LOVASTATIN IMPROVES FLUORIDE-INDUCED HYPERCHOLESTEROLEMIA IN ALBINO RATS

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1 69 69 LOVASTATIN IMPROVES FLUORIDE-INDUCED HYPERCHOLESTEROLEMIA IN ALBINO RATS Snjy S Krn, Snjy B Pnvr, Rupl A Vsnt, AVRL Nrsimhchry Vllbh Viyngr, Ini SUMMARY: Fluorie (F) is n environmentl contminnt in mny countries n, the chronic intke of F lters the boy physiology, especilly for lipi n ntioxint metbolism. Since F is reporte to cuse hypercholesterolemi leing progressively to hyperlipiemic conitions n people resiing in enemic F res re likely cnites for hypercholesterolemi n criovsculr iseses, the present work ws esigne to investigte the efficcy of stnr rug lovsttin in F-inuce hypercholesterolemi. A comprison ws me, in rts, between F- expose, high cholesterol iet, n F-expose + high cholesterol iet nimls with specil reference to the utility of lovsttin in normlizing the plsm n tissue lipis n the ntioxint profiles. The results inicte tht both F-expose n F- expose + high cholesterol iet nimls registere higher lipi profiles with reuction in ntioxint sttus. However, the F-expose + high cholesterol iet fe nimls were more ffecte thn the F-expose nimls. Lovsttin ministrtion cuse significnt ecline in lipi profiles n improve the ntioxint sttus of the F-expose n F-expose + high cholesterol iet nimls significntly besies being effective in the high cholesterol iet rts. Thus the results inicte tht lovsttin might be consiere for treting hypercholesterolemi in F enemic res. However, it ws note tht sttins my hve limite or no efficcy in reucing overll mortlity n crry significnt verse effect risks incluing for muscle pin, memory impirment, reuce energy, n exertionl ftigue. Keywors: Antioxint sttus; Hypercholesterolemi; Lovsttin; Oxitive stress; Sttins. INTRODUCTION Fluorie (F) relte humn helth problems hve become globl spnning severl ntions n continents. Higher mounts of F intke bring bout severl metbolic ltertions by intercting with vrious cellulr processes n isrupting the ntioxint efense systems in the boy. 1,2 It is lso well known tht F cts s hypercholesterolemic gent s it increses plsm n tissue lipis in the lbortory mmmls. 3,4 The present experiment ws esigne to investigte the effects of lovsttin, ( stnr ntihypercholesterolemic rug) on the plsm n tissue lipi profiles n on the ntioxint sttus of F-expose n F-expose + high cholesterol iet fe lbortory rts. MATERIALS AND METHODS Animls: Colony bre femle Chrles Foster rts weighing ( g) were use for the present investigtion. The nimls were house iniviully with free ccess to wter with mesure fee, in well ventilte (26±2ºC, humiity 62%) niml house fcility of the Deprtment of Biosciences, Srr Ptel University, Vllbh Viyngr, uner nturl photoperioic conitions. The experimentl work ws crrie out following the guielines of Institutionl Animl Ethics For corresponence: Dr AVRL Nrsimhchry, Lbortory for Animl Sciences, Deprtment of Biosciences, Srr Ptel Min, Vtl Ro, Stellite Cmpus, Srr Ptel University, Vllbh Viyngr , Gujrt, Ini; E-mil: nrsimhchry@yhoo.com

2 70 70 Committee (MoEF/CPCSEA/ Reg. 337/2001) n pprove by the Institutionl Committee for niml reserch. After 10-y pttion perio, 42 nimls were rnomly segregte into seven groups of six rts ech: norml control (NC), hypercholesterolemic control (HC), hypercholesterolemic nimls trete with lovsttin (HLS), F control (FC), F-expose nimls trete with lovsttin (FLS), F-expose nimls fe hypercholesterolemic iet (FHC) n F-expose hypercholesterolemic iet fe nimls trete with lovsttin (FHLS) (Tble 1). Tble 1. Experimentl groups n tretment protocol. (Fluorie exposure: 100 ppm of fluorie [ mg of soium fluorie in 1 L of rinking wter]; Inuction of hypercholesterolemi: 1 g % of cholesterol n 0.15 g % of bile ci ws e to the commercil iet; Lovsttin tretment: 0.08 g of lovsttin ws incorporte in 1 kg of the hypercholesterolemic