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Page 1: 50089153
BRITISH.AMERICAN TOBACCO COMPANY LIMITED ~--~ 13 October 1994 Professor J T Satonen, Research Institute of Publ/c Health University of Kuopio P.O.B. 1627 70211 Kuopio Finland Dear Professor Salonen, Re: Antiozidant supplementation in atherosclerosis prevention Thank you for submitting your research proposal to British-American Tobacco. This was discuss~ at a recent meeting of BAT scientists and raised much interest. We receive many research proposals, and as with other grant awarding orgadsations, we seek expert advice to assist us in their evaluation. I have therefore sent your research proposal to two independent consultants, who will of course treat it in strict confidence. Once we have received the reviewers comments, we will be better placed to make a decision re~'arding the funding of your project, and will be in touch with as soon as that decision has been made. Your sincerely. Dr Linda Rudge Smoking Issues Department MILLBANI/ KNO~=~ GRI~EN STAIN1E$ MIDDLF.SHX TWIB IDY FAX Ol784 455 000 TI~LEX Z7.~8415 a^TTOS c T~.LI~PHO.~E OlT84 460 400 A ~E!~I~ER OF THE BAT ]NOt:$TRIE.¢ CROVP |~CCRPOt~ATEP I~ LO~.DO.~ NO ;'4974 C.D c~ ".C LJ'~ BATCo document for Legal Services • Health Canada 30 May 2000
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KUODbO NC Ref. UNIVERSITY OF KUOPlO Finland Research Institute of Public Health September 9, 1994 c.JP. ! • "" ~_ : BAT Scientific Research Group Attn: Dr. Sharon Boyse Attn: Dr. Christopher Procter Regd Office, Mi!lbank, Knowle Green Staines TWI8 IDY U.K. Dear Dr's Boyse and Procter: I approach you to inquire the possibility to get funding for a study that in my view has relevance for the tobacco industry. The study is a randomized double-blind placebo-controlled trial to test the preventive effect of vitamins C and E and their combination in smokers and nonsmokers against atherosclerosis, hypertension and cataracts. The study will also address the issue, whether the atherosclerosis and cataracts promoting effect of smoking can be counteracted by these antioxidants, which is our assumption. If that hypothesis would be verified, that would imply that smoking would do no harm (to arteries) in persons who have a sufficient intake of these antioxidants. In fact, this is the only anti- oxidant trial being planned or underway, that will compare the effect between smokers and nonsmokers. The study will take five years (1995 - 99) and its total cost will be approximately $ 500,000 per year, totalling $ 2.5 million. We have now a research grant forn the Academy of Finland that covers approximately a half of the total cost of the study, and a smaller part will come from the University of Kuopio. Oy Ferrosan Ab will donate the antioxidant supplements needed. We are looking for the lacking $ 200,000 per year for five years. Is there any chance that BAT could participate in the funding of this study? I enclose a copy of the study protocol, which is strictly confidential. I would be happy to meet with you to discuss these issues further. Yours sincerely, Jukka T. Salonen, MD, PhD, }£SCPH Professor of Academy of Finland Unrvees~ of K~. P.O.B. 1627. 70211 KUO~O. ~,n~. TEL. 971-162 211 Fax: ]58-71-162 936 ~j'~ C~ Co "-D ~J7 ~D BATCo document for Legal Services • Health Canada 30 May 2000
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Study protocol version September 7, 1994 Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) - A randomiz~ double-masked ptacebo--conn'olled 2x2 factorial trial concerning the effect of vitamin E and C supplementation in the prevention of athemscterotic progression, blood pressure elevation and eye lens opacities in smoking and nonsmoking men and postmenopausal women Principal Invesdgazor: Jukka T. Salonem MD, PhD, MScPH, Professor of the Academy of Finland and Professor of Epidemiology, University. of Kuopio Fax: 358 - 71 - 162 937 or 162 936 O~er iavc~ig=ors C" zioi~Ix~ical order) and their rcspousibuities': lagemar BjOrkbc-m. MD. PttD, Professor of Clhfical Chem/su3, (Katoliaska Insciru~. Stockholm. S~ede~): Planning and teporci.~ ,,'n~msuremem of cholesterol ox/datior, products) Herman Esterbaue.-. F--_D. Professor of Bioch~'mis~. fUniversi~ of Gr-az. Austria): Planning and reporting (I.DL oxidation). Pemi Happens, ME). R~e.aw.h associate: Co-proj~ officer. David Jacobs, PhD, P:-.'ofe~ot of Epki~'miology (University of Minnesota, MN, USA): Planning, st~istical analysis, reporting. Ma~kaa ~ M:D, Research A3sociate: Mea~ of exhaled pe~ane. George A. Kapian. P"..D. Direaor (Humaa Population Laboratory, Sta~ of California Health Deparnment, Berkeley., CA): P[a~.~. reporting. Ju.~i ~. MD, RzD. Resear.h A.csoc~e of Finnish Academy: Co-pmje~ officer. Heikki Korpela. MD. PkD, DVM, A.~tstant Professor in Preventive Medicine: Co-projea officer. Jorma Kw~p~ P-.D. Di.~=or, Laboratory of Food chemis~y, Food Resem-~ las~ru~e, Agricultural research Cemer of F~--!aad: M=~ar~nem of chohmerol oxidation products. Timo l..akka, MD. P'-..D. Act~g A.~istant Professor of prevemive mediciae: plmmmg and reporfiag (physical aaivi~,' ). back-up for clinic.xl examinadon.~. Leena ~e'~.. DrPH srudemt. Reseam.h associate: Planaing and repor-~g, a~es.smc.at of smokiag stage. BATCo document for Legal Services : Health Canada 30 May 2000 O O O
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LoRa Maijala. Mcd St.: The role of physical ~-tivi .ry in free radical stress. Minna Ma.'~.'uncn, MD, Rescarr.h a.ssociatc: Hypertension. Timo McL~-Kctcla, PhD, Acting Professor of Pharmacology' {Umvcrsi .ty of Ta.rnpere): Mca,sur~mem of LDL hy~ope.~xi~cs, con.sultadon in LDL-TRAP mc, asu~mem. Kfi.sRina N vyssSncn, MSc, Clinical Chemist: Measur~mcm of vi~. lipids and lipid oxidation. Aie_xa~cr N. Orekhov, PhD, Biochemist (Inadratc of experimental Cardiology, National Card/ology Rcsealr,21 C.~. Moscow, Russia): Planning and mponing, measurements of immun~ cholesterol and a~'ogesicity of Elina PurkkaiR. MSc. Biochemist: Mcastmm~m of susc~dbility of LDL to oxidanon mxl I..DL-TRAP. Hmr~ E. Poulsen, MD, Phl), Pharmacologist, Profit, or of Toxicology and En~tal Mcd~ne ~i of Pharmacology, University of Copenhagen): Me.asuremcmt of urinm7 DNA oxid,~on produc~. URa ;RLsm~. MPH, Nuu-itionist: food r~cvrdings. Kimmo Ron.k~ncn, MPh, Computer analyst: D=a analysis. Harri RouhiaJncn. MD. P~., Ophtaln~logir,: Planning and r-'q)or'dng, cyc Ions opackics. ~, M.D, Ophtalmologi~: ~ ~xl r~por~ng, na~sum~zm of ~¢ l~s op=:it~. Yadon~, MD, PhD, Resear~ Fellow of F~ ~: Plmu~.g and reporl~tg, ukr4sonogr~hic mscssm~t of athcm~crosis. Se~. " ~. MSc, Chemist: Selenium and mercury measummcms. Rob=~ Se].,~: (California Insrimm of Technology, ~ CA): So~ for amomau~ re~R~ of ultr'asound Jot4:2 Takaia. MD, PhD, Pmfvssor of Gcnc~ Prg~cc: Hylxn'umsion Tamkanen, Med. St.: Gender d~Tenmccs in risk factors for athemsderofic progzession, Tc:'valtama. PhD, Resea_mh investig~o~ Hemochrumatosis gear.tics. Mm'kku T~ MD, FaD, Acting Professor of Ophr, zlmology: Planning tnd reporting, ~¢ tom opa~tic,. Tomi-Ptk.ka Tuomainem. MD. Research associ=e: Iron status, hemockmmatmis. l~kko Tuomfle.hto. MD, PhD. E.pidemiologist, Researr.h Professor (National Public Health In~ of Ymlam~: Planning and repotting (Hype:tcasion). V~i-P~kk~ V~lkoaen. Mcd.St.: Fr~ radicals in hyp~umsion. Yll-Hctmmla, MD, Ph.D, settior Rm~m-..h Fellow of F~ Aca6cmy: Planning and rt~rting, na:~sm'~mcms of genetic factors in lipid oxidation. ~mfibod~es ~w.ins.' ozidi~ LDL. "M~:~ of t~ study Brt:mp wtm, sut ~ =rt mx ~ ~ f'rcms tim b~ of I[,tm@~. P.O~ 1 ~. ~t ] ~. F~. C~ms ef tt~ts proto~i or =my. Imrt 0f = m=y em b~ remit avt~abk to =~/oe= e.t=qx tt= ¢o=~m~ ~ ~e ~ ~ ~ tip===:== wm~u¢ = perm£tr~n of Proft=~r £dea==. "11~ revietmn (edit tl=m a====med ~re) ef am study F:opo~ ~rd mmm ~ti~ ¢=~ ee ~k=m~." it ~t=r re~mg. Notre of dae urlput~=lmd datta Iz~ammd i= ttti~ pmme.ol m=y Im =. BATCo document for Legal Services : Health Canada 30 May 2000 C
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Study summar'y The objective cf this climcal-epidemiological stud)' is to investigate the effect of the reduction of free :-adical stress by antioxidam supplementation in the prevention of athero- sclerotic progression, blood pressure elevation and cataract formation in man. The specific aim is to test the hypotheses that supplementing repatar smokers and nonsmokers (men and women) with either RR.R(d)--c~-tocopherol or ascorbic acid will retard the progression of atheroscterosis, reduce the incidence of atherosc!erosis related symptoms, and slow down the elevation of blood pressure and the progression of eye lens opacities. The design of the study is a 2x.2 factorial double-masked placebo-controlled randomized clinical trial. The mbjects will be regularly smoking (:>5 cigarettes/d) or nonsmoking men am:l post-menopausal women aged 50-69 years with serum cholesmml concentration ~ 5.0 mmo]/l. Smoking and nonsmoking men and women a'e, separately randomized (in 4 strata) for 3 years into o~ of the following tre2tments: (I) 200 nag (272 IU vitamin E) of d-~-tccopheryl acetate daily, (2) 500 mg of as~rbic acid daily, O) both antioxidams,'or (4) placebo. The sample size is 640 subjects at en~." and over 549 subjects are expected to complete the 3-year follow-up, providing a power of over 0.85 at type I error of 0.05 and 25% treatment effect on atherosclerofic progression at 3 years. The outcome measurements are: 1) Mean maximal inmna-modia thickness of common carotid artery walls by high-resolution B-mode ulwa~m, graphy (the primary, study outcomeXevery 6 months), 2) Ath.,,rosclerosis-relarad symptoms (chest pain. claudieationXanmmlly), 3) Blood pressu~ (every, 6 months), 4) Eye lens opac~es by ophtalmologist zssessme~." (twice), 5) Plasma c~-tccop, herol and ascorbic acid concenn'adons (ammally), and 6) Mcasuremenr.s of whole blood glutathione (GSH), exhaled pcmat~ (in a subsample), plasma cholesterol oxidation products (in a subsample), total radical scavenging antioxidative capacit'y (TRAP) of LDL, plasma mm-LDL (mildly oxidized LDL), cholesterol eonmnt of circulating immune complexes, atherogenic potential of serum, the susceptibility to oxidation of VLDL and LDL, and u_r'im_ry excretion of DNA oxidation products (in a subsample), pending on fundi_~. The stad~cal analysis will estimate the independent and inmm~ve effects of vitamins C and E. Interactions according to (dependence of treatment effects oO gender and smoking status will also be analyzed. Statistical analyses based also on nonexperimental designs will be carried out in the study cohort. These concern (1) the role of lipid peroxidadon, antioxidants in general and pro-oxidants in atherosclerotic progression, hypertemion and cataract, (2) genetic factors in LDL oxidation. (3) the dietary and other behavioral determinants of ~utathione status aud lipid peroxidation and (4) gender differences in risk factors for athemselerotic pro m'ession, hypertemion and cata_ra~. The recruitme~ of the subjects was carried out in 1993 and the spring of 1994. the screening of suborns was started in August, 1994. Entry of the subjects to the 8-week lead- in phase will start September I, 1994. The subjects will Ix: emcred into the randomized double-masked treatment phase between October 1994 aud Scplcmber, 1995. The last subject will complete the. study by the end of September, 1998. Chemical and other m~en~, statistical analys,.-s and reporting will Ix completed by thc end of 1999. BATCo document for Legal Services : Health Canada 30 May 2000 O O co P~
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I. Specific Aims of the Study The ge~ral objective of this study is to investigate the effect of the reduction of free radical stress by anti~xidant suppiemen.'~tion in the prevention of atherosclerodc progression. hypertension and c.z.:.~-acts. The proposed srddy is a multidisciplmary randomized double-masked placebo-controlled factorial 3-year real to investigate the effect of antioxidant supplementation on atherosclerotic pm~ssion, hypertension and cataracts and the hypothesized pathways of the preventive eff~ts. Ttae specific study h.~theses are ~ follows: The supplementation of regularly smoking ard nonsmoking men and postmenopausal women aged 50-69 years with either d-~-ttr,~pherol or ascorbic acid will retard the I) Progression of atherosclerosis in the common carotid arteries (the primary outcome), 2) Atherosclerosis-related symptoms (chest pain, claudication), 3) Elevation of blood pressure, and 4) Development ef eye lens opacities. The preventive eff_,~'ts of the antioxidative vitamins are assumed to be consequences of the reduction of free rascal stress in cellular membranes of the arterial endothelium and the corneal cells and of ~he reduction of oxidation of lipids in membranes of these cells and of circulating as well as arterial subintimal lipids. For the basis of ~ sample size caladation 20-30 per cent treatment effects (reduction in atherosclerotic pm~ession) with each treatment alone are assumed. This effect size is considerably less than that ctu'renfly seen in trials testing the effects of lipid lowering agents on athemsclerotic p~ion. The eff~'ts of d,-tocopherol and ascorbic acid are asstnned ~ent and at P_ast additive. Only scarce data are available to estimate the power to &'t~t effects in omer outcome measures, but clinical observations are comisram with 3-year change of this ma~tude in smokers and nonsmokers of this age. All outcome measures (except #2) will be ~.-smnfitative, continuous variables to ensure maximal statistical power. The proposed study is the first of its kind. Besides that k will be the f~'t study testing the prevention of ather~sclerotic progression by antioxidants, it is unique in addressing several major aging-related degenerative disease simultaneously. The study will eon=ibum to test the theories of the role of free radical stress in the etiology of atheroselerosis, hypertension and camsacts. In addition to a test of the andoxidam hypothe,.¢¢~, the goal of this study is to ascertain the relative contributions of the two major natural a~oxidams, vitamin E and ascorbic acid. This ~ present~ a unique opportunity to test the role of antioxidant supplementation in ~e preventic, n of the progressio='of age-related degenerative changes using a high-ink population: It is also the only study being planned to test the effects of vitamin C and to ~mpare the preventive effeas of antioxidants between smokers and nonsmokers. Sizaificandy, it involves inteadisciplimry collaboration with investigators in Finland, United Star.~, Denmark. Sweden. Austria axxt Russia. 2. Background and Significance 2.1.~ne~ Declines in mor~ity rates at the older ages and decreased fertility have led to large changes in the age rtrucmre of the populations of most developed cotmtries, with an increasing pmpo~on of the population being composed of the elderly. Because of the prevak=x= of most degenerative "'diseases ~,ith increasing age, ttmre are cone, eros BATCo document for Legal Services : Health Canada 30 May 2000
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that ~e may be wimessing the begin.-~,~.g of a pandemic of age-related disease and disability. Whe~er or not this occurs v,'il!, to a 12rge exten',, depend on the ability of the scientific commvmiW to suggest effective preventive measu.res which will decrease the burden of disease in the later years. Wl'die there t'~,~s been some progress in the development of preventive approaches to the problems prevalent k,-1 old age, most of-ran these approaches have not been based on an appreciation of the tmdc.-lying biology, of aging. The:'e are many' theories as to the uaderlymg biologic mechanisms which account for age- r~late~ diseases, and there is currendy insufficient information to specify the correct approach. However, one approach which has considerable biologic rationale and great potomial with respect to in~-entiom is based on the cumulative damage associated with c.h~nic exposure of ~es to free radical stre~)s with inc'm, asing age. 2.2. Free radical damage and agiag-reiated degenerative processes Aging tias been clef'meal as the accumulation of changes responsible for the sequential alterations that accompany advancing age and associated progressive increases in the chance of d~ and death (I-tarmzn 1992). According to another definition, aging is the progressive postmamrity strucua-al and compositional changes, as well as the declines in f~mction and adaptability." of an organism with consequem increased susceptibility to disease and probability of dca~ (Porta 1988). The free radical theory of aging postulates that an important cause of aging is fr~ radical reactions and that these reactions may be involved in production of the aging-associated ctmnges related to the envirvtanem, disease and the intrinsic aging process fflarman 1992). Even though studies of the effect of ionizing tadiazion on living_ organisms, life span cxpcrimcn~ and studies implicating free radical reactions in the pathogencsis of specific diseases provide some support for thc free radical theors.' of aging (Cutler 1986. Harman 1992.), no experimental studies in humans testing the thcoD" have been conducted. Most prevalem public health problems in thc developed world are manifestations of aging- related deterioration of physiological functions. Coronary heart disease (CI-.ID) and most of the ~rcbrovascular disease are consequem.es of thickening of the a_nmJal wall and athc~sclerosis. Hypertension may' arise as a consequence of atherosclcrosis, or otherwise, and may accelerate the atherosclerotic process, CI-K) and all forms of stroke (Salonen et al. 1952a. Salonen and Saloncn 1990). Cataracts develop as a consequence of lens opacities over increasing age. Several, if not all aging-related changes in the human body have been suggested to bc consequences of a chronic exposure of ~ to con~miag free radical attacks. The human tissues most susceptible to the oxidative damage are the ~ endothelium and the lens of the eye. Degenerative processes in these tissues are believed to a major extent to depend on free radical damage. The ext~m of the oxidative d~tmagc in tlac body is determined by the mount of promoters of oxidative proccss~ such as transition mctals (eg. iron, copper. mercury) and the availabili~" of antioxidative defencc sy. stems. Oxidative damage in the arterial cndothelium leads m endothelial injury and the development of atherosclerosis (Ross 1986, Steinberg et al. 1959). In the eye lens, oxidative stress causes opacities and eventually cataracts, ~e m, ost common cause of blindness. ~..wI C ,,2 L.- BATCo document for Legal Services • Health Canada 30 May 2000
