58

AtherosclerosisandAntioxidants

YehoshuaLewis

Abstract:

Cellularlipidoxidationisaknowncauseforthecascadeleadingtotheformationoflipidladenfoam cells, which can cause of atherosclerosis.While statins and antioxidants have recentlycome under question in the amelioration of atherosclerosis, Flavonoids have recently beentoutedasapowerfulantioxidantandsuppresserofatherosclerosis.Thispaperwillattempttoshow why statins and vitamin E have come under scrutiny, and how the desired effects ofFlavonoidscanbeattributedtotherole itplays in increasedparaoxonase-1activity(aknownanti inflammatoryassociatedwithHDL),decreasedC-Reactiveproteinactivity,and increasednitricoxide(NO)inendothelialcellsamongotherfactors.

Introduction:Coronaryarterydisease(CAD)istheleadingcauseofdeathintheUnitedStateseveryyear.Accordingtothe Center for Disease Control and the National Center for Health Statistics, data for the year 2004indicatethatheartdiseasekilled652,486people(U.S.DEPARTMENTOFHEALTHANDHUMANSERVICES,2008).Thisisnearly100,000morethanthesecondleadingcauseofdeathintheUnitedStates,cancer.Atheroscleroticplaquebuild-up inthearteries isthecauseofCAD,andhasbeentheprimaryfocusofscientists in search of a cure for CAD.Needless to say,much research has been done on finding thecausesofheartdiseaseanddevelopingdrugsthatinhibititsdevelopment.Otherthenpharmacologicalproducts,thereisaverylargebodyofresearchintomorenaturalremediesforatherosclerosis,includingantioxidants such as vitamin E that have been touted as providing protection against lipid oxidation.Recently, however, there is a growingbodyof evidence that statins andantioxidant supplementationmaynotbebeneficialasthought.Incontrast,Flavonoids,byoperatingundermechanismsdifferentthenantioxidantsandstatins,offersnewhopeinthetreatmentofatherosclerosis.

CoronaryArteryDisease:WhiletherearemanyfactorsthatcontributetoCAD,thereareanumberoffactorsspecificallyrelatedtoatherosclerosisthatplayakeyroleinitsdevelopment.Atherosclerosisisaprogressivediseaseofthe

arteriesandistheresultofplaquebuildupinthearteriesdrivenbytheuptakeofcholesterolinarterywalls(MedlinePlus,2007).Lipidsarenotwater soluble,andmust relyon lipoproteins,produced in the liverandsmall intestines, in order tobe transported in theblood.By combininglipidswithlipoproteins,thelipidsareabletobetransportedeithertotheliver for elimination, or from the liver for the production of steroidhormones, bile salts, and for cell membrane repair. The two majorlipoproteins involved in these functions are low-density lipoproteins(LDL), and high-density lipoprotein (HDL); with LDL being responsible

Figure1.Dissectedaortawithatheroscleroticlesions(Ewing,1972)

YehoshuaLewis,BS’08graduatedTouroCollegewithadegreeinBiology.HeiscurrentlyastudentatTouro’sSchoolofHealthSciencesPAprogram.

59

lipid transport from the liver, and HDL being responsible for transport of lipids to the liver forelimination(Tortora&Derrickson,2006).

PlaqueformationbeginswiththeaggregationofexcessLDL in the inner layerofarterialwallswhere the lipoproteins are subject to oxidation. In response, substances released by endothelial andsmoothmusclecellsattractmacrophagesthatingesttheoxidizedLDLformingplaque.Somuchso,thatthe macrophages take on a foamy appearance under the microscope (foam cells). Additionally,lymphocytes (T cells) follow the macrophages into the arterial wall and increase the streaks in thearterialwalls.Subsequently,themacrophagescausethemigrationofthemiddle layerofthearterytothe surface of the plaque thus separating it from the bloodstream, but also narrowing the arteriallumen.Intheeventthatthecapovertheplaquebursts,theTcellsstimulatethefoamcellstoproducetissue factor (TF), which ultimately leads to blood clot formation and possible obstruction of thecoronary artery, resulting in myocardial infarction (MI). Other factors currently being considered byresearchersincludeC-reactiveproteins,whichbindtodamagedcellsandassistinphagocytosis,clottingfactors, and the amino acid homocysteine that promotes platelet aggregation (Tortora& Derrickson,2006).