iet; W: wter; FW: fluorie wter; CD: commercil iet, HCD: high cholesterol iet; LS: lovsttin) Group Tretment weeks W FW CD HCD LS Norml control (NC) Hypercholesterolemic control (HC) Hypercholesterolemic nimls trete with lovsttin (HLS) Fluorie control (FC) F expose nimls trete with lovsttin (FLS) F expose nimls fe hypercholesterolemic iet (FHC) F expos e hypercholes terolemic iet fe nimls trete with lovsttin (FHLS) Hypercholesterolemi ws inuce by ition of cholesterol (1 g%) n bile ci (0.15 g%) to commercil iet (Kevl Agro Inustries, Vor) 5. F toxicity ws inuce by exposing the rts to 100 ppm of F ( mg/l of NF) through rinking wter. 1 The high ft iet ws supplemente with lovsttin t the rte of 0.08 g/kg. 6 Bloo cholesterol levels were monitore from the fourth week onwrs t intervls of one week up until the en of the experiment. Animls showing 1.5 fol increse in bloo cholesterol levels (compre to the NC group) were consiere hypercholesterolemic t the en of 7 week perio. The nimls were eprive of foo n wter overnight, n scrifice t the en of 10 th week using ether nesthesi. Bloo, liver, kiney tissues, n fecl mtter were collecte immeitely n kept frozen until nlyze. Bloo ws collecte by cric

3 71 71 puncture into EDTA cote tubes n plsm ws seprte fter centrifugtion t 3000 rpm n store t 20ºC for further nlysis. Anlyticl Proceures: PLASMA AND HEPATIC LIPID PROFILES: Plsm totl cholesterol (TC), high-ensity lipoprotein cholesterol (HDL-C) n triglyceries (TG) were mesure by stnr kits (Eve s Inn Dignostics, Ini) n the plsm totl lipi (TL) content ws estimte by the sulphophosphovnillin metho. 7 Low-ensity lipoprotein cholesterol (LDL-C), very low-ensity lipoprotein cholesterol (VLDL-C), n therogenic inex (AI) were clculte. 8 The heptic TL ws extrcte in chloroform: methnol (2:1) n estimte by grvimetry. 9 The sme extrct ws use for the estimtion of TC n TG contents by the respective kits (Eve s Inn Dignostics, Ini). HEPATIC HMG-COA REDUCTASE AND BILE ACID PROFILE: Heptic hyroxymethylglutryl-coenzyme A reuctse (HMG-CoA reuctse, EC ) ctivity ws mesure in terms of rtio of HMG-CoA to mevlonte 10 s HMG-CoA reuctse ctivity is inversely proportionl to the rtio of HMG-CoA/ mevlonte. Bile ci content ws estimte using vnillin-phosphoric ci regent. 11 FECAL LIPIDS AND BILE ACID CONTENT: Fecl totl lipi (TL) content ws estimte by the sulphophosphovnillin metho. 7 The fecl cholesterol n bile cis were extrcte in lkline-methnol meium n estimte. 11,12 PLASMA, HEPATIC, AND RENAL TISSUE LIPID PEROXIDATION AND ANTIOXIDANT PROFILES: Lipi peroxition ws etermine by the thiobrbituric ci (TBA) ssy. 13 Totl scorbic ci n reuce glutthione contents were estimte using methos of Schffert n Kingsley 14 n Jollow et l. 15 Superoxie ismutse (SOD; EC ), ctlse (CAT; EC ) n glutthione peroxise (GPx; EC ) ctivities were mesure by stnr methos Ferric reucing bility of plsm (FRAP) ws mesure in the plsm by the metho of Benzie n Strin. 19 Sttisticl nlysis: Dt re presente s men±sem. One-wy nlysis of vrince (ANOVA) with Tukey s significnt ifference post hoc test ws use to compre ifferences mong groups. Dt were sttisticlly hnle by Grph P Prism 3.0 sttisticl softwre. P vlues <0.05 were consiere sttisticlly significnt. RESULTS AND DISCUSSION The present investigtion clerly shows tht lovsttin significntly improves the lipi n ntioxint profiles of hypercholesterolemic nimls (both F-expose n F-unexpose) inicting its ntihyperlipiemic, ntiperoxitive n ntioxint properties even in F-inuce metbolic ltertions in lbortory rts. F-exposure is reporte to increse the plsm lipi n cholesterol levels leing to hyperlipiemi. 1,4,20 It is clerly seen tht when HC nimls (fe on high cholesterol iet) were expose to F through rinking wter, the plsm lipi profiles were further elevte. In ition, these nimls lso exhibite significnt ecline in plsm HDL-C levels long with lowere FRAP vlues.

