control group.
Fig. 3. Ubiquinol-10 in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. Ubiquinol-10 was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *PB 0.05 versus corresponding value before feeding; §P B0.05 versus control group.
highest levels of a-tocopherol were measured in the group supplemented with vitamin E. In contrast to a-tocopherol, plasma g-tocopherol did not reveal a clear tendency towards increased levels during the study. Moreover, plasma levels of g-tocopherol were significantly lower in ubiquinone-10- and vitamin E-fed animals when compared to controls after 6 months (data not shown). In contrast to tocopherols, plasma levels of both aand b-carotene tended to decrease throughout the study. b-Carotene significantly decreased in the control and ubiquinone-10 group after 6 months of supplementation (Fig. 2). a-Carotene behaved similarly, showing a significant decrease in the control animals after 6
months (data not shown). Interestingly, no significant reduction of b-carotene was observed in the probucolfed group. The vitamin E-fed rabbits showed only a trend towards lower b-carotene levels. Similarly to carotenes, plasma levels of ubiquinol-10 tended to decrease during the study. Significant decrease in ubiquinol-10 levels was observed in the control group after 3 months of supplementation (Fig. 3). The vitamin E-fed group also showed significantly lower concentrations of ubiquinol-10 after both 3 and 6 months. The decline of ubiquinol-10 in the vitamin E-fed rabbits was so pronounced that its level in this group was significantly lower than that measured in the control animals after 6 months. On the other hand,
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feeding with probucol seemed to be able to partially prevent a decrease in ubiquinol-10, since it kept the plasma level of this antioxidant significantly higher when compared to controls after 3 months. Supplementation with ubiquinone-10 also prevented a decrease in plasma levels of ubiquinol-10. In fact, a slight increase in plasma ubiquinol-10 was observed in this group. Plasma levels of ubiquinone-10, an oxidation product of ubiquinol-10, showed a between-group distribution which approximately mirrored that measured for ubiquinol-10 (data not shown). Plasma levels of hydrophilic antioxidants SH-groups and bilirubin were much less dependent on the antioxidant supplementation than those of their lipophilic counterparts. At the beginning of the study, total plasma SH-groups and bilirubin were 3589 67 and 46.0 97.8 mM, respectively (averaged for all 40 animals). Throughout the study, these levels did not significantly change in any of the groups (data not shown), except that SH-groups increased by 14.7% (P =0.02) in the vitamin E-fed rabbits after 3 months. Nor was there any significant difference found in either SH-groups or bilirubin levels between the feeding groups at any of the three time-points studied (data not shown). Supplementation with probucol resulted in its accumulation in rabbit plasma to a level of 7.339 3.77 and 9.3194.96 mM after 3 and 6 month feeding, respectively.
3.1.3. Fatty acids Hyperlipidaemic WHHL rabbits had higher plasma levels of fatty acids than those typically measured in normolipidaemia [37]. Total PUFAs reached 1419 20, MUFAs 63 913 and SFAs 85912 mg/dl at the begin-
ning of the study (averaged for all 40 animals). These levels did not significantly change in any of the rabbit groups throughout the study (data not shown). However, when individual fatty acids were expressed as a percentage of total fatty acids, significant effects became evident (Fig. 4). In the control and vitamin E-fed animals, PUFAs significantly decreased after 6 months of supplementation. When PUFAs were subdivided into their n-3 and n-6 constituents, it was found that n-6 PUFAs made up about 97% of total PUFAs in rabbit plasma and were mainly responsible for the decrease in total PUFAs (data not shown). No significant difference in plasma fatty acids were found between the feeding groups at any of the three time-points studied, independent of how fatty acids were expressed (data not shown).
3.1.4. Oxidizability In accordance with data previously reported for human plasma [33], the absorbance increase of diluted rabbit plasma oxidized with 50 mM Cu(II) was characterized by three consecutive phases, namely the lag-, propagation and decomposition phases of oxidation (Fig. 5). To quantitatively describe plasma oxidizability, the lag-phase and propagation-phase duration as well as the maximum rate of oxidation in the propagation-phase were calculated [38]. It was found that in all feeding groups, the lag-phase (Fig. 6A) and propagation phase (data not shown) were prolonged during the study and the maximum rate of oxidation within the propagation phase (Fig. 6B) was decreased. For the control animals, these findings correlated with the increase in plasma a-tocopherol (r= 0.39 and r= 0.40, P= 0.03 and 0.02, for the lag-phase and propagation phase, respectively).
Fig. 4. PUFAs in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. PUFAs were measured using capillary gas chromatography. *PB 0.05 versus corresponding value before feeding.
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Fig. 5. Kinetics of Cu(II)-induced oxidation of plasma of WHHL rabbits fed with antioxidants for 6 months. The curves with the median value of the lag-phase duration are shown for each feeding group. The numbers at the curves denote the lag-phase duration (min). The rabbits were fed as described in Fig. 1. Rabbit heparin plasma was diluted 150-fold by PBS and oxidized at 37°C in the presence of 50 mM Cu(II). Plasma oxidation kinetics were assessed as an increase in the absorbance of the samples at 234 nm.
The changes in the plasma oxidizability parameters were more pronounced in the antioxidant-fed, than in control, animals. Supplementation with probucol or, less pronounced, vitamin E resulted in the significantly longer lag- and propagation phases and lower maximum rates of oxidation compared with controls at the same time-points. The decrease in plasma oxidizability observed in the ubiquinone-10-fed animals did not reach significance when compared with control animals. It may therefore be concluded that the activity of antioxidants towards plasma oxidation by Cu(II) decreased in the order probucol\ vitamin E\ ubiquinone-10. In contrast to Cu(II)-induced oxidation, oxidation catalyzed by lipoxygenase was much less affected by the antioxidant supplementation. The plasma oxidation rate was found to be significantly lower only in ubiquinone-10 and vitamin E-fed animals than in controls, when measured after 6 months of supplementation (data not shown).
3.2. Extent of aortic atherosclerosis Extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio averaged for 14 cross sections equally distributed over the whole aorta. Supplementation with probucol for 12 months was found to significantly delay atherogenesis as shown by the lower intima-to-media ratio (Table 2). No reduction in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits. To estimate the potential predictive value of oxidation parameter measurements in plasma for the devel-
opment of atherosclerosis, the oxidation values measured after 0, 3 and 6 months were correlated with the extent of atherosclerosis measured after 12 months. Plasma levels of probucol, ubiquinol-10, g-tocopherol, a-carotene, PUFAs as well as the duration of the lag-phase and propagation phase of Cu(II)-induced oxidation were found to negatively correlate with the intima-to-media ratio. PUFAs revealed significant correlations when measured at the beginning of the study (r= − 0.35, PB0.05), probucol (r= −0.65, PB0.05) and ubiquinol-10 (r= −0.37, P B0.05) when measured after 3 months, g-tocopherol (r= − 0.44, PB 0.01) and a-carotene (r= − 0.37, P B 0.05) when measured after 6 months. Duration of the lag-phase and propagation phase of Cu(II)-induced oxidation negatively correlated with the intima-to-media ratio when measured both after 3 months (r= − 0.31, PB 0.05, and r= −0.38, PB 0.05, respectively) and 6 months (r= −0.31, PB 0.05, and r= −0.44, P B 0.01, respectively) of the study. All of these parameters, except ubiquinol-10, also revealed significant negative correlations with the intima thickness (data not shown).