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2.3. Atherosderosis Atherosclerosis is a degenerative process in the arteries which is characterized in its early phase by the accumulation of lipid and proliferation of smooth muscle cells and consequent thickening of the hatimal and medial layers of the arterial wall. The oxidative modification of low density. [ipopmmn (LDL) is assumed to increase iu athcrogenicir," (Sminberg et al. 1989, 1992, Salonen eta/. 1992a). The oxidative modifi- cation of LDL in the human arterial wall is influenced by the availability of amiorddative mechanisms as Well as the presence of transit.ion.metals such as iron and copper, which act as ca~ysts of oxidation. Among. the key physiological antioxidams inhibi6ng the oxidative modification of LDL arc vitamin E, ascorbic acid and fr~ radical scavenging sysmms including selenium-dependent glutathione peroxJdase (Gey 1986, Halliwdl and Gum'ridge 1989, Bruckdorfer I990, Saloncn e.. al. :98ga, 1991b, Estcrbauer et al. 1992). The~ is some epidemiological data which is consmcnI with this. We have shown in a 2-year follow-up study of 126 inca that both low serum selenium level and low plasma vitamin C conccnwation are associamt with acceleram:l progression of common carotid athemsclemsis (Salooen ct al. 1991a). We have alto found that the presence of oxidized LDL, as expressed as aumanfibodica against oxidized LDL, associates with an accelerated progression of athemsclerosis (Salonen et al. 1992a, 1992b, 1993a, see section 3.3. for derails). In our pmsp~tive population study in easmm F~h men, high s~red levels of iron, a gxcnt catalyst of lipid peroxidar.ion, assodat~ wi~ excess risk of CHD (Salonen et al. 1992c). is some experimental evidence of the importan~ of oxidation of lipids in the etiology, of atherosclcrosis (see St~inbcn'g et al. 1989, Stc~ et al. 1992). Pmbucol, a drug_ with antioxidative properties, and BHT, a vitamin E r~embling antioxidam, have prevented the development of atherosclerosi~ in WHHL and cholesterol-fed rabbits (Carew et a/. 1987, Steinberg eta/. 1989, Bj6rkhem et ~. 1991). Also vitamin E has attenuated the development of early athcrosclerotic lesions in Wamnabe rabbits (Williams et al. 1992) and monkeys (Verlan=~cri and Bush 1992). We observed in a pair-matched, randomize, placebo-<:onn-olled clinical trial a platctet activity, reducing effect of a combination of a- tocopherol, fl-caromnc, a.scorbic acid and selenium (Salom'n e~ al 1991b). In two recently r~ort~l large American cohort studies, the reported use of vitamin E supplemen~ a_esociated with a reduced risk of coronary events (Smmpfer et al. 1993, Rimm et al. 1993). In the Nurses' Health Study in 87,245 women aged 34-59. free of previous CHD, vitamin E supplement u.~rs had a 36% lower risk of CHD (Smrapfer .'t al. 1993). In the Health Professionah FoUow-up Study, ~ on 45,720 men aged 40-75 with no history of CVD~ vitamin E supplement using men had 24% lower risk of CI-L'D event than non-users (Rimm et al. 1993). Even though statistical adjustment was made for ,,~vcra'. coronary ri~k factors, it is impossible to exclude the possibility ~hat vi~am E suppl:ment users were mor~ health conscious than non-u.~rs and had also other, possibly un'measu.,~ cha~c~ri~cs or behaviors that put them at a lower coronary ri:k:- In another recent size.able cohort mldy, the National Heal~ and Nutrition Examimfion Survey (NHA=\'E.S I) epidemiologic Follow-up Study, men and women respectively, with the highest vi~ C intakes ( > 50 mg/d and regular vitamin C containing supplements) had 45% and 7.5% lower CHD morality than subjects with the lowest vitamin C intake (<50 mg]d)(Enstmm et al. 1992). In the 12-year follow-up of the Prospective Basel Study, a low level of both plasma vitamin C and caro[ene associaa~d with 2-fold risk of CHD (p=O.{~22)(E~hhol.z~ a al. 1992). q~ 0 0 BATCo document for Legal Services : Health Canada 30 May 2000
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In the KIHD (see Salonen et ai. 1992¢ for study design and measurements), low plasma concenu'-ation of ascorbate, measured from fresh samples (Parviainen et al. 1986, Nyyss6nen et al. 1988, Parviaknen and Salonen 1990), associated with increased risk of AMI in 1605 men free of CHD a.: enwy (unpublished dataL Vitamin C deficient men (plasma ascorbate < 2.0 mg/l or II /~moVI) had a 2.7-fold (p<0.01) risk of AM] ha a Cox model adjusting for age. examimtion year and season, cigareae-yea,"s, serum apolipopmtein B, triglyceride. HDL cholesterol and ferritin concenwatious, systolic blood p~, diabetes, blood hemoglobin, blood leukocyte count, alcohol intake a~ maximal oxygen uptake, ha a populahon-based case-conta'ol study in Scoti-,-~,.:2~ plasma concentradom of both vitamin E and C were associated with increased r;_sk of angina Ixaxoris (R.iemersma et al. 1991). In the reeemly published F~ lung c2.,xe: prevention trial, the supplementation of smo/dng men with either 50 nag of dl-a-tocoptzryl acetate or 20 mg of B-carotene daily for 5-8 years did not reduce either CI-H) or cerebrovascular mortality signific, anfly, although there was a nonsignificant trend towards reduced CKD (by 5 %) and ischemic stroke (by 16%) mortality in men who received a-tocopheryl acetate (The Alpha-Tocopheml. Beta Carotene Cancer Prevention Group 1994). 2.4. Hypertension There is some evidence in favor of a role of oxidauve stress m the etiology of hypertension (Nakazono et al. 1991). The fimction of the endothelium derived relaxing factor, niu-ic oxide, is dependent on the redox balance in the arterial wall (Vane et al. 1990). We have observed in a cross-sectional popul~on s'~dy an izxlqxndem association betw~n lowered serum levels of selenium and ase0rbic'acid and elevated levels of resting_ blood p~ both in normotemive and in hypertensive men (Salonen et al. 1988b). As a pilot study for the presently proposed stud)', we carried OUt a clinical trial to investigate the eff~t of antioxidant supplementation on blood pressure taxi on the susceptibility to oxidation of plasma lipids. The subjects were 40 regularly smoking middle- aged men who were randomized either to supplememation with a combination of 200 nag of d-~-tm.opberol, 400 mg of ascorbic acid, 30 mg of fl-carotene and 100 microg of organic selenium daffy or to double-mask~ placebo for three months. The mean systolic blood prts~tre decreas~ by 12.5 mmHg (S.E. 2.5) in the sxtpplemented group and by 5.2 mmHg (S.E. 1.9) in the pta~bo group (mlpubiished data. see section 3.5. for details). The difference between groups in the blood pmssu,"e ch,~ge was stati~cally significam tp=0.027 in t-test) and the blood pressure reduction correlazed strongly with the increase 0f plasma ascorbic acid cow2ntration. This sugge~xs that amioxidam supplementation can lower blood p~, at least in regularly smoking men. There is mppordng evidence from other population smdie~ and small uncontrolled u'ials (I-lemiI~ 1991, Salonen 1991, Trout 1991, Simon 1992). We also observed an associador between high stored iron levels and elevated blood pressure (Salonen et al. 1992c). There are, however, no previous controlled trials concerning the specific effects of the reduction of oxidative stress by supplementation with vitamins C and E on blood pressure in humans. 2.5. Cataract Cataracts are the leading cause of blitxlness m man (Robertson et al. 1989). The world Health Organization ~ted that almost a ~ of all blindness cases are caused by cataracts (Ce.mmi and Ctab~ 1986). With aging, a aumbe.t of d~gene.tative chang~ occur ia C (D G', BATCo document for Legal Services : Health Canada 30 May 2000
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8 the lens. These lead to lens opacities and eventually to cataract formation. The lens is subject to oxidative stress and becomes less capable of handling such stress with aging. Free radical mediated chemical modification has been suggested as one mechartism of cataract formation 0hriegand et al. 1984, Cerami and Crabbe 1986. Taylor 1989, 1992, Spector 1991). In two major prospective population st-tidies from Harvard cigarette smoking associated with an increased risk of cataract m men (Christen et al. 1992) and in women CHankinson et al. 1992). Several anticataractic agents have also antioxidative activity (Woollard et al, 1990). Biochemical and epidemiologic evidence suggests that c~-tocopherol and ascorbic acid provide, protection to the tens against oxidative stress causing cataracts (Harding and van H eyningen 1987, Robertson et al. 1991, Jacques and Chylaek 1991, Varma 1991, Taylor 1992). A role of sit~-speeific, metal-cataly-zed oxidation has been hy- po~ in lens aging and cataract formation (Garland 1990). In a recently published nested case-control study low serum concentrations of ,-,-tocopherol and ascorbic acid were risk factors for end stage senile cataract (Knekt et al. 1992). In a case-cone'ol study, supplementary vitamin C and E but not B-e.arotene associated with 50% reduced cataract risk (Robertson et al. 1991). In the Physicians' Health Study, the use of multivitamin supplements was ~ociated with reduced risk of cataracts and cataract extractions in 22,071 men, who were free of cataracts at baseline (Seddon et al. 1992.). In the Finnish lung_ cancer prevention trial, supplementa',ion with a-tocopherol (50 nag/d), 8- carotene (20 rag/d), or a combination of these antioxidants improved visual acuity in a subgroup of 1,200 smoking, men aged 50 to 69 years (Teikari 1992). Several authors have suggested a clinical trial in cataract prevention with vitamins C and E (Robertson et al. 1991). 2.6. Justification of the Proposed Study Neither prima-E,." nor secondary, preventive studies in atherosclerosis, hypertension or cataract formation have been conducted concerning antioxidant supplementation. In the Finnich alpha-tocopherol - beta-carotene (ATBC) :ancer prevention study, no preventive effect on either the primary study outcome - lung cancer incidence - or on CHD or stroke mortality, was observed by either antioxidam. This was a surprising finding, in fight of evidence from both ~ and epidemiologic stud~ suggesting preventive effect. However, the inmrventionmay have been simply too little (only 50 nag of dl-a-tocopheryl acetate daily) and too ham to establish effects on clinical events. After the controversy in findings of the previous clinical trials, k is all the more important to carry out a conclusive study testing the amiatherogenic effect of vitamin antiorddants in humans. Cardiovascular event represent a late phase of the "dherosclerotic disease. Prevention of the progression of the disease at an earlier phase make more sense both from the pathophysiologic and from the public health pek'~pc~ve. Even though we recognize that the population b-~.~d approach (Rose 1985) will have the greatest effect on the prevention of aging-related degenerative ~, in the first clinical studies on the impact of amioxidant supplememation a strategy based on intervention in high- risk populations is preferable. A trial i, hig~-r~.sk L.-,dividuals can be done in a smaller sample size and with a shorter duration tL~--atriak in-a representative population sample. A trial in high-risk persons, based on assessments of fl~e development of atherosclerosis over time (progressiorzJregression) will serve as a specific test. of the hypothesis concerning the role of lipid peroxidation in atherogenesis. Also, there are no previous triats concerning, the C j7 C J BATCo document for Legal Services " Health Canada 30 May 2000
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effects of antioxidam supplementation on the development of aging-related hypertemion and eye lens opacities. The Kuopio area in Eastern Finland offers a number of unique advantages as a site for the present proposed study: (1) The occurrence of aging-related degenerative diseases is very high in Eastern Finland. Eastern Finnish men and postmenopausal women have the highest recorded incidence of IHD (Keys 1980. Tuomilehto et al. 1992)-. (2) The average dietary, intakes of vitamins C and E, B-carotene and selenium are relatively low (Sa]onen et al. 1988a, 1991c, Parvi-,inen and Salonen 1992). Unlike in the U.S.A., the use of antioxidant suoplements is relatively rare (Berg et al. 1991). Only 6% of men and women aged 55-64 t~ selenium or other mineral supplements, the most commonly used antioxidam product in Finland. Because of th~ common use of antioxidant supplements in the U.S.A, subjects for a s~ study would be bard to fred in th~ U.S.A. and the lob-term compliance would be difficult to ~. Also, the regular ~ of aspirin is ve~" ra~ in Finland ('Berg et al. 199I). At least in some U.S. populations of th~ order of 50 % of people are taking aspirin regularly (Stampfer et al. 1993, Rimm ~ al. 1993). As aspizin is an effective annoxidant this would dilum any observed trcatmcm effects of other antioxidants. (3) The Im,"ticipadon rams in studies in caz~ovasazlar ~ are exceptionally high: e.g. between 80 and 90 % with one ~ of a self-a~mred questionnaire ($alonen et al. 1981, $a/onen 1988). Ea~m Finns have excepdoml]y good compliance record in previous clinical t~ls. In our previous "KAPS" study (see below for d~tils) the drop-out ra~ was below 10% during three yeats. (4) Tbe population is stable, which, together wit.h high willingn~s to participam in car- diOV~ar $'l:udies, minimiT,~ losses to fOl.IOW-L~. (5) T'ne population is homogenous in terms of ethaicity and economic weU-farc. This reduces confounding__ of findings. (6) All Finnich citizens have a unique personal i:tentificadon code, and comp~ na- tional rsgdstrics of death certificams, hospital discharges, drugs for chronic diseases (such as hypertension) and addresses etc. which are lmpt by the Finnish governmental agencies. Universe'-based re~,.aw.h groups an: atlowed to u~ze these registries. (7) There is virtually no ittiteracy in Finland. This is an advantage in all questionmi~- based methods. (8) An established laboratory, at the Research Instimt~ of Public Health, University of Kuopio is available for lipid oxida;Jon aud other demrminarions. (9) A methodology, for the assessment of carotid athemsclerosis with ukrasonography has been developed in our previous sradies (see Sa/onev 1988, Salonen and Salonen 1990, 1993). The modern high-resolution ultrasonography of arterial wall provides a ~ method to a.ss~s the effect of antioxidant supplementation on the process of athcrosclemsis itself. In addition, arerial ultrasonography is noninvasive, safe and inexpcusive. (10) Methods to assess lens opacities have been tested and used at the Reseax~ Institam of Public Health, University. of Kuopio in two earlier studies ~, KAPS, see below). Because of the above memioned characteristics, eastern Finns are optimal subjects for a trial in the prevention of athemsclemtic progression with anfioxidant supplementation and the Research Institute of Public Health, University of Kuopio, is an optimaJ site to conduct the study. Also. the investigator group has a long-term experience in studies concerning antioxidants and cardiovascular disease (see cg. Salonen et al. 1981, 1982a, 1982b, 1985, -t98ga. 19ggb. 1991a, 1991b, t992a. 1992b, Karpe~ 1.990, F_stm-baa~ e~ al. 1987~ 1990, LY~ ,,.D Lr'. BATCo document for Legal Services : Health Canada 30 May 2000