4 72 72 Lovsttin ministrtion not only reuce the plsm n heptic lipi profiles in the HC group but lso in the FC n HFC groups. It is well ocumente tht while lower levels of HDL-C re inictive of n increse risk for coronry hert isese, n increse in HDL-C levels is consiere beneficil. 21 With lovsttin ministrtion to the HC, FC, n FHC groups the triglyceries n LDL-C levels were lowere n HDL-C levels were elevte. Thus lovsttin ppere to normlize the plsm n heptic triglycerie levels (Tbles 2 n 3). Tble 2. Plsm lipi profiles n FRAP vlues of experimentl groups (Vlues re men±sem in mg/l (n=6) prt from those for FRAP which re in mmoles/ml; TL: totl lipis; TC: totl cholesterol; TG: triglyceries; HDL-C: high-ensity lipoprotein cholesterol; VLDL-C: very low-ensity lipoprotein cholesterol; LDL-C: low-ensity lipoprotein cholesterol; Al: therogenic inex; FRAP: ferric reucing bility of plsm; NC: norml control; HC: hypercholesterolemic control; HLS: hypercholesterolemic nimls trete with lovsttin; FC: fluorie control; FLS: F expose nimls trete with lovsttin; FHC: F expose nimls fe hypercholesterolemic iet; FHLS: F expose hypercholesterolemic iet fe nimls trete with lovsttin) Prmeter NC HC HLS FC FLS FHC FHLS TL TC TG HDL-C VLDL-C LDL-C AI FRAP ± ± ± ± ± ± ± ± ±21.24 ±26.63 b ±23.74 ±17.02 bc ±59.90 c ± c ±3.97 ±2.23 b ±3.47 ±2.81 c ±5.08 ±2.83 b ±2.77 ±2.04 b ±3.75 ns ±1.90 ±2.49 c ±3.94 b ±3.31 ±1.03 ±0.9 ±0.73 ±0.45 b ± ±0.55 ±0.41 b ±0.38 ns ±0.75 b ±0.49 c ± ±4.65 ±2.70 b ±3.31 ±2.72 c ±3.89 c ± ±0.24 ±0.11 b ±0.14 ±0.11 c ±0.12 b c ± ±0.21 ±0.62 b ±0.84 ±0.51 c ±0.54 b ±0.32 p<0.05 compre with NC; b p<0.05 compre with HC; c p<0.05 compre with FC; p<0.05 c ompre with FHC; ns not significnt. Exposure of rts to high cholesterol (through iet), F, n both F n high cholesterol iet mrginlly influence the ctivity of HMG-CoA reuctse n resulte in n increse in heptic bile ci content when compre to controls. Aministrtion of lovsttin to these nimls resulte in significnt increses in HMG-CoA ctivity (Tble 3). Lovsttin hs been shown to inuce HMG-CoA reuctse ctivity in rt heptocyte smooth enoplsmic reticulum (SER) n it ws suggeste lovsttin cuses the formtion of smooth enoplsmic reticulr (SER) whorls in rt heptocytes. 22 In stuy on norml humn subjects, lovsttin ministrtion (20 40 mg twice y) ws shown to inuce HMG-CoA ctivity in mononucler leukocytes. 23 With this stuy, Stone et l., 23 conclue tht the effect of lovsttin on LDL-metbolism is inversely relte to the homeosttic control of

5 73 73 sterol synthesis through HMG-CoA reuctse ctivity. Further, Ymuchi et l., 24 emonstrte tht when 0.1% lovsttin with low cholesterol iet ws fe to lbortory rts, there ws seven fol increse in HMG-CoA reuctse ctivity. However, the response in the enzyme ctivity ws not significntly higher when the rts were fe high cholesterol iet. In the light of these observtions, the presently note increses in HMG-CoA ctivity clerly point to the homeosttic mechnism in sterol synthesis in the HC, FC, n HFC groups trete with lovsttin. These observtions lso inicte tht s the heptic n fecl bile ci content increse, the plsm cholesterol levels ecrese n the HMG-CoA ctivity increses s compenstory mechnism (Tbles 3 n 4). Tble 3. Heptic lipi