Statins:In order to treat atherosclerosis,

scientists have focused their attention onreducing the amount of cholesterolavailable in the bloodstream for oxidationwiththeuseofstatindrugs.

In the 1960’s (Endo, 1992) scientistsdiscovered that when cholesterol iscompletely eliminated from the diet, theliver takes over and produces up to 82%percent of the required cholesterol.However,whencholesterol isaddedtothediet the synthesis of cholesterol is almostcompletely suppressed. They discoveredthat the production or suppression ofcholesterol was in response tomodulationby HMG-CoA reductase which modulatesthe inhibition of the Mevalonate pathwayofcholesterolsynthesis (Figure2).Throughmodulating this enzyme theywere able toalter the production of cholesterol. Thuswas born the first statin drugs. However, therearemedical researchers who challenge the claim that statin drugs actually lower the risk factors formyocardial infarction (MI). In a scathing review of studies purporting to show that by lowering

Figure2.Mevalonatepathwayofcholesterolsynthesis(ChemistryDaily,2007)

60

cholesterolwith statin drugsmortality rates are decreased, Kauffman (Kauffman, 2007) cites a studycarriedoutinNewYorkCityofagroupof2,277peoplewiththemedianageof76whowerestudiedforaperiodoftenyears.Whathefoundwasthat“Thechanceofdyingwastwiceasgreatinsubjectswiththe lowest quartile of total cholesterol (TC) or LDL-C levels, compared with those in the highestquartile”. Kauffman goes on to cite another studywhich shows that formen aged 35-57 “all cause”mortality rates increased for those with a TC of

61

early 1930’s by Fritz Haber and Josef Weiss and proceeds as follows: Recalling that 1%-15% of O2respired by mammals undergoes the superoxide anion (·O2−) state, we can propose the followingreactions:

Fe3++•O2−→Fe2++O2,

FollowedbytheFentonreaction:

Fe2++H2O2→Fe3++OH−+•OH,

Foranetreactionof:

•O2-+H2O2→•OH+HO-+O2.

Hydroxylradicalsareshortlivedinvivo,(Approx10-9s)andarehighlyreactive(Yanetal.,2005).Theyalsocannotbeenzymaticallyremovedbecauseof itsshorthalf lifeandtendstoreactextremelyquicklywithwhateverisinitsvicinityincluding:carbohydrates,nucleicacids,lipids,andaminoacids.

ThelimitationsofthisprocessareinherentinthatitrequiresIronIIIinordertoproceed,andtheblood plasma protein Transferrin is very effective at scavenging free iron, making it unavailable forreduction.However,ifthereisabuildupofironeitherduetodietaryIronoverloadoranyothermultipleofdiseases,itcanoutstriptheabilityofTransferrintoperformproperlyandresultsintheproductionoffreeradicals(Fouad,2008).

Antioxidants:Astheoxidationoflipidsformakeystepintheformationofatherosclerosis,itshouldalsolead

to the conclusion that “antioxidants” offer protection against lipid oxidation. However, the moreresearch that is being done, themore it becomes clear that exogenous sources of antioxidants havequestionable effects in vivo. The field of antioxidants is very broad and can be the subject of manypapers,butjusttoscratchthesurface;antioxidantscanbebrokendownintometaboliteandenzymaticcategoriesaswellaslipidsolubleandwatersoluble.Thedifferencebetweenthemistheirmechanismaswellassiteofaction.Forexample: lipidsolubleantioxidantsact incellmembranes,andwatersolubleantioxidants act in the blood plasma and cytoplasm. Lipid soluble antioxidants include vitamin E andcoenzymeQ(Q10).Watersolubleantioxidantsincludeascorbicacid(vitaminC),glutathione,lipoicacid,uric acid, and polyphenols (Flavonoids, tannins). Enzymatic antioxidants include the previouslymentionedSOD,catalase,aswellasglutathioneamongothers(Sies,1997).