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l0 1991, Balla e~ al 1~!. Orc~hov et al. 1986, t988, 1989. 1990, 1991a-e). All coinvestigators and :onsultants have collaborated with the PI, professor Jukka T. Satonen m previous studies and ways to communicate effectively (bimet. fax, conference calls etc.) have b~n worked c,u; in eaz!ier collaboration. 3. Preliminary Studies 3.1. Studies of Atherosclerotic Progressioa Wc (Professor Jukka T. Satonen and cc#orkers) have investigated risk factors for the progression of common carotid athemscl:~sis for ~everal years. We use high-resolution B- mode ultrasonography to assess atherosclerosis. This methodology is superior to angiography, which provides information only on the obstruction of the arterial lumen but not about the layers of the ar~rial wall. Early atheroscterosis is detectable as thickening of the intimat and m~,ial lavers of the vessel wall. Obstruction of the lumen and changes in blood flow appear at a much more progressed stage and are only dcmzctable later (see e.g. Ross 1986, Steinberg et al. 1989). In addition, arterial ultrasonography is safe and inexpensive. In the K/HD srady (with George A. Kaplan as a co-PD, we have studied the intra- and inmr-observer variabiliD' of B-mode ultrasonographic ~ent of common carotid armry (CCA) atheroscterosis, its preflietive value in terms of the association with the risk of CUD (Salonen and Salom-a 1991a), risk factors fer carotid and femoral attm'osclerosis, and predictors of the progression of CCA atherosclerosis (Saloren and Salonen 1990). On the basis of our experie,'-'-'-'-'-'-'-'-~ and findings, the ultrasohographic assessmcm of CCA atherosclerosis appears a feasible. ~tiable. valid and cost-eff~--'tive method for both population studies and clinical trials of atherosclerosis pro m'ession and regression (see Salonen and Salonen 1993 for more detailed pvzsentation). Our ftr~ cohort study concerned 128 randomly sampled Eastern Finnish men examined in 1987 and 1989 in the Kuopio Isc~c Heart Disease Risk Factor Study 0GI-~). in which an ultrasonographic assessment of atherosclemsis was carried out for a total of over 1300 subjects (Saloncn and Salonen 1991a, 1991b, 1993, Saloncn et al. 1991a). The maximal thickness of the intima-media complex (IMT) of CCA far wall was assessed with B-mode ultrasonography by one observer (RS) twice in 24 months. The strongest risk factors for atherosclerotic [nx~,ession were age, cigarette pack- years and S-LDL-cholesteml 0..DLC). S-LDLC was a risk factor only in men with serum COl)Pea" above median, and this synergism was stronger in men with low serum seleniums. Also serum copper and selenium (inversely), plasma vitamin C (inversely), platclet aggega- bility and blood le-.kocvte count were independem, although weaker, determinants of athcrc>- genesis. The "Kuopio Atherosclerosis Prevention Study" 0LAPS) is a randomized clinical trial concerning the effects of LDLC lowering on th,: progression of carotid and peripheral athemsclerosis (Saloncn ct al. 1992b, 1992.d, 1993a). This trial is also analyzed like a non- experimental follow-up study with repetitive measurements of disease outcome to investigate the impact of lipids, lipid peroxidation and antioxidants on atherosclcrotic progression. Six hundred and six eligible men were invited to a double-masked placebo-controlled 3-year trial concerning the eff~t of pravasmtin (40 nag daffy) trearment on the progression and reg- ression of carotid and femoral atherosclemsis. Eligible men who agreed to participate were given placebo and dietary" advice to lower LDLC. Of these, 447 men whose serum LDLC remained at least ".0 mmoL:l and total serum cholew..crol was below 7.5 mmol/l after the 2 112 month diet~.' advice period and who did not have spec.if.u~ ~ons, were 0 (=3 o3 .,.,.o ,42, BATCo document for Legal Services • Health Canada 30 May 2000
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entered into the K.~,PS and under'went annuz! ultra~nographic examination of carotid and femoral arteries. The -'ntry of subjects was cor~.pleted in September. 1990 and the clinical phase of the study w:Li be completed by the end of September. 1993. The working grou..- at the Research Insti~Jte of Public Health, University of Kuopio. is currend.v running the KA.PS study and has acquired an expertise in the execution of large clinical u-ials. Methccs to measure athero,.cterosis ultrasonographically, to assess eye leas opacities, and most of the chemical methods to be usc-d in the presently proposed study have been developed, validated and thoroughly tested in me KAPS study. Also the whole support staff has been train~ for years in KAPS. 3.2. Studies on Measurement Precision and Reproducibility. of Ultrasound Technical measurement precision: We carried ou, z small study to determine the actual precision of the m~ent of distances betweetr interfaces similar to those in the arterial wall (Salonen et al !993). A wedge phantom was constructed at the Cali.fomia Institute of Technology, (by Robert Seized which had six gaps of different, known size. The width of the gaps could be v~,'-ied by add,_ng thin shingles between two plastic blocks which were screwed together. T~',..e distances ranged from 0.2565 mm to 1.5342 ram. At the Resea.~h Institute of Public Health, Universi .ty of Kuopio, we used a Biosound Phase 2 scanner (same as plant~ for the p,,-eposed study) to meas'--"-e dis~ between the inmrfaees in the phantom. We varie~ the width of the gaps by adding 0no by one up to four 0.002 inch (appr. 0.05 ram) thick shingles. Thus, we ~ 30 ~ between two ultmsonogr~_, hic in~.,-faczs. The scanning was recorded on a S-VHS VCR. One of our ul~ rachniciam did six comeeutive measurements for each known width using a mi~mputer-base~ videoimage digitizer and morphometry software. The average of the true distances was 0.998 mm and that of the 180 m-,as, a.n~d distances 1.005 ram. The mean of the differences between all 180 actual and obse~'ed dL~'tances ~ 0.008, range -0.I05 to 0.140. SD 0.052 m-. and the 95% confidence ime.--val (CI) for the mean difference +/- 0.008 ram. The m ~ez.u of the absolute value of the difference, often used as an estimator of measu.rement precision in this context, was 0.040 ram. The Pearson's correlation coefficiem for the concordance between the true and all ~ values was 0.993. As the mean of several distance measurements is most often ~ in the ~ent of IMT, we also cal :'eulzmd the same estimates using the 30 means of the 6 measurements for each disunce. The'=. the mean diff~ I:~tw~n me acuaal and observed distances was 0.008 ram. range -0.071 mm to 0.115 ram, SD 0.040 ram. and the 95% CI for the mean difference was +/- 0.014 ram. The mean absolute value of the differeax:e was 0.032 ram, median 0.02_3 mm =d 95% CI +/- 0.009 ram. The correlation between ume and measured differences was 0.~7. The experiment was repeated once by the san~ observer in Kuopio and the results were almost idenrdcal. A similar experiment in the California Institute of Technology. with a Toshiba SSH 140A ultrasoun6 sy.ctem gave very similar results (m~mem precis-on 0.02 mm using auton~6c edge detection as proposed for the present study). On the basis of o'~ phantom studies, distances similar to IMT can be measured with B- mode ultrasound syr--~as having, an axial ~soluzion of 0.2-0.4 mm at a precision of the order of 0.02-0.05 ram. A typical IMT is 1.0 mm and the changes in 2-4 years observed in follow-up studies a~ 0.2-0.6 ram. Bas~ on our experiments, these sort of changes can be measa.u'ed in individ'.:al subjects with current B-mode ultrasound systems. O O:3 G BATCo document for Legal Services • Health Canada 30 May 2000
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12 Reproducibility: To estimate the reprodu:ibilit3" of our ultrasound measurements, we (R.S.) re~..ated the IMT measurements in 6,3 subjects (see, Salonen et al. !992a) twice from the videorecordings of the original ~atmings. The remeasurements were supervised by representat!ves of the Finnish Academy. The reader (R.S.) was blinded with regard to the identities of subjects and whether she was reading a baseline or a follow-up scanning. The results of this second rereading of our ultrasound scannings were identical with the ones presented in our publication (Salonen e: al. 1992a): None of the original cases and controls would have switched goup on the basis of either the first or second remeasuremenr.s. The Pearson's correlation ~3cient:for the concorda~.c in the 2-year change of L-MT between the origiml measuremems ",ml rtmea.mrements was 0.97 for the first and 0.98 for the second remea_varemems. The mean absolute value for the difference between the original and the second repeat m~ (for 2-year change) was 0,04 ram. This, the mean m~ment error, is an empirical estimate of the measurement predsion. In another sample of 4I men, the rote of w.amaing and videotape reading as sources of intraobserver variabiliD' was invesr2gated (Salonen and Satonen 1993). The random ~ment variabilit)" due to scanning was a little larger than that due to videotape reading (correlation 0.98 between ret~dve scanni~*s and 0.99 for repetitive rea. dings). 3.3. Oxidation of Lipids and Atherosderotic Progression Oxidative mcx£i_fication of LDL 0-DL) renders k immunogenic and autoantibodies to epitopcs of oxidized LDL (Ox-LDL) have been shown. To demrmine whether these antib(xfies are related to atherosclerotic pro~'cs~ion we compared serum triter of auto- antibodies to malondialdehyde (MDA)-modified LDL and native LDL in baseline sera of 30 eastern Fi~nlch men with aceelerated 2-year pro Lwession of carotid atherosclerosis and 30 age-matched controls without pro~'ession (Salonen et al. 1992a). IgG antibody timr was determined by solid phase RIA. Neither group had SlX~ific antibody binding to native LDL and a riter was defined as ann'body-binding to MDA-LDL/binding to native LDL. Cases had significantly higher titer to MDA-LDL (2.67 vs. 2,06, p=0.003). Cases also had greater tm)tx)rfion of smokers (37 vs. 3%), higher LDLC (4.2 vs. 3.6 retool/L), and higher serum copper cotr.emrarion (1.14 vs. 1.04 mg[L). Even a.~ter adjusting for ~ variables and baseline atherosclerosis severity, in a mtdtivadate logistic model, the difference in antibody remained significaat (p=0.031). Thus. the tit~r of auto-antibodies to MDA-LDL was an indc.lx'mient predictor of the progression of carotid atlmrt~lerosis in F~nni.~b men. "I'Imse data provide furtber support for a role of oxidatively modified LDL in atherogcmsis. In a second study, conducted ix coUaboraticrn wi*..h professor Esterbauer, we investigated the relationship between the presence of autoantibodics against Cu-oxidized LDL and the progression of carotid and femoral athcrosclerosis in 212 cairn Finnich men with serum LDLC > .#,.0 mmol/l (Salonen e: al. 1992b. 1993a). The maximal IMT in CCAs and femoral atxries was measured twice in 12 months using high-resolution (I0 MHz) B-rood, ultrasonography (Biosound Phase 2). Ox-LDL-antibodies were determined by ELISA. In covariancc models adjusting for age, smoking, serum LDLC and triglyceride concentrations, triglyceride and protein content of LDL, systolic blood px'emax~, and the 12-month change in serum LDLC, the 12-month increase of IMT (mean of all 6 arterial segments measured) was significandy greater in men with elevated (mean 0.29 ram, SD 0.15 ram) as compared to men with borderline (0.17 ram. SD 0.18 ram) and low titer (0.17 mm, SD 0.13 mm)(p=0,002 for hetcrogenciry~. These data suggcst~ that Ox-LDL-antibodics associate kJ~ C G O2 t_r" BATCo document for Legal Services : Health Canada 30 May 2000
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13 with accelerated atherosclerotic progression in hypercholesterolemic men and provide fun.her evidence for the role of in vivo oxidation of LDL in atherosclerosis. 3.4. Vitamin C and Blood Pressure We observed in a population study in Eastern Fir.nish men an association between low plasma ascorbate and serum selenium concentrations and elevated blood pressure (Salonen et al. 1988b). The inverse relation.ship of plasma vitamin C and selenium levels with blood pressure has also been reported from other cross ~...tional.smdies and.in small trials (Salonen 199 I). To furor test the blood pressure lowering effect of amioxidants we conducted a randomized double-masked clinical trial testing the effect of combinod supplementation with ascorbic acid, selenium, a-tocopherol and B-c.a~tene on blood pressure in normotensive subjects (Salonen et al. 1994). Forty, men from Kuopio, Easmm Finland, aged 30-58 years smoking regularly bctw~n 15 and 40 cigarettes daily were randomly allocated either to antioxidant supplementation or control, twenty men in each group. Half of the subjects are smokers as smokers have lowered plasma antioxidant levels. Men with any conditions or medications influencing blood pressure or oxidative stress were excluded. There were neither any reRtsals nor drop--outs dm'ing the trial. The supplementation comprised two tablets daily, each containing 200 nag of slow release ascorbic acid, 50 micrograms of organic selenium, !00 mg of d-a-tocophcryl acetate and 15 nag of ~-caxotcne. Identically appearing placebo tablets were given to the control group. The mbjccts were advised not to use any other vitamin or mineral supplements and to keep their health habits unchanged during, the 3-month study period. Plasma antioxidant levels and blood pressure were m~ at the beginning and at the end of the double-masked period after a fast of 12 hours and abstinence from smoking_ of one hour. Plasma ascorbic acid and dchydroascorbic acid (DHA) were determined by a I-IPI.,C method (Parviainen et al. 1986) and serum selenium by atomic absorption spectrometry (Saloncn et al. 198ga). Blood pressure was measured by a physician using a s'tatxiard mercury sphygmomanometer. Two ~nts were done within five minmcs after a rest of 15 minutes in sitting position. Means of :hcse two mcas',:rements were used in the data analysis. The baseline mean (range)- in all 40 subjects was 140.8 (110-180) mmHg for systolic and 84.5 (57-I02) mmHg for diastolic blood pressure. The mean systolic blood pressure w~as reduced by 12.5 mmHg (i)<0.001 for change) in the supplemented and by 5.2 mmHg (p<0.01 for change) ~n the placebo group (unpublished data). The net effect (change in anfioxidant grot:p minus change in placebo group) of antioxidant supplementation on systolic blood pressure was a reduction of 7.4 mmHg (95% CI 1.1-13.6 mmI-Ig, p--0.027). There was also a significant decline of 3.3 mmHg (p<0.01 for change) in the mean diastolic blood pressure in the supplemented and no change in the placebo group (net effect 2.9 mmHg, 95% CI -1.5-7.3, p=0.206). Plasma ascorbatc rose by 45% (p<0.001 for change) and serum selenium by 20% (p<0.001 for chang C, in tie supplemented group, whereas there were no changes in the placebo group. In both groups combined (n=40), the r=zluction of systolic blood pressure correlated with the increa.~ of both plasma ascorbatc ~r=9.35, p=0.028) and serum selenium (r=0.21, p=0.184) concentrations. C:) O C~ ,,£) r',,2 BATCo document for Legal Services - Health Canada 30 May 2000
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14 The table below shows the mean baseline values and changes of blood pressure and plasma antioxidam :oncentratiom before and during 3-month study period in 20 antioxidant supplemented men md 20 placebo men ("Based on t-tests comparing changes between groups (n--40). Antioxidant supplemented Placebo Before Change Before Change p-value* Systolic BP (mmHg) 143.1 -I2.5 138.6 -5.2 0.027 Diastolic BP (mm'n,g) 85.8 -3.3 83.2 -0.4 0.206 P-ascorbate ~mu"l) 54.4 24.5 43.6 -1.6 < 0.001 S-selenium ~gfl) 11 ! .7 22.6 112.2 0.9 < 0.001 We have suggest~ that as~rbic acid and other anfioxidants could lower blood pressure by enhancing the availability of pronacyclin, a lo",.al vasedilater and possibly by inhibiting the synthesis of thrombzxane A,., a potent local vasoconstrictor (Salonen et al. 1988b). More recently it has been shown that oxidative mess, specifically sutx:mxide radical inactivates endothelittm-deriv~, relaxing factor (nitric oxide) and that mpemxide racfic..als may play a critical role in the ~thogenesis of hypertension ('Nakazone et al. 1991). 3.5. Peroxidation of LDL LDL is not only rich in choles~l but ".,.1so in polyunsaturated fatty acids (PUFAS) susceptible to oxi .--d~ion by lipid peroxi -clarion. The 1300 molecules of PUPAS (mainly 18:2), contained on ave.~e in the lipids of an LDL particle with a molecular weight of 2.5 million, are protec~ against oxidation by a variety of Iipophflic amioxidants. Of these by far the most impor-ant one is ~-tocopheml, its average comem i¢ 6-12 molecules per LDL .particle. The other substances in LDL with anfioxida.~ activity are: E-carotene, c~-¢arotene, gamma-tocopherot, lycopene, cryptoxamhin, camaxanthi~ phytofluene and ubiquinol-lO. Each of them is p~t in amounts of only about 1/20 to I1300 of c~-tocopherol. If LDL is exposed to an oxidative stress, the antioxidants prevent oxidation for a certain time (= lag- phase or oxidation re.s~), during which they are consumed with ¢x-tocopherol f'ust and fl-t.amtene as the last one (for review see Esterbauer et al. 1990, 1991). Oxidation of LDL is now considered to be a crucial step in the pathogenesis and progression of athemsclerosis (for review see Steinberg et al. 1989, 1992) and various lines of research suggesz that antioxidantssuch as ~tamin E, .ascot'ba~, carotenoids, ubiquinol-10 and possibly others play a significant role in protection of LDL and prevention of atherosclerosis. Professor ~uer's group has d~veloped an in vitro assay to determine the oxidation resi~,.ace of LDL iv- a reproducible manner (Esterbauer et al. 1990, 199I, Dieber-Rotheneder et al. 1991). The assay to measure oxidation resirumce of LDL has been used by professor Salonen's group and by others to compare the amioxidant efficacy of natural and synthe-~c amioxidams. Profes,~r ~auer has collaborated with professor Salonen also in e,a,Sier studies (see eg, Salonen et al 1992b, 1993a). tan CD CD m tJ" BATCo document for Legal Services • Health Canada 30 May 2000
