Abstract
The present study investigated the effects of antioxidant vitamins along with atorvastatin and atorvastatinniacin combination on diet-induced hypercholesterolemia in rats. High cholesterol diet produced a significant increase in the serum total cholesterol, LDL-C, VLDL-C, TG, atherogenic index and decrease in HDL-C and HDL/LDL ratio. The lipid peroxidation and oxidative stress were significantly high in the hyperlipidemic control group. Atorvastatin improved atherogenic index but not the HDL/LDL ratio whereas atorvastatin-niacin combination improved both atherogenic index and HDL/LDL ratio. However, both atorvastatin and atorvastatin-niacin did not affect antioxidant status significantly. Co-administration of vitamin-E and vitamin-C along with atorvastatin and atorvastatin-niacin have improved serum lipid profile, prevented lipid peroxidation and improved antioxidant status. Addition of β-carotene along with lipid lowering drugs did not show additional benefits on serum lipid profile, lipid peroxidation and antioxidant status. Atorvastatin, atorvastatin-niacin combination when added with anti-oxidant vitamins, increased reduced glutathione level but did not affect MDA level, SOD and catalase activity in the liver tissue. Administration of both vitamin-E and vitamin-C along with atorvastatin-niacin therapy produced a significant improvement in the lipid profile as well as antioxidant status. Addition of β-carotene along with atorvastatin-niacin-vitamin-E-vitamin-C combination improved lipid profile but improvement was not as marked as observed with atorvastatin-niacin-vitamin-E-vitamin-C combination. The same beneficial effects of atorvastatin-niacin combination on lipid profile were not observed when it was combined with anti-oxidant vitamins especially β-carotene. The pro-oxidant role of β-carotene may be responsible for this effect
Keywords: hypercholesterolemia, lipid peroxidation, antioxidants, vitamin-E, vitamin-C, atorvastatin
Introduction
Hypercholesterolemia is one of the major risk factors for coronary artery disease (CAD) and atherosclerosis. It is the LDL that plays a crucial role in the atherogenesis [1]. It is the oxidative modification that imparts an atherogenecity to LDL [2]. Fatty streaks develop in response to specific phospholipids contained in LDL that become oxidized as a result of exposure to the oxidative waste of the artery wall cells [3, 4]. It has been shown that lipid peroxidation is involved in the oxidative modification of LDL [5, 6]. The lipid peroxidation starts only after the depletion of natural antioxidants such as vitamin-E, vitamin-C, β-carotene, etc. in the body [7]. This was supported by the fact that the low serum levels of antioxidant vitamins are associated with high risk of CAD [8, 9]. Antioxidant vitamins prevent lipid peroxidation both in vivo and in vitro [10, 11]. Numerous epidemiological evidences support the beneficial role of the dietary antioxidant vitamins [12-14]. However some studies have questioned the beneficial role of antioxidant vitamins [15, 16]. Atorvas-tatin-niacin therapy improves lipid profile but has no significant effects, if any, on antioxidants status. It has been observed that co-administration of antioxidants with atorvastatinniacin therapy suppress the response of such therapy to HDL-C [17]. Hence, the present work was undertaken with the aim to study the effects of anti-oxidant vitamins in combination with atorvastatin and atorvastatin-niacin on diet -induced hyperlipidemia in rats.
Material and methods
Animals
Healthy rats (Sprague-Dawley strain) of either sex weighing 180-220 g were divided into different groups each of six (Table 1). Normal group received standard pellet diet (Pranav agro industries, Vadodara, India). All other groups received a high cholesterol diet along with respective treatments. Animals were treated for seven days. Water was made available ad libitum. The study was approved by the institutional animal ethics committee established in accordance with committee for the purpose of supervision and control of experiments on animals (CPCSEA) [18].
Table 1.
