Research Article, J Proteomics Enzymol Vol: 6 Issue: 2
Effects of Vitamin D Combined with Aspirin or Atorvastatin on Plasma Lipid Profiles and Lipid Peroxidation in Triton-XInduced Hyperlipidemia in Rats
Adedapo DA* and Ogunfowora OO
Department of Pharmacology, University Of Ibadan, Nigeria
*Corresponding Author : Adedapo Deborah A
Department of Pharmacology, University of Ibadan, Nigeria
E-mail: [email protected]
Received: December 07, 2017 Accepted: December 19, 2017 Published: December 21, 2017
Citation: Adedapo DA, Ogunfowora OO (2017) Effects of Vitamin D Combined with Aspirin or Atorvastatin on Plasma Lipid Profiles and Lipid Peroxidation in Triton-X-Induced Hyperlipidemia in Rats. J Proteomics Enzymol 6:2. doi: 10.4172/2470-1289.1000131
Vitamin D deficiency as an independent cardiovascular risk factor has since been associated with increased risks of cardiovascular events. The present study assessed the modulatory effects of posttreatment of vitamin D alone and with aspirin or atorvastatin on triton-X-induced hyperlipidemia in rats. Forty-nine (49) Wistar rats were divided into seven experimental groups of seven per group. Group A, control negative group, received no treatment. Group B-G received triton (400 mg/kg) to induce hyperlipidemia. Groups C, D and E were post-treated with vitamin D only (200 IU/kg), aspirin only (1 mg/kg), atorvastatin only (10 mg/kg) respectively. Groups F and G were post-treated with vitamin D in combination with either aspirin or atorvastatin. Results obtained showed increased MDA( an indicator of lipid peroxidation) levels in group B animals [rats that received triton (400 mg/kg) only and not treated with any drug] by 77.4% and an elevation of low density lipoprotein (LDL) by 65.8% when compared with control negative group(p< 0.05). Similarly, high density lipoprotein (HDL) deceased in this group of rats that received triton only (p> 0.05). Vitamin D (200IU/kg), aspirin (1mg/ kg) and atorvastatin (10mg/kg) did not significantly (p> 0.05) alter total cholesterol TC, TG, HDL, LDL and malondialdehyde (MDA) levels respectively when administered alone. However, Vitamin D plus aspirin or atorvastatin treated animals reduced triton-induced lipid profile and MDA, although not statistically significant (p> 0.05). In conclusion, this present study suggested vitamin D possesses lipids and lipid peroxidation lowering activity. Thus, vitamin D supplementation could offer chemoprevention in this condition.
Keywords: Vitamin D; Triton; hyperlipidemia; lipid peroxidation
In the diversity of cardiovascular diseases, atherosclerosis and or hypertension are the most common . Both physiological and morphological changes that alter cardiovascular function lead to increased risk of cardiovascular disease, even in healthy asymptomatic individuals . Although cardiovascular disease usually affects older adults, the antecedents of cardiovascular disease, notably atherosclerosis, begin in early life, making primary prevention efforts necessary from childhood . There is also increased emphasis on preventing atherosclerosis from occurring or preventing the progress of atherosclerosis, by modifying risk factors, for example by healthy eating, exercise, and avoidance of smoking tobacco . WHO (2011) stated that cardiovascular diseases are the number one cause of death globally. An estimated 17.3 million people died from cardiovascular diseases in 2008, representing 30% of all global deaths (WHO, 2011). Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke (WHO, 2011) . The number of people, who die from cardiovascular diseases (CVD), mainly from heart disease and stroke, will increase to reach 23.3 million by 2030 . Vitamin D is classically known for its role in bone metabolism by modulating calcium homeostasis and ensuring physiologic calcium absorption by the gut [7,8]. It was recently reported that Vitamin D deficiency, CVD and related risk factors are highly prevalent worldwide and frequently co-occur . This has led to extensive research on vitamin D as a potential influencing factor in the pathogenesis of several chronic non-skeletal diseases including infectious or autoimmune diseases as well as cancer or CVD . Animal studies have revealed that the biologically active metabolite of vitamin D-1,25-dihydroxy-vitamin D (1,25[OH]2D) can modulate various processes involved in the pathogenesis of CVD . Also, numerous observational studies, prospective meta-analyses, as well as some interventional studies have addressed the possible linkage of vitamin D deficiency and the development of CVD and its risk factors . While it seems plausible that vitamin D deficiency can be considered a surrogate marker for poorer health status, most notably observed in patients with chronic diseases, including cardiovascular risk factors and CVD, it remains to be proven whether vitamin D itself can directly impact on cardiovascular outcomes . Presently, there is paucity of data on the effect of vitamin D supplementation on the lipid profile to elucidate the mechanisms between vitamin D and lipids . Although, evidence abounds of potential benefits of vitamin D, but, its therapeutic roles in specific heart related conditions is unknown . The solution of Triton X-100 has successfully been used to induce hyperlipidemia in rats in previous studies, and it was chosen as the hyperlipidemic model due to its convenience, reproducibility and availability . The present study investigated the anti-atherogenic effects of vitamin D alone and in combination with atorvastatin or low dose- aspirin in rats in order to ascertain if vitamin D supplementation can significantly alter atherogenic process as well as the probable mechanisms that may be involved.
Materials and Methods
Drugs and chemicals
Atorvastatin 10mg (Lipitor) produced by Pfizer was purchased from CCD Pharmacy in Ogudu, Lagos state Nigeria. Dispersible aspirin (Acetylsalicylic acid) 75 mg tablets was purchased from M&A Pharmchem Ltd. and Vitamin D3 (Cholecalciferol) 400IU tablet was obtained from Nature Made. Triton-X-100, iso-octylpolyoxy ethylene phenol formaldehyde polymer was purchased from Sigma-Aldrich chemical Co. (USA). Total cholesterol (TC), Triglyceride (TG) assay kits were obtained from Randox Laboratory (Crumlin, U.K). All other chemicals and reagents used were of analytical grade.
Forty nine Wister Albino adult rats (of either sex) with average weight of about 200g were obtained from the Animal House Unit of the Faculty of Basic Medical Science, University of Ibadan. The animals were housed under standard laboratory condition in plastic cages at 25°C (light and dark cycles were maintained). The animals were left for an initial adaptation period of 7 days before any experimental adaptation and supplied with commercially available standard pellet diet and water ad libitum. Animal experiments were conducted strictly according to ethical guidelines for use of animals in scientific research as stated in International Guiding Principles for Biomedical Research Involving Animals .
Forty nine (49) albino rats weighing between 180 g and 220 g of both sexes were used for the work. They were randomly distributed into seven groups of 7 rats per group. Group A, control negative group received distilled water. Group B, control triton group was administered 400 mg/kg triton-X orally (p.o) for 30 days to make them hyperlipidemic. Group C, Vitamin D only, were administered 400 mg/kg triton-X p.o. for 30 days and then treated with Vitamin D (200 IU/kg) for 15 days. Group D, Aspirin only, received 400 mg/kg triton-X p.o. for 30 days plus aspirin (1 mg/kg) for 15 days. Group E, Atorvastatin only, was administered 400 mg/kg triton-X p.o. for 30 days plus atorvastatin (10 mg/kg) for 15 days. Group F (Vitamin D and Aspirin) received 400 mg/kg triton-X p.o. for 30 days plus vitamin D (200 IU/kg) and aspirin (1 mg/kg) for 15 days. Group G (Vitamin D with Atorvastatin) was given 400 mg/kg triton-X p.o. for 30 days plus treated with vitamin D (200IU/kg) and atorvastatin (10 mg/kg) for 15 days.
Collection of blood samples for assays
The animals were fasted for 24hrs after the last day of treatment, after which blood sample was collected via retro-orbital sinus into heparinized bottle before the animals were sacrificed and animals were sacrificed by cervical dislocation. The anticoagulant contained blood samples were centrifuged at 4200 rpm for 5 minutes to obtain the clear supernatant (i.e. serum) from which all biochemical assays were carried out.
Serum total cholesterol (TC) and Triglyceride (TG) concentration were estimated following the principle described by Trinder (1969) using commercialkits obtained from Randox Laboratories Ltd (Crumlin, UK). High Density Lipoprotein (HDL) was estimated according to Warnick and Albers (1978) while serum low density lipoprotein (LDL) was calculated using Freidewald formula (1972). Atherogenic index was also determined using the formula described by Haglund et al. (1991). Lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method of Varshney and Kale (1990).
Statistical analysis of data
All data were expressed as mean ± standard error of mean (SEM). Significant differences among the group were determined by oneway analysis of variance (ANOVA) and student T-test using the statistical analysis programme for social sciences (SPSS). Post hoc testing was performed for inter-group comparisons using the least significant difference (LSD) (Levin, 1991). Results were considered to be significant at p ≤ 0.05.
There was an elevation of total cholesterol (TC) and Triglyceride (TG) in triton (400 mg/kg) treated rats when compared with control distilled water group, this was however, not statistically significant (Table 1). Vitamin D (200 IU/kg), aspirin (1 mg/kg) and atorvastatin (10mg/kg) did not significantly (p>0.05) reduce TC and TG levels when administered alone. Also, animals post-treated with Vitamin D plus aspirin (1 mg/kg) or atorvastatin (10 mg/kg) co-administration (200 IU/kg) for 15 days after triton-induced hyperlipidemia showed decreased levels of TC and TG though not statistically significant (p>0.05) when compared with triton group, No difference between aspirin or atorvastatin plus Vitamin D treated animals and vitamin D group as observed in this study.
|A||Control (DW, 10 ml/kg)||51.0 ± 5.2||31.8 ± 5.8|
|B||Triton (400 mg/kg)||59.5 ± 11.9(-16.7)a||41.0 ± 16.8(-28.9)a|
|C||Vitamin D (200IU/kg||54.0 ± 8.3(-5.9)a||23.8 ± 4.3(25.2)a|
|D||Aspirin (1 mg/kg)||51.3 ± 2.7(-0.59)a||31.7 ± 7.7(0.3)a|
|E||Atorvastatin Vitamin D (200IU/kg)||54.0 ± 8.0(-5.9)a||34.5 ± 8.5(-8.5)a|
|F||+Aspirin (1 mg/kg)||57.8 ± 9.3
(2.9)b(-7.0)c(-13.3)a (46.8)b (8.4)c (31.4)a
|21.8 ± 5.6
(46.8)b (8.4)c (31.4)a
|G||+ Atorvastatin (10 mg/kg)||55.0 ± 13.1 (7.6)b(-1.9)c(-7.4)a||32.8 ± 8.9 (20.0)c (-22.0)c (-3.1)a|
Table 1: Effect of Vitamin D and Aspirin on serum total cholesterol (TC) and triglycerides (TG) levels in normal and triton-treated rats.
Table 2 shows a decrease of high density lipoprotein (HDL) in triton (400 mg/kg) treated rats by 7.6% when compared with control distilled water group (p>0.05). Vitamin D (200 IU/kg), aspirin (1 mg/kg) and atorvastatin (10 mg/kg) when administered alone showed elevated HDL levels when compared with triton (400 mg/kg) group, this is however not significant (p>0.05). Though not significant (p>0.05),there was an increase in HDL level for aspirin or atorvastatin plus Vitamin D treated animals when compared with triton only treated group. Triton-induced rats showed significant (p<0.05) elevation of low density lipoprotein (LDL) when compared with control distilled water group. Aspirin, Vitamin D or atorvastatin animals when administered alone did not significantly alter LDL levels. Also, Vitamin D plus aspirin or atorvastatin co-administration decreased LDL (p>0.05) levels by 33% and 28% respectively when compared with triton treated rats.
|A||TREATMENTS||26.6 ± 6.1||19.6 ± 4.0|
|B||Control (DW, 10 ml/kg)||19.0 ± 2.0(7.6)a||32.5 ± 9.7*(-65.8)a|
|C||Triton (400 mg/kg)||27.0 ± 6.8(-1.5)a||25.0 ± 3.6(-27.6)a|
|D||Vitamin D (200IU/kg Aspirin (1 mg/kg)||23.7 ± 4.1(10.9)a||21.3 ± 3.8(-8.7)a|
|E||Atorvastatin Vitamin D (200IU/kg)||25.5 ± 9.5(4.1)a||21.5 ± 0.5(-9.7)a|
|F||+Aspirin (1 mg/kg)||32.0 ± 8.0 (-68.4)b(-18.5)c(-20.3)a||21.5 ± 2.6 (33.8)b (14.0)c (-9.7)a|
|G||+ Atorvastatin (10 mg/kg)||25.3 ± 6.7 (-33.2)b(6.3)c(4.9)a||23.3 ± 5.3
Table 2: Effect of Vitamin D and Aspirin on serum high density lipoprotein (HDL) and low density Lipoprotein (LDL) levels in normal and triton-treated rats.
Table 3 shows a significant increase (p<0.05) of lipid peroxidation (Malondialdehyde, MDA) in triton (400 mg/kg) treated rats by 77.4% when compared with control distilled water group. Although, Vitamin D (200 IU/kg), aspirin (1 mg/kg) and atorvastatin (10 mg/ kg) did not alter MDA levels when administered alone on comparison with control distilled water group, the drugs decreased (P>0.05) MDA when compared with triton treated group. Also, aspirin or atorvastatin plus Vitamin D treated animals reduced (p>0.05) tritoninduced MDA 18.2% and 12.7% respectively. Atherogenic index was reduced across all treatment groups, though there was only a significant (p<0.05) reduction in the aspirin plus Vitamin D group.
|A||Control(DW,10ml/kg)||4.0 ± 1.0||3.1 ± 0.5|
|B||Triton(400mg/kg)||6.4 ± 2.2(-60.0)a||5.5 ± 0.6*(-77.4) a|
|C||Vitamin D (200IU/kg)||3.5 ± 0.9(12.5)a||3.4 ± 0.6(-9.7)a|
|D||Aspirin(1mg/kg)||4.4 ± 1.7(-10.0)a||3.0 ± 0.2(3.2)a|
Vitamin D (200IU/kg)
|4.7 ± 2.5(-17.5)a||3.5 ± 0.7(-12.9)a|
|F||+ Aspirin (1mg/kg)||2.4 ± 0.6**
|4.5 ± 0.8
|G||+ Atorvastatin (10 mg/kg)||3.9 ± 0.6
|4.8 ± 0.6
Table 3: Effect of Vitamin D and Aspirin on serum Atherogenic Index (AI) and Malondialdehydelevels in normal and triton-treated rats.
Vitamin D deficiency has emerged as a potential risk factor for cardiovascular diseases [22-24]. We investigated on the antiatherogenic effects of vitamin D alone and in combination with atorvastatin or low dose- aspirin in rats. Hyperlipidemia was achieved through administration of the solution of Triton X-100 as previously reported by . In epidemiological studies of the general population, lower vitamin D levels have been associated with cardiovascular diseases, hypertension, and diabetes. High density lipoprotein cholesterol (HDL) and low density lipoprotein (LDL) cholesterol are surrogate markers of cardiovascular risk such as coronary artery calcification and carotid intima medial thickness [24,25]. From the result obtained, there were increased plasma lipid profiles and lipid peroxidation. This indicates the suitability of Triton X-100 to induce hyperlipidemia with increase of VLDL secretion by the liver accompanied by a strong reduction of VLDL and LDL catabolism . In this study, Vitamin D was selected to screen for its anti-hyperlipidemic activity in Triton X-100 (100 mg/kg) induced hyperlipidemic rats in the presence of asprin and atorvastatin as standards during a post-treatment. There is a large body of research from clinical studies in humans indicating that low levels of serum 25-hydroxy vitamin D are associated with atherosclerosis [27-31]. There are however contrasting reports showing evidence that vitamin supplementation has no effect on vascular disease mortality or allcause mortality . From the result obtained, total cholesterol and triglyceride increased in triton (400 mg/kg) treated rats (Table 1), although, insignificantly. Animals post-treated with aspirin and atorvastatin plus Vitamin D co-administration following tritoninduced hyperlipedemia showed decreased levels of TC and TG. Also, high density lipoprotein (HDL) decreased insignificantly in triton (400 mg/kg) treated rats (Table 2). Vitamin D administration and coadministration with aspirin and atorvastatin increased HDL, though not significantly when compared with hyperlipedemic triton treated rats. Triton-induced rats showed significant elevation of low density lipoprotein (LDL) when compared with control distilled water group. Atorvastatin plus Vitamin D co-administration decreased LDL when compared with hyperlipedemic triton treated rats. Tritoninduced hyperlipedemia presented significantly elevated MDA level, indicating the occurrence of lipid peroxidation and also an increase in atherogenic index was seen. Vitamin D, Aspirin and Atorvastatin at the doses used in this present study did not alter Malondialdehyde (MDA) levels when administered alone. However, aspirin or atorvastatin plus Vitamin D insignificantly depleted elevated MDA levels in treated animals. Atherogenic index was reduced across all treatment groups. Co-administration of atorvastatin and Vitamin D significantly reduced atherogenic index. It has been demonstrated that 1α,25-dihydroxy vitamin D3 reduced macrophage adhesion and migration as well as foam cell formation in monocytes isolated from type 2 diabetic patients . This was evident with the aid of a mouse model of vitamin D deficiency facilitated atherosclerosis . Further evidence showed that calcitriol treatment on atherosclerosis development was achieved from an oral calcitriol treatment which decreased the production of pro-inflammatory chemokines and reduced amount of inflammatory effector cells in atherosclerotic plaques . Evidence of similar link between vitamin D, T cell modulation, and atherosclerosis has also been published in humans . Thus, our present study further strengthen the anti-atherogenic effects of vitamin D alone and in combination with atorvastatin or low dose- aspirin in rats, although, the probable mechanisms that may be involved still require further investigation.
- Kelly BB, Fuster, Valentin. Promoting Cardiovascular Health in the Developing World: A Critical Challenge to Achieve Global Health. Institute of Medicine Washington, D.C: National Academies Press 2010.
- Dantas AP, Jimenez-Altayo F, Vila E (2012) Vascular aging: facts and factors. Front Physiol3: 1-2.
- McGill HC, McMahan CA, Gidding SS (2008) Preventing heart disease in the 21stcentury: implications of the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Circulation 117: 1216-1227.
- Lewington S, Whitlock G, Clarke R (2007) Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths. Lancet 370: 1829-1839.
- World Health Organization. Global atlas on cardiovascular disease prevention and control. Geneva 2011.
- Mathers CD, Loncar D (2006) Projections of global mortality and burden of disease from 2002 to 2030. PLoS Medicine 3: 442.
- Holick MF (2007) Vitamin D deficiency. N Engl J Med357: 266-281.
- Bouillon R, Carmeliet G, Verlinden L, van Etten E, Verstuyf A, et al. (2008) Vitamin D and human health: Lessons from vitamin D receptor null mice. Endocr Rev 29: 726-776.
- Katharina K, Andreas T, Nicolas V, Thomas P, Martin G et al. (2013) Vitamin D and Cardiovascular Disease. Nutrients5: 3005-3021.
- Souberbielle JC, Body JJ, Lappe JM, Plebani M, Shoenfeld Y, et al. (2010) Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice. Autoimmun Rev 9: 709-715.
- Wang L, Song Y, Manson JE, Pilz S, März W, et al. (2012) Circulating 25-hydroxy-vitamin D and risk of cardiovascular disease: A meta-analysis of prospective studies. Circ Cardiovasc Qual Outcomes5: 819-829.
- Forman JP, Scott JB, Ng K, Drake BF, Suarez EG, et al. (2013) Effect of vitamin D supplementation on blood pressure in blacks. Hypertension61: 779-785.
- Eugenia CM, Amparo FP, William BG, María MM (2013) Vitamin D status and hypercholesterolemia in Spanish general population. Dermatoendocrinol5: 358-362.
- Zittermann A, Iodice S, Pilz S, Grant WB, Bagnardi V (2012) Vitamin D deficiency and mortality risk in the general population: A meta-analysis of prospective cohort studies. Am J Clin Nutr 95: 91-100.
- Ghule BV, Ghante MH, Saoji AN, Yeole PG (2006) Hypolipidemic and antihyperlipidemic effects of Lagenariasiceraria (Mol.) fruit extracts. Indian J Exp Biol 44: 905-909
- Council for International Organization for Medical Science (CIOMS). The International Guiding Principles for Biomedical Research Involving Animals. Geneva 1985.
- Trinder P (1969) Quantitative determination of triglyceride using GPO-PAP method. Ann Clin Biochem 6: 24-27.
- Warnick GR, Albers JJ (1978) A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol. J Lipid Res 19: 65-76.
- Freidewald WT, Levy RI, Frederickson DS (1972) Estimation of the concentration of low density lipoprotein choles- terol in plasma, without use of the preparative ultracen- trifuge. Clin Chem 18: 499-502.
- Haglund O, Luostarinen R, Wallin R, Wibell L, Saldeen T (1991) The effect of fish oiltriglycerides, cholesterol, fibrinogen and malondialdehyde in humans supplemented with vitamin E. J Nut 121: 165-169.
- Varshney R, Kale RK (1990) Effect of calmodulin antagonist on radiation induced lipid peroxidation in microsomes. Int J RadiatBiol 58: 733-743.
- Ulbricht TL, Southgate DA (1991) Coronary heart disease: seven dietary factors. Lancet 19: 985-992.
- Zittermann A, Schleithoff SS, Koerfer R (2005) Putting cardiovascular disease and vitamin D insufficiency into perspective. Br J Nutr 94: 483-492.
- Vacek JL, Vanga SR, Good M, Lai SM, Lakkireddy D, et al. (2012) Vitamin D deficiency and supplementation and relation to cardiovascular health. Am J Cardiol 109: 359-363.
- Robinson AB, Thierry-Palmer M, Gibson KL (2012) Disease activity, proteinuria, and vitamin D status in children with systemic lupus erythematosus and juvenile dermatomyositis. J Pediatr 160: 297-302.
- Lin J, Yang R, Tarr PT, Wu PH, Handschin C, et al. (2005) Hyperlipidemic effects of dietary saturated fats mediated through PGC-1β coactivation of SREBP. Cell 120: 261-273.
- Reis JP, von Mühlen D, Michos ED, Miller ER 3rd, Appel LJ, et al. (2009) Serum vitamin D, parathyroid hormone levels, and carotid atherosclerosis. Atherosclerosis 207: 585-590.
- Cheraghi Z, Poorolajal J, Hashem T, Esmailnasab N, Irani AD (2012) Effect of body mass index on breast cancer during premenopausal and postmenopausall periods: a meta-analysis. PloS one 7: 51446.
- Gunta SS, Thadhani RI, Mak RH (2013) The effect of vitamin D status on risk factors for cardiovascular disease. Nat Rev Nephrol9: 337-347.
- Wang G, McCain ML, Yang L, He A, Pasqualini FS, et al. (2014) Modeling the mitochondrial cardiomyopathy of Barth syndrome with iPSC and heart-on-chip technologies. Nat med 20: 616-623.
- Qin XF, Zhao LS, Chen WR, Yin da W, Wang H (2015) Effects of vitamin D on plasma lipid profiles in statin-treated patients with hypercholesterolemia: A randomized placebo-controlled trial. Clin Nutr 34: 201-206.
- Ponda MP, Huang X, Odeh MA, Breslow JL, Kaufman HW (2012) Vitamin D may not improve lipid levels: a serial clinical laboratory data study. Circulation 126: 270-277.
- Ceglia L (2009) Vitamin D and its role in skeletal muscle. Curr Opin Clin Nutr Metab Care12: 628.
- Weng S, Sprague JE, Oh J, Riek AE, Chin K, et al. (2013) Vitamin D deficiency induces high blood pressure and accelerates atherosclerosis in mice. PLoSOne8: 54625.
- Takeda M, Yamashita T, Sasaki N, Nakajima K, Kita T, et al. (2010) Oral administration of an active form of vitamin D3 (calcitriol) decreases atherosclerosis in mice by inducing regulatory T cells and immature dendritic cells with tolerogenic functions. Arterioscler Thromb Vasc Biol 30: 2495-2503.
- Yadav AK, Banerjee D, Lal A, Jha V (2012) Vitamin D deficiency, CD4+CD28null cells and accelerated atherosclerosis in chronic kidney disease. Nephrol 17: 575-581.