4. Discussion Three chemically different antioxidants, namely probucol (15 mg/kg body weight), ubiquinone-10 and vitamin E (both 4.3 mg/kg body weight), were examined for their ability to influence oxidation parameters and prevent atherosclerosis in homozygous WHHL rabbits, when administered at these pharmacological doses.
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Plasma cholesterol levels increased significantly in all animals during the study. This reflects the expected hypercholesterolaemia in WHHL rabbits and is in accordance with the data of others [39]. The high
levels of cholesterol in WHHL rabbits induce the development of severe atherosclerosis. For their part, triglyceride levels did not show a clear tendency to increase.
Fig. 6. Duration of the lag-phase and maximal oxidation rate measured within the propagation phase of Cu(II)-induced oxidation of plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. Rabbit heparin plasma was diluted 150-fold by PBS and oxidized at 37°C in the presence of 50 mM Cu(II). Plasma oxidation kinetics were assessed as an increase in the absorbance of the samples at 234 nm. *P B0.05, **PB 0.01 versus corresponding value before feeding; §PB 0.05, §§PB 0.01 versus control group. Table 2 Extent of aortic atherosclerosis in WHHL rabbits fed with antioxidants for 12 monthsa
Intima thickness (mm) Media thickness (mm) Intima-to-media ratio
Control group (n = 10)
Ubiquinone-10-fed group (n= 10)
Vitamin E-fed group (n = 10)
Probucol-fed group (n = 10)
0.3499 0.100 0.2219 0.023 1.809 0.62
0.37590.109 0.215 9 0.027 1.9290.64
0.371 90.081 0.206 90.027 2.05 90.70
0.321 90.079 0.236 9 0.027 1.41 9 0.38*
a The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. Each feeding group consisted of ten animals. The extent of aortic atherosclerosis in rabbits was assessed after 12 month feeding and averaged for 14 cross sections equally distributed over the whole aorta. * PB0.05 versus control group.
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The plasma levels of the lipophilic antioxidant a-tocopherol increased during the study in all feeding groups. This was obviously due to the fact that this antioxidant was present in small amounts in the diet of all animals. In contrast, a-carotene, b-carotene and ubiquinol-10 tended to decrease throughout the study. Low values of b-carotene and ubiquinol-10, but not a-tocopherol have been previously reported in humans with hyperlipidaemia and coronary heart disease [24 – 26,40]. This may reflect either accelerated consumption of these antioxidants during lipoprotein oxidation in the arterial wall or their diminished supply in the diet. Since all the rabbits were fed with the same diet except for differences in added antioxidants, the latter possibility can be excluded. It could therefore be possible that some oxidants, such as hypochlorite [41], peroxynitrite [42] or nitric oxide [43], whose mechanisms of action are not yet fully characterized, might selectively deplete lipoprotein antioxidants in vivo. Moreover, plasma levels of a-carotene, ubiquinol-10 and g-tocopherol negatively correlated with the extent of atherosclerosis measured several months later. This might reflect the value of these lipophilic antioxidants, especially acarotene and g-tocopherol [26], as predictors of atherogenesis. Similarly to antioxidants, plasma PUFAs decreased in control rabbits during the study. Since PUFAs are major substrates of lipid peroxidation, it seems valid to assume, that in our study their consumption occurred in parallel to the progression of atherosclerosis. The decrease in the amount of PUFAs was concomitant with the decrease in plasma oxidizability by Cu(II) observed in all study groups. The PUFAs and the duration of the lag- and propagation phase of Cu(II)induced oxidation of plasma also revealed negative correlations with the extent of atherosclerosis. Thus, both of these parameters are related to the oxidative resistance of plasma in vivo. However, although correlations between each of the plasma parameters mentioned above and the extent of atherosclerosis reached significance, all of them were relatively weak. This suggests that in order to adequately assess the oxidation-related risk of atherosclerosis, several oxidation parameters should be measured. Supplementation of rabbit diets with ubiquinone-10 or vitamin E resulted in no measurable reduction of aortic atherosclerosis. Ubiquinone-10, in its reduced form, is an important lipophilic antioxidant in plasma [28,44]. It has been suggested to be effective against atherosclerosis [20], although no data is available about its antiatherosclerotic activity in animal models. Similarly, yet quantitatively more abundant is vitamin E, a well-known major lipophilic antioxidant in humans [45]. Beneficial effects of high levels of vitamin E intake on the risk of cardiovascular diseases have been frequently reported (see [20,46] for review). Vitamin E has
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also been shown to retard atherogenesis in animal models, when given at high doses [7–9]. However, no antiatherosclerotic effect has been found in animal studies performed with low doses of vitamin E corresponding to less than 1 g daily in humans [12,15]. On the other hand, vitamin E at low doses decreased atherosclerosis in hypercholesterolaemic hamsters [16,17]. In our study, both vitamin E and ubiquinone10 were given to WHHL rabbits at a dose of 4.3 mg/kg body weight which is equivalent to about 300 mg daily in humans. Our data show the inability of pharmacological doses of these antioxidants to inhibit atherosclerosis in this animal model. The inefficiency of low-dosed ubiquinone-10 as an antiatherosclerotic agent could be related to its insufficient accumulation in plasma lipoproteins. Indeed, no increase in plasma ubiquinol-10 was found following ubiquinone-10 supplementation. Therefore, it could be argued that the dose of ubiquinone-10 was not sufficient to increase stationary plasma concentration of ubiquinol-10 and, accordingly, its concentration in the aorta. This suggests that the rate of exogenous ubiquinol-10 supply may have been lower than the rate of its endogenous consumption. On the other hand, supplementation with ubiquinone-10 delayed a decrease in plasma PUFAs when measured after 3 and 6 months feeding. This indicates that a certain level of lipoprotein protection against oxidation was achieved after the supplementation with ubiquinone-10. However, this level was not high enough to significantly retard atherosclerosis. It seems that in order to achieve this protective effect, a threshold level must be reached. Interestingly, supplementation with ubiquinone-10 had a hypotriglyceridaemic effect in our study. This previously unknown activity might also be protective against atherogenesis. The same explanation (insufficient accumulation in plasma lipoproteins) might be true for vitamin E, since plasma a-tocopherol levels were only 35% higher in the vitamin E-supplemented compared to control rabbits after 6 months of feeding. Interestingly, supplementation with vitamin E resulted in the lowest plasma ubiquinol-10 values and vice versa. This suggests the existence of a coregulation mechanism in their metabolism and/or absorption, and highlights potential problems of supplementation with one alone. It appears that, for vitamin E and ubiquinone-10 to be effective in WHHL rabbits, they should be either higher dosed or given together in order to prevent their antagonistic decrease in plasma lipoproteins. Ubiquinol-10 is a coantioxidant for vitamin E and prevents it from becoming a prooxidant in vitro [47]. In the absence of coantioxidants vitamin E can develop a prooxidant activity [48,49]. This phenomenon may be related to the inefficiency of vitamin E in decreasing atherosclerosis in our study. This additionally argues for the combined