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15 Drs. }acobs and Belcher did an experiment in which they incubated isolated LDL (from I0 middle-aged non.smoking healthy men and women) preconditioned with hemin and H:O: with porcine aortic endothelial cells (P.M~C). before diet was supplemented with vitamin E, after 2 weeks of supplementation, and 2 weeks after s-~pplementadon was ended. The vitamin E supplement was 800 I.U., except for a [owe: amount in one subject. ,-,-tocopherol per apoB molecule doubled, then renwned to baseline with supplementation and its withdrawal. Similarly, LDL resistance to oxidation as measured by the lag phase more than doubled and then returned to baseline. Percent of the PAEC killed by incubation with preconditioned isolated LDL was 43 .% before supi.,iemea~tion, 3 % after supplementation and r~turncd to 40 % after vitamin E was v:.thdrawn. The pattern of reduced, then increased cytotoxicity was seen in ever)" subject, i.e a single subject who took 800 I.U. for only 3 days, the cytotoxicity was 52 % before supplementation and after its withdrawal, and 27 % following the brief supplementation. 3.6. Chemical Modifications of LDL, Lipoprotein Immune Complexes and Their Role in Atherogenesis Smooth muscle cells cultured from human atherosclerotic plaque have been used for the study of antioxidant effects on intracellular lipids and proliferative activi~" by Dr. Orekhov and coworkers. BHT and a-tocopherol significantly reduced cholesteryl esters and thymidine incorporation into cells (Orekhov ¢t al. 1986). We can conclud~ that antioxidants possess ~lerotic effect reducing atherosclero~ic Kmnifesmtions at the arterial cell level. Blood serum or plasma of patients w~ athczoscl=-rosis stimulates cholesterol accumulation in ~ smooth muscle cells of normal human aorta (Chazov et al. 1986, 1988, Orekhov et al. 1988, 1990). Besides. atherogenlc serttm Stimulates other atherosclerosis-related processes, namely: proliferative activiry and extrace~ular matrix synthesis (Orekhov et al. 1988, 1990). LDL isolated from athcroge.~',c serum, like whole serum, stimulates cholesterol accumul~on, proliferation, extraceUular matrix synthesis, while native LDL of healthy subjects possesses no effects (Orekhov ct al. 1988, 1990, Tertov et al. 1989). Atherogenic LDL isolated from paticms' blood is a modified lipoprotcin with low sialic acid conics, high eteetronegative charge, small size, high density,, a.lm~d prou,-in structure and lipid -composition (Orek.hov ctal. 1989, 1991a, 1992, Te,-'tov er. al. 1990a, 1992a, 1992b). The oxidation of multiple modified dcsialylated LDL in freshly isolated atherogenic LDL of patients is more pronounced in the presence of Cu"-ions (Tertov et al. 1992a, 1992b). Desiatylat~! LDL has higher level of oxysterols and lower amounts of c~-tocopherol and 8- caromne as compared to native LDL (Terror et al. 1992b). In tlae blood multiple modified LDL forms circulating immune complexes with autoantibodies against desialylated LDL (Orekhov et al. 199Ib, 1991c, 1991d, Orekhov 1991, Chekhov and Tertov 1991a, 1991b, Tertov et al. 1990b, 1990c). The content of lipopromin in circulating immune complexes is ve:y-good discriminator of atherosclerosis (Orekhov et al. 1991c, 1991d, Orekhov 1991). 3.7. Lipid peroxidation and eye lens opacities In the KAPS trial cohort of over 400 hyperchole~-terolemic men in eastern Finland. men who had high plasma concentrations of dehydroasc~,."bate, an indicator of oxidative stress in vivo. and LI)L ~ acid ~,xmtcat bad ~ceacra~. _~'ogression of eye leas opar.it~ 0 o BATCo document for Legal Services • Health Canada 30 May 2000
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16 (unpublish~ datal. This observation provides support to the hypothesis that oxidative stress and lipid peroxidation play a role in the formation of cataracts. 4. Research Design and Methods 4.1. Stud)' Hypotheses The study is designed to test the hypothesis that the supplementation of 50-69 -year old smoking and nonsmoking men and postmenopausal women with either 200 mg of vitamin E or 500 nag of vitamin C daily or both will re,ard the progression of common carotid atbetusclerosis, will retard the elewafion of blood pressure or lower blood pr~surc and will slow down the pro~ionn of eye lens opacities in three years. No gender diff~re~c~ in treatment effcats are hypothesized. The treatmem effects are expected to be larger among smokers than in nonsmokers for atherosclerosis and cataracts and possibly for h~ion. 4.2. Study Design This is a clinical randomized double-masked trial with two by two factorial design. The study consists of an 8-week dietary counselling and placebo lead-in phase and a 3-year double-masked phase, for which the subjects are randomly allocated to one of the following I/talaaaen~: 1) d-ct-tocophcrol only, 2) ascorbic acid only, 3) both d-a-tocoptxa'ol and ascorbic acid, and 4) placebo. The purpose of the lead-in phase is (1) to screen out the subjects with potentially bad compliance, (2) to provide smoking cessation services and (3) to give dietary advice to lower serum LDL cholesterol for those who have elevated serum cholesterol levels (> 6.5 retool/l). The aim is to minimize changes in the amount of smoking and dietary as well as blood lipids ~ the do~ble-masked phase. The rationale for providing dietary advice prior to ra.~domizadon is to ~ dietary changes during the double-masked sandy period. They could be induced by dietary assessments unless dietary councclling has aJxcady been provided. Secondly, for ethical reasons it is necessary to provide advice to salllrar.ed fat intake for subjects with high serum chol~l. The reduction of satttrat~ fatty acid (SAFA) intake is not, however, assumed to affect lipid per'oxidation and will thin not eff~'t lipid peroxidadon measurements. The subjects aged 50-69 will be randomiz~ into the trial in four strata: I) smoking_ men, 2) nonsmoking men, 3) smoking postmeno~usal women, and 4) nonsmoking postmenopausal women. The aim is that each stratum will be of equal size. If it will be impossible to recruit enough smoking women, the mamm of smoking men will be increased respectively. The outcome m~nts will be carried oat ard~ffferent'frequencies. The ultrasonographic assessment of carotid ~d'mries will be carried out at baseline and semJannually (7 times). Atheroselerotic symptoms will be assessed annually. Blood pressure m~ents will be done semiannually. Ophtalmologieal assessment will be done twice. Most trot not ell cbemi~ tests will also be pe.rform~ anm~lly. To balanc~ C~ BATCo document for Legal Services : Health Canada 30 May 2000
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17 variations, the subjects will be en,.ered into the r.~.al over a period of 12 months. An interim analysis of effects on chemical measurements of fr~ radical stress and lipid peroxidation will be carried out after one year of treaunem. 4.3. Study visits The subjects will visit the study center (Re ~se,~"ch Institute of Public Health, University of Kuopio) at six months' intervals. At each visit, the adherence to the study lxearment is assessed by counting the returned capsules. A new supply of vitamins is given for the next six months. 4.4. Experimental Treatment In the prevention of atberosclerosi~, hypertemion and cataracts, ,,,-tocopherol aad ascorbic acid are biologically the most relevant natu.~ antioxidants (see Gaby et al. 1991, SIater and Brock 1991). a-tocopherol is the principal amioxidam in human LDL and ceLlula~ membranes (Chow 1991, Esterbauer et al. 1991, 1992, Janero 1991a, 1991b, 1992, Berry. 1992, Jialal and Scaccini 1992). Both vitamia E and ascorbic acid have been implicated in the prevention of atherosclerosis (Steinberg et al. 1992), and cataracts (Taylor 1992). Also, ascorbic acid regenerat~ vitamin E in vivo and thus synergistically enhances the anfioxidadve efficacy, of a given dose of vitamin E (Sam et al. 1990). Both vitamin E ('Dic~zr-Rotbeneder et al. 1991, Princen et al. 1992, T~lal and Grundy 1992, Sies et al. 1992) and vitamin C (Jiaial ctal. 1990, Harats et al. 1990, Frvi 1991, Sies e~ al. 1992,, Retsky et al. 1993) inhibit the oxidation of LDL. Ames and cowork~rs (Fr~i et al. 1989, 1990) and Frei (1991) have reported ",hat ascorbic acid is the fn's~ antioxidam consumed during lipid peroxidation in plasma and detectable lipid peroxidadon starts only after all ascorbate has been co~ed complemly. They have even concluded that only ascorhat~ can prevent the initiation of lipid l~roxidation. For these reasons, the antioxidant tr~ment will cons~ of a-tocopherol and ascorbic acid. two nam.ral and safe anfioxidants (see s~rion 5.4 for review). In the NI-ILBI workshop on "Antioxidants in the Prevention of Human Atherosclerosis" dm'ing September 5-6, 1991 k was concluded that "a trial of vitamin C, vitamin E. and 3- carotene in a 2 x 2 x 2 factorial design would be the best approach at this Rme" (Steinberg ~al. 1992). The daily dose of antioxidams will be 200 mg of d-a,-tocopheryi acetate (272 I'U vitamin E) and 500 mg of ascorbi¢, acid. Both will be given in two tabW, s daily. The daily doses have teen selected on the basis of our experiments concerning the effects of a-tocopherol and a:;corbic acid on the susceptibility of LDL to oxidation. A maximal effect is reached at these doses, whereas higher doses do not provide any appreciable additional effect. Also, to ensure that the findings will be useful from the public health point of view, we are using the smallest dose possible which we think will achieve the desired beneficial effects without side effects. No side effects have been reported in previous clinical trials using these doses. The nana~ (XRR) isomer of vitamin E will be used for two reasons: First, only the RRR isomer is deposited in the liver. Secondly, the results of th~ study will be generalizeable to dietary vitamin E. • A slow release tablet containing 250 mg of vitamin C will be used. Kinetic studies by Dr. Metsa-Ketel~i indicate that when the tablet is taken with meal, vitamin C is released for more 12 hours i.txi that tl~ plasma ~ c,o.r¢.e,n,tmti~ ~ P~'val~ for over 17_ hot~ c~ Co ,,.D U'I G', BATCo document for Legal Services • Health Canada 30 May 2000
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18 (measurements not done for longer time). This dosing will ensure a nearly constant plasma ascorbate level over the whole day. The treatment scheme will be single dummy: the subjects take one tablet at a time, which contains either vitamin E (d-a-tocopheryl acetate) or vitamin C or both or placebo with both the morning and the evening meal (one tablet twice daily). Addkional pilot studies have been carried out during the fall 1993 and spring 1994 testing the effects of various doses of a-tocopheryl acetate and slow release ascorbic acid on lipid oxidation and the kinetics of these antioxidants in humans. fl-carotene will not be included in the experimemal, .t."..~...atment-beeause it scavenges only singlet oxygen trot not the most aeuve oxygen radicals such as the hydroxyl radical. Also, the evidctr.e concerning the ability of fl-c.a~tm~ to inhibit LDL oxidation is controversial. In two recent clinical trials large doses of fl-earotenc did not reduce the susceptibility of LDL to oxidation ~n et al. 1992; Witznma et al. 1993). Also, in the F~ lung cancer prevention trial, subjects who received fl-r.a~tenc had an increased ine~ of total cancers and no reduction in either CI-ID or stroke mortality (The Alpha-TocopheroI, Beta Carotene Cancer Prevention Study Group 1994). Finally, the bright orange colour of B- carotene would create a problem in the blinding. 4.5. Power Analysis." and Sample Size The primary endpoim of this 2x2 factorial trial is the progression of carotid at~msclcrosis, opcrationalized as the- ilincar trend of-change in the mean IMT (arterial wall intima-mcdia thickness) over time (semiannual exar,,-imtions)..The mean and SD of IMT by age in our previous studies in Eastern Finlaxxl is presented below. Age (years) Mean (SD) I'WIT LNTI; Rznge IMT incr/yr 54 0.98 0.38 0.51-4.00 0.06 60 1.15 0.54 0.54-4.09 0.09 70-74 1.4 0.7 0.7 -5.3 no data From: Saloncn and Salonen 1991c, Saloncn et al. 1993b. Less data from previous studies coneemi~ women a~,~ available. In the Cardiovascular Health Study (O'Lcary e~ al. 1993) the mean IMT in the common carotid armries was of the order of 10% smaller in women than in men aged between 65 and 90 years. The mean ammal increase of common carotid hM'l" in regular smokers was 0.i I mm in a cohort of 100 men aged 42 to 60 years and 0.20 mm in smoking or hypercholesterolemic men aged 60 years (Salonen and Salonen 1990), which will bc tbc average age of the subjects of this study. This was recently confirmed in the 4.-year follow-up of almost 1000 middie-aged men (Salonen and Salonen 1993). As the cross-sectional age-specific mean IMT is similar in men and in postmenopausal women, it is assumed that also the progression ram will be similar. The slope over time will be used to characmrize each individual, and averaged within each treatment group for assessment of treatment effects. An overall analysis of variance will be conducted with slope as the dependent variable to assess differences among the 4 treatment o 0 co BATCo document for Legal Services : Health Canada 30 May 2000
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19 groups (placebo. vitamin E alone, ascorbic acid alone, vitamin E and ascorbic acid). Quality control and side effect measures for other important variables will be monitored annually. Sample size is based on a two sample t test, comparing any pair of treatments. If the variance of a slope is a:b, then the sample size per gender-risk factor stratum required for all 4 treatment groups to detect the difference 6 in average slope between groups is: N = 2(Za + Zfi)z a2~//5: Z= is chosen to be 1.96 for a two-tailed test ~ a Type I error of 0.05, and Z~ is chosen to be 1.04 for a power of 0.85. On the basis of our on-going studies (KIItD, KA.PS), the variance of slope over ~me was estimated to approximate 0.014 mm2/yr~. In the e,xpericnce of Ro~rt Seizer, the use of automated edge detection software in IMT measurements and repeating the IMT measuremen~ semiannnally (i~-tead of ~...~lly) will approximately halve the variance, i.e. to 0,007 mm'/yr:. Using the same automated edge detection software as will be used in the presently proposed study, Blankenhorn, Seizer and coworkers (1993) observed in the CLAS trial a statistically hi~aly significant (p<0.0001) treatment effect (which was only 14%) in three years in the increase of common carotid IMT in as few as 78 Sample size requixed in each group, cl~pen~ on anticipated treatment effect. Based on previous studies, we assume that the placebo group will have an average slope of CCA IMT of 0.20 ram/yr. The sample size per gender-smoking stratum required for all 4 treatment groups is then " Treatment effect (% reduction, slope under treatment) %. 0.15 mm/yr 30 %, 0.14 mm/yr 2O2 140 The goal for the sample size selected for the study is 640 (e.a~ctad/y 160 in each smman), based on the following table, assuming that smokers who quit are kept in the study and analysis and assanning 5 % dropout per year. In our earlier similar trial in 447 hypereholesmmlemic men aged 45-64 years (KAPS, see above), the drop-out was less than 50 men in three years, corresponding, to appr. 3% per year. In another similar trial in heavy smokers (see Salonen et al. 1989, Palloncn ctal. 1992), the annual drop-out has been below 5 %. This initial sample size ~ a fina! sample size of over 550 in three years, adeqtmm to detect as small as a 20 % ~catment effect at a very high power for all four strata combined and a treatment effect of 25 % for men and women separately, and for smokers and nonsmokers scparately. Projected available sample size. by" year of study, assuming 5% dropout (including smoking quitters in analysis): Smdv year Sample size 0 64O 1 608 2 578 3 549 (.y- C u t...'- C:,: BATCo document for Legal Services : Health Canada 30 May 2000