profiles, HMG-CoA reuctse, n bile ci content of experimentl nimls (Vlues re men±sem in mg/g (n=6); TL: totl lipis; TC: totl cholesterol; TG: triglyceries; HMG- CoA reuctse ctivity is inversely proportionl t o the rtio of HMG-CoA/mevlonte; NC: norml control; HC: hypercholesterolemic control; HLS: hypercholesterolemic nimls trete with lovsttin; FC: fluorie control; FLS: F expose nimls trete with lovsttin; FHC: F expose nimls fe hypercholesterolemic iet; FHLS: F expose hypercholesterolemic iet fe nimls trete with lovsttin) Prmeter NC HC HLS FC FLS FHC FHLS TL TC TG HMG-CoA reuctse* Bile ci ± ± ± ± ± ±1.62 ±1.44 b ±1.62 ±1.78 c ±1.89 ± ±2.02 ±3.10 ±1.67 ±1.76 c ±5.05 ± ±3.57 ns ±3.25 ns ±8.57 ns ±3.96 ns ±19.55 ns ±3.47 ns ±0.60 ns ±2.13 b ±0.89 ns ±1.39 c ±1.03 c ± ±0.82 ns ±0.91 ±0.03 ns ±0.04 c ±0.82 ns ±0.15 c p<0.05 compre with NC; b p<0.05 compre with HC; c p<0.05 compre with FC; p<0. 05 c ompre with FHC; ns not significnt. Tble 4. Fecl totl lipis, totl cholesterol, n bile ci content of experimentl nimls (Vlues re men±sem in mg/g (n=6); TL: totl lipis; TC: totl cholesterol; NC: norml control; HC: hypercholesterolemic control; HLS: hypercholesterolemic nimls trete with lovsttin; FC: fluorie control; FLS: F expose nimls trete with lovsttin; FHC: F expose nimls fe hypercholesterolemic iet; FHLS: F expose hypercholesterolemic iet fe nimls trete with lovsttin) Prmeter NC HC HLS FC FLS FHC FHLS TL TC Bile ci ± ± ± ±1.52 ns ±0.66 ns ±0.33 ns ±0.57 ns ±1.20 ns ±0.33 ns ±2.39 ±1.54 ±0.76 ns ±1.97 ns ±1.34 bc ± ±1.95 ±2.79 ±4.40 b ±3.51 ns ±2.08 c ±2.02 c p<0.05 compre with NC; b p<0.05 compre with HC; c p<0.05 compre with FC; p<0. 05 c ompre with FHC; ns not significnt.

6 74 74 HC, FC, n FHC nimls exhibite significntly higher lipi peroxition in plsm, heptic, n renl tissues with reuction in enzymtic n nonenzymtic ntioxint profiles. Lovsttin tretment to these rts ecrese tissue lipi peroxition long with significntly elevte ntioxint ctivities (Tbles 5 n 6). Tble 5. Lipi peroxition n non-enzymtic ntioxint profiles in plsm, heptic, n renl tissues of experimentl groups (Vlues re men±sem (n=6); LPO: lipi peroxition; TAA: totl scorbic ci; GSH: reuce glutthione; NC: norml cont rol; HC: hypercholest erolemic control; HLS: hypercholesterolemic nimls trete with lovsttin; FC: fluorie control; FLS: F expose nimls trete with lovsttin; FHC: F expose nimls fe hypercholesterolemic iet; FHLS: F expose hypercholes terolemic iet fe nimls t rete with lovst t in) Group LPO (nm MDA/g) TAA (µg/g) GSH (mg/100g) Plsm Heptic Renl Plsm Heptic Renl Plsm Heptic Renl NC 55.5 ± ± ± ± ± ± ± ± ±0.12 HC HLS FC FLS FHC FHLS ±4.37 ns ±0.42 ±1.90 ±0.48 ns ±1.01 ns ±0.40 ±0.14 ±0.16 ± ±0.72 ns ±0.32 ns ±0.82 b ±0.58 b ±2.10 ns ±0.55 b ±0.11 b ±0.15 b ±0.11 b ±5.64 ns ±0.84 ±1.20 ±1.02 ns ±1.03 ns ±0.36 ±0.15 ±0.09 ± ±5.80 ns ±1.46 ±0.86 ±0.60 c ±1.26 ns ±0.18 ±0.14 ±0.14 c ± ±4.75 b ±1.13 bc ±0.78 ±0.19 ±1.08 ns ±0.84 ±0.28 bc ±0.11 ±0.23 b ±4.1 ±0.74 ±0.93 ±0.50 ±2.01 ns ±0.54 ±0.13 ±0.16 ±0.16 ns p<0.05 compre with NC; b p<0.05 compre with HC; c p<0.05 compre with FC; p<0.05 compre with FHC; ns not s ignificnt. Ascorbic ci is well-known ntioxint tht ecreses F toxicity in the boy. 25 Lovsttin tretment significntly increse the scorbic content in plsm n renl tissues of the HC, FC, n FHC rts lthough heptic tissue i not register ny vrition. Superoxie ismutse converts superoxie ricls into less hrmful proucts like hyrogen peroxie n ecrese superoxie ricl concentrtion wheres ctlse reuces the hyrogen peroxie n provies protection to tissues. 