VitaminE isapotentantioxidantandsomeresearchhasshownthat itcanregulateHMG-CoAreductase(Paletal.,2003;Parker,1993).VitaminEisthenameforagroupoffourtocopherolsandfourtocotrienols,ofwhichthemost interestingtoscientists is theα-tocopherolvarietybecauseof itshighbioavailability.VitaminEisthemainlipid-solubleantioxidantinthebodyanditworksincellmembraneswhereitpreventsthepropagationoffreeradicalreactions(Herrera&Barbas,2001).

BecausevitaminEislipidsoluble,whenabsorbedvitaminEispackagedinChylomicrons(largelipoprotein particles) that are produced in the absorptive cells in the small intestine. They are then

62

secretedviaexocytosisintolactealsofthelymphaticsystemanddeliveredintothecirculatorysystematthejunctureofthethoracicductandtheleftsubclavianwheretheyaretransportedtotheliver.Attheliver, lipoprotein lipase (LPL) induces theunloadingofsomeof thechylomicrons intotheextrahepatictissue and the remainder chylomicrons transports the tocopherols into the liver. Here, “α-tocopheroltransferprotein"incorporatesα-tocopherolintoverylowdensitylipoprotein(VLDL)theexcessofwhichisexcretedintothebileduct.Theα-tocopherolbindedVLDListhentransportedintocirculationwhereitisconvertedtoLDLagainbytheactionofLPL.Theα-tocopherolisthentakenupbytheendothelialcellmembranebytheuptakeofLDLviacellreceptors(Herrera&Barbas,2001).

Onceinthecellmembrane,α-tocopherolcanactasaterminatortolipoproteinoxidationchainreactionasfollows:

Where“I”istheinitiatorand“LH”isthefatty acidandL•isformedfromthefattyacid

I+LH→L•+IH[1]

Propagationcontinuesasfollows:

L•+O2→LOO•[2]

LOO•+LH→LOOH+L•[3]

(SeeFigure3)

Terminationoccurswhentocopherol“TO”breaksthechainreaction:

LOO•+TOH→LOOH+TO•[4]

The tocopheroxyl radical then reacts withanother peroxyl radical to form tocopherylquinine (Q10) and thereby arresting the chainreaction(Wolf,2005).

As with almost every system in the body, vitamin E does not function in isolation but is part of anantioxidant network in which vitamin E can be synthesized from vitamin C and thiol redox cyclesinvolving glutathione and lipoic acid. (See attachment). Vitamin C has pro-oxidant properties as wellbecauseof itsability to reducemetal ionsvia theFenton reactionwhichcanbea sourceofoxidativestress.However,thisisthoughttobeofminorsignificanceascomparedwithitsantioxidantproperties(FREI,1999).

Ontheotherfront,researchhasshownthatvitaminE,specificallytocotrienols,canmodulateHMG-CoAreductasesimilarwaytothatdonebystatinsasdiscussedpreviously.(Parkeretal.,1993)

Figure3.Thefreeradicalmechanismoflipidperoxidation

63

Anumberof studiesshowedthiseffect inmammals. Inonestudyhyperlipidemicpigsgivenadiet rich in tocotrienols showed a marked decrease in plasma concentrations of cholesterol,apolipoproteinB(requiredintheformationofLDL),thromboxaneB2andplateletfactor4(bothrequiredforbloodcoagulation),indicatingaprotectiveeffectonendotheliumandplateletaggregation(Qureshietal.,1991).Therehavebeensomeconflictingresults insimilartrials inhumans;however,thosemaybeattributed toplasma levelsof tocotrienolsandnot to theefficacyof the compound (Packeretal.,2001).ItwouldthusseemthatbyingestingvitaminEonewouldbeabletotakeconcretemeasuresinarrestingthepropagationofatherosclerosis.