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2O This sample size has very. high power for analysis of any treatment versus any other treatment, combining all four strata. The powe.- for a 25 % treatment effect with n = 578 (combining strata) is over 0.80 and for 30 % is over 0.90. Alternatively, the study has high power for within gender comparison of vitamin, E (vitamin E alone plus vitamin E plus ascorbic acid) versus no vitamin E (placebo plus ascorbic acid alone); or for ascorbic acid versus no ascorbic acid. A 25 % effect is detected with high power within gender, for an)' treatment group compared with any other treatment group. For any single split (men only, women only, smokers only, nonsmokers only, combined vitamin E treatments or combined vitamin C treatments) power is over 0.70 for 30 ~. effect. The magnitude of the treatment effect of lipid lowering ageats on atherosclerotic progression has been of the order of 50%. In animal experknents in rabbits and in monkeys, the size of the effect of antioxidative compounds (probucol, BI-YI', vitamin 12) has varied between 30% and 60% and has been of the same magnitude as that of lipid m~on by diets or by drugs. Thus, the assumption of a mtatmem effect size of 25 % is, if anything, very conservative and can toleram sizeable dilv~on of the treatment effect due to drop-in (antioxidant comcumpzion) in the placebo groups. Sample size considerations for eye opacity measures In the baseL~ examination of 447 KAPS subjects (see above) the mean Lensmeter reading_ was 21.5 and SD 6. At alpha=0.05 two tailed, power=0.85, the requirement for all 4 groutrs assuming test retest r=0.7 is Difference Number in 4 groups O. 75 2.30 1 130 1.25 83 1.5 .58 1.75 43 Thus there is 85 % power to detects any di~ercnc~ of 1.50 or greater between pairs of ~ sex-risk factor ~. 4.6. Nonexperimental Studies of Predictors of A~erosderotic Progression and other Aging-related D%~,ue.,-ative Changes Besides a clinical trial, the study will be u "ulized as a nonexlmSmenml cohort study to investigate the role of lipid peroxidation, antioxidanm in general and pro-oxidants in atheroselerotic progression. Statis~cal analyses of predicto~ of adm'osclerotic progession will be analyzed after the completion of the 1-, 2- and 3-year follow-up periods. Antioxidants to be smdiod are plasma #-c.arotcnc, ubiquinol-10 and selenium. Serum ferritin, an indicator of body iron stores, will also be investigated. Amoa~'bcxlies against oxidized LDL will be assayed in serum samples drawn at the baseline examimn'on. 4.7. Dietary. and physical activity studies A e-day food record wilt be carried out three'times. The records will be received by a • I~ who will ~~ in an ~ A ~rd,,w~ baw~k i~i!~ti~g~3odel o MD BATCo document for Legal Services • Health Canada 30 May 2000
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21 portions will be bo.--.~wed for each subj~t. These books am the ones that were used in the "Serti" study. Alco~oi consumption will be assessed additionally with a 12-month frequency " questionnaire. Leisu~-time physizal activity will be assessed at baseline and anm~ly using the 12-month physical activity hi_no~ questionnaire, previously extensively tested in the KIHD study (See eg. Lakka et al. 19'34). The purpose of me dietary and physical activity measurements is to (I) To study diem.z; dcmrminanB of lipid pcroxichtion, atherosclertic progression, hypermnsion and cau~-act progression, (2) To study the role of physical activi~" with ~ u: atherosclerotic progression and hypertension, (3) To enable the ~caI control (using covariance models) of possible incidental parallel changes in the diet and physical activitT, ¢s'pcx:iaUy in the imak~ of the supplemcauxi and other antioxidants, and (4) to study the di~ determinants of blood glutathione stares and lipid perox.idadon. 4.8. Inclusion Criteria The subjects will.' bc men and posuncnopausal women aged 50-69 years who arc at high oxidative str~ becaa~ of regular smoking (~ 5 cigaretms/day) or mild hypctr.holesmrolemia, defined as serum cholesterol of at least 5.0 mmoFl. Prcmenopansal women will be ~x~.h~d bemuse of the antioxidan~ action of cstroggns (Sugioka et a.1. 1987, M~crc et al. 199D. The rationale for selecting r=gular smokers as study subjects is as follows: Smokers and smokers have a chronically ~ exposure to fr~ radical stress (see eg. Ludwig et al. 1982, Law et al. !991). For this reason they have an acceleratcxi progression of athcrosclcrosis, ~ incidence of hypermnsion and lens opacities. This rtxiuces the requital sample si~. Subjects with low scrttm cho!esmrol (< 5 mmolfl) ar~ excluded because they have slow progre~ion of ta!harosclcrosls and possibly, elevated risk of ~r. Smola~ and, r~l,~iveAy ~, h~lesmrolemics also have reduced levels of a,- mcoplm-ol, asc.orbic add Lud other amioxidams due to ~ mc~bolic consumption (s~ eg. Salonea et ~I. 1991b). The majority of smokers and hypercho[es~rolemics have amioxidam ~ficie:~-y, which e~_hances t~ expecmd Izeannem effect of an~oxidam st~pplement~fion. The r'~ionale for including :two diffe.mm ~ of high-risk subjects is m increase the ge~bilhy of findings. Tim s~ti~.,'..afion into-smokers and nonsmokers also enables the c~a.d.son of the Irmm~em effects between smokers and nonsmokers, For ethical reasons, mr.h participant will be ~vis~ to stop smoking on entrI into the study. Otherwise .no efforts will be made to change the participants smoking l~its. An assessment of the stage of smoking c~.sation process (s~ Pallomn et al. 1992,~ will be c~t~ried out before entry, to the study. Smoker~ who am in the "action stage" of quk~£ug smoking proc.~ ~dll be excluded. Thus, smokers who are in either the "prcconmmplarion" or in the "contet~tation° stage will be accepted. On past experience, it is expected that at tha most 3 % of Fardcipants wilI stop smokiag spontaneously each year. Subjects who quit smoking after randomization will bc kept in the study according to randomization. As noted above, separate randomization will be done for smoking and nonsmoking men and women (in 4 re'am). M1 analyses will be carried out using 4--way covadance models (by two treatments, &e gender and smoking) allowtag for all interactions (sec section 4.9. for dmails). CD CO ,.,.9 (3", BATCo document for Legal Services : Health Canada 30 May 2000
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4.9. Exclusion Criteria 22 Subj~ts with any of the following conditions will not be entered into the trial: I) 2) 3) 4) 5) 6) 7) 8) 9) 10) Prcmenopausc or regular oral estrogen substitution therapy in women. Life threatening illness such as cancer, Regular antioxidant intake, Regular intake of acctosalicylic acid, Regular intake of any other drug with antioxidative properties, Scvc-:~ obesity (BMI > 32 kg/m:), Insulin dc1~ndcnt (type I) diabetes, Cataracts ex-tract~ bilamrally making opacity assessment impossible, Any condition that limits the mobility of the subject making study visits impossible, Other disease or condition that would worsen the adherence to the mcam~ments or treanncm. 4.10. Recruitment and Screening of Subjects The subjects will be t~craited by thr~ ways: (i) marled s~If-adminism~ qucstionmire, (2) newspaper advcrfiscm~ms, and (3) from our previous trials (KAP$, TVTT). The questionaire contains questions concerning demographic background, smoking, health stares and medicadons. The sampling frame is the whole population of the city. of Kuopio and adjar.ent rural municipalities, aged 50-69 years at the beginning of 1994 (born in 1925- 44). This population includes approximately over 30,000 persons. These will be mailed a screening questionnaire. Only those willing to pm'ficipate axe asked to remra the qu~onnaire. Based on our own previous exlxa'icnce with more dmanding qucstionmircs, aI~n'oximately 20,000 persons are c,xpected to mann the questionm.irc. Tim proportion of r~nzla~ smokers in the city of Kuopio was in 1991 27.2% in men and 7.4% in women aged 50-69 years (Berg ct ai.1991). On this basis on the order of 3800 smokers (3000 nmn and 803 women) are exlaected to be fotmd. The prevalence of hypcrcholcsterokania (serum cholesmrol ;~ 7.5 mmol/l) is approximately 10 %. Thus, on the order of 2000 hypcn:holcsterolemics arc expected to be found in the screening. Of smokers and nonsmokers, 640 persons (320 men and 320 womc-~_, s0% smokers, 50% nonsmokers) will be recruited. • Approximately 900 potential subjects will be invRcd to a screening visit, which includes an inmrview concerning smoking, medical history and medications, an examination by a physician, and the measurement of serum chok~tcrol concentration. C x£) BATCo document for Legal Services • Health Canada 30 May 2000
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4.11. Measurements 4.11.1. Outcome Measurements The outcome measurements arc: I. 2. 3. 4. 5. 6. The mean (over right and left) Lntima media thickness of CCA walls (see below), Atherosclerosis-related symptoms (chest pain, claudicadon)(Rose et al. 1982), Re~dng blood pressu~ (s(m below), Eye lens opacities (se~ below), Plasma a-tocopberol and ascorbic acid concentrations (see below), Total free radical s'trt~ (in all subjects or in a subsample, pending on Eroding): 6.1. Urinary. excretion of DNA oxidation products (Loft et al. 1992)(only twice), 6.2. Pentane excretion in exhal~ air (in a subsample, see below), 7. Antioxidative capacity (TRAP) of LDL (see below), 8. Mcasurrments of lipid pcroxidadon: 8.1. Plasma concentration of ~ally modified LDL by FPLC (see below), 8.2. Suscepu'bility of LDL to oxidation (dien¢ formation, when incubated in presence of Cu-ions (Estm'bau~r ct al. 1989) and separately, wi~ hacmin and hydrogen peroxide)(Balla et al. 1991), 8.3. Cholcsmrol oxidation products (in a subsamDle, s~ below), 8.4. Plasma/urinary malondialdehyde (sec below), 8.5. Cholesterol coment of circulating immune complexes (Tertov et al. 1990a), 9. Athemgenicity. of serum (Orekhov et al. 1988)(in all subjects or in a subsample), I0. Blood Icuk~ count and plasma fibrl.noger., concentration (Clauss 1957), and 11. Serum fcrrirln concentration. The following_ table presents a flow chart of subject visits, tasks of different observers and timing of the measurements. 23 C:) C~ G'x t'x2 BATCo document for Legal Services : Health Canada 30 May 2000
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CO -I 0 e~ 0 e- 3 ~D e-ql,. 0 r"- I r~ ii -r m 0 N 0 0 ! I ' wr'lr ~Vd~0,n ..~,,,,,.,,,,~ ..... , Sm~kl~ INII~ QI • Fa~1% by I~.N~. 'l'lh:i, mc mmlOckn ~eeo~ • • liP. Hfl. 4v~ t . -. - Oru~ Q~ I IJh~mumd ~lKtmkS I~'~ Lmb~lo~ L • S'. LDL'. ~OL. ih~ q~d TO, P.leco4bite, i toeo~(~, #Ilk~, ly¢openo. ~ ¢e~4e~. 6 GSH. V~)L~L~ Ore. eOIIltI~qCI IO h4mk% tde~d GoNe, I~h, CO • ,.,~.~.,t~.T,~,;: ~o~.~ o.. ,..~.,.,,.. ,. C,,. ~'-,.,,,*,o~.,, -.h.,.,, ~,..,...,,... T,,,y.,,,c, 0.,,, ~,,~,,.~..,:,A ~o,.. s .,.!!.'..~I-o~.,,k,m. hd,-mwc~v 1( X I~ewV~O Xo , ,, *2 0 O !~1 10 , i . , ,,i P( X X Xl X X X X X X X X X X X X X K X X X~ X X X X X X I.~.olmple Subeinple X X M ITS4, X X X X X7 I ' X mlen~ Ihl the mlMurilnlml W Ime~lmtlon II done foe la lulblecll. ' I'Jt olvtn it ,| ml~lth villi ~ teklm bKIi. ihlKkl4 in(I ~I~nl~llitd l! 0 mmtlh villi. FII~ Iii1, vlll|l ltl|-IIIIldlllolYell q~llllIlllllel WI ~11 IIIlilild ~l|OIl the villi. 30 X X X x '] • . t X ...... ,,,,, X X X~ 24 3I x X X X X X Z9_q 16 00c5
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25 The justification ,or the choice of measurements proposed and descriptions of the measurement met.beds are presented in the following section. I. Ultraso.no~raohic Assessment of Carotid Arte.~, Intima-Media Thickness This is the main outcome measure of the study. Equipment: The ultrasound system us,.'~ is ~e Biosound Phase 2 sc.anncr (Biosound, Indianapolis, LN, USA) equipped with a 10 M~.z annular array.transducer. The scan converter enables ~ of the images during re, arming and distance and area meamrements using software driven cursors (calipers) with a digital display in 0.1 mm increments. The calibration of distance m~cnts is ~ periodically against a ~-ue plmmom. The scanning procedures will be videotaped with PAL S-VHS Panasonic AG 7330 VCR. Images with measurements am printed with Sony UP 811 videoprinter. Two identical systems will be used in the study. The aim is to do the scannings with a single system, the second one will serve as a back-up. Observers: Three ultrasound technicians (AM, PV, Jr) trained in arterial scanning several months prior m the study will carry out the scannings. Dr. Riina Salonen will be a back-up person for cases of absenmism. The ai~ is that all sc~nnings for each subject will be carried out by the same observer) An experienced physician (Riitm Salonen) will bc the supervisor for the scannings. As this is s double-masked study, the observers will be bRndcd as m the allocation of the subjects into u'eauncms. ~mming Cnnaging) Procedure:' The al~-asonograpbic.scanning of common carotid arteries (CCA) is performed afzer a supine res;. of I0 mimm~ while the subjgct is lying in the supine position. The head is rotated 45 degrees away from the side being scanned. Both longitudinal and cross-segtioml images are displayed for the whole length of the vessels to be scanned. Three distinct angles of interrogation (projections) will be used: the antero- lateral, the lateral and the posterolateral. The emphasis in tbe ~ will be on a 2 cm distance longfimdimlly just proximal of the carotid bulb. The ulna,sound examination and blood pressure ~ents lasts on the ave, rage for 60 miming. The proximal end of the carotid bulb will be ~ in frt~-n flames with the software- driven cursors. .' Classification of severity, of atheroscierosis: During the scanning, the observer will also be classify the subjects according to the configuration of the lumen-indma h~rface and O~e occurrence of mineralizations in common carotid m-'~'ries. Toe classification system is same that we used previously in elderly men in the Seven Cotmn-i~ Study (Salonen R. et al. 1994). Atherosclcmtic lesions will be scored accc~ng to two ~ent classifications. First, the arterial wall confimzration is classified into four categories, separately for the right and the left carotid arteries, concerning the most severely aflb~md site each side: I) a smooth arterial wall surface, 2) an uneven arterial wall surface, 3) a protrusion to the lumen, and 4) a large protrusion ( > 25 % of lumen diameter). For the statistical analysis, a person is defined to have an ultrasonographic non-mineralized athcroma when hc or she has either a~ uneven arterial wall surface or a lumenal prou-asion or b) a maximal IMT of 1.5 mm or more (ha re.al-timc measurements) in either the right or ~ lch carotid anew' sysmm. BATCo document for Legal Services • Health Canada 30 May 2000 o o ~c
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26 Second, the occ.un-ence of mineralizatio.,'ts will be classified separately for the right and the left carotid artery as follows: I) no mine."alizatiorts. 2) a single mineralization, and 3) multiple mineralizations. For the analysis, a person is defined te have a ml.narzlization4f he or she has at least a single mineralization in either the right or left caroud armry system. An athemsclemtic lesion is defined to be present, if there is either a non-mineralized atheroma or a mineralization as defined above. Videorecordings: The scanning will be started with a dia~ostic examination of enme accessible carotid thr~, to Rod tbe most severe soft and hard lesions. These sites will be documented on tbe VCR. Secondly, the distal CCA ~-i.U Ix: scanned from tlm three defm~ angles. Whole this scanning will be recorded. T'.~c ~ of each subject will be recorded on a single metallic 180-minute S-V'I-IS videotape. Measurement of IMT from Videotapes: All 134T m~ents (both baseline and follow-up) from videotapes will be made at t-txecl sites: the far wall of the distal 2 can of both tb.s right and the left CCA, just proximal of the bulb. Ultrasound Image Analysis: Computeranalysis of ultrasound images to measure IM'r wilt be performed with a 486/50 PC computer with a .540 Mb hard disk and Dam Translation DT 2861 video frame grabber interfaced to a Panasonic AG 7355E VCR. The "Pmsou~" software, developed by Robert Seizer, will be used. Automated Boundary Dcu~on: A thr~e step edge detection process will deal with variable densities and discontixn~es in intima-media boundary. First the operator idcnl~cd the approximate location of the blood-inRma interface at four points using a mouse. A smooth curve fit to these points serves as a guide to the edge tracking algorithm. Secondly, a seargh will be made along paths perpendicular to this curve for points of maximum intensity change, conditionally labelled as edges. For each edge, both the edge coordimms and the gradient value will be stored. The third SL-'p is tO compare the gradient valu~ for each comiitional edge to the maximum gradient value of all conditional edges of the inlcfface and to eliminate points with gradiem valtms le,s than a p.,welecmd fraction of the maximum value. A similar process will be used to d~terminc ~r~ coordinates of the mcdia/advcnfitia echo. IMT will be demm)Jo~ in the ~ 20 mm--~the CCA as the average diffe~w.e at 100-200 points be:wcen infima/Ittmen and mcdia/aa,entida int~ace. Measurement Variability: Intra- amt inter-operator variability in the scannings and computer measuremcnt of will be assessed. All three observers will scan 10 subjects twice at two weeks' interval. The videotapes from the 20 scannings will be read by all three observers. Calibrations: .An Icm vertical line will be insertad during the baseline and 36-month seannings to enable the vertical calibration of the measurements. The horisontal calibration is made by using the horisontal 1 em markings in the Biosound screen. In addition, a tissue phantom will be scanned and videotaped periodically. 2. A~erosclerosj.c-relat~ symptoms Chest pain (angina pea'tom) and claudicatiov will be zssessed annually according to the London School of Hygiene Interviews (Rose et al. 1982). BATCo document for Legal Services : Health Canada 30 May 2000 c~ C ,.c LF C~