Animal groups and respective treatments.
| Animal groups | Treatments |
|---|---|
| Group 1 | Normal (Vehicle only) |
| Group 2 | Hyperlipidemic control |
| Group 3 | Atorvastatin |
| Group 4 | Atorvastatin + vitamin-E |
| Group 5 | Atorvastatin + vitamin-C |
| Group 6 | Atorvastatin + β-carotene |
| Group 7 | Atorvastatin + vitamin-E + vitamin-C |
| Group 8 | Atorvastatin + β-carotene + vitamin-C |
| Group 9 | Atorvastatin + β-carotene + vitamin-E |
| Group 10 | Atorvastatin + Niacin |
| Group 11 | Atorvastatin + Niacin + vitamin-E |
| Group 12 | Atorvastatin + Niacin + β-carotene |
| Group 13 | Atorvastatin + Niacin + vitamin-E + vitamin-C |
| Group 14 | Atorvastatin + Niacin + β-carotene + vitamin-E + vitamin-C |
Drugs administration
Atorvastatin (1.4 mg/kg, as suspension in 1% CMC), niacin (250 mg/kg, as solution in distilled water), vitamin-E (60 mg/kg, as solution in olive oil), vitamin-C (80 mg/kg, as solution in distilled water) and β-carotene (40 mg/kg, as solution in arachis oil) were administered orally by gavage, once in a day in the morning (9.00 to 10.00 a.m.) to respective groups for 7 days.
Diet-induced hyperlipidemia
Method of Blank [19] with modification was used to produce diet-induced hyperlipidemia. Briefly, normal group received standard chew diet and all other groups received high cholesterol diet consisting of – standard Pellet diet 92%, cholesterol 2.0 %, cholic acid 1 % and coconut oil 5% for seven days. The standard pellet diet (Pranav Agro Industries, Vadodara.) consisted of crude protein (22.06%), crude oil (4.04%), crude fiber (4.0%), Ash (10.0%) and sand silica (0.15%). The standard pellet diet supplies energy of 3620 Kcal/kg.
Reagents and chemicals
Cholesterol, sodium cholate, vitamin-E, vitamin-C, niacin were purchased from Kemphasol, Bombay; β-carotene was purchased from HiMedia Laboratory Ltd, Bombay. Atorvastatin was received as gift sample from Zydus research centre, Ahmedabad, India. All other chemicals and reagents were of analytical grade.
Blood collection and biochemical estimation
The animals were treated for 7 days. At the end of experimental period, the rats in each group were deprived of food overnight but not the water and sacrificed. The blood was collected by retro-orbital puncture technique and serum was separated. The serum total cholesterol (TC), triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) were estimated using commercially available kits (Bayer diagnostic (P) Ltd. India). Very low density lipoprotein-cholesterol (VLDL-C) was calculated as TG/5. Low density lipoprotein-cholesterol (LDL-C) levels were calculated using Friedewald′s formula [20]. The Atherogenic index was calculated using formula – Atherogenic Index (AI) = (VLDL-C + LDL-C)/ HDL-C. The liver tissues were collected, washed thoroughly in normal saline, bloated and preserved at -40°C for further analysis. The liver homogenates were prepared in trishydrochloride buffer. They were subjected to protein [21], malondialdehyde (MDA) [22], superoxide dismutase (SOD) [23], catalase [24], and reduced glutathione (GSH) [25] estimation.
Statistical analysis
All values were expressed as Mean ± SEM. The statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tuckey's multivarient test. The value of p less than 5% (p< 0.05) was considered statistically significant.
Results
There were no significant differences in food intake among various groups. There was significant increase in serum TC, TG, LDL-C, VLDL-C in hyperlipidemic control group compared to normal group. The HDL-C as well as HDL to LDL ratios was significantly decreased in hyperlipidemic control. This was evidenced by increased levels of atherogenic index. Atorvastatin significantly reduced serum TC, TG, LDL-C and VLDL-C levels compared with the hyperlipidemic control and the atherogenic index declined significantly. The combination of atorvastatin with vitamin-E and vitamin-C showed significant beneficial effects on serum lipid profile. Only this combination showed additional benefits as compared with statin alone. When β-carotene was added to this combination, the beneficial effects of vitamin-E and C disappeared (Table 2).
Table 2.
Effects of various treatments on serum lipid profile of diet-induced hyperlipidemic rats.
| Groups (n=6) | TC (mg/dl) | TG (mg/dl) | HDL-C (mg/dl) | LDL-C (mg/dl) | VLDL-C (mg/dl) | HDL/LDL Ratio | Atherogenic Index |
|---|---|---|---|---|---|---|---|
| Group 1 | 64.55 ± 2.99 | 57.49 ± 3.55 | 24.71 ± 1.39 | 28.33 ± 2.11 | 11.51 ± 0.71 | 0.87 ± 0.05 | 1.61 ± 0.20 |
| Group 2 | 308.74 ± 30.62* | 121.34 ± 16.93* | 10.20 ± 1.70* | 276.31 ± 29.77* | 24.27 ± 3.50* | 0.04 ± 0.01* | 29.47 ± 1.96* |
| Group 3 | 164.60 ± 9.89** | 72.65 ± 4.12** | 12.72 ± 1.33 | 135.50 ± 2.51** | 14.53 ± 0.82** | 0.09 ± 0.04 | 11.79 ± 0.25** |
| Group 4 | 132.32 ± 7.04** | 94.06 ± 4.79 | 19.22 ± 2.10 | 89.29 ± 6.31** | 15.80 ± 0.95 | 0.22 ± 0.02 | 5.47 ± 0.35**‡ |
| Group 5 | 143.34 ± 6.78** | 107.58 ± 14.33 | 17.65 ± 1.65 | 104.18 ± 6.62** | 21.51 ± 1.39 | 0.17 ± 0.02 | 7.12 ± 0.49**‡ |
| Group 6 | 199.10 ± 20.92** | 92.24 ± 17.17 | 10.51 ± 2.89 | 170.14 ± 24.00** | 18.44 ± 3.34 | 0.06 ± 0.03 | 17.94 ± 0.95** |
| Group 7 | 62.45 ± 2.01**‡ | 67.05 ± 3.15** | 25.98 ± 1.12** | 23.06 ± 1.87**‡ | 13.41 ± 0.79** | 1.13 ± 0.05** | 1.40 ± 0.24**‡ |
| Group 8 | 152.29 ± 16.33** | 57.03 ± 9.03** | 24.86 ± 3.46** | 91.16 ± 12.60** | 11.41 ± 1.86** | 0.27 ± 0.11 | 4.13 ± 0.42**‡ |
| Group 9 | 173.49 ± 14.13** | 65.34 ± 6.25** | 22.23 ± 4.40** | 115.56 ± 43.57** | 13.07 ± 1.25** | 0.19 ± 0.39 | 5.79 ± 1.02**‡ |
| Group 10 | 93.04 ± 5.38** | 95.61 ± 1.43 | 16.17 ± 1.36 | 57.74 ± 6.44** | 19.12 ± 0.22 | 0.28 ± 0.05** | 4.75 ± 0.49**‡ |
| Group 11 | 94.53 ± 1.96** | 69.36 ± 3.74** | 23.07 ± 1.33** | 57.06 ± 2.51** | 14.40 ± 0.78** | 0.40 ± 0.04** | 3.10 ± 0.25**‡ |
| Group 12 | 200.82 ± 41.42 | 94.91 ± 14.33 | 20.14 ± 5.74 | 161.69 ± 42.29* | 18.98 ± 0.87 | 0.12 ± 0.07 | 8.97 ± 0.75** |
| Group 13 | 63.53 ± 1.87**† | 68.04 ± 3.93** | 25.80 ± 1.02** | 24.09 ± 1.47** | 13.60 ± 0.78** | 1.07 ± 0.08** | 1.46 ± 0.22**‡ |
| Group 14 | 94.86 ± 4.33** | 71.69 ± 3.68** | 19.28 ± 0.55 | 65.16 ± 3.57** | 14.35 ± 0.82** | 0.30 ± 0.03 | 4.12 ± 0.80**‡ |
All values represent Mean ± SEM from six rats. Statistical analysis was carried out using One Way ANOVA followed by Tukey's test. The value of P < 0.05 was considered statistically significant.
compare with control group
compared with the hyperlipidemic control group
compared with the atorvastatin
compared with all other groups.
Compared to atorvastatin alone, the atorvas-tatin-niacin significantly improved serum lipid profile. When this combination was supplemented with vitamin-E and C, maximum improvement in serum lipid profile was observed. However, when β-carotene was added to this combination, no additional benefits were observed, in fact increase in the atherogenic index was found. The atorvastatinniacin with β-carotene decreased the beneficial effects of atorvastatin-niacin on serum lipid profile (Table 2).
MDA is a marker of lipid-peroxidation. There was significant lipid-peroxidation in hyperlipidemic control group as indicated by increased MDA levels compared with normal group (Table 3). Atorvastatin alone and in combinations with different vitamins significantly reduced liver lipidperoxidation. Atorvastatin–vitamin-E–vitamin-C combinations showed highest inhibition of lipidperoxidation. Atorvastatin-niacin combination significantly reduced lipid peroxidation when compared with hyperlipidemic control group (Table 3). However, atorvastatin produced significant inhibition of lipid peroxidation compared with atorvastatin-niacin combination. Atorvas-tatin-niacin combination when combined with different anti-oxidant vitamins did not show any additional benefits. The atorvastatin-niacin-β-carotene has significantly reduced lipid peroxidation compared with both atorvastatin alone and atorvastatin-niacin.
Table 3.
Effects of various treatments on the lipid peroxidation and the anti-oxidants status of hyperlipidemic rat livers.
| Groups (n=6) | MDA (nmol/mg protein) | SOD (U/min/mg protein) | Catalase (U/min/mg protein) | GSH × 10-3 (μg/mg protein) |
|---|---|---|---|---|
| Group 1 | 1.85 ± 0.18 | 6.13 ± 0.67 | 167.20 ±14.00 | 933.00 ± 60.80 |
| Group 2 | 45.70 ± 18.40* | 13.98 ± 0.87* | 3.60 ± 0.10* | 357.60 ± 12.46* |
| Group 3 | 3.62 ± 0.08** | 15.15 ± 0.46 | 4.50 ± 0.63 | 458.40 ± 8.21 |
| Group 4 | 30.8 ± 4.30** | 4.77 ± 0.22**‡ | 221.00 ± 18.00**‡ | 398.70 ± 27.10 |
| Group 5 | 7.80 ± 0.79** | 4.58 ± 0.25**‡ | 162.00 ± 12.00**‡ | 363.00 ± 16.80 |
| Group 6 | 1.51 ± 0.20** | 5.98 ± 0.59**‡ | 45.50 ± 5.82*‡* | 718.20 ± 19.20** |
| Group 7 | 1.12 ± 0.06** | 4.20 ± 0.21**‡ | 277.12 ± 21.14** | 812.04 ± 22.48**‡ |
| Group 8 | 1.21 ± 0.07** | 6.48 ± 0.53**‡ | 147.40 ± 14.50**‡ | 1049.12 ± 59.77**‡ |
| Group 9 | 1.47 ± 0.14** | 7.57 ± 0.48**‡ | 116.90 ± 29.10**‡ | 1330.10 ± 82.20**‡ |
| Group 10 | 183.1 ± 12.20** | 25.87 ± 1.45 | 5.75 ± 0.49 | 480.60 ± 32.10 |
| Group 11 | 176 ± 3.20** | 23.41 ± 2.34 | 6.44 ± 0.63 | 640.00 ± 34.68** |
| Group 12 | 2.18 ± 0.51**‡ | 7.57 ± 0.47 | 49.70 ± 20.30** | 975.40 ± 23.60** |
| Group 13 | 176 ± 30.00 | 76.47 ± 3.02 | 8.80 ± 0.29 | 935.30 ± 36.12** |
| Group 14 | 125.5 ± 30.00**‡ | 147.50 ± 9.43 | 17.94 ± 1.73** | 365.30 ± 21.12 |
All values represent Mean ± SEM from six rats. Statistical analysis was carried out using One Way ANOVA followed by Tukey's test. The value of P < 0.05 was considered statistically significant.
compared with normal group
compared with hyperlipidemic control group
compared with atorvastatin-niacin.
SOD levels were significantly increased in hyperlipidemic control group as compared with normal group. Atorvastatin alone did not affect SOD levels. However, in combination with different vitamins it significantly reduced SOD levels in liver tissues (Table 3). Atorvastatin-Niacin did not decrease liver SOD activity and even showed increase. When atorvastatin-niacin was combined with different anti-oxidant vitamins, increase in SOD activity was observed instead of reduction. Highest increase in SOD activity was observed with atorvastatin-niacin-β-carotene-vitamin-E-vitamin-C (Table 3).
Catalase levels were significantly increased in hyperlipidemic control group when compared with normal group. Atorvastatin alone did not affect catalase activity. However, in combination with different anti-oxidant vitamins, atorvastatin significantly increased catalase activity. Highest improvement in catalase activity was achieved with atorvastatin-vitamin-E-vitamin-C combination (Table 3). Atorvastatin-niacin did not increase catalase activity. When supplemented with β-carotene or vitamin-E-vitamin-C-β-carotene, atorvastatin-niacin combination significantly increased the catalase activity (Table 3).
Due to oxidative stress, there was significant decrease in reduced glutathione levels in hyperlipidemic control groups. Atorvastatin when given in combination with more than one vitamin significantly increased the reduced GSH levels. Highest increase in the GSH level was observed with atorvastatin-β-carotene-vitamin-E combination (Table 3). Atorvastatin-niacin did not affect liver reduced glutathione levels. However, when combined with vitamin-E, β-carotene, vitamin-C, it significantly increased GSH levels. When all the vitamins were given with atorvastatin-niacin, no additional improvements in GSH levels were observed (Table 3).
Discussion
Cholesterol homeostasis in the body is maintained by the balance between cholesterol biosynthesis, and its metabolism. The cholesterol biosynthesis is controlled by the rate limiting enzyme-HMG CoA Reductase. Atorvastatin blocks this enzyme and thereby prevents cholesterol biosynthesis. In the present study, the cholesterol lowering effects of atorvastatin (mainly LDL-C) can be attributed to HMG CoA reductase inhibition. Niacin by limiting lipolysis in adipose tissue, decreasing esterification of hepatic TG, and increasing the activity of lipoprotein lipase reduces serum TG and TC levels. The combination of atorvastatin-niacin caused and additive reduction in serum total cholesterol, LDL-C, TG, VLDL-C levels [26].
The inhibition of lipid peroxidation observed with atorvastatin might be secondary to lipid lowering effect as well as its antioxidant effects [27]. However, the drug alone did not improve SOD, catalase activities, and GSH levels in the present study. The combinations of atorvastatin with other vitamins significantly inhibited lipid peroxidation due to their anti-oxidant properties [28, 29]. However, its combination with more than one vitamin did not show any additional inhibition in the lipid peroxidation. Atorvastatin alone did not affect SOD and catalase activities in liver tissues. The combination of atorvastatin with vitamin-E, vitamin-C, or β-carotene, improved liver SOD and catalase activities. Highest improvement was observed with vitamin-E-vitamin-C combination. Supplementation of β-carotene did not show any additional benefits. The GSH levels were significantly increased when atorvastatin was combined with more than one vitamin compared with its combination with individual vitamins. This might be attributed to their sparing effects. Vitamin–C has been found to increase intracellular glutathione levels by sparing effect [30].
The atorvastatin-niacin alone and in combination with vitamins inhibited lipid peroxidation. However, inhibitory effects on lipid peroxidation were not as beneficial as observed with atorvas-tatin-vitamins combinations. Atorvastatin – niacin –vitamins combinations showed high SOD activities and low catalase activities compared with atorvastatin-vitamins combinations. Since, SOD is a stress protein; its levels were increased during oxidative stress [31]. It may be likely that antioxidant vitamins in combination with Atorvastatin-niacin induce oxidative stress. Studies have shown that excessive vitamins in the body may act as pro-oxidant leading to oxidative stress [32].
In conclusion, antioxidant vitamins when given with Atorvastatin additionally improve lipid profile, inhibit lipid–peroxidation, and improve antioxidant status. More than one vitamin with Atorvastatin did not show any additional advantage. When Atorvastatin-niacin combination was used with vitamins, the beneficial effects on lipid profile, lipid peroxidation and anti-oxidant status were not as marked as with Atorvastatin– vitamins combinations. When more than two vitamins were used with Atorvastatin–niacin, it may lead to oxidative stress. Hence, vitamins in combination with lipid lowering drugs must be used with care.
References
- 1.Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low-density lipoprotein that increases it atherogenecity. N Engl J Med. 1989;320:915–924. doi: 10.1056/NEJM198904063201407. [DOI] [PubMed] [Google Scholar]
- 2.Parathasarathy S, Santanam N, Auge N. Oxidized low density lipoprotein: a two faced janus in coronary artery disease? Biochem Pharmacol. 1998;56:279–284. doi: 10.1016/s0006-2952(98)00074-4. [DOI] [PubMed] [Google Scholar]
- 3.Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, Shih DM, Van Lentern BJ, Frank JS, Demer LL, Edwards PA, Foglemen AM. The yin and yang of oxidation in the development of the fatty streaks. New Engl J Med. 1996;16:831–842. doi: 10.1161/01.atv.16.7.831. [DOI] [PubMed] [Google Scholar]
- 4.Terpstra V, Bird DA, Steinberg D. Evidence that the lipid moiety of oxidized low density lipoprotein plays a role in its interaction with macrophage receptors. PNAS. 1998;95(4):1806–1811. doi: 10.1073/pnas.95.4.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Heinecke JW. Free radical modification of low density lipoprotein: mechanism and biological consequences. Free Rad Boil Med. 1987;3:65–73. doi: 10.1016/0891-5849(87)90040-2. [DOI] [PubMed] [Google Scholar]
- 6.Jargons G, Hhoff HF, Chisolm GM, Esterbauer H. Modification of human serum low-density lipoprotein by oxidation, characterization and pathophysiological implications. Chem Phys Lipids. 1987;45:315–336. doi: 10.1016/0009-3084(87)90070-3. [DOI] [PubMed] [Google Scholar]
- 7.Esterbauer H, Jargons G, Quehenberger O, Koller E. Auto-oxidation of human low-density lipoprotein: loss of polyunsaturated fatty acids and vitamin-E and generation of aldehydes. J Lip Res. 1987;28:495–509. [PubMed] [Google Scholar]
- 8.Ramirez J, Flowers NC. Leukocyte ascorbic acid and its relationship to coronary heart disease in man. Am J Clin Nutr. 1980;33:2079–2087. doi: 10.1093/ajcn/33.10.2079. [DOI] [PubMed] [Google Scholar]
- 9.Reimersma RA, Wood DA, Macintyre CCH, Elton RA, Gey KF, Oliver MF. Low plasma vitamin-E and C increased risk of angina in Scottish men. Ann NY Acad Sci. 1989;570:291–295. doi: 10.1111/j.1749-6632.1989.tb14928.x. [DOI] [PubMed] [Google Scholar]
- 10.Gey KF, Moaer UK, Jordan P, Stahelin HB, Eichholzer M, Ludin E. Increased risk of cardiovascular disease at suboptimal plasma concentrations of essential antioxidants: an epidemiological update with special attention to carotene and vitamin-C. Am J Clin Nutr. 1993;57:787s–797s. doi: 10.1093/ajcn/57.5.787S. [DOI] [PubMed] [Google Scholar]
- 11.Reaven PD, Khouw A, Beltz WF, Parathasarathy S, Witztum JL. Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin-E but not by beta-carotene. Arterioscler Thromb. 1993;13:590–600. doi: 10.1161/01.atv.13.4.590. [DOI] [PubMed] [Google Scholar]
- 12.Donaldson WE. Atherosclerosis in cholesterol fed Japanese quill: evidence for amelioration by dietary vitamin E. Poult Sci. 1982;61:2097–2102. doi: 10.3382/ps.0612097. [DOI] [PubMed] [Google Scholar]
- 13.Hodis HN, Mack EJ, LaBree L, Cashin-Hemphill L, Sevanian A, Johnson R, Azen SP. Serial coronary angiographic evidence that antioxidants vitamin intake reduces progression of coronary artery atherosclerosis. J Am Med Assoc. 1995;21:1849–1854. [PubMed] [Google Scholar]
- 14.Stephens NG, Parson A, Shields PM, Kelly F, Cheesman K, Mitchinson MJ. Randomized controlled trial of vitamin E in patients with coronary disease: Cambridge heart antioxidants study. Lancets. 1996;347:781–786. doi: 10.1016/s0140-6736(96)90866-1. [DOI] [PubMed] [Google Scholar]
- 15.Gaziano JM, Hatta A, Flynn M, Johnson EJ, Krinsky NJ, Ridker PM, Hennekens CH, Frei B. Supplementation with beta-carotene in vivo and in vitro does not inhibit low-density lipoprotein oxidation. Atherosclerosis. 1995;112:187–195. doi: 10.1016/0021-9150(94)05414-e. [DOI] [PubMed] [Google Scholar]
- 16.Zhang SH, Reddick RL, Avdievich E, Surles LK, Jones RG, Reynolds JB, Quarfordt SH, Maeda N. Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E deficient mice. J Clin Invest. 1997;99:2858–2865. doi: 10.1172/JCI119479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Cheung MC, Zhao X-Q, Chait A, Albers JJ, Brown BG. Antioxidant supplements block the response of HDL to simvastatin-niacin therapy in patients with coronary artery disease and low HDL. Arterioscler Thromb Vasc Biol. 2001;21:1320–1326. doi: 10.1161/hq0801.095151. [DOI] [PubMed] [Google Scholar]
- 18.Committee for the Purpose of Supervision and Control of Experiments on Animals, editor. CPCSEA guidelines for laboratory animal facility. Indian J Pharmacol. 2003;35:257–272. [Google Scholar]
- 19.Blank B, Pfeiffer FR, Greenberg CM, Kerwin JF. Thyromimetics II: The synthesis and Hypocholesterolemic activity of some b-Dimethylaminoethyl esters of Iodinated Thyroalkanoic acids. J Med Chem. 1963;6:560–563. doi: 10.1021/jm00341a021. [DOI] [PubMed] [Google Scholar]
- 20.Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol without the use of the preparative ultracentrifuge. Clin Chem. 1972;18:499–502. [PubMed] [Google Scholar]
- 21.Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin Phenol reagent. J Bio Chem. 1951;193:265–275. [PubMed] [Google Scholar]
- 22.Okhawa H, Ohisi N, Yagi K. Assay of lipid peroxides in animal tissues by thiobarbituric reaction. Anal Biochem. 1979;95:351–353. doi: 10.1016/0003-2697(79)90738-3. [DOI] [PubMed] [Google Scholar]
- 23.Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Boil Chem. 1972;247:3170–3175. [PubMed] [Google Scholar]
- 24.Aebi H. Catalase. Methods of Enzymatic Analysis. In: Bergmayer HU, editor. New York and London: Academic Pres; 1974. pp. 673–677. [Google Scholar]
- 25.Beutler E, Duron O, Kelly B. Reduced glutathione estimation. J Clin Med. 1963;61:882–885. [PubMed] [Google Scholar]
- 26.Guyton JR, Capuzzi DM. Treatment of hyperlipidemia with combined niacin-statin regimens. Am J Cardiol. 1998;82:82U–84U. doi: 10.1016/s0002-9149(98)00955-2. [DOI] [PubMed] [Google Scholar]
- 27.Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y, Kitakaze M, Liao JK. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest. 2001;108:1429–1437. doi: 10.1172/JCI13350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Huang HY, Appel LJ, Croft KD, Miller ER, 3rd, Mori TA, Puddey IB. Effects of vitamin C and vitamin E on in vivo lipid peroxidation: results of a randomized controlled trial. Am J Clin Nutr. 2002;76:549–555. doi: 10.1093/ajcn/76.3.549. [DOI] [PubMed] [Google Scholar]
- 29.Porkkala-Sarataho E, Salonen JT, Nyyssönen K, Kaikkonen J, Salonen R, Ristonmaa U, Diczfalusy U, Brigelius-Flohe R, Loft S, Poulsen HE. Long-Term Effects of Vitamin E, Vitamin C, and Combined Supplementation on Urinary 7-Hydro-8-Oxo-2'-Deoxyguanosine, Serum Cholesterol Oxidation Products, and Oxidation Resistance of Lipids in Nondepleted Men. Arterioscler Thromb Vasc Biol. 2000;20:2087–2093. doi: 10.1161/01.atv.20.9.2087. [DOI] [PubMed] [Google Scholar]
- 30.Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Bio Chem. 1994;269:9397–9400. [PubMed] [Google Scholar]
- 31.McCord JM. Is superoxide dismutase a stress protein? Stress proteins in inflammation. In: Burbon R, Rice-Evans, Blake D, Winrow C, editors. London: Richelieu Press; 1990. pp. 125–134. [Google Scholar]
- 32.Edge R, Truscott TG. Pro-oxidant and antioxidant reaction mechanism of carotene and radical interaction with vitamin E and C. Nutrition. 1997;13:992–998. doi: 10.1016/s0899-9007(97)00346-8. [DOI] [PubMed] [Google Scholar]