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supplementation with vitamin E and its coantioxidants, as has been recently proposed [46]. Differences in the antiatherosclerotic activity of vitamin E in animal models have been also ascribed to the differences in plasma levels of cholesterol. It has been shown that vitamin E becomes less efficient when plasma cholesterol increases [16]. Thus, the absence of the antiatherosclerotic action of ubiquinone-10 and vitamin E found by us, can be explained by the fact that WHHL rabbits represent a highly hypercholesterolaemic, compared to the doses of the antioxidants, model of atherosclerosis. In such hyperlipidaemic models, the proatherosclerotic role of increased plasma lipids can be expected to outweigh the antiatherosclerotic role of decreased lipoprotein oxidation, in comparison with their relationship at normal lipid levels. Probucol is another well-known antioxidant which has been shown to retard atherogenesis in animal models [4–6]. However, high doses of probucol corresponding to about 10–20 g of daily intake in humans have been typically used in these studies. Kleinveld et al. [12] found no antiatherosclerotic effect in the only animal study performed to date with a low dose of probucol corresponding to about 500 mg daily in humans. It is therefore possible that such low doses may be insufficient to inhibit atherogenesis in WHHL rabbits. The antiatherosclerotic effect of probucol has been previously reported to be associated with its antioxidative action. In our study, probucol accumulated in plasma and developed substantial antioxidant activity as shown by the reduced plasma oxidizability by Cu(II). Furthermore, it was able to prevent a decrease in plasma levels of ubiquinol-10, carotenes and PUFAs. In our study, the lag-phase of Cu(II)-induced plasma oxidation was prolonged by about 870% after feeding with 15 mg/kg body weight probucol. This effect is more pronounced than those reported by Kleinveld et al. [12] who used lower doses of probucol (about 7 mg/kg body weight daily) and observed a 52% prolongation. The action of probucol as an antiatherosclerotic agent has also been reported to be accompanied by its lipid-lowering activity [4 – 6]. However, no lipid-lowering effect of low-dosed probucol was observed in our study, which is in accordance with the data of others [12]. This indicates that probucol primarily influenced atherogenesis by an oxidation-related mechanism. However, it has been shown previously that the antiatherosclerotic activity of probucol does not always correlate with its antioxidative activity in plasma lipoproteins [50]. Other mechanisms, such as inhibition of interleukin 1 secretion [51,52] and decreased expression of adhesion molecules [53], might then be also involved. It seems reasonable to assume that in order to retard atherogenesis, probucol (or any other antioxidant) must be given at the early stage of disease, at a threshold dose and for a sufficient period of time. This assump-
tion is in line with the absence of the beneficial effect of probucol (about 7 mg/kg body weight daily for 3 years) observed in patients who already had femoral atherosclerosis at the beginning of the treatment [14]. A dose sufficient to achieve a protective effect in WHHL rabbits seems to be at least 15 mg/kg body weight, if the treatment is initiated at the early stage of disease i.e. at the age of 5 3 months, and is performed for at least a year. Too low dosage or too short duration of the treatment could explain negative results obtained by others for this animal model [12,50,54]. Under conditions of milder hyperlipidaemia (such as those typically observed in humans), lower doses may be efficient, if the treatment is started early enough. It can be concluded that in order to be efficient against atherosclerosis, an antioxidant must be given at a dose which is directly related to the level of hyperlipidaemia. In summary, our data indicate that a pharmacological dose of probucol retards atherogenesis in WHHL rabbits, most likely by an oxidation-related mechanism. In contrast, ubiquinone-10 and vitamin E given at a slightly lower dose are ineffective. Since the potential capacity of antioxidants in atherosclerosis can be underestimated in hyperlipidaemic animal models, the efficiency of low-dosed ubiquinone-10 and vitamin E should be further studied under normolipidaemic conditions. In addition, a group of oxidation-related parameters of plasma, such as lipophilic antioxidants, PUFAs and oxidizability, can be measured as biochemical markers to assess the risk of atherogenesis.
Acknowledgements We thank Mrs. Doris Arndt for excellent technical assistance and Dr. David Evans for critical reviewing of this manuscript. This study was supported by the grant Klinische Forschergruppe Gr 258/10-1, Deutsche Forschungsgemeinschaft.
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The effect of pharmacological doses of different antioxidants on oxidation parameters and atherogenesis in hyperlipidaemic rabbits Sirus Djahansouzi, Jan Hinrich Braesen, Kai Koenig, Ulrike Beisiegel, Anatol Kontush * Biochemisches Labor, Pa6. 39, Medizinische Kern- und Poliklinik, Uni6ersita¨tskrankenhaus Eppendorf, Martinistraße 52, 20246 Hamburg, Germany Received 30 November 1999; received in revised form 31 March 2000; accepted 7 April 2000
Abstract The oxidation hypothesis of atherosclerosis implies that antioxidants are able to inhibit lipoprotein oxidation in the arterial wall and thereby retard atherogenesis. Since most of the animal studies performed have used very high doses of antioxidants, it is to date unknown whether antioxidants are effective antiatherosclerotic agents when given in pharmacological doses. Here we addressed this question using homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits as an animal model of atherosclerosis. The rabbits were divided into four groups, each consisting of ten animals. They received either a standard diet or a diet containing 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. After 12 months, the extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio in 14 cross sections equally distributed over the whole aorta. To evaluate the antioxidant effects of the diet, lipophilic and hydrophilic antioxidants, lipids, fatty acids and plasma oxidizability were measured after 0, 3 and 6 months of feeding. We found that supplementation with probucol significantly decreased aortic intima-to-media ratio compared to controls. The antiatherosclerotic action of probucol was accompanied by its beneficial action on plasma oxidizability and some plasma antioxidants. No decrease in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits, despite the fact that both antioxidants decreased plasma oxidizability and ubiquinone-10 increased the plasma levels of antioxidants. Taken together, these data suggest that pharmacological doses of probucol retard atherogenesis in WHHL rabbits by an antioxidant mechanism, while ubiquinone-10 and vitamin E at these dosages are ineffective in this highly hyperlipidaemic model. The measurement of some oxidation-related parameters in plasma, such as lipophilic antioxidants, polyunsaturated fatty acids and lipoprotein oxidizability, may be useful in assessing the risk of atherogenesis in humans. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Lipoprotein oxidation; Antioxidants; Watanabe heritable hyperlipidaemic rabbits; Probucol; Vitamin E; Ubiquinone10
1. Introduction The oxidation hypothesis of atherosclerosis implies that the oxidation of low density lipoprotein (LDL) in the arterial wall plays an important role in atherogenesis [1,2]. Since LDL contains a variety of antioxidants able to inhibit its oxidation, increasing the antioxidant content of LDL should be able to retard atherogenesis. The antioxidant content of LDL can be easily increased by dietary supplementation [3]. Accordingly, supple* Corresponding author. Tel.: + 49 40 428034449; fax: +49 40 428034592. E-mail address: [email protected] (A. Kontush).