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27 3. Resting blood pre.ssure The measurement of blood pressure is done became it is hypothesized to rise less rapidly or even decrese in subjects supplemented with anfioxidants. Resting blood pressure (BP) will be measured at eveQ" 6-monthly visit manually by the project assistants who are well trained public health nurses using the standard mercury sphygmomanometers. This measurement will take place in the sitting position, three m~mems at 5 minutes' intervals. In addition, the resting BP in the supine position w~ be measured semiannually during the ultrasound examinations. Systolic and diastolic BP, me.m-, arterial pressm~ and heart rate will be measured during the ultrasotmd scanning simukaneo~ly with two separate automated systems. Each of these functions on the basis of diffemm principle to measure blood pressure noninvasively. All these systems also record the heart rate. (1) the Paramed 9300 device, every three minu~s. ~ Paramed 9300 device works on the basis of the principle of d/fferemial audiomeu-/c sensor. Two different cuff sizes are used, the same cuff for each subject on repetitive visits. The cuff is placed on the right arm of the subject. (2) Ohmeda 2300 Fiaapres Blood Pressttre Monitor which measures blood pressure continuously for 15 minutes from th~ finger and is based on the vascular unload/rig technique (volume clamp method). The ~at protocol is as follows: the amomamd me~atu-Lm~nts will be started right after the subject has laid down. They will oumim~ during the initial lO-mim~ rest, duriag the st:azmi~ and during a 5-taim~ rt~t period aft~ th, scatmi~. In the rams-deal aml.v~, the means of the 3-tainum readings from (1) will be used. Separate analysis will be based on the comiauous BP monitoring (3). In this analysis, both the mean systolic and diastolic BP over 15 minutes, the maximum and m/numum as well as the SD will be used. 4. Eye lens opacities. One of the study hypotheses is that the development of eye opacities and clinical cataracts will be retarded by antioxidanz supplementation. The ophthalmological examination includes best corrected visual acuity, biomicrosoupy, appianation tonometry, b~ ~py through a dilated p~il and grading of the lens opacities by two methods. The whole examination will be carried out by ophthalmologist (P.R.), who has tzsed the proposed methods in an earli~ trial of the same magaimde, the KAPS (Rouhiaima et al. 1993). First the Lens Opacities Cla.~ificadon System ti (LOCS ID is based on standardized reference tables, where the lens status is evaluated by the slit-lamp e:~tion. Grading of the lens is made though dilated pupils; nuclear colour and opalescence, cortical and posterior subc.xpsular opacities are assessed separately. LOCS n has been shown to be an acauate, rapid, reliable and reproducible lem opac~ies classification method (C"hylack et al. 198ga, 19ggb, 1989. Leske et al. 1988). Second, ~ Lens Opacity Meter 701 (LOM; Interzcag. Switz~'laM) will be used to quandf3' lem opacities. LOM mcasm~ the sway Light produc~ by an opacified lens. It emits a beam of modulated light into the eye and the scanered light is received by a sensor which converts it into an electrical impulse. LOM has been shog'n to be a quick and reproducible method, and it has been recommemled for populadon smdi~ (Flammer et al. 1987, De Namle et al. 1988, Costagliola et al. 1989). BATCo document for Legal Services : Health Canada 30 May 2000 O o (j- G', CT,
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28 5. Plasma c~-tocophe~l and ascorbic acid concentrations To monitor the adherence to treatment and the bioavailabiliD" of supplemented antioxidams, plasma a-tocopherol and ascc:'bic acid concentrations will be determined annually (4 times). ~-tocopheml will be extracmd from heparin plasma samples with hexane and determin~ with reversed phase HPLC. Rex:luced ascorbic acid in beparin plasma is stabiliz~ with mctaphospboric acid immodiamly after plasma separation. Ascorbic acid is detetmiu~ with a high performance liquid chromatographic method (Pari::h~n et al.- ).-98~r).-Dehyd.roascorbic acid is redur.~ by homocysmine to ascorbic acid and aft~., t~luction :he total ascorbic acid is determined. Dehy~rbic acid is mcaatr~ as the difference between total and reduced ascorbic acid conc~n~tions. 6. DNA oxidation products (m~ only at baseline and at the end of the doublc-mask~ period) The supplementation with aadoxidams is assumed to r~uce the total fr~ radical ~ in the body. and more or less proportionately equally the oxidative s'tmss in the arterial ~xlothclium and cells of the eye lens. To show that the hypotbefizcd treatment effects of andoxidants arc mcdiarad thtt~ the reduction of fr~ radical ~ in the target organs, it is necessary to mcasu~ the total oxidative stress.' The 'udmry excr~on of DNA oxidation products is a scmitive and specific measure of the t0tkl oxidative stze~ in the body. It also ~y reflects the ram of aging of fi.s~es~ to the extmat that deg~ve changes are causod by oxidative strem. This measurements cnaSles us to estimate how much the oxidation stress can be overcome by zndoxidant s',~ppl~menmtion and what role this will have with regard to disease outcomes. 24-hour urine samples will be coU~ted twice (at haselin¢ and at the end of the study) of all subjects. The m~t og DNA oxidation products will be carried out either for all subjects or for a mbsample, dqg'nding on the capacity and fiux~. In addition, 24-hottr ttriae will be collected and DNA oxidation products are me.asu~d in a sttbsample (minimum t0 s~bjects per stratum, total n=40 subjects) at study moath 6 to study the short-t.m-m effects of antioxidant supplements. In vivo oxidative DNA damage is assessed by measuring urinary 8-hydroxy-2- deoxymmnosinc by high-pc-rfotmm.,ce liquid chromatography with el~-'ttt>-cbemical d~tccdon (Loft et al. 1992). The measu~ments of DNA oxidation products will be carded out in Professor Poulsen's laboratory, in Copenhagen. 7. Exhaled penmne Pentanc is produced during_ oxygen radical induced lipid peroxidadon and can be quandtatcd from alveolar breath. Exhaled pentane will be measured three times in a subsample of 40 subjects (10 from each stratum) at study months 0. 6 and 36. Exhaled air (20 l) is f'LrSt collecmd in a t~flon bag and then adsorbed by a column with activated caxton matrix (Carbotrap 300, Supelco Inc.). Water steam from exhaled air is removed by a helium flow through the carbon mau-Lx before gas chromatographic analysis. Pentane is desorbcd from carbon coltu-nn by warming the column up to 280°C and analysed -usin~ gas ~ ma¢~ ~'. .... 0 0 0", BATCo document for Legal Services • Health Canada 30 May 2000
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29 8. Whole blood e_!umt.hione Glutathione (GSH) is an important intraccllular antioxidant, which may be influenced by antioxidam supplementation. It is also affect~ by o'.her nuu-,'ients. The whole blood GSH is mainly determined by erythrocyte GS-'6. bu. is sL::pi;.er to measure. Also. it is less affected by the treatment of the samples. GSH will be measured annoy for all subj~,.s. Whole blood tom1 glutathione will be determined as follows. Total giutathione (GSH) of blood is meantr~ w~kly from acidified blood with an enzymatic method (Grif-fith 1985). Reduced glutathione reacts non-enzymatically with dithio-bis(2-nitrobenzoic acid) to yield glutathioncdisulphide, which is then reduced enzymaticaUy by glutathione reducusc and NADPH to rcgencrat~ GSH. This cyclic ldnctic reaction is followed spectrophotomcn'ieaUy (Hitachi U-2000, Tokyo, Japan). 9. Total pc,,'oxvi-.,-adical trappin~ antioxidant pr,'ametcr (TRAP) of LDL. The reduction of pcroxidation of LDL is assumed to be mediated through an increase in the an:.ioxidative capacity of LDL. LDL-TRha° is a ".:ncasurc of the overall antioxidativc capacity, of LDL. LDL-TRAP will be assessed twice (at baseli~ -_nd at the end of the study) for all subjects. The precipitated LDL fraction is exuaeted with choloform: methanol and divided for dctermimrlons of organic phosphorus and for a chemil~ assay of chainbreaking antioxidants. The sample is exposed to pcmxyl ,--adicals prcxluced by the thermal decomposition of 2.2-azobis (2,4-dimcthylvalcronitrile). The rate of peroxyt radical production in the thermal decomposition of AM'VN is followed by the luminol cnha~cd chemil~nce. The addition of an antioxidant to the reaction mixuu-e extinguishes the chemilumJnesc.~nce and its duration has a linch- correlation to the radical trapping capability.- of the sample. D-a-tocophcrol will be used as a .qandard. The duration of the extinction caused by I nmol of D-a--tocopherol is about 1100 s=c. Normal human LDL has total radical-trappLng capability of 16.9+/--0,9 (SiC,n=39) mmol per mole of organic phosphorus. If haman LDL conr~in¢ 700 molecules of ohosphoiipids per LDL particle this antioxidam capacity can be achieved with the tocophcrol composition of 7 molecules per LDL particle. We have found that 75% of this TRA/%m. is due te toeophcrols and 25% due to other LDL- incorporated antioxidants. A supplementation of daily dose of 600 mg of a-toeophcrol for four weeks elevates the TRAP, ~L to 35.2+/-2.1 (SE,n=5) pmol/nmol phospholipids. 10. M,:asurements of lipid peroxidation I0. I. Plasma concentration of minimally modified (ram) LDL Whereas hcavyly oxidized LDL, such as aldehyde-modified LDL's (see Saloncn et al. 1992a) is clea.,~ quickly from the c~ulation by t~- - liver (and is thus not measurable), the half-life of mildly oxidized LDL (~y m~,.d I.DL or mm-LDL) in blood is longer. For this reason, the plasma conccntrati.Ja of mm L.DL reflects the oxidation of LDL in vivo (see Avogaro e'. al. 1988). MIn-LDL will be measured at least twice (at basetinc and at the end of the study) for all mbjects azxt. " .acgentximg on funding, also at stttcly months 12 and .24 ....... CD CD ,,.O (7", (22 BATCo document for Legal Services • Health Canada 30 May 2000
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30 LDL is separated from plasma by a very rapid ultracentrifugational method using the Beclar.an table-top centrifuge. The electmnegativ~ subfraction of LDL, called mm-LDL, is determined from ultracentrifugally separated plasma LDL by a liquid chromatographic technique (Vedie ym.199t, Cazzolato ym. 1991). The LDL sample is injected into the FPLC system fPhan'nacia, Uppsala. Sweden) and mm-LDL is separated in an ninon-exchange column and detected by a UV-monitor. Mm-LDL is expressed as percentages from the total plasma LDL, 10.2. Susceptibility of VLDL and LDL to oxidation In addition to the m~ent of in vivo or, Jdizcd LDL, the susceptibility and resistance to oxidation of VLDL aad LDL will bc measured. The advantage of these measurements is thz.t they are quick and relatively inexpensive. These techniqu~ are also becoming standard methods in studies concerning lipid peroxidadon. While LDL-TRAP is an assessment of the antioxidadve capacit." of LDL, entirely determined by LDL antioxidants, the oxidation resistance of LDL is additionally ale"cement on the physico-chemical structure and content of LDL, e.g. the fatty" acid composiuon (PUFA's, MUFA's, SAFA's), phospholipid composition and relation of free to esterified cholesterol. Susceptibility." of V'LDL-LDL to oxidation in the presence of copper (at least twice): VLDL and LDL are scparar~l together from EDTA plasma using one st~ gradient ultracennfifugadon. Because EDTA blocks the lipopmtcin reaction with Cu, EDTA is removed from the VI.,DI.,-LDL fraction by using small gel filtration PD-10 columns (Pharm~ia, Uppsala, Sweden). The reaction is star-ted by adding C~ chloride into the VLDL-LDL fraction and conjugated diene formation in kinetic reaction is followed ~hotometrically fl-Iitaehi U-2000 speetrophotometcr with an enzyme kinetic calculation pro un-arn). The time of lag phase anJ the maximum velocity of the reetion are (Estcrbauer et al. 1989). Susceptibility of VLDL-LDL to oxidation in the presence of heroin and byda-ogen peroxide (annually): VLDL and LDL are sepa.ramd together from EDTA plasma using one gradient ul~gadon. The VLDL-LDL fraction is incutmted with hernia ami hydrogen peroxide. Oxidative inm,-'a~ons between heme, lipoprot~n particles and peroxides lead to degradation of the home ring_ and consequent release of heine iron, which further accelerates heine degradation (BaIIa et al. 1991). The oxidadon of lipoproteins and the degradation of heine is measured kit~ticaIly in a Labsystems microtiter plate reader (Labsystems, Espoo, Pinland). Tae time of lag phase, and the maximum velocity, of the reaction are deter. 10.3. Cholesterol oxidation producu (in a subsample) Cholesterol is the most ubiquitous unszmrated fa: in humans, pro~- to oxidation. The products of cholesmrol oxidation are the most toxic'products of LDL pemxidation. They are in part derived from the diet, in part synthesiz~ in lipid pemxidation in vivo. Even though they are rapidly cleared from the circulation by liver, we have fotmd measurable concentrations in human plasma. COPS will be measured by gas chromatography mass-spectrometry in a sample of 20 subjects from each of the four treatment group, totalling 80 subjects, both at baseline and at the 12-month follow-up. o 0 (Do BATCo document for Legal Services : Health Canada 30 May 2000
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31 Blood will be collec'~.c.d into a tube containing burylhydrox~oluene, which prevents in vitro oxidation of lipids. Serum is separated and lipids are extracted with chloroform:methanol mixture (2:1). The solution is filtered and water is separated. The filtrate is dried with scx:lium sulphate and evaporated dry in a romvapor at 40°C. The residual fat is saponified overnight. Then the sample is exu--acted into dicthylether, washed with KOH and water and evaporated tc dryness. T, .the. residue, pyridine and Sylon BTZ reagents are added to form u'imethyl- ,q ~.'.her d~tlv~tivcs of the cholesterol oxides. The sample is txansfe.n~ into an autosaml;iC: am.r,o~e which is filled with nitrogen. Th~ sample is stored rdrigerat~i unRl ~he gas c~- ..na.~raphy-~.cs sp~tromcu-y is performed (Park and Addis, 1985). The separation of ~'lmethyls~yl e.t~r "b,riva~iv~ of the cholcsmml oxid~ is carded oat on a HP-I column using an Hewlctz Pac~.-.:i mode! 5890 gas chromatogaph and 5970 series mass scl=tiv¢ detector. The calibration run is performed for following chok:~'ml oxides: 7- a-hydrox'ycholcsterol, 7-fl-hydroxycholcsmrol, 7-1mu)chole.stcrol, cholcsta-3fl,5a-6~-triol, 2.5-hydroxycholcs~rol. cholcs~rol-5a,6a-epox3de and cholcsterol-5B,6fl-epoxide. The quantification of cholcs~rol oxides takes place by calculating the ratios of areas of the sample peaks and the inmmal standard, and comparison of the.so values m those of tbe cah'bration values. 10.4. Plasma malor~d~Id=l~ycl= :~:" ' ,* ) Malondialdehydc is a major end-product of thc oxid,~on of polyunsatttrated fatty ~Is. It is a sm.mia~ mcasurgmc~, a "gold .~oda_rd" of lipid pcroxidatlon. MI)A will be with HPLC in professor Esmrbauer's laboratoD" ir,'Austria. I I. Cholesterol concenn'adon of cir~ulatiuR immune comulexes Oxidative modifications of LDL render k immunogenic. An inexpensive method to measure serum concenn'~on of modified LDL is to separam all seram immune complexes and to measure ~ cholestm-ol conrrm of these. This method has been developed by a highly group in Moscow. Serum "immune cholcs~rol" will be measu_r~ twice (at baseRne and at the end of the study) either for all subjec~ or for a subsample, l~".ding on funding. The serum "immu~ chol~'mml" com;#.~fiun wfiLbe de~ by sed~ ch'culafing immun~ complexes using 2.5 % PEG 6C,00, and the total cholcsmrol comcm will be measu.r~ using the CHOD-PAP method (Terror et al. 1990). 12. Athero~enicitv of ~eram The main pathophysiologic mechanisms by which oxidized LDL promotes ath~rogcncsis is the accumulation of cholesterol in the ar~rial c~lls. The potenual of serum to induce cholesterol accumulation can be mcasured. The athcrogeniciry of serum will be measured twice (at baseline and at the end of the study) for all subjects and in a subsample at six months. The atherogenir,." of serum will be determined using primary culture of human aortic cells. The cells are isolated from grossly normal human aortic intima by dispersion with 0. 1% collagem.~. On the 7th day of p~ culture, the ceils are incubated in in the ~ of 40 % se.ram to ~ msu~i for. 24 k. c~rol ~.~_t~f~d __ 0 cD Co m -.,,,j BATCo document for Legal Services • Health Canada 30 May 2000