However, a recent Meta analysis by The Cochrane Collaboration - of studies proving theremedialeffectsofvitaminEaswellasotherantioxidants-hasnotsupportedthoseresults.Notonlydidtheingestionofexogenousantioxidantsnotprovetobebeneficialbuthasalsoshowntobedetrimental.The study included 232,550 participants who were “randomised to antioxidant supplements (beta-carotene,vitaminA,vitaminC,vitaminE,andselenium)versusplaceboornointervention”.“Atotalof17,880of 136,023participants (13.1%) randomised to antioxidant supplements and 10,136of 96,527participants(10.5%)randomisedtoplaceboornointerventiondied.Intheanalysesofthetrialswithlowriskofbias,beta-carotene,vitaminA,andvitaminEsignificantly increasedmortality.”Thestudy flatlystatesthatthereisnoevidencetosupportantioxidantsupplementation.Thisstudybringsintoquestionthewidespreaduseofantioxidantsasaremedialand/orprophylacticcompoundforoxidativediseases(Bjelakovicetal.,2008).

Flavonoids:Increased consumption of fruits and vegetables is associatedwith reduced incidences of CAD

andotherdisease. The causeof thishasbeenattributed inpart, toantioxidant Flavonoidspresent inthesefoods.ThehypothesisinthiscaseisthatbecauseaftertheconsumptionofFlavonoidsthereisamarked increase in the antioxidant properties of blood plasma and it can play a direct role in thepreventionof lipidoxidation (Lotitoa&Frei,2006).Aclearexampleof this canbeseen in theFrenchparadox,wheredespitetheFrenchconsuming largeamountsofredmeatthatarerich inunsaturatedfats theystilldevelop lower instancesofdeathfromCADthenAmericans.ThishasbeenattributedtotheirconsumptionofredwinewhichisrichinFlavonoids(Ferrières,2004).

MichaelAviramoftheTechnionfacultyofmedicinehasdoneextensiveresearchinthefieldofpomegranatepolyphenols(Flavonoids),andhasmadeaveryimportantcontributiontothefield.Amongthe points studied threewill be focused on: Antioxidant properties of blood plasma, paraoxonase-1(PON-1)1levels,andaorticstenosis-allaftertheingestionofPJ.Inonestudy(Aviram,etal.,2000),bothhealthy human males and atherosclerotic E-deficient (E0) mouse subjects were fed PJ for variableperiods of time. The objective of the study was to determine what effect PJ consumption had onlipoproteinoxidation,aggregationandretention;macrophageatherogenicity;plateletaggregation;andatherosclerosis.Theresultsofthestudywerestriking.Inhumans,PJconsumptionresultedinadecreasein LDL susceptibility to aggregation (-11% ex vivo) and retention, also observed was serum PON-1increasedactivityby20%andthatitalsohadaprotectiveeffectagainstLDLoxidation.Humanplasmain1 Paraoxonase1(PON1)isananti-inflammatoryenzymelocatedonHDL.