mentation with different antioxidants, such as probucol [4–6], vitamin E [7–9] and butylated hydroxytoluene (BHT) [10], retards atherogenesis in animal models, such as Watanabe heritable hyperlipidaemic (WHHL) [4–7] or cholesterol-fed [8–10] rabbits. However, to obtain these effects, all of these antioxidants were given in amounts clearly exceeding those typically used in humans [11]. In most studies, the antioxidant content of the rabbit diet was approximately 0.5 –1% wt/wt, corresponding to about 10–20 g of daily intake in humans (assuming a daily dietary intake of 100 g and a weight of 3.5 kg for rabbits and a weight of 70 kg for humans).
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 5 1 0 - 4
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Influence of pharmacological doses of antioxidants, i.e. those not exceeding 1 g daily in humans, has not yet been extensively studied in animal models. Supplementation of WHHL rabbits with 0.025% wt/wt (equivalent to about 500 mg daily in humans) of probucol or vitamin E for 6 months did not result in a statistically significant change in aortic atherosclerosis when compared with control animals [12]. A slightly higher dose of probucol (0.05% in a diet) also failed to reduce atherosclerosis in cholesterol-fed rabbits [13]. Supplementation of atherosclerotic patients with 500 mg probucol a day was also ineffective in retarding the development of femoral atherosclerosis [14]. Similarly, only a trend towards decreased atherosclerosis was found in cholesterol-fed rabbits supplemented with 10 mg vitamin E/kg body weight (equivalent to about 700 mg daily in humans) [15]. However, vitamin E at lower doses retarded aortic atherosclerosis in hypercholesterolaemic hamsters [16,17]. Differences in the antiatherosclerotic activity of vitamin E have been reported to be determined by plasma levels of cholesterol, with a lower activity at higher cholesterol levels [16]. The ability of vitamin E to affect atherogenesis has also been assessed in large-scale randomized trials in humans. In the CHAOS study, a reduced risk of cardiovascular death and non-fatal myocardial infarction was found when vitamin E was given at a dosage of 400 or 800 IU/day [18]. However, there was no beneficial effect on the rate of cardiovascular disease by supplementing vitamin E at a dose of 50 mg/day in the ATBC study [19]. In most prospective observational and nested case-control studies, a beneficial effect of high levels of dietary vitamin E intake on the risk of cardiovascular disease has been reported (see [20] for review). In contrast, ubiquinone-10, another potent lipophilic antioxidant, seemed to be effective in decreasing the rate of cardiovascular disease when given at a dose of 200 – 400 mg/day [21]. These data clearly indicate that the question of antiatherosclerotic efficiency of pharmacological, i.e. those appropriate to humans, doses of antioxidants remains equivocal. In the present study we investigated whether pharmacological doses of chemically different antioxidants influence atherogenesis under conditions of extreme hyperlipidaemia in homozygous WHHL rabbits. The rabbits were fed with a standard diet alone or a diet containing either 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily (equivalent to about 300 mg ubiquinone-10 and vitamin E and 1 g probucol in humans). After 12 months feeding, the extent of aortic atherosclerosis was assessed. To ensure efficient uptake of the antioxidants, their plasma concentrations were measured. Plasma levels of antioxidants and other oxidation-related parameters have been implicated as clinically important
markers for the development of atherosclerosis [22 –26]. To estimate the predictive value of these measurements and to evaluate antioxidant effects of the diet, lipophilic antioxidants, hydrophilic antioxidants, lipids, fatty acids and oxidizability in vitro were measured in plasma at the beginning of the study and after 3 and 6 months of supplementation.
2. Methods
2.1. Animals and diets Forty homozygous WHHL rabbits bred at the animal facilities of the University Hospital of Hamburg were divided into four groups of ten animals matched according to sex and litter at the age of 3 months. The control group was given an Altromin Standard Rabbit Diet 2110 (Altromin International, Lage, Germany) which contained 0.006% wt/wt vitamin E. The supplemented groups received the standard diet containing added antioxidants vitamin E, ubiquinone-10 or probucol. Antioxidants were added to the diet either by Altromin International (a-tocopherol and probucol) or as a solution in diethyl ether followed by rigorous mixing and overnight drying to remove the ether (ubiquinone10). The diets were given in a fashion such that the first portion of the daily food intake contained the body weight-adjusted dose of the supplement and the rest contained the standard chow (total 100 g). The ubiquinone-10-supplemented group received 4.3 mg/kg body weight ubiquinone-10 (HPH, Hamburg, Germany), the vitamin E-supplemented group 4.3 mg/kg body weight DL-a-tocopherol acetate (Sigma, Deisenhofen, Germany) and the probucol-supplemented group 15 mg/kg body weight probucol (Sigma, Deisenhofen, Germany) daily for 12 months (assuming an average rabbit body weight of 3.5 kg). The doses were chosen to approximately match pharmacological daily doses used in humans, i.e. 300 mg ubiquinone-10 and vitamin E and 1 g probucol. Dietary consumption and weight gain did not differ among the four groups of animals during the study period.
2.2. Blood sampling Fasting blood samples were obtained from the lateral ear vein at the start of the study and after 3 and 6 months of supplementation. Blood was taken into heparin- or ethylenediaminetetraacetic acid (EDTA)-containing tubes (15 IU heparin or 1.6 mg EDTA/ml blood; Sarstedt, Nu¨mbrecht, Germany). Plasma was obtained by centrifugation of the blood at 4°C for 10 min and stored at −80°C under nitrogen until assayed.