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32 with standard cultural medium without human serum addition. After incubation, intra~llular lipids are extracted and total cellular protein determined by Lowry. et al. method. Cholesterol content of control cellg is taken for 100% and chol~terol content of ceils incubated in the presence of se.'-~:n to be tested is expressed as % of control values (Orek.hov et al. 1988). 13. Blood leukocyte count and plasma fibrino_ge.n con~n.trafion_ The blood leukocyte count and the pl -a:,nna fib~-inogen conccnnation will be measured to confirm or refute unpublished findings from previous trials concerning the effect of vitamin E on blood leukocytes and plasma fibrinogen. Blood cell coun~ (RBC, WBC), hemoglobin and eryflm~xc indices will be as.w.ssed annually. Plasma fibrinogcn will be m~ at least twice (at baseline and at the end of the study) and possibly also at study months 12 and 24, pcndi~ on funding. 14. Serum femtin concentration Serum ferritin concentration will be detemaiaed a: baseline and at the end of the study. The rationale for this measurement is the interaction betw~n vitamin C and iron: ascorhate enhances iron absorption and on the other hand, l~gi~ body iron stores may make vitamin C a pro-oxidam file,bert 1994). Thus, k is assumed "~,.~t the antioxidative, antiatherogenic, anl2hypermnsive and anticarar~togenic effectsof vi:amir. C will be greater in subjects who have low serum ferritin concentrations (<200 t~g/l) at haseline. It is likely that serum ferritin levels tend to increase during the study in the vitamin C groups. Scmma samples for the assays is kept in -20"C until determined in batches with a radioimmunoassay (Amersham International, Am~, IRK). 15. Serum selenium and. hair mercu~ Selenium is a cofaaor in the glutathi~,nc peroxidase, an enzyme scavenging hydrogen peroxide in the hnm~n body. Mercury is a common transition metal, which acts as a catalyst of lipid pcroxidation. Scr=n selenium and the hair content of mercury will be meammd at baseline and at the end of the study with HA-atomic absorption spectrometry. 4.11.2. Other Measurements Serum tom2. LDL and HDL cholesterol and triglycerides are measured annually to enable the stati~cal control for changes in blood lipid levels effectiag atlm~sclerosis progression, due w changes in diet and other habits. Cholesterol and trigtycerides are determined enzymatically in an automatic anal.v-~r (Kone, Espoo, Finland). HDL eholesmml is measured after magnesium chloride dexnan sulphate p.~.cipitation. Serum LDL cholesterol concentration will be measured using PVS precipimdtm. As the)" are potential confounding factors, diet, ~'-cohol consumption, smoking, physical activity, the use of an>" medications and anthropomemcs will be asses.~ annually. Diet will be assessed by' a 12-month frequency ~onna~, vslidated against food records in our previous studies. Leisure time physical activity wiL be assessed by a 12-month physical activity history, which has been modified from the Taylor Questionnaire and validated in our previous studies ~ and Salonen 1992a, 1992b, I.,akka et al. 1994). Measm'ements of O O C:3 ".,,3 BATCo document for Legal Services : Health Canada 30 May 2000
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33 anthropometrics include height and weight to derive B/vfl, body fat percent by infrared method (Futrex-5000) and waist and hip circumference. Plasma B-carotene concentration, plasma co-e.-.zyme Q 10 concentration, serum ferritin concentration and autoandbodies against oxidized LDL (Salonen et al. 1993a) will be assessed at baseline. The purpose of these determinations is to allow the investigation of their relationship to the outcome measures and to emble their statistical control as potential confounders. ~-ca.,'otene and lyeopene will be m~ simultaneously with c~-tocopherol with reversed phase HPLC. Ubiquinol-10 (coenzyme Q10) is determined at least at baseline from plasma in its oxidized and reduced forms with an '.--r_PLC method (Takada et. al. 1984, Lang et al. 1986). Pending on famding, flavonoids will be measured with I-n>LC at least at baseline. After hexane and ethanol extraction ubiquinol is injecmd into a reversed phase HPLC system and the concentration is detected by an electrochemical deteaor. DNA will be collected for assays of hemochromatosis and other genes regulating free radical reactions ~nd lipid peroxidation. Hair sample will be coliect~ at baseline for mercury measurement and serum sample for selenium ~ents. The intake of the major nutrients will be assessed annnally by 4-day food recordings a.s explained above. Lekqxre-time physical activity, will be assessed twice with the KII-ID 12- month LTPA questionmire. Several extra pl~crr~, serum a:x:l blood san~ples will be collected a~ frozen at all annual laboratory, visits. 4.11.3. Blood sampling All blood samples will be drawn between 8:00 ~ 10:00 am after a fast and abstinence from smoking for at least 12 hours and abstinence from alcohol for at least 3 days. The subjects are also instructed to avoid m'enuous excsretse during the previous 24 hours. 4.11.4. FoUow-up of Deaths and Morbid Event..5 Deaths in the study population will be obtained from the compu~ mtioml death certificate registry, which is acce~ible to the investigators, Hospitalizations will be obtained from the computerized hospital discharge registry. Clinical diagnoses will be confu-med from hospital records. All coronary events will be recorded in a prospectively fttw.tioning myocardial infarction regi.~try using uniform a prio~ defined diagnostic ~ (see Saloneu et al. 1981, 1992c, Tuomilehto et al 1992). The evems are monitored for safety reasons. Also, even though the power of the study is subop~m21 in the estimation of the separate effects of a-tocopbcrol and ascorbic acid on morbidity and mortality, the effect of both treatments combined on the incidence of all corora,'y events and acute myocardial infarctions as well as total mortalit3." will be analyzed. 4.12. Statistical Methods The primary study h.~vothesis is that the supplementation with either cE-tocopherol or with ascorbic acid will rednce the rate of atberosclerotic progression (defined as a smaller mean increa~ of far wall IMT) in CCAs in the subject population by 25 % in three years. The change will be estimated with the linear slope of the mean of the integrated IMT -- -men~n~ e,~,ethe ~ ted the left CCA. - .... 0 0 o3 .,.0 LJ"I BATCo document for Legal Services • Health Canada 30 May 2000
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34 Secondary sr,,dy h.~'po:hescs arc concerned with the effects of antioxidam supplementation on the sccondaD study outcomes: the elevation of blood pressur~ (development of hypertension), and the d:velopment of eye lens opacities (and cataracts). Randomization wilt be. balanced within four strata. Strata are formed b v gender and smoking. Baseline chara:teristlcs and demography will be assessed by linear models for continuous variables (such as age a.:'l weightl and iugistic-regression models for disc~te variables. Both models will include effect~ for strata and the stratum-by-treatment inmracdon. Any heterogeneity bctw~n the treatment groups will be investigated for its potential confounding or modification of r:.eatment effects. Outcome variables derailed above will be analy-z~ by linear models and appmp:iatc camgorical data methods. Both n~thods of analysis will s'd'atify by g--~er and risk factor. Treatment interactions with the sn'afify, ing variables will be assemed by ~ inter'acdon effects in the models. If inmractions are found to be nonsi~canz, then tests and esti~dom will be pooled over all four strata. Primary. analysos wil/be carried out using the principle of intendon-to-treaL Stadsdca/ significance will be defined as p < 0.05 for a two-sided test. Supporting analyses of per- protocoI or adherence-tc,-a~atment populations will be performed. Analyses of covariance with time-dependent co~riatcs will be investigated. Additional analyses of subgroups and r~'e~sion analyses will be undertaken. The statistical analysis concerning, the disease our.:omes will be carded out by using maximum likelihood analysis for covariance (A/'!COVA) for repeated measures of the SAS software (PROC MIXED). Factors (all binary.) in-the analysis will be (I) vitamin E (yes vs. no), (2) vitamin C (yes vs. no), (3) gender (men vs. women), and (4) enn'y risk faczor (smoking vs. h vperchole~mlemia). "Fne uov~.ates will be sclecraxi outcomc-speci_fically on the basis of the stren~h of association between each disease outcome and each covariate. All variables with a partial smndardiz~ m m'ession coefficient of 0.10 or higher will be entered as a covariatc. Secondly. a variable has to differ bctwcen 4 treatment groups (in 2-way ANOVA) sitmificandy (~ p< 0.05). Potential confounders considered for en=y ar~ dietary factors (including alcohol imake), amount of smoking, physical activity, medications, scram lipids, ferritin and other anfioxidams ('beside.s vitamin E and C). First, a samramd linear model with all possible main effects and inmractior~ will be fitmd. Secondly, a r~aced ('optimal') model will be developed which will include all main effects and only those inmractions that were simificant (a: p<0.05) in the saturated modcl. For instance, ifaU inmractions concerning_ gender am nonsignificant, mmtment effects are considered homogenous in both me,,, and women. For each disease outcome, separate models will be fitmd. To analyze the role of chemical measurements (~xidative stress, LDL-TRAP, lipid peroxidafion) at mec~ mediating the preventive effects of vitamins E and C, changes (for urinary DNA oxidation products) and linear slopes (for lipid peroxidation mcasuremems) will be entered into the optimal covariancc model as additionai covariates. The reduction in the prvportion explained by treatment of each disease outcome variable is an estimate of the role of oxidative stress and lipid pemxidarion measurements as mediating pathways in the preventive effect. The proportion explained is estimated by the proportionate reduction in the regression coefficients for the treauneut term (vitamin E, vitamin C) after the mediating variable is added m the model. Tn.= p, ~ponions explained by oxidative stress and lipid pemxidation va.nables (individua!ly and jcln.fly) are estimates of the role of these mechanisms in the etiologs_ • of ~ch disease outcome. The mechardsms (fr~ radical stress, serum nitrams, lipid pemxidation) mediating the --- .hypoOsem~ blood pTessere k~vermg_ effe,as of antioxidams will be amiyz~ _,;,,,.a.,.|y. The 0 0 BATCo document for Legal Services : Health Canada 30 May 2000
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35 linear trend over 6-mon'~!y measurements (mean of a!l measurements each time, see above~ of both systolic and sepe.-amly, diastolic blood pres.sure as the dependent variable, the proportion of its variance explained ar~ the proportion of blood pressure lowering effect of antioxidants explained by the following covariates will be analyzed: serum nitrate concenu'adon, total free radical stress (u~-na-'3' DNA cxidadon products), lipid peroxidauon measurements and the lirear trend m car'C"¢ IMT...The last analysis aims to answer the question, whether the h.~:-.'.~thesized anRh~pe.-,ensive effect of antioxidams is due to a reduction of arterial IM't" and consequently, reduced wall rigidity, or whether it is independent of at',anal ~-a.ll thickness. A similar analysis will be carried out for -,all di.sea~ outcome variables. It is assumed that tl~ changes in oxidative ~ and lipid peroxida~on mmsu~ments will explain a major proportion of the e~ difference bcnvcen tr~tment ~mups in the linear trend in eamdd IMT, blood pressure and eye lens opacities ovc.x-e.,.e: Clinical events (see above)will be surnmarizeJ a...,,d rates will be assessed for treatment diffcrenc, s. These analyses will include life table and Cox proportional hazards' models. Rates of adverse events and laboratory, abnormalities will be summa.dz~ and examined by similar methods. Periodi= inmrim am.lyses will be exccmad and pre~nted to a Data and Safety Monitoring Committee. Resul~ will be prr.semed to the Committee by its Statistical Rcprt~ntatlve (David Jacobs). EveD" atmmpt wili be made to maintain the integrity of the treatment blind. 4.13. Data Handling A randomization sch~-_,fle will be prepared by Dr Jacobs, with stratification by gender, and balancing after ere'c,.; 2. ~ or 6 (in hap -baza~ .¢equetr.e) study recruits. Dr. Jacobs will pml)are sequenced randomization envelopes, wk-~-:h will be opened in sequence in Finland as people are randomized..An a",.cessio,-, log will be maLmained, including name, ID, date of randomization and assigned tr~tment group. A copy of the in progress accession log will be monthly to Dr. Jacobs for auditing. A participation log file will be created in Finland, and used to create, under Dr. Jacobs' superAsion, the monthly rtxmdtment and r~'ntion rtlmrt. Data en~ and iaidal f'tle creation will be dc, nc in Finland. SAS will be used for data management and to crtmm data dictionaries. A sept, -am file will be crated for each data form or other data sourr2 (i.e. laboratory). Dr. Jacobs will carry out analyses pertinent to stndy conduct and pro~. for both the Principal Investigator and the Data Safety Monitoring Committee. 4.14. Quality" Control A carefully written ~ fo:lowexi protocol will bc the main quality assurance. The principal investigator or other qualified staff will. fcom time to time, observe clinic operation and make suggestions to correct any deficiencies which might exist in carrying out the protocol. In addition, thee are many laboratory.', t~hnician mediated and questionnaire items which will be subject to qualir,." control assesm'.ent. All laboratory measures will be reassessed by periodic blinded duplicate samples. Wherever possible, laboratory stability, will be assessed by periodic analyses of frozen pools or other standard materials, depending on the test. T~hnician mediated tests will also be monitored. Blood pressure taking adherence to protocol will be monitored by a supcrvisor using a double stethoscope. Ultrasonography -rereading. wilt be pedorme~. In • small mmp~ a~,i~ #.atotid uaxasmmd ~_witt 0 0 4~ BATCo document for Legal Services : Health Canada 30 May 2000
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36 be performed twice, for comparison. Eye lens opacit~ measurement and questionnaire items (chest pain, claudication) will be repeated in a small sample. Where multiple technicians are taking the same measuremem, resulting average values will be compared between them, on the assumption that techrdcians are randomly allocated to participams, and should make similar measuremenu. Quali~ conu'ol reports will be available at 6 month inmrvals both for the investigators and the data safe~, monitoring com.~ttee. The following table presenLs between-batch coe,~.cien:s of variation (CV) for some chemical me, asurements m be ca.~ed ou~. "n *he laboratc.D' of the applicant insdmte. VLDL-LDL oxidation "-'- the pre~nce of copper - lag time, sec - maximal velocity (abs/min) VLDL-LDL oxidation in the presence of heroin and H20: - lag time, sec - max. velociP,' a-tocopherol, l~mo'Jl 3-caroten, ~mot/l Ascorbic acid,/~mol/l Insulin. mU/l n Mean CV (%) 9 5533 11.2 9 0.0011 10.9 11 11 " 5 • 5 I0 I0 Glucose, mmoL/l ~0 Ferrkin, ttg/l 20 9.6 14.8 °. 2,6.9 6.2 1.0 10.6 88 7.2 I 1.3 13.5 39.8 4.3 110.0 6.8 14.2 2.8 52 6.4 172 6.0 490 10.9 Chol~.~rol (total), mmol/l 49 7.51 I.I HDL-cholesmrol. mmol/l 14 0:71 2.8 LDL-cholesteroL mmoUl I 1 3.20 4.4 Triglyccrides, mmoi/l 36 1.06 2.0 4.15. Time Table The recruitment of the subjects was carried out during the fall of 1993 and the spring of 1994. Entry. of the subjects to the lead-in phase wi11 start in August, 1994. The subjects wilI be entered into the randomiz~ double-masked treatment phase between October 1994 and September. 1995. Thus. all subjects will have sin.ned the randomized trealmcm by the end of October, 1995. Then. r, hey will complete the study by" the end of September, 1998. Chemical and other measurements, stati~cal analyses and rep~-.,-dng will be completed by the end of 1999. L.n CD CD C~ LY3 L,n BATCo document for Legal Services • Health Canada 30 May 2000
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5. Human Subjects 5.1. Description of Subjects The subjects of the study will be volunteers from a larger screening survey (see above). Six hundred and forty, men or posunenopausal ~,omenwill be entered in the study. Age range will be 50-69 years at entry. Any persons with any severe disease or condition that might interfere with the effect of antioxidant supplementation will be excluded. All subjects will be caucasian, as there are no si~ -:.ficznt racial r,'2nodties in Eastern Finland. 5.2. Sources of Research Material All information will be gathered for research purposes only. 5.3. Recruitment of Subjects 37 Subjects will be recruited by mailing a seLf-adminismred questionnaire to all citizcm of the city. of Kuopio, Eastern Finland, aged 50-69 years Information on smoking, other health habits, medical history, medications and the use of vitamins and antioxidants will be collected, Eligibility will be judged on the basis of these dam, laboratory tests as well as based on clinical examination by physicians. The wil "lingnc~ to participate in the study will be asked in the mailed questionnaire and the tim} c~nsent on the visit to physician. Written information expl~inlng the study will be provided. A written consent will be asked sever'a] weeks after providing the information about the study. 5.4. Potential Risks The amioxidant supplementation involves no risks to the subjects. There is no known allergy to the natural, physiological vitamins d-a-tocopherol aad ascorbic acid. These vimm~r~, used at reasonable dose~, have no'known toxic side effects, either (Steinberg et al. 1992). The measurements include ~ venous blood samples. This will be always done by registered laboratory nurses (who are licensed to &-'aw blood samples in Finland). The imravenous glucose tolerance test will be performed by a physician in laboratory conditions. Vitamin E Numerous studies ]nave shown that vitamin E has low toxicity and that o.,-al intake at multiples of the Recommended Die~ Allowance of 15 IUs produces no significant side effects in normal individuals. In 1988, an extensive literature search of all research data since 1975 concerning the safety of oral vitamin E ~a~ppternentation was undertaken. In a report citing_ 45 studies involving animals aad human subjects, Bendich and Machlin (1988) concluded that the aeu:e (single dose) and chronic (.,'¢petitive dose) toxieities of oral vitamin E intake are ve~ low. The stron~-e=- evidence .f vi~ninE's low toxicity, was found in six double-masked, placdxr-contmlled studies with daily intake of 600 to 3200 IU for three weeks to six months, where very. few side effects were observed and no specific side effect was consistently seen. Before 1975. no controlled l.'~,..g-term Studies had been reported on the possibte toxicity of vitma-hn E to ~. ~, ~he rego~ of ie~ rigotmw m~kim -. ,.E ¢3", BATCo document for Legal Services : Health Canada 30 May 2000