64

one case showedadecrease in lipidplasmaperoxidationof ≤33%. In E0mice, a 90%decrease in LDLoxidationwasobservedaswellasa20%decreaseintheuptakeofLDLbyperitonealmacrophages.Alsoobserved was a 44% decrease in atherosclerotic lesions and foam cells as compared with water fedmice.Aviramet al., attributes theprotective antioxidant effectof PJ to increased cellular glutathionecontent, and states that the reduction of atherosclerotic lesions is directly related to the antioxidantproperties of PJ. Another study by Aviram of the long term effects of PJ consumption - in humansubjectswithcarotidarterystenosis(CAS)-oncarotidlesionsandchangesinoxidativestressandbloodpressurehad remarkable results.After 1 year control subjects showeda9% increase in intima-mediathickness(IMT)asopposedtothePJgroupthatshoweda30%decreaseinIMT.PON-1activityincreasedby83%,serumLDLbasaloxidativestatewasreducedby90%,copperioninducedoxidationwasreducedby59%andtotalantioxidantstatus(TAS) increasedby130%.Again,AviramattributestheseresultstotheantioxidantcharacteristicsofPJpolyphenols(Aviram,etal.,2004).

JaneHigdon, Ph.D. of The Linus Pauling Institute atOregon StateUniversity takes a differenttack and claims that because the bioavailability of Flavonoids is very low, its antioxidant propertiescannot be attributed to the antioxidant properties proved in vitro. For example, thebioavailability ofFlavonoidsinvivois100-1000timeslowerthanthebioavailabilityofvitaminCorglutathioneandthushasa very small antioxidanteffect.However, it is available in concentrations thateffect cell signalingproteins. “Numerous studies in cell culture suggest that flavonoids may affect chronic disease byselectively inhibitingkinases” including those related togrowth factors.Higdonhypothesizes that it isthisproperty thatprotectsagainstCADbydecreasing inflammation,decreasingvascularcelladhesionmolecule expression, increasing endothelial nitric oxide synthase (eNOS) activity, and decreasingplateletaggregation(Higdon,2005).Anotherstudy(Lotito&Frei,2006),attributestheprotectiveeffectof Flavonoids not directly to its antioxidant activity, but rather to the consequence of increased uricacid.2What isclearfromallthesestudies isthat it isnottheantioxidantpropertiesofFlavonoidsthatdirectly protect against CAD but rather to a whole host of activities including: increased nativeantioxidants suchasglutathione,uricacid,modifiedcell signalingpathways, increasedPON-1activity,andincreasedNOlevels.

PossibleRisksofAntioxidantsvs.Flavonoids:Sothenwhatreallydoesaccountforthedifferences inFlavonoidsandforexamplevitaminE?

Whatmaybeatplayisasfollows:

VitaminEaswellashavingantioxidantpropertiesisapro-oxidantaswell(Bowryetal.,1992).Forexample:insteadoftheterminationreactionbytocopheroltotheoxidativechainreactionoflipidsasafinalstep:

LOO•+TOH→LOOH+TO•[4]

Weinsteadform

2 Recentstudieshaveimplicatedhyperuricemiaasbeingatrueriskfactorindevelopmentofcardiovasculardisease(JingFang&MichaelH.Alderman,2000).However,nearlyhalfofthebodies’antioxidantactivityisfromuricacidandinmoderationmayplayapositiverole.

65

TO•+LH→TOH+L•[5]

Thiswillthenfeedintothechainreaction

L•+O2→LOO•[2]

LOO•+LH→LOOH+L•[3]

Onestudy(Bowry,Ingold,&Stocker,1992)hasfoundthatinaqueoussolution,strikesbetweenthestableTO•andotherradicalswere foundto takeupwardof17minutes,a time longenoughthateven the most stable radical will find “`something to do’” i.e. propagate the chain reaction. TheterminationofthisreactionmustthenrelyontwoendogenousantioxidantsvitaminC,asdemonstratedby the thiol redox cycles (Appendix 1), and ubiquinol (Q10H2, a reduced form of Q10) in the electrontransportchain.WhatcanbeinferredfromthisisthatbyingestingvitaminEthebodysetsoffacascadeofendogenousantioxidants, inorder tominimize thedamagebeing incurredbyvitaminE.While thebodydoeshavethemechanismsofscavengingTO•howcanmakingmoreofthembyingestingvitaminEbeadvantageous?Essentially there isadelicatebalanceofelectronradicals thatarepasseddownacascade until they can be neutralized to a non reactive end product, and by adding more electronradicalsintheformofα-tocopherolitmayhavetheeffectofbeingcounterproductive.

