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Protonated nanostructured aluminosilicate (NSAS) reduces plasma cholesterol concentrations and atherosclerotic… Sivak, Olena; Darlington, Jerry; Gershkovich, Pavel; Constantinides, Panayiotis P; Wasan, Kishor M Jul 28, 2009

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ralssBioMed CentLipids in Health and DiseaseOpen AcceResearchProtonated nanostructured aluminosilicate (NSAS) reduces plasma cholesterol concentrations and atherosclerotic lesions in Apolipoprotein E deficient mice fed a high cholesterol and high fat dietOlena Sivak1, Jerry Darlington2, Pavel Gershkovich*1, Panayiotis P Constantinides3 and Kishor M Wasan*1Address: 1Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada, 2AMCOL International Corporation, Hoffman Estates, Illinois, USA and 3Biopharmaceutical and Drug Delivery Consulting LLC, Gurnee, Illinois, USAEmail: Olena Sivak - olenas777@hotmail.com; Jerry Darlington - jerry.darlington@amcol.com; Pavel Gershkovich* - pgershko@interchange.ubc.ca; Panayiotis P Constantinides - pcon@amcol.com; Kishor M Wasan* - kwasan@interchange.ubc.ca* Corresponding authors    AbstractThe aim of this work was to assess the effect of chronic administration of protonatednanostructured aluminosilicate (NSAS) on the plasma cholesterol levels and development ofatherosclerotic lesions in Apolipoprotein (ApoE) deficient mice fed a high cholesterol and high fatdiet. Apolipoprotein E (ApoE) deficient mice were divided into the following treatment groups:protonated NSAS 1.4% (w/w), untreated control and 2% (w/w) stigmastanol mixed with high-cholesterol/high-fat diet. Animals were treated for 12 weeks, blood samples were withdrawn every4 weeks for determination of plasma cholesterol and triglyceride levels. At the end of the study theaortic roots were harvested for assessment of atherosclerotic lesions. NSAS at 1.4% (w/w) andstigmastanol at 2% (w/w) treatment groups showed significant decreases in plasma cholesterolconcentrations at all time points relative to the control animals. The lesion sum area in 1.4% (w/w)NSAS and 2% (w/w) stigmastanol groups were significantly less from the control animals. Inconclusion, in this study, the effectiveness of chronic administration of protonated NSAS materialin the reduction of plasma cholesterol levels and decrease in development of atheroscleroticlesions was demonstrated in Apo-E deficient mice model.IntroductionElevated plasma cholesterol levels have been associatedwith increased risk of atherosclerosis and coronary arterydisease [1]. Although competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)cholesterol absorption inhibitors as an alternative/adju-vant method of treating hypercholesterolemia [1]. Themain agents are plant sterols, plant stanols and ezetimibe.However, since these agents are reported to be absorbedinto blood circulation, there is a potential for systemicPublished: 28 July 2009Lipids in Health and Disease 2009, 8:30 doi:10.1186/1476-511X-8-30Received: 17 July 2009Accepted: 28 July 2009This article is available from: http://www.lipidworld.com/content/8/1/30© 2009 Sivak et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 5(page number not for citation purposes)reductase (statins) are the gold standard in the treatmentof hypercholesterolemia, there is a long history of use ofadverse effects [2-5]. Potential candidates for non-absorb-able agents for inhibiting gastrointestinal absorption ofLipids in Health and Disease 2009, 8:30 http://www.lipidworld.com/content/8/1/30cholesterol would be the naturally occurring aluminosili-cates clays (Figure 1) [6]. A calcium montmorillonite clayhas been reported to be safe and effective in reducing ofexposure to aflatoxin by adsorption mechanism inrodents and humans [7,8]. We have previously reportedthat protonated nanoscaled aluminosilicate compoundefficiently inhibited the intestinal absorption of choles-terol following acute administration in a rat model [[9];See Additional File 1]. It was unclear, however, what willbe the effect of NSAS in the chronic administration onplasma cholesterol levels and on development of patho-physiology of atherosclerosis [10]. Thus, the current studywas designed to elucidate the effect of chronic administra-tion of protonated form of NSAS to Apolipoprotein-E(Apo-E) deficient mice on plasma cholesterol concentra-tions and formation of aortic atherosclerotic lesions.MethodsMice, C57B1/6 B6.129P2-ApoE™1UNC, 4 week old, withhomozygous deletion of the ApoE gene (apolipoprotein Eknock-out) were purchased from Jackson Laboratories,USA. The Apo-E deficient mice model has been usedextensively, since these mice develop severe hypercholes-terolemia and atherosclerotic lesions similar to thoseobserved in humans [10,11].The protonated NSAS