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involving I0.06~ subjects indicated only a 0.8% incidence of minor side effects. This extremely low incidence of side effects would be expected to occur in an untreated group and indicates that vitamin E is extremely well tolerated. In addition, research has documented that the~ is no evidence of mutagenJcit3:i teratogenicit3.' or carcinogenJcit)'. Vitamin C 38 For vitamin C, the only safeP," risk suggesr~i is the potential increase of iron absorption. However, in a review of 24 studies based on 1412 subjects Bendich and Cohen (1990) concluded that "ascorbic acid did not increase the incidence of "high" iron absorbers" and that data indicau:d no change in the dism'bufion of iron absopfion values by ascorbic *,'id doses ~mr than I00 mg/d. It has been claimed that ~estion of vimml. C in excessive qu~a~zities can conn-itmm to kiclney stone formation (conditio~ and re~usx~ sctu'vT, v'itamin Bt~ dcsmaction, mutagenici~, ~ c~:q)per utilization, abnormal psychological function, iron overload, transient diarrhea, and laboratory, testing errors). Only the latter two effects appear to be sup~rmd by some published literature (Hornig and Moser 1981, Rivers 1987). High intakes of ascorbic acid were postulated to conmbut~ to kidney stone formation because oxalatc, a compo. ~nt of stones, is produced during catabolism of the vitamin and bceattse vitamin C may acidify urine. However, oxalar~ formation appears to be a saturable system (Schmidt = al. 1981), and in hyperoxalurics, oxalat~ that is produced from vitamin C is insignificant in comparison with oxalatc from ~ sources (Hagler and Herman, 1973). In addition, mamertms studies have shown no imlication that high doses of vitamin C do, in fact, increase the incidetx~ of kidney stone formation (Sclamiclt et al. 1981, Sut~n ctal. 1983, Hoffer 1985, Erden ctaI. 1985, Singh et al. 1988). Incidences of "rebound scurvy-', the appearance of scorbutic symptoms following the withdrawal of high-<lose vitamin C intake, have been largely anecdotal. There has been no experimental evidence for such an effect in humans (I-Iomig and Moser 1981, Gersmr and Moscr 1988). Guinea pig studies in this area have been negative or inconclusive (Rivers 1987). The finding that large amoums of ascorbic add destroyed vitamin Bu was shown to be an artifact of the expcrimetml method (Marcus et al. 1980). The assertion that vitamin C is mutagenic also resulted from an error in in vitro methodology (Norkns et al. 1983). Controlled studies have shown no support for claims that high ascorbie acid intake impairs psy. cbological performance (Pasc.oe and Stone 1984) or COFper absorption (Jacob et al. 1987, Miine et al. 1988). Because adverse effects of oral vitamin C supplementation have not been convincingly demonsuated, even at intakes as high as 10-20 g/day, a safe upper limit c.aJmot be defined. 5._¢. lh-ocedure~ for Protecting the Subjects The laboratory." of the Research Imtirate of Public Health is situated beneath the Kuopio University Hospital In the unlikely ca~ of any emergency, emergency medical team is available or a subjc,.--t can bc tramfcrred to intcmivecare in a couple of minutes. Also, the physicians of the Rcsm~h Institute are well trained in emergency care such as cardiopulmonar3" resuscitation. O O (2o ,,L3 BATCo document for Legal Services • Health Canada 30 May 2000
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39 Data and Safer,' Monitoring CommitTee A Data and Safer,.- Monitoring Committee will be established for the study. The purpose of the committee is to monitor adverse events among the study subjects. The Data Safety. Monitoring Comminee will consist of a chair and two other scientists. To ensure the blind of investigators in Kuopio who arc ca.rry.ing out the field work, dr. hcobs will air.end meetings and he will present inte.'im data t~ the commmee. The Data Safety Monitoring committee periodically reviews dam and has the right to r~v, ommend changes in study design and U'canncnt, including termin~on of a trmnn~_t arm...q'h~m .would be a formal vot~ (among the three commitme members), with Dr. Jacobs advice. Such a nmommendation then goes to d~c principal investigator and the steering commitmc consisting of the PI and thnm project officc'rs for final action. The chairperson of the safety monimcing commitme will be professor Jussi K. Hummcn, MD, PhD, D~r of flac National Public He.alth Institute of Finlm~ and co-PI of the large ATBC lung ~r prevention u'ial. Tncrc will be two additional mcmbem in the committee. The Dam safety committee will make its decisions in a executive session which is not a1~xlcd by r.~ invcsti~mrs. 5.6. Benef'rts for the Subjects - All information con=rod during the study will bc given to the subjects at the end of the study. This includes .rhc results of all clinical and ~cal ~ and ~cms. The physicians of the Rcseamh ~ will talc-, cam of all medical issmm r~luiring medical anention during the cnm~ study. This is dole, besid~ to the subjects' own good, also to ensn~ a high compliance. Also, all r~'mits will be provided anti.smoking education and dietary advice to lower serum LDL cholesmrol. 6. Vertebrate Animals V~tebram animals ~.~ not involved in the study. 7, Consultants and Collaborators and their Role in the Study. I. Division of Epidemiologv, University of Minnesota, MN: David hcobs, Phl), Professor of Epidcmiology: Generation and auditing of randomization, inmrprc~tion of statistical analysh done in Finland and co- investigator rcp~ve of the Dam and ,Safety. Monitoring Committee, participation in reporting of reaults. Dr. Iacobs is a Professor in the Division of Epidemiology, School of Public Health, University. of Minnesota, and obtained his Ph.D. in Mathematical Stmis'tics from The Johr~ Hopkins University in 1971. His ~ since then has focused primarily on ca=diovascular cpidemiology and use of biomcuic methods. His mseamh work Ires ~,m~b/_d~,~t and ..~-.,,~n Acxkd lu:imacRy.by.lhc CD 0 C~ Co BATCo document for Legal Services • Health Canada 30 May 2000
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4O Institutes of Health. He has participated in several randomized, double masked clinical trials, including the Coromr3" Drug Project. He has extensive data analysis experience. He has for the past 12 years been Evaluation Director and co-principal investigator of the Minnesota Heart Health Prom'am. Since 1984 he has been principal, then co-principal investigator of a study of evolution of cardiovascular risk in young adults (CARDIA). Hi.~ recent work centers on the elevated risk for nonatherosclerotic diseases at low levels of blood cholesterol. He co-chaired an NI-K.BI sponsored conference on this topic in 1990. He has worked with Dr. Belcher and others or~ the relation of oxidation ofLDL .to development of atherosclerosis. 2. H,r~.. Population Laboratory, State of California Health Deoartment, Berkeley, CA: George A. Kaplan, PhD, Director: Planning of the study, and participation in the reporting of results especially regarding the aging aspects. Dr. George A. Kaplan is Chief of the Human Population Laboratory, California Department of Health Services. Dr. Kaplan is an internationally weLl known epidemiologist with inmrests in aging, social epidemiology, and cardiovasaflar disease. He has conn'ibuted ~ paI>~rs ~ the role of primary. ~d s~axlary prevention in the ekicrly, and ~:**ntly published a chapr.cr on methodologic issues in the cpidemiologic study of the elderly. His oarrem ~ and activities involve a nttmber of studies examining primary and secondary prevention of functional problems in the elderly, and the prevention of age- and disease-related declines in functioning. Dr. Kaplan regularly acts as an advisor to the National I.n~tut~ of Aging, the National Heart, Lang, and Blood Institute, the National Center for Health Statistics, the Buck Center on Aging, and other agencies and foundations. He asxl Dr. Saloncn have collaboratad on rcse.arch since the early 1980's, and are csatre~y conducting the Knopio Ischacmic Heart Disease Risk Factor Study (KIKD) in Finland. . Jet Propulsion Laboratory, California Institute of Technology. Pasadem. CA: Robert H. Sclzar, Group Supervisor: Consultant in computerized me, asu~ment of ulna.sound videor~.ordings, participation in the reporting of the results. Robert Seizer is one of the few experts in the world in the computerized reading of vidcorecordings of ultrasonographic examinations. He has writmn software for this purpose for the Rescar~.h Institute of Public Health, University of Kuopio and will continue to provide support for the software. He has previously worksd with leading scientists in athcrosclcrosis research such as prefcssor David Blanketthom from Los Angeles. Robe~ SeLzcr and professor Salcnen have previously collaborated in studies conce~ the assessment of the measurement precision of uhrasonog, raphic measurement of atherosclerosis (scc section 3.3. for details). 0 o co BATCo document for Legal Services • Health Canada 30 May 2000
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4. Institute of BiocY.¢mistrv, University of Graz, Austria: Hcrmann Esm,~,,~ucr, PhD, Professor of Biochemis~T: Planning, participation in ~ reporting of resul~s, consultant in the assessment of LDL oxidation and autoantibodics against oxidized LDL. Professor EsL.,'rbaucr is inm,,-nationally very well known and is one of the leading cxpcm in the ~n'y of lipid oxidauon m the world. 41 . Ins~Aw- of ~cl~al ~iolo~,"¢. Nadonal Cardiology Research Center. Moscow, Russia: Alexander N. Omkhov, PhD, Biochemist: Planning and r~pordng, measurement of modified LDL forms and LDL conmnt of immune complexes. Dr Alcxand~. Omkhov and his working group at t2~c Insdmtc of Expcrimcnml Ca.~ology, National Cardiology ~h Ccnmr, Moscow', Russia has a long-term ~cnc~ in ~ com~rning a~-~'ogcniciP/of lipid oxidation products and is world famous i~ this ate. Dr. Omkhov is presently collaborating with profcssor Salonen in KIHD and KAPS studies. . Department of ~colog'v, Universi~ of Cooenha~en: Hcntik E. Poulsm, M:D, Phanzmcclogist: Mcasur~mcn: of DNA oxidauon products. Dr. Poulsen is :Lssociam Professor in Toxicology and Environmental Medicine at the I)~arnn~nt of Pharmacology, Univcmity of Copcnl~gcm I:)cmzmrk. The research is in d~lopmcnt of methods to estimate oxidarlvc ~ on DNA ~d othcr cellular components and to estimate the capacity of enzym~ that activates xenobiotics to harm~ men.liras, for use in biochemical and molecular cpidcmJology. 7. Department of Biomedicine, University of Tampcre, Finland: Timo Mcts~-Kcm-~, Phi), Assistant Professor, Biochemist: Measurement of LDL- TRAP. Dr. Mcts,:-~ is a highly regzrd~ lipi¢, oxidation chemist who has co~ramd with professor Saloncn in several earlier sm~, most notably in thc KII-ID and KAPS. Scppo Yli-Hcrmaaia, MD, Phl), Senior r~search fellow of F~nni~h Academy: Measu~mcms of generic factors in EDL oxidation (an add-on study funded by the Finnish A~'). Dr. Yl~-Hcr~ata is a young, very talientcd and already internatiomlly regarded ~.iemst. ~ ~,~a ,a ~ mmaiag wi.m ~ ~.in_~ .! 0 o Co o BATCo document for Legal Services : Health Canada 30 May 2000
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Also he has collaborated with professor Salonen in previous studies concerning the role of lipid oxidztion in atherosclerosis. 42 Department of E~id.emiolo~v and Health Promotion, National Public Health Institute of Finland, Hetsir~i. Finland: Jaakko Tuomilehto, MD, PkT.;, EpidemJologist. Research Professor: Planning and reporting_, consultant in glucose :~..~.~i~-~e~aboli.~n.. , Professor Jaakko Tuomilehto is an internationally known expert in the field of epidemiology and prevention of hypertension. He has been centrally involved in several multinatioml studies of hypert..'~nsion (Tau-opean Working Party of the Hypertension in the Elderly, MetoproloI Atherosclerosis Prevention of Hypertensives, Systolic HypertemSon in the Elderly in Europe, WHO Community Control Programme for Hypertension, WHO Hypefinsulinemia and Glucose Intolerance as a Risk Factor of Cardiovasaxlar Disea~ in the Population of Mauritius, where the national rates for cardiovascular mortality and the prevalence of glucose intolerance are highest recorded in the world today). Dr. Tuomilehto has collaborated with professor Salonen for almost 20 years in a number of stndies, most notably in the North Karelia Proj~---'t. ~" ,., -':. Iea~rat0ry..0f Food. chemistry, Food Research ~re. Am'iculmral research Cem~ of Finland (Jokioizea) - Iorma Kumpulainen, PhD, Laboratory director Dr. KumpulaJnen is a well known trace element chemist and expert in free radical stress and lipid pemxidadon. All collaborators arid consultants in the present study have worked with professor Salom-n and his group for years in earlier stndies. As the study is an inmrdi.~iplinary one, k requires experts in different fields of science. Methods to collaborate have been established in previous collaboration. These inclu~ the use of I) Conferez~ calls, 2) Bimet and fax. and 3) Annual investigators' meetings: The transfer of d~-p-frozcn blood, serum, plasma and urinary samples by air freight between collaborating, c,.-amrs has been tested in earlier collaboration. BATCo document for Legal Services - Health Canada 30 May 2000 O C~ L~ Co
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43 Co-Investigators at the Applicant Institution (University of Kuopio) (In alphabetical order): Jussi Kauhanen. MD, PhD, Research A.~sociauc of the F~h Academy: Planning and reporting. Ce-project officer. Dr. Kauhanen is a very tallented ar~ already experienced young cardiovascular ¢pidemlologist and a long-term coworker of professor Saloncn. Marjatta Kantota, MPh, Chemist: Selenium determinations Marjatta Kamola is an experienced trace element chemist who has previously collaborated for several years with professor Salonen in studies concerning the role of selenium and toper in atherosclerosis and Ct~ (see sections 2.3. and 3.1. for details). Heflcki Korpela. M.D, PhD, DVM, Assistant Professor in Preventive Medicine: Co- Project Officer Heildd Korpela, MD, PhD, DVlvi, is an Assistam Profemor of Preventive Medicine at the University. of Kuopio, Finland and he has been a member of Society for Free Radical Re.scazch since 1982. He has investigated the role of lipid peroxidation and antioxida~ in cancer, pre-eclampsia, multiple sclerosis and liver diseases. His current specific imctest is in the role of free radicals, lipid peroxidation and antioxidants in atherosclcrosis and coronary heart disease. T'tmo Lakka, M'D. Epidemiologist: Clinical subj~t management. Dr. Lakka is a very tallentcd cardiovascular cpidemiologist and a smdem and long- term coworkcr of professor Saloncn. He is an expert in the mcasm'~ment of physical activy. Lotta Maijala, Med St.: The role of physical activity in free radical stress. Lotta Maijala is a young talented medical student who is also studying in the MD- Phl) program of the University of Kuopio. Minna Marttunea. MD, Research associate: Hypertension. Minna Martmnen. MD, has me.enfly graduated from the medical school of the UniversiD' of Kuopio has has juat s'axtcd h.--r-doctoral work. P Kristiina Nyyss~nen, MPh, Clinical Chemist: Mcasummcm of vitamins, lipids and cholesmrol oxidation products Kristiina Ny~'ss0ncn is a very" talemcd and innovative young chemist who has a su'ong back~ound in chromatom'aphic memods. She has concentrated in the last few CD BATCo document for Legal Services : Health Canada 30 May 2000
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years in measurements concerning iipids and lipid oxidation. She has worked with professor Salonen for approximately four years in the KAPS study. EEna PorkkaIa. MPh, Biochemist: Measurement of susceptibility of LDL to oxidation 44 Etina Porkkala is a young and tallented biochemist student who is working with Kristiina Nyyss6nen and professor Salonen in KAPS with rtspomibility in the measurement of susceptibility of LDL to oxidation. Ulla Ristonmaa, MPH, nmritionist: food mco~li.~s Ulla Ristonmaa is a young nutrkionist who has previous research experience, in food recordings and GSH metabolism. Kimmo Ronkai.en, MPh, System analyst: Dam analysis K.immo Ronkainen has worked with professor ,Saloren for approximately I0 years at the P,.~ea~h Institute of Public Health, University of Kuopio. He is experienced and lmowlcdgable in data mamgemem znd statistical data analysis. Hath Rouhiainea, MD, PhD, Ophfl~lmologist: Planning and r~porting, eye leas