C-reactiveproteinandPON-1Bycontrast,Flavonoidsareeffectivenotbyactingasantioxidantsbutthroughmodulatingthe

inflammatoryresponseofoxidizedcells.

TherelationshipbetweenC-reactiveprotein(CRP)andFlavonoidsillustratesthispoint.CRPisfoundtobeelevatedduring inflammatoryresponsebythebody,specifically,macrophagesandTcellsrelease Interleukin-6 (IL-6) as a pro inflammatory to stimulate a response to tissue damage. CRPproductionisthenpartoftheacute-phaseresponsetomostformsofinflammation,infection,andtissuedamage. CRP levels by themselves cannot beused asmarker for atherosclerosis, but theyhavebeenpositivelycorrelatedwithCAD(Pepys&Hirschfield,2003).FlavonoidshaveaninverserelationshipwithCRP. One study using demographic data (Chun et al., 2008) found that the greater consumption ofFlavonoidrichfoods,thelowertheconcentrationsofserumCRPthusclearlyindicatingthatFlavonoidsaffect inflammatoryresponse. AnotherstudyhasalsofoundaninverserelationshipbetweenlevelsofCRP and levels of PON-1 expression. They found that “Higher levels of CRP seem to be generallyassociated with low levels of PON1 activity” (Macknessa et al., 2006). Which should lead to theconclusionthattheatheroscleroticprotectionassociatedwithFlavonoids isnotrelatedtoan inherentantioxidantactivitybutrathertootherfactorsasmentioned.

Anotherstudyalsofoundthatpolyphenols(i.e.Flavonoids)acutelyenhancednitricoxidebioactivity.NOis necessary to maintain vasodilatation - the lack of which is implicated in increased risk ofcardiovasculardisease(Anteretal.,2004).

66

Conclusions:In comparing the protective effects on atherosclerosis associatedwith statins, vitamin E, and

Flavonoids,therearemanyquestionsastotheabilityofstatinsandvitaminEtoaffordrealprotectionagainst atherosclerosis. Thedata inmany cases implicates these very compounds increasedmortalityrates. By contrast, Flavonoids have shown themselves able to boost the bodies’ native antioxidantsincludinguricacidandglutathione,whileatthesametimereducingtheinflammatoryresponsetotheuptakeofoxidizedlipids.Itcouldbethatmotherreallyknewbestallalong“anappleaday–reallydoes–keepthedoctoraway”. (Applepealshavebeenfoundtoconsistof40%flavonolsbyweight (Łata&Tomala,2007)).

WorksCited:

Anter,E.,Thomas,S.R.,Schulz,O.M.,Shapira,E.,Vita,J.A.,&Keaney,J.F.(2004).ActivationofEndothelialNitric-oxideSynthasebythep38MAPKinResponsetoBlackTeaPolyphenols.THEJOURNALOFBIOLOGICALCHEMISTRY,297(45),46637–46643.Aviram,M.,Dornfeld,L.,Rosenblat,M.,Volkova,N.,Kaplan,M.,Coleman,R.,etal.(2000).Pomegranatejuiceconsumptionreducesoxidativestress,atherogenicmodificationstoLDL,andplateletaggregation:studiesinhumansandinatheroscleroticapolipoproteinE–deficientmice.AmericanJournalofClinicalNutrition,71(5),1062-1076.Aviram,M.,Rosenblat,M.,Gaitini,D.,Niteck,S.,Hoffman,A.,Dornfeld,L.,etal.(2004).Pomegranatejuiceconsumptionfor3yearsbypatientswithcarotidarterystenosisreducescommoncarotidintima-mediathickness,bloodpressureandLDLoxidation.ClinicalNutrition,23(3),423-433.Bjelakovic,G.,Nikolova,D.,Gluud,L.,Simonetti,R.,&Gluud,C.(2008).Antioxidantsupplementsforpreventionofmortalityinhealthyparticipantsandpatientswithvariousdiseases.CochraneDatabaseofSystematicReviews,2.Bowry,V.,Ingold,K.,&Stocker,R.(1992).VitaminEinhumanlow-densitylipoprotein.Whenandhowthisantioxidantbecomesapro-oxidant.TheBiochemicalJournal,288(2),341-4.ChemistryDaily.(2007,Jan4).HMG-CoAreductasepathway.RetrievedMay27,2008,fromTheChemistryEncyclopedia:http://www.chemistrydaily.com/chemistry/HMG-CoA_reductase_pathwayChun,O.,Chung,S.,Claycombe,K.,&Song,W.(2008).SerumC-ReactiveProteinConcentrationsAreInverselyAssociatedwithDietaryFlavonoidIntakeinU.S.Adults.TheJournalofNutrition,138,753-790.