26,27 In the present stuy, both SOD n CAT ctivities were foun to be lowere in the HC, FC, n FHC groups s compre to the NC nimls. Lovsttin tretment to these nimls resulte in notble increse in the SOD n CAT ctivities of plsm, liver, n kiney tissues. It hs been shown tht reuction in tissue GSH coul be the result of reuce synthesis or elevte egrtion of GSH by oxitive stress in hypercholesterolemic n/or fluorotic conitions. 1,28,29 In the present context, lowere GSH levels coul be ue to inuction of hypercholesterolemi n/or exposure to F which ws reverse with the ministrtion of lovsttin. Glutthione peroxise (GPx) utilizes glutthione

7 75 75 to ecompose H 2 O 2 n/or other orgnic hyroperoxies to less/non-toxic proucts. 30 A significnt ecline in the ctivities of GSH n GPx in plsm, liver n kineys of HC, FC, n FHC nimls ws note; with lovsttin the levels of both GSH n GPx improve (Tbles 5 n 6). Tble 6. Enzymtic ntioxint profiles in plsm, heptic, n renl tissues of experimentl groups (Vlues re men±sem (n=6); SOD: superoxie ismutse; CAT: ctlse; GPx: glutthione peroxise; NC: norml control; HC: hypercholesterolemic control; HLS: hypercholesterolemic nimls trete with lovs ttin; FC: fluorie cont rol; FLS: F expose nimls trete with lovsttin; FHC: F expose nimls fe hypercholesterolemic iet; FHLS: F expose hypercholesterolemic iet fe nimls trete with lovsttin) Group SOD (U/mg protein) CAT (nm H 2O 2 ec ompos e/sec /g) GPx (U/mg protein) Plsm Heptic Renl Plsm Heptic Renl Plsm Heptic Renl NC ± ± ± ± ± ± ± ± ±0.53 HC HLS FC FLS FHC FHLS ±0.78 ±0.09 ±0.16 ±2.3 ±4.0 ±2.12 ±0.09 ns ±0.51 ±0.21 ns ±0.60 ±0.11 ±0.24 b ±2.5 b ±4.5 ±4.3 b ±0.65 ns ±1.41 b ±0.24 ns ±0.59 ±0.17 ±0.18 ±1.40 ±2.70 ±0.98 ±0.04 ns ±0.84 ±0.38 ns ±0.47 c ±0.09 c ±0.18 ±4.42 ±4.40 ±2.3 c ±0.48 ns ±0.81 c ±0.46 ns ±0.87 ±0.17 b ±0.16 ±0.8 ±1.6 ±2.91 b ±0.98 ns ±0.62 ±0.59 ns ±1.05 ±0.15 c ±0.16 ±1.63 ±5.25 ±1.79 ±0.81 ns ±0.19 ±0.48 ns p<0.05 compre with NC; b p<0.05 compre with HC; c p<0.05 compre with FC; p<0.05 compre with FHC; ns not s ignificnt. In conclusion, the present work revels the potentil of lovsttin to normlize the lipi n ntioxint profiles in F-expose, high cholesterol fe nimls. Therefore, it is surmise tht lovsttin coul be use to mintin the norml rnges of lipi n ntioxint profiles in people living in F enemic res suffering from hypercholesterolemi. However, note of cution is lso pproprite. Gøtzshe, he of the Noric Cochrne Centre, note tht sttin shoul be tken by ptient to reuce their risk of ying, not to improve some lbortory vlues. 30 He consiere tht tking sttin my reuce the risk of ying from hert isese but increse the risk of ying from some other cuses. Gøtzshe note tht Cochrne review of eight sttin trils showe reuce llcuse mortlity of 16% with 2.8% of the tril prticipnts ying. This correspone to Number Neee to Tret (NNT), the inverse of the risk ifference, in orer for one person to obtin benefit of 222 (1/(2.8% 2.35%). 31,32 Only one of the trils tht provie t on ll-cuse mortlity ws publicly fune. He note tht lrge, publicly fune tril, the ALLHAT-LLT tril, which ws not inclue in the review becuse more thn 10% of the ptients

8 76 76 h pre-existing criovsculr isese, i not fin reuction in mortlity, risk rtio 0.99, 95% confience intervl 0.89 to ,33 The pper with the tril results gives the risk rtio s 0.96, 95% confience intervl 0.89 to Sie effects inclue muscle pin 31 n memory impirment. 34 Cses of myopthy n rhbomyolosis with cute renl filure seconry to myoglobinuri hve been reporte with HMG-CoA reuctse inhibitors. 31 Sttins my cuse heptic injury n rise the bloo glucose. Golomb et l. foun tht 20% of men n 40% of women tking sttins experience worsening in either energy or exertionl ftigue. 35 ACKNOWLEDGEMENTS Thnks re ue to the He, Deprtment of Biosciences, Srr Ptel University, for proviing the fcilities require for the present work. The finncil ssistnce in the form of Project Fellowship to SSK from University Grnts Commission, New Delhi, Ini, n Reserch Associteship to RAV from DST-PURSE Progrm, Srr Ptel University, Vllbh Viyngr, Ini re grtefully cknowlege. REFERENCES 1 Strunecká A, Ptock J, Blylock RL, Chinoy NJ. Fluorie interctions: from molecules to isese. Current Signl Trnsuction Therpy 2007;2: Brbier O, Arreol-Menoz L, Del Rzo LM. Moleculr mechnisms of fluorie toxicity. Chem Biol Interct 2010; 188: Gruck-Mmczr, E, Birkner E, Ksperczyk S, Ksperczyk A, Chlubek D, Smujlo D, et l. Lipi blnce in rts with fluorie-inuce hyperglycemi. Fluorie 2004;37: Vsnt RA, Kotiy DR, Bhole KL, Nrsimhchry AVRL. Therpeutic benefits of glibenclmie in fluorie intoxicte ibetic rts. Fluorie 2010;43: Visviy NP, Nrsimhchry AVRL. Hypocholesterolemic n ntioxint effects of Glycyrrhiz glbr (Linn) in rts. Mol Nutr Foo Res 2006;50: Mohn-Kumri HP, Dhle MA, Niu AK, Vijylkshmi G. Antioxint effect of re moul rice in hypercholesterolemic Wistr mle rts. Cell Biochem Funct 2011;29: Frings CS, Fenley TW, Dunn RR, Queen CA. Improve etermintion of totl serum lipis by sulpho-phospho-vnilln rection. Clin Chem 1972;18: Frieewl WT, Levy RI, Frerickson DS. Estimtion of the concentrtion of low-ensity lipoprotein cholesterol in plsm without use of the preprtive ultrcentrifuge. Clin Chem 1972;18: Folch J, Lees MS, Stnley GHS. A simple metho for the isoltion n purifiction of totl lipies from niml tissues. J Biol Chem 1957;226: Ro AV, Rmkrishnn S. Inirect ssessment of hyroxymethylglutryl-coa reuctse (NADPH) ctivity in liver tissue. Clin Chem 1975;21: Snell FD, Snell CT. Colorimetric methos of nlysis. Vol 4. New York: D Vn Nostrn Compny, Inc.; Klek HD, Stellr F, Kruis W, Pumgrtner G. Detection of increse bile ci excretion by etermintion of bile ci content in single stool smples. Clin Chim Act 1984;140: Ohkw H, Ohishi N, Ygi K. Assy for lipi peroxies in niml tissues by thiobrbituric ci rection. Anl Biochem 1979;95: Schffert RR, Kingsley GR. A rpi, simple metho for the etermintion of reuce, ehyro-, n totl scorbic ci in biologicl mteril. J Biol Chem 1955;212: Jollow DJ, Mitchell JR, Zmpglione N, Gillette JR. Bromobenzene-inuce liver necrosis. Protective role of glutthione n evience for 3,4-bromobenzene oxie s the heptotoxic metbolite. Phrmcology 1974;11:

9 Kkkr P, Ds B, Viswnthn PN. A moifie spectrophotometric ssy of superoxie ismutse. Inin J Biochem Biophys 1984;21: Aebi H. Ctlse. In: Bergmeyer HU, eitor. Methos of enzymtic nlysis. Vol. 2. 2n e. New York: Acemic Press; pp Flohé L, Günzler WA. Assy of glutthione peroxise. Methos Enzymol 1984;105: Benzie IFF, Strin JJ. The ferric reucing bility of plsm (FRAP) s mesure of ntioxint power: the FRAP ssy. Anl Biochem 1996; 239: Vsnt RA, Nrsimhchry AVRL. Allevition of fluorie-inuce heptic n renl oxitive stress in rts by the fruit of Limoni ciissim. Fluorie 2011;44: Wilson PW, Abbott RD, Cstelli WP. High ensity lipoprotein cholesterol n mortlity. The Frminghm hert stuy. Arteriosclerosis 1988;8: Singer II, Scott S, Kzzis DM, Huff JW. Lovsttin, n inhibitor of cholesterol synthesis, inuces hyroxymethylglutryl-coenzyme A reuctse irectly on membrnes of expne smooth enoplsmic reticulum in rt heptocytes. Proc Ntl Ac Sci U S A1988;85: Stone BG, Evns CD, Prigge WF, Dune WC, Gebhr RL. Lovsttin tretment inhibits sterol synthesis n inuces HMG-CoA reuctse ctivity in mononucler leukocytes of norml subjects. J Lipi Res 1989;30: Ymuchi S, Linscheer WG, Bech DH. Increse in serum n bile cholesterol n HMG- CoA reuctse by lovsttin in rts. Am J Physiol Gstrointestinl n Liver Physiology 1991;260 (4 Pt 1):G Susheel AK. Fluorosis mngement in Ini. Current Science 1999;77: Robinson BH. The role of mngnese superoxie ismutse in helth n isese. J Inherit Metb Dis 1998;21: Brioukhnov AL, Netrusov AI. Ctlse n superoxie ismutse: istribution, properties, n physiologicl role in cells of strict nerobes [review]. Biochemistry. 2004; 69: Kushik T, Shym R, Vts P, Suri S, Kumri MML, Shrm PC, et l. Glutthione metbolism in rts expose to high-fluorie wter n effect of Spirulin tretment. Fluorie 2001;34: Suzumur K., Kshr E, Wng Y, Chien K, Inoue M Decrese turnovers of glutthione n scorbic ci in wtnbe heritble hyperlipiemic rbbits. J Nutr Sci Vitminol (Tokyo) 46: Freemn BA, Crpo JD. Biology of isese; free ricls n tissue injury. Lb Invest 1982;47: Gøtzsche PC. Dely meicines n orgnize crime: how big phrm hs corrupte helthcre. Lonon: Rcliffe Publishing; pp. 47-8, Tylor E, Wr K, Moore THM, Burke M, Dvey Smith G, Css JP, Ebrhim S. Sttins for the primry prevention of criovsculr isese [review]. Cochrne Dtbse Sys Rev 2011;1:CD Upte in: Tylor F, Huffmn MP, Mceo AF, Moore TH, Burke M, Dvey Smith G, et l. Cochrne Dtbse Sys Rev 2013;1:CD Mrgolis KL, Dvis BR, Bimbrige C, Ciocon JO, Cuyjet AB, Drt RA, et l.; ALLHAT Collbortive Reserch Group. Long-term follow-up of moertely hypercholesterolemic hypertensive ptients following rnomiztion to prvsttin vs usul cre: the Antihypertensive n Lipi-Lowering Tretment to Prevent Hert Attck Tril (ALLHAT- LLT). J Clin Hypertens (Greenwich). 2013;15(8): oi: /jch Epub 2013 Jun Grveline D. Lipitor : thief of memory. Sttin rugs n the misguie wr on cholesterol. Avilble from: USA: Dune Grveline MD: Golomb BA, Evns MA, Dimsle J, White HL. Effects of sttins on energy n ftigue with exertion: results from rnomize controlle tril. Arch Intern Me 2012;172: Copyright 2014 The Interntionl Society for Fluorie Reserch Inc Eitoril Office: 727 Brighton Ro, Ocen View, Dunein 9035, New Zeln.