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2.3. Measurement of plasma parameters
2.4. Extent of aortic atherosclerosis
Plasma total cholesterol and triglycerides were determined by commercially available enzymatic kits (Boehringer-Mannheim, Mannheim, Germany). Lipophilic antioxidants a-tocopherol, g-tocopherol, a-carotene, b-carotene and ubiquinol-10 (in its reduced and oxidized form) were quantified in EDTA plasma samples by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection as described elsewhere [27,28]. Ubiquinol-9, ubiquinone-7 and d-tocopherol were used as internal standards. Plasma concentrations of lipophilic antioxidants were normalized to plasma lipids and expressed as pmol/(mg plasma cholesterol+triglycerides). Probucol was measured in EDTA plasma using HPLC with UV detection at 242 nm after extraction with diethyl ether as described elsewhere [29]. Total sulfhydryl (SH)-groups and bilirubin were measured as plasma hydrophilic antioxidants. SH-groups were determined spectrophotometrically at 412 nm after their reaction with dithionitrobenzene [30]. Bilirubin was measured at 530 nm after its reaction with ethyl anthranilate in dimethyl sulfoxide [31]. Fatty acid composition of plasma was characterized by capillary gas chromatography as described elsewhere [32]. Tricosanoic acid was used as an internal standard and t-butyl hydroxytoluene as an antioxidant. The total amount of fatty acids was calculated as the sum of polyunsaturated fatty acids (PUFAs, consisting of linoleic, linolenic, g-linolenic, eicosadienoic, eicosatrienoic, arachidonic, eicosapentaenoic, docosadienoic, docosatetraenoic, docosapentaenoic and docosahexaenoic acids), monounsaturated fatty acids (MUFAs, consisting of palmitoleic, oleic, vaccenic, eicosaenoic and docosaenoic acids) and saturated fatty acids (SFAs, consisting of myristic, palmitic, stearic, arachidic and behenic acids). Susceptibility of plasma lipoproteins to oxidation was characterized as in vitro oxidizability of heparin plasma [33]. To measure the oxidizablity, plasma samples (20 ml) were diluted 150-fold with phosphatebuffered saline (PBS), pH 7.4, containing 0.16 M NaCl. PBS was treated with Chelex 100 ion-exchange resin (Bio-Rad, Munich, Germany) to remove transition metal ions. PBS-diluted plasma was oxidized by CuSO4 (50 mM) or soybean lipoxygenase (25 U/ml). The oxidation was performed in a spectrophotometrical cuvette at 37°C and the change in sample absorbance at 234 nm was monitored. The absorbance was measured every 5 min over a period of 20 h. The level of conjugated dienes in plasma samples has been shown to correlate with other indices of lipid peroxidation and to be indicative of the oxidizability of plasma lipoproteins [33 – 35].
At the end of the 12-month study period, the rabbits were anesthesized through the lateral ear vein by a thiobarbituric acid derivate. This was followed by an intracardial exsanguination and overdose of the same derivate. Subsequently, a complete autopsy was carried out with special attention paid to quick dissection of the aorta from the left subclavian artery to the bifurcation and then the whole system of large and middle sized arteries, followed by other organ sampling. The aortas were cleaned of excess adventitial tissue, rinsed with saline buffer and quickly divided into 14 segments equally distributed over the aorta, each about 1 cm in length. The segments were alternatively asseverated in neutral buffered 4% formaldehyde for 12 h or flash frozen and kept in saline buffer for biochemical analyses. The seven formalin fixed aortic segments were routinely dehydrated and paraffinized and the endings were brought to the bottom of the resulting paraffin blocks after transverse bisection in an upright position, resulting in 14 cross sections equally distributed over the whole aorta. Serial sections were performed and stained histochemically according to routine methods and Masson-Goldner-ElasticaKossa combined stain as described elsewhere [36], as well as immunohistochemically with the monoclonal mouse antibodies HHF35 (Enzo Diagnostics, New York, USA) concentrated as delivered for smooth muscle cells, and RAM11 (Dako, Hamburg, Germany) diluted 1:50 for macrophages using the ABC-DAB peroxidase technique (Vector Laboratories, Burlingame, USA). Morphometrical analyses were carried out with computer-assisted measurements using a Zeiss unit supported by Kontron software KS400 (Zeiss/Kontron, Eching, Germany). The thickness of the intima and media were measured perpendicular to the internal elastic lamina at 0.5 mm intervals on all 14 cross sections by the same operator in a blinded fashion. The extent of atherosclerosis was assessed as the intima thickness, media thickness as well as the ratio of intima to media thickness (intimato-media ratio) for each of the 14 cross-sections and presented as means for all cross sections of each animal.
2.5. Statistical analysis Differences between groups fed with different diets were analyzed by Mann –Whitney U-test. Differences between different time-points within the same group were analyzed by Wilcoxon’s matched pairs test. Pearson correlation coefficients were calculated to evaluate relationships between variables. All results are presented as means9 SD.
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3. Results
3.1. Plasma parameters
3.1.1. Lipids As expected, WHHL rabbits had already developed severe hyperlipidaemia at the beginning of the study, i.e. at the age of 3 months. At this time-point, plasma total cholesterol and triglyceride levels reached 7579 71 and 260964 mg/dl, respectively (averaged for all 40 animals). Plasma cholesterol levels further increased significantly in all study groups after 3 months of supplementation and remained elevated during the following 3 months (Table 1). Plasma triglycerides increased significantly only in the vitamin E- and probucol-fed rabbits after 6 months of supplementation. Low significance of the differences in plasma lipid levels was partly due to their high variability within the same feeding group, as indicated by high S.D. values.
There was no significant difference in plasma cholesterol levels between the feeding groups at any of the three time-points studied, indicating that no cholesterol-lowering effect of probucol or vitamin E was observed. In contrast, plasma triglycerides were significantly lower in the ubiquinone-10-supplemented animals than in the controls after 3 months feeding.