67

Davis,K.J.(1995).Oxidativestress:theparadoxofaerobiclife.BiochemicalSocietySymposia(61),1-31.Endo,A.(1992).ThediscoveryanddevelopmentofHMG-CoAreductaseinhibitors.JournalofLipidResearch,33,1569-1582.Ewing,E.P.(1972).(CentersforDiseaseControlandPrevention)Retrieved527,2008,fromhttp://en.wikipedia.org/wiki/Image:Atherosclerosis%2C_aorta%2C_gross_pathology_PHIL_846_lores.jpgFerrières,J.(2004).TheFrenchparadox:lessonsforothercountries.Heart,90(1),107-111.Fouad,T.(2008).FreeRadicals,Types,SourcesandDamagingReactions.RetrievedMay27,2008,fromDoctorsLounge:http://www.doctorslounge.com/primary/articles/freeradicals/freeradicals6.htmFREI,A.C.(1999).DoesvitaminCactasapro-oxidantunderphysiologicalconditions?TheFASEBJournal,13,1007-1024.Herrera,E.,&Barbas,C.(2001).VitaminE:action,metabolismandperspectives.JournalofPhysiologyandBiochemistry,57(2),43-56.Higdon,J.(2005,April19).MicronutrientInformationCenter-Flavonoids.RetrievedMarch27,2008,fromLinusPaulingInstitute-MicronutrientResearchforOptimumHealth:http://lpi.oregonstate.edu/infocenter/phytochemicals/flavonoids/Holbrook,T.F.(2000).Oxidants,oxidativestressandthebiologyofageing.Nature,408,239-247.JingFang,M.,&MichaelH.Alderman,M.(2000).SerumUricAcidandCardiovascularMortality.JAMA-TheJournalofTheAmericanMedicalAssociation,283,2404-2410.Kauffman,J.M.(2007).MisleadingRecentPapersonStatinDrugs.JournalofAmericanPhysiciansandSurgeons,Volume12(Number1),7-9.Koppenol,W.(2001).TheHaber-Weisscycle–70yearslater.RedoxReport,6(4),229-234.Łata,B.,&Tomala,K.(2007).Applepeelasacontributortowholefruitquantityofpotentiallyhealthfulbioactivecompounds.Cultivarandyearimplication.JournalofAgriculture&FoodChemistry,55(26),10795-802.Lotito,S.,&Frei,B.(2006).Consumptionofflavonoid-richfoodsandincreasedplasmaantioxidantcapacityinhumans:cause,consequence,orepiphenomenon?FreeRadicBiology&Medicine,41(12),1727-46.