material was prepared as previouslyreported [[9], See Additional File 1]. Briefly, the crudeNSAS was dried to 10% moisture content and the particlesize was reduced by a passing through a 200 mesh (74μm) screen. The base NSAS was then purified by a previ-ously reported method [12] and mixed with deionizedwater using a blender at 11,500 rpm for 5 min. As a resultof the purification process essentially all of the exchange-able surface cations were replaced by sodium ions.Sodium NSAS sample was pumped through two lab-scaleion-exchange columns filled with hydrogen-loaded resinexchange beads to protonate the montmorillonite.All animals used in this study were cared for in accordancewith the principles promulgated by the Canadian Councilin Animal Care and the University of British Columbia.The research adhered to the "Principles of Laboratory Ani-mal Care" (NIH publication #85-23, revised in 1985).The Apo-E deficient mice (Jackson Laboratories, USA)were divided into the following treatment groups: proto-nated NSAS 1.4% w/w, untreated control and 2% w/wstigmastanol. All animals received high-cholesterol/high-fat diet (45 kcal% fat) (Research Diets Inc., USA) for theduration of the study. The tested active compounds wereincorporated into the diet. All animals were treated for 12weeks. Blood samples were withdrawn from saphenousSchematic presentation of montmorillonite structureFigure 1Schematic presentation of montmorillonite struc-ture. Montmorillonite is a layered silicate with the property of adsorbing organic substances on its external surfaces or within its interlaminar space. The hydration of the clay induces swelling, which is mostly attributed to the increase in d (or c) dimension.Page 2 of 5(page number not for citation purposes)vein every 4 weeks and at the end of the study for determi-nation of total plasma cholesterol and triglyceride levels.Lipids in Health and Disease 2009, 8:30 http://www.lipidworld.com/content/8/1/30At the end of the study the animals were sacrificed andaortic arch was harvested for histopathology assessmentof atherosclerotic lesions in control animals and in groupsthat showed statistically significant differences in theirplasma lipid profile relatively to the control group. Tissuesurrounding the aorta including all fat were trimmed, andfrozen in liquid nitrogen. The aorta was cut in 3 consecu-tive slices (10 μmol/L) 5 mm above the aortic root. Slideswere stained with Oil-Red-O, Movat's Pentachrome andHematoxylin-eosin. An independent pathologist, blindedto the treatment groups scored the lesion formation basedon cumulative atherosclerotic exposure area (sum area).Statistical analyses were performed using one-wayANOVA followed by Dunnett multiple comparisons test.Results were expressed as mean +/- SEM, p < 0.05 indi-cated a significant difference between groups.Results and discussionAll animals based on physical appearance did not appearto have any deleterious effects from administration ofNSAS at 1.4% w/w or stigmastanol at 2% w/w. The activityand behaviour of the animals were similar between allgroups and consistent with the ApoE deficient phenotype.The baseline of the body weight was 19 g (average) andafter 12 weeks was 33.2 (average). As a result of similarfood and water consumption (data not shown) during allperiod of study, the body weight increased by the sameamount over the experimental period in all groups (treat-ment and control) (Figure 2). The average food intake forall treatment and control groups throughout the durationof study was 3.14 g per day (Figure 3).The total plasma cholesterol concentrations throughduration of the study in different treatment groups areshown in Figure 4. The treatment by protonated NSAS at1.4% w/w and stigmastanol at 2% w/w resulted in a sig-nificant decrease in plasma cholesterol levels at 4, 8 and12 weeks of the study compared to untreated controls.This cumulative reduction in plasma cholesterol levelsover the duration of the study is reflected by a statisticallysignificant difference in histopahotogically evaluated totalarea covered by atherosclerotic lesion in the 1.4% w/wNSAS and 2% w/w stigmastanol treatment groups com-pared to untreated controls (Figure 5). The change inplasma triglyceride levels through duration of the study indifferent treatment groups is shown in Figure 6. There areno statistically significant differences in total triglyceridelevels as well as in change in triglyceride levels from thebaseline between all treatment groups, at all time points,relatively to the control animals. This observation, inaddition to the fact that there were no changes in bodyweight (Figure 2) and food intake (Figure 3) throughoutthe study indicates that it is unlikely that protonated NSAScompound affects significantly the intestinal absorptionof triglyceride which is the main component of lipids inthe diet. When the food intake in figure 3 is plotted ascumulative intake over time for each group no statisticallysignificant differences are observed (data not shown).Thus the effect we observe in Figure 4 (plasma cholesterollevels) and Figure 5 (atherosclerotic lesion area) is due tothe effect of the compounds.In conclusion, in this study we have demonstrated theeffect of protonated NSAS material on reducing plasmaBody weight (Mean +/- SEM) from the beginning of the treat-ment (week 0) in the Apo-E deficient mice treated with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated c ntrol (n = 8)Figure 2Body weight (Mean +/- SEM) from the beginning of the treatment (week 0) in the Apo-E deficient mice treated with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated control (n = 8). No statistically significant differences between treat-05101520253035400 2 4 6 8 10WeekMice Body Weight, [g]Control 1.4% protonated NSAS 2% StigmastanolWeekly food in-take (Mean +/- SEM) from the beginning of the treatment (week 0) in the Apo-E deficient mice treated with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w sti mas-anol (n = 7) vs. untreat d c ntrol (n = 8)Figu  3Weekly food in-take (Mean +/- SEM) from the begin-ning of the treatment (week 0) in the Apo-E deficient mice treated with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated control (n = 8). No statistically significant differences 0123456Week 1Week 2Week 3Week 4Week 5Week 6Week76Week 8Week 9Week 10Week 11Week 12Food Intake [g]Control 1.4% protonated NSAS 2% StigmastanolPage 3 of 5(page number not for citation purposes)ment and untreated control groups were observed. between treatment and untreated control groups were observed. SEM were not shown in this figure.Lipids in Health and Disease 2009, 8:30 http://www.lipidworld.com/content/8/1/30cholesterol concentrations and the development ofatherosclerotic lesions in Apo-E deficient mice model fol-lowing chronic administration. The chronic administra-tion of protonated NSAS material on cholesterolabsorption seems not to affect the intestinal absorption oftriglyceride, as demonstrated by absence of changes inplasma triglyceride concentrations and lack of differencesin body weight and food intake. The shown significanteffect of supplementation of high fat/high cholesterol dietwith protonated NSAS material on the formation ofatherosclerotic lesions is particularly important, since thefinal aim of cholesterol-lowering treatment is reduction ofthe development of atherosclerosis. Although the effect ofprotonated NSAS material on inhibition of cholesterolabsorption following acute administration was demon-strated previously [[9], See Additional File 1], and the ben-eficial effect of chronic administration on plasmacholesterol levels and atherosclerotic lesions formation inApo-E deficient mice was demonstrated in this study, themechanism of cholesterol absorption inhibition is stillnot completely clear. In addition, a dose-response studywith protonated NSAS would be required to confirm itsactivity. Future mechanistic studies aimed to elucidate themechanism(s) by which the protonated form of NSASmaterial inhibits the intestinal absorption of cholesterolwill be needed.Total plasma cholesterol concentrations (Mean +/- SEM) fr m the beginning of the treatmen  (week 0) to the end of t eatment (week 12) in the Apo-E defici nt mice treat d with 1.4% w/w n = 8) of p tonated NSAS, 2% w/w s igmastanol(n = 7) v . untreated c ntr l (n = 8)Figure 4Total plasma cholesterol concentrations (Mean +/- SEM) from the beginning of the treatment (week 0) to the end of treatment (week 12) in the Apo-E defi-cient mice treated with 1.4% w/w (n = 8) of proto-nated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated control (n = 8). Statistically significant differ-ences for both 1.4% w/w protonated NSAS and 2% w/w stig-mastanol at 4, 8 and 12 weeks compared to untreated controls (*p < 0.05 vs. untreated controls and **p < 0.01 vs. untreated controls) were observed; one-way ANOVA fol-lowed by Dunnett multiple comparisons test02004006008001000120014001600Week 0 Week 4 Week 8 Week 12TC concentration, mg/dlControl 1.4% protonated NSAS 2% Stigmastanol `  *   *   ** * * * *  * *Total area covered by atherosclerotic lesion (Mean +/- SEM) in ApoE-deficient mice treated with 1.4% w/w (n = 8) of pro-ton ted NSAS, 2% w/w stigmastan l (n = 7) vs. untreated c tr l (n = 8)Figure 5Total area covered by atherosclerotic lesion (Mean +/- SEM) in ApoE-deficient mice treated with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated control (n = 8). Statistically significant differences for both 1.4% protonated NSAS and 2% w/w stig-mastanol compared to untreated controls (*p < 0.05 vs. 020004000600080001000012000GroupTotal area covered by atherosclerotic lesionControl 1.4% protonated NSAS 2% Stigmastanol**Total plasma triglyceride concentrations (Mean +/- SEM) fr m the beginning of the treatmen  (week 0) to the end of t eatment (week 12) in the Apo-E defici nt mice treat d with 1.4% w/w (n = 8) of protonated NSAS, 2% w/w s igmastanol(n = 7) v . un eated c ntr l (n = 8)Figure 6Total plasma triglyceride concentrations (Mean +/- SEM) from the beginning of the treatment (week 0) to the end of treatment (week 12) in the Apo-E defi-cient mice treated with 1.4% w/w (n = 8) of proto-nated NSAS, 2% w/w stigmastanol (n = 7) vs. untreated control (n = 8). No statistically significant dif-ferences between treatment and untreated control groups were observed.050100150200250300350400Week 0 Week 4 Week 8 Week 12TG concentration, mg/dlControl 1.4% protonated NSAS 2% stigmastanolPage 4 of 5(page number not for citation purposes)AbbreviationsNSAS: nanostructured aluminosilicate; ApoE: apolipopro-tein E; TC: total cholesterol; TG: triglyceride.untreated controls) were observed; one-way ANOVA fol-lowed by Dunnett multiple comparisons test.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Lipids in Health and Disease 2009, 8:30 http://www.lipidworld.com/content/8/1/30Competing interestsThe authors declare that they have no competing interests.Authors' contributionsOS carried out all aspects of the animal studies, includinganalysis of plasma lipids. JD participated in the design ofthe study and provided the NSAS material. PG partici-pated in the design of the study and performed some ofthe aspects of the animal studies, data analysis and writingof the manuscript. PC participated in the design of thestudy. KW participated in the design of the study, dataanalysis and writing of the manuscript.Additional materialAcknowledgementsThis study was funded by AMCOL International Corporation.References1. Wang DQ: Regulation of intestinal cholesterol absorption.Annu Rev Physiol 2007, 69:221-248.2. Sudhop T, von Bergmann K: Sitosterolemia – a rare disease. Areelevated plant sterols an additional risk factor?  Z Kardiol 2004,93:921-928.3. Assmann G, Cullen P, Erbey J, Ramey DR, Kannenberg F, Schulte H:Plasma sitosterol elevations are associated with an increasedincidence of coronary events in men: results of a nested case-control analysis of the Prospective Cardiovascular Munster(PROCAM) study.  Nutr Metab Cardiovasc Dis 2006, 16:13-21.4. Patel MD, Thompson PD: Phytosterols and vascular disease.Atherosclerosis 2006, 186:12-19.5. Florentin M, Liberopoulos EN, Elisaf MS: Ezetimibe-associatedadverse effects: what the clinician needs to know.  Int J ClinPract 2008, 62:88-96.6. Cai Y, Meng XF, Cao YX, Lu H, Zhu SF, Zhou LZ: Montmorilloniteameliorates hyperthyroidism of rats and mice attributed toits adsorptive effect.  Eur J Pharmacol 2006, 551:156-161.7. Afriyie-Gyawu E, Mackie J, Dash B, Wiles M, Taylor J, Huebner H,Tang L, Guan H, Wang JS, Phillips T: Chronic toxicological evalu-ation of dietary NovaSil clay in Sprague-Dawley rats.  FoodAddit Contam 2005, 22:259-269.8. Phillips TD, Afriyie-Gyawu E, Williams J, Huebner H, Ankrah NA,Ofori-Adjei D, Jolly P, Johnson N, Taylor J, Marroquin-Cardona A, XuL, Tang L, Wang JS: Reducing human exposure to aflatoxinthrough the use of clay: a review.  Food Addit Contam Part A ChemAnal Control Expo Risk Assess 2008, 25:134-145.9. Gershkovich P, Darlington J, Sivak O, Constantinides PP, Wasan KM:Inhibition of intestinal absorption of cholesterol by surface-modified nanostructured aluminosilicate compounds.  JPharm Sci 2009, 98:2390-2400.10. Lukic T, Wasan KM, Zamfir D, Moghadasian MH, Pritchard PH: Dis-in apolipoprotein E-deficient mice.  Metabolism 2003,52:425-431.11. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG,Rubin EM, Breslow JL: Severe hypercholesterolemia andatherosclerosis in apolipoprotein E-deficient mice createdby homologous recombination in ES cells.  Cell 1992,71:343-353.12. Clarey M, Edwards J, Tsipursky SJ, Beall GW, Eisenhour DD: Methodof manufacturing polymer-grade clay for use in nanocom-posites.  US Patent No. 6050509 2000.Additional file 1Inhibition of Intestinal Absorption of Cholesterol by Surface-Modified Nanostructured Aluminosilicate Compounds. This study demonstrated the ability of surface-modified nanostructured aluminosilicate (NSAS) compounds to reduce dietary cholesterol intestinal absorption in a rat model.Click here for file[http://www.biomedcentral.com/content/supplementary/1476-511X-8-30-S1.pdf]yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 5 of 5(page number not for citation purposes)odium ascorbyl phytostanyl phosphate reduces plasma cho-lesterol concentrations and atherosclerotic lesion formation

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