3.1.2. Antioxidants a-Tocopherol, a major lipophilic antioxidant in rabbit plasma, increased during the first 3 months of the study in all feeding groups (Fig. 1). The increase in plasma a-tocopherol was not associated with the increase in plasma lipids, since the effect was seen when a-tocopherol concentration was normalized to the lipids. After both 3 and 6 months of supplementation, a-tocopherol levels remained significantly elevated in all groups, except ubiquinone-10-fed rabbits who had significantly less a-tocopherol in their plasma compared with the controls, after 6 months. As expected, the
Table 1 Increase in total cholesterol and triglyceride levels in plasma of WHHL rabbits fed with antioxidants for 3 and 6 monthsa Group
Control Ubiquinone-10 Vitamin E Probucol
Increase in total cholesterol (% of the initial level)
Increase in triglycerides (% of the initial level)
0–3 months
0–6 months
0–3 months
0–6 months
15.69 10.5* 15.89 10.1* 12.999.7* 7.498.9*
3.6 9 26.5 8.0 9 14.1 3.1 915.4 1.89 14.0
17.8 942.6 −13.2919.6§ 13.9 9 38.4 24.1 941.4
35.0 958.1 7.1 954.6 31.7 9 35.7* 42.7 950.5*
a The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. Each feeding group consisted of ten animals. * PB0.05 versus corresponding value of plasma cholesterol before feeding. § PB0.05 versus control group.
Fig. 1. a-Tocopherol in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. a-Tocopherol was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *PB0.05, **PB 0.01 versus corresponding value before feeding; §PB 0.05 versus control group.
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Fig. 2. b-Carotene in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. b-Carotene was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *P B0.05, **PB 0.01 versus corresponding value before feeding; §P B0.05, §§PB 0.01 versus control group.
Fig. 3. Ubiquinol-10 in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. Ubiquinol-10 was measured in EDTA plasma samples using reversed-phase HPLC with electrochemical detection. *PB 0.05 versus corresponding value before feeding; §P B0.05 versus control group.
highest levels of a-tocopherol were measured in the group supplemented with vitamin E. In contrast to a-tocopherol, plasma g-tocopherol did not reveal a clear tendency towards increased levels during the study. Moreover, plasma levels of g-tocopherol were significantly lower in ubiquinone-10- and vitamin E-fed animals when compared to controls after 6 months (data not shown). In contrast to tocopherols, plasma levels of both aand b-carotene tended to decrease throughout the study. b-Carotene significantly decreased in the control and ubiquinone-10 group after 6 months of supplementation (Fig. 2). a-Carotene behaved similarly, showing a significant decrease in the control animals after 6
months (data not shown). Interestingly, no significant reduction of b-carotene was observed in the probucolfed group. The vitamin E-fed rabbits showed only a trend towards lower b-carotene levels. Similarly to carotenes, plasma levels of ubiquinol-10 tended to decrease during the study. Significant decrease in ubiquinol-10 levels was observed in the control group after 3 months of supplementation (Fig. 3). The vitamin E-fed group also showed significantly lower concentrations of ubiquinol-10 after both 3 and 6 months. The decline of ubiquinol-10 in the vitamin E-fed rabbits was so pronounced that its level in this group was significantly lower than that measured in the control animals after 6 months. On the other hand,
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feeding with probucol seemed to be able to partially prevent a decrease in ubiquinol-10, since it kept the plasma level of this antioxidant significantly higher when compared to controls after 3 months. Supplementation with ubiquinone-10 also prevented a decrease in plasma levels of ubiquinol-10. In fact, a slight increase in plasma ubiquinol-10 was observed in this group. Plasma levels of ubiquinone-10, an oxidation product of ubiquinol-10, showed a between-group distribution which approximately mirrored that measured for ubiquinol-10 (data not shown). Plasma levels of hydrophilic antioxidants SH-groups and bilirubin were much less dependent on the antioxidant supplementation than those of their lipophilic counterparts. At the beginning of the study, total plasma SH-groups and bilirubin were 3589 67 and 46.0 97.8 mM, respectively (averaged for all 40 animals). Throughout the study, these levels did not significantly change in any of the groups (data not shown), except that SH-groups increased by 14.7% (P =0.02) in the vitamin E-fed rabbits after 3 months. Nor was there any significant difference found in either SH-groups or bilirubin levels between the feeding groups at any of the three time-points studied (data not shown). Supplementation with probucol resulted in its accumulation in rabbit plasma to a level of 7.339 3.77 and 9.3194.96 mM after 3 and 6 month feeding, respectively.
3.1.3. Fatty acids Hyperlipidaemic WHHL rabbits had higher plasma levels of fatty acids than those typically measured in normolipidaemia [37]. Total PUFAs reached 1419 20, MUFAs 63 913 and SFAs 85912 mg/dl at the begin-
ning of the study (averaged for all 40 animals). These levels did not significantly change in any of the rabbit groups throughout the study (data not shown). However, when individual fatty acids were expressed as a percentage of total fatty acids, significant effects became evident (Fig. 4). In the control and vitamin E-fed animals, PUFAs significantly decreased after 6 months of supplementation. When PUFAs were subdivided into their n-3 and n-6 constituents, it was found that n-6 PUFAs made up about 97% of total PUFAs in rabbit plasma and were mainly responsible for the decrease in total PUFAs (data not shown). No significant difference in plasma fatty acids were found between the feeding groups at any of the three time-points studied, independent of how fatty acids were expressed (data not shown).
3.1.4. Oxidizability In accordance with data previously reported for human plasma [33], the absorbance increase of diluted rabbit plasma oxidized with 50 mM Cu(II) was characterized by three consecutive phases, namely the lag-, propagation and decomposition phases of oxidation (Fig. 5). To quantitatively describe plasma oxidizability, the lag-phase and propagation-phase duration as well as the maximum rate of oxidation in the propagation-phase were calculated [38]. It was found that in all feeding groups, the lag-phase (Fig. 6A) and propagation phase (data not shown) were prolonged during the study and the maximum rate of oxidation within the propagation phase (Fig. 6B) was decreased. For the control animals, these findings correlated with the increase in plasma a-tocopherol (r= 0.39 and r= 0.40, P= 0.03 and 0.02, for the lag-phase and propagation phase, respectively).
Fig. 4. PUFAs in plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. PUFAs were measured using capillary gas chromatography. *PB 0.05 versus corresponding value before feeding.
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Fig. 5. Kinetics of Cu(II)-induced oxidation of plasma of WHHL rabbits fed with antioxidants for 6 months. The curves with the median value of the lag-phase duration are shown for each feeding group. The numbers at the curves denote the lag-phase duration (min). The rabbits were fed as described in Fig. 1. Rabbit heparin plasma was diluted 150-fold by PBS and oxidized at 37°C in the presence of 50 mM Cu(II). Plasma oxidation kinetics were assessed as an increase in the absorbance of the samples at 234 nm.
The changes in the plasma oxidizability parameters were more pronounced in the antioxidant-fed, than in control, animals. Supplementation with probucol or, less pronounced, vitamin E resulted in the significantly longer lag- and propagation phases and lower maximum rates of oxidation compared with controls at the same time-points. The decrease in plasma oxidizability observed in the ubiquinone-10-fed animals did not reach significance when compared with control animals. It may therefore be concluded that the activity of antioxidants towards plasma oxidation by Cu(II) decreased in the order probucol\ vitamin E\ ubiquinone-10. In contrast to Cu(II)-induced oxidation, oxidation catalyzed by lipoxygenase was much less affected by the antioxidant supplementation. The plasma oxidation rate was found to be significantly lower only in ubiquinone-10 and vitamin E-fed animals than in controls, when measured after 6 months of supplementation (data not shown).
3.2. Extent of aortic atherosclerosis Extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio averaged for 14 cross sections equally distributed over the whole aorta. Supplementation with probucol for 12 months was found to significantly delay atherogenesis as shown by the lower intima-to-media ratio (Table 2). No reduction in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits. To estimate the potential predictive value of oxidation parameter measurements in plasma for the devel-
opment of atherosclerosis, the oxidation values measured after 0, 3 and 6 months were correlated with the extent of atherosclerosis measured after 12 months. Plasma levels of probucol, ubiquinol-10, g-tocopherol, a-carotene, PUFAs as well as the duration of the lag-phase and propagation phase of Cu(II)-induced oxidation were found to negatively correlate with the intima-to-media ratio. PUFAs revealed significant correlations when measured at the beginning of the study (r= − 0.35, PB0.05), probucol (r= −0.65, PB0.05) and ubiquinol-10 (r= −0.37, P B0.05) when measured after 3 months, g-tocopherol (r= − 0.44, PB 0.01) and a-carotene (r= − 0.37, P B 0.05) when measured after 6 months. Duration of the lag-phase and propagation phase of Cu(II)-induced oxidation negatively correlated with the intima-to-media ratio when measured both after 3 months (r= − 0.31, PB 0.05, and r= −0.38, PB 0.05, respectively) and 6 months (r= −0.31, PB 0.05, and r= −0.44, P B 0.01, respectively) of the study. All of these parameters, except ubiquinol-10, also revealed significant negative correlations with the intima thickness (data not shown).
4. Discussion Three chemically different antioxidants, namely probucol (15 mg/kg body weight), ubiquinone-10 and vitamin E (both 4.3 mg/kg body weight), were examined for their ability to influence oxidation parameters and prevent atherosclerosis in homozygous WHHL rabbits, when administered at these pharmacological doses.
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Plasma cholesterol levels increased significantly in all animals during the study. This reflects the expected hypercholesterolaemia in WHHL rabbits and is in accordance with the data of others [39]. The high
levels of cholesterol in WHHL rabbits induce the development of severe atherosclerosis. For their part, triglyceride levels did not show a clear tendency to increase.
Fig. 6. Duration of the lag-phase and maximal oxidation rate measured within the propagation phase of Cu(II)-induced oxidation of plasma of WHHL rabbits fed with antioxidants for 3 and 6 months. The rabbits were fed as described in Fig. 1. Rabbit heparin plasma was diluted 150-fold by PBS and oxidized at 37°C in the presence of 50 mM Cu(II). Plasma oxidation kinetics were assessed as an increase in the absorbance of the samples at 234 nm. *P B0.05, **PB 0.01 versus corresponding value before feeding; §PB 0.05, §§PB 0.01 versus control group. Table 2 Extent of aortic atherosclerosis in WHHL rabbits fed with antioxidants for 12 monthsa
Intima thickness (mm) Media thickness (mm) Intima-to-media ratio
Control group (n = 10)
Ubiquinone-10-fed group (n= 10)
Vitamin E-fed group (n = 10)
Probucol-fed group (n = 10)
0.3499 0.100 0.2219 0.023 1.809 0.62
0.37590.109 0.215 9 0.027 1.9290.64
0.371 90.081 0.206 90.027 2.05 90.70
0.321 90.079 0.236 9 0.027 1.41 9 0.38*
a The rabbits were fed with a standard diet or a diet containing 4.3 mg ubiquinone-10, 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. Each feeding group consisted of ten animals. The extent of aortic atherosclerosis in rabbits was assessed after 12 month feeding and averaged for 14 cross sections equally distributed over the whole aorta. * PB0.05 versus control group.
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The plasma levels of the lipophilic antioxidant a-tocopherol increased during the study in all feeding groups. This was obviously due to the fact that this antioxidant was present in small amounts in the diet of all animals. In contrast, a-carotene, b-carotene and ubiquinol-10 tended to decrease throughout the study. Low values of b-carotene and ubiquinol-10, but not a-tocopherol have been previously reported in humans with hyperlipidaemia and coronary heart disease [24 – 26,40]. This may reflect either accelerated consumption of these antioxidants during lipoprotein oxidation in the arterial wall or their diminished supply in the diet. Since all the rabbits were fed with the same diet except for differences in added antioxidants, the latter possibility can be excluded. It could therefore be possible that some oxidants, such as hypochlorite [41], peroxynitrite [42] or nitric oxide [43], whose mechanisms of action are not yet fully characterized, might selectively deplete lipoprotein antioxidants in vivo. Moreover, plasma levels of a-carotene, ubiquinol-10 and g-tocopherol negatively correlated with the extent of atherosclerosis measured several months later. This might reflect the value of these lipophilic antioxidants, especially acarotene and g-tocopherol [26], as predictors of atherogenesis. Similarly to antioxidants, plasma PUFAs decreased in control rabbits during the study. Since PUFAs are major substrates of lipid peroxidation, it seems valid to assume, that in our study their consumption occurred in parallel to the progression of atherosclerosis. The decrease in the amount of PUFAs was concomitant with the decrease in plasma oxidizability by Cu(II) observed in all study groups. The PUFAs and the duration of the lag- and propagation phase of Cu(II)induced oxidation of plasma also revealed negative correlations with the extent of atherosclerosis. Thus, both of these parameters are related to the oxidative resistance of plasma in vivo. However, although correlations between each of the plasma parameters mentioned above and the extent of atherosclerosis reached significance, all of them were relatively weak. This suggests that in order to adequately assess the oxidation-related risk of atherosclerosis, several oxidation parameters should be measured. Supplementation of rabbit diets with ubiquinone-10 or vitamin E resulted in no measurable reduction of aortic atherosclerosis. Ubiquinone-10, in its reduced form, is an important lipophilic antioxidant in plasma [28,44]. It has been suggested to be effective against atherosclerosis [20], although no data is available about its antiatherosclerotic activity in animal models. Similarly, yet quantitatively more abundant is vitamin E, a well-known major lipophilic antioxidant in humans [45]. Beneficial effects of high levels of vitamin E intake on the risk of cardiovascular diseases have been frequently reported (see [20,46] for review). Vitamin E has
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also been shown to retard atherogenesis in animal models, when given at high doses [7–9]. However, no antiatherosclerotic effect has been found in animal studies performed with low doses of vitamin E corresponding to less than 1 g daily in humans [12,15]. On the other hand, vitamin E at low doses decreased atherosclerosis in hypercholesterolaemic hamsters [16,17]. In our study, both vitamin E and ubiquinone10 were given to WHHL rabbits at a dose of 4.3 mg/kg body weight which is equivalent to about 300 mg daily in humans. Our data show the inability of pharmacological doses of these antioxidants to inhibit atherosclerosis in this animal model. The inefficiency of low-dosed ubiquinone-10 as an antiatherosclerotic agent could be related to its insufficient accumulation in plasma lipoproteins. Indeed, no increase in plasma ubiquinol-10 was found following ubiquinone-10 supplementation. Therefore, it could be argued that the dose of ubiquinone-10 was not sufficient to increase stationary plasma concentration of ubiquinol-10 and, accordingly, its concentration in the aorta. This suggests that the rate of exogenous ubiquinol-10 supply may have been lower than the rate of its endogenous consumption. On the other hand, supplementation with ubiquinone-10 delayed a decrease in plasma PUFAs when measured after 3 and 6 months feeding. This indicates that a certain level of lipoprotein protection against oxidation was achieved after the supplementation with ubiquinone-10. However, this level was not high enough to significantly retard atherosclerosis. It seems that in order to achieve this protective effect, a threshold level must be reached. Interestingly, supplementation with ubiquinone-10 had a hypotriglyceridaemic effect in our study. This previously unknown activity might also be protective against atherogenesis. The same explanation (insufficient accumulation in plasma lipoproteins) might be true for vitamin E, since plasma a-tocopherol levels were only 35% higher in the vitamin E-supplemented compared to control rabbits after 6 months of feeding. Interestingly, supplementation with vitamin E resulted in the lowest plasma ubiquinol-10 values and vice versa. This suggests the existence of a coregulation mechanism in their metabolism and/or absorption, and highlights potential problems of supplementation with one alone. It appears that, for vitamin E and ubiquinone-10 to be effective in WHHL rabbits, they should be either higher dosed or given together in order to prevent their antagonistic decrease in plasma lipoproteins. Ubiquinol-10 is a coantioxidant for vitamin E and prevents it from becoming a prooxidant in vitro [47]. In the absence of coantioxidants vitamin E can develop a prooxidant activity [48,49]. This phenomenon may be related to the inefficiency of vitamin E in decreasing atherosclerosis in our study. This additionally argues for the combined
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supplementation with vitamin E and its coantioxidants, as has been recently proposed [46]. Differences in the antiatherosclerotic activity of vitamin E in animal models have been also ascribed to the differences in plasma levels of cholesterol. It has been shown that vitamin E becomes less efficient when plasma cholesterol increases [16]. Thus, the absence of the antiatherosclerotic action of ubiquinone-10 and vitamin E found by us, can be explained by the fact that WHHL rabbits represent a highly hypercholesterolaemic, compared to the doses of the antioxidants, model of atherosclerosis. In such hyperlipidaemic models, the proatherosclerotic role of increased plasma lipids can be expected to outweigh the antiatherosclerotic role of decreased lipoprotein oxidation, in comparison with their relationship at normal lipid levels. Probucol is another well-known antioxidant which has been shown to retard atherogenesis in animal models [4–6]. However, high doses of probucol corresponding to about 10–20 g of daily intake in humans have been typically used in these studies. Kleinveld et al. [12] found no antiatherosclerotic effect in the only animal study performed to date with a low dose of probucol corresponding to about 500 mg daily in humans. It is therefore possible that such low doses may be insufficient to inhibit atherogenesis in WHHL rabbits. The antiatherosclerotic effect of probucol has been previously reported to be associated with its antioxidative action. In our study, probucol accumulated in plasma and developed substantial antioxidant activity as shown by the reduced plasma oxidizability by Cu(II). Furthermore, it was able to prevent a decrease in plasma levels of ubiquinol-10, carotenes and PUFAs. In our study, the lag-phase of Cu(II)-induced plasma oxidation was prolonged by about 870% after feeding with 15 mg/kg body weight probucol. This effect is more pronounced than those reported by Kleinveld et al. [12] who used lower doses of probucol (about 7 mg/kg body weight daily) and observed a 52% prolongation. The action of probucol as an antiatherosclerotic agent has also been reported to be accompanied by its lipid-lowering activity [4 – 6]. However, no lipid-lowering effect of low-dosed probucol was observed in our study, which is in accordance with the data of others [12]. This indicates that probucol primarily influenced atherogenesis by an oxidation-related mechanism. However, it has been shown previously that the antiatherosclerotic activity of probucol does not always correlate with its antioxidative activity in plasma lipoproteins [50]. Other mechanisms, such as inhibition of interleukin 1 secretion [51,52] and decreased expression of adhesion molecules [53], might then be also involved. It seems reasonable to assume that in order to retard atherogenesis, probucol (or any other antioxidant) must be given at the early stage of disease, at a threshold dose and for a sufficient period of time. This assump-
tion is in line with the absence of the beneficial effect of probucol (about 7 mg/kg body weight daily for 3 years) observed in patients who already had femoral atherosclerosis at the beginning of the treatment [14]. A dose sufficient to achieve a protective effect in WHHL rabbits seems to be at least 15 mg/kg body weight, if the treatment is initiated at the early stage of disease i.e. at the age of 5 3 months, and is performed for at least a year. Too low dosage or too short duration of the treatment could explain negative results obtained by others for this animal model [12,50,54]. Under conditions of milder hyperlipidaemia (such as those typically observed in humans), lower doses may be efficient, if the treatment is started early enough. It can be concluded that in order to be efficient against atherosclerosis, an antioxidant must be given at a dose which is directly related to the level of hyperlipidaemia. In summary, our data indicate that a pharmacological dose of probucol retards atherogenesis in WHHL rabbits, most likely by an oxidation-related mechanism. In contrast, ubiquinone-10 and vitamin E given at a slightly lower dose are ineffective. Since the potential capacity of antioxidants in atherosclerosis can be underestimated in hyperlipidaemic animal models, the efficiency of low-dosed ubiquinone-10 and vitamin E should be further studied under normolipidaemic conditions. In addition, a group of oxidation-related parameters of plasma, such as lipophilic antioxidants, PUFAs and oxidizability, can be measured as biochemical markers to assess the risk of atherogenesis.
Acknowledgements We thank Mrs. Doris Arndt for excellent technical assistance and Dr. David Evans for critical reviewing of this manuscript. This study was supported by the grant Klinische Forschergruppe Gr 258/10-1, Deutsche Forschungsgemeinschaft.
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