68

Macknessa,B.,Hinea,D.,McElduffb,P.,&Mackness,M.(2006).HighC-reactiveproteinandlowparaoxonase1indiabetesasriskfactorsforcoronaryheartdisease.Atherosclerosis,186(2),396-401.McGraw-Hill'sAccessScience.(n.d.).Superoxidechemistry.Retrieved527,2008,fromEncyclopediaofScienceandTechnologyOnline:http://www.accessscience.com/abstract.aspx?id=669650&referURL=http%3a%2f%2fwww.accessscience.com%2fcontent.aspx%3fid%3d669650MedlinePlus.(2007,March30).MedlinePlusMedicalEncyclopedia.(U.S.NationalLibraryofMedicine&theNationalInstitutesofHealth(NIH))RetrievedMay27,2008,fromMedlinePlus:http://www.nlm.nih.gov/medlineplus/ency/article/000171.htm#Causes,%20incidence,%20and%20risk%20factorsPacker,L.,Weber,S.U.,&Rimbach,G.(2001).MolecularAspectsof{alpha}-TocotrienolAntioxidantActionandCellSignalling.JournalofNutrition,131,369S-373S.Pacher,P.,Beckman,J.S.,&Liaudet,L.(2007).NitricOxideandPeroxynitriteinHealthandDisease.PhysiologicalReviews,87(1),315-424.Pal,S.,Thomson,A.M.,Bottema,C.D.,&Roach,P.D.(2003).α-TocopherolmodulatesthelowdensitylipoproteinreceptorofhumanHepG2cells.NutritionJournal(2).Pepys,M.B.,&Hirschfield,G.M.(2003).C-reactiveprotein:acriticalupdate.TheJournalforClinicalInvestigation,11(12),1805-1812.Qureshi,A.,Qureshi,N.,Hasler-Rapacz,J.,&Weber,F.e.(1991).Dietarytocotrienolsreduceconcentrationsofplasmacholesterol,apolipoproteinB,thromboxaneB2,andplateletfactor4inpigswithinheritedhyperlipidemias.AmericanJournalofClinicalNutrition,53,1042S-1046S.Ravnskov,U.,Rosch,P.,Sutter,M.,&Houston,M.(2006).Shouldwelowercholesterolasmuchaspossible?BritishMedicalJournal,332,1330-1332.Parker,R.A.,Pearce,B.C.,Clark,R.W.,Gordon,D.A.,&Wright,J.J.(1993).Tocotrienolsregulatecholesterolproductioninmammaliancellsbypost-transcriptionalsuppressionof3-hydroxy-3-methylglutaryl-coenzymeAreductase.THEJOURNALOFBIOLOGICALCHEMISTRY,268(15),11230-11238.Runge,M.S.(1999).THEROLEOFOXIDATIVESTRESSINATHEROSCLEROSIS:THEHOPEANDTHEHYPE.TRANSACTIONSOFTHEAMERICANCLINICALANDCLIMATOLOGICALASSOCIATION,110,119-130.

69

Sies,H.(1997).Oxidativestress:oxidantsandantioxidants.ExperimentalPhysiology,82,291-295.Tortora,G.J.,&Derrickson,B.(2006).Disorders:HomeostaticImbalances.InPrinciplesofAnatomyandPhysiology(11ed.,pp.726-727).JohnWiley&Sons,Inc.U.S.DEPARTMENTOFHEALTHANDHUMANSERVICES.(2008,April11).NCHS-Fastats-LeadingCausesofDeath.(CenterforDiseaseControl)RetrievedMay27,2008,fromNationalCenterforHealthStatistics:http://www.cdc.gov/nchs/FASTATS/lcod.htmWolf,G.(2005).TheDiscoveryoftheAntioxidantFunctionofVitaminE:thecontributionofHenryA.Mattill.TheJournalofNutrition,135,363-366.Yan,E.B.,Unthank,J.K.,Castillo-Melendez,M.,Miller,S.L.,Langford,S.J.,&Walker,D.W.(2005).Novelmethodforinvivohydroxylradicalmeasurementbymicrodialysisinfetalsheepbraininutero.JournalofAppliedPhysiology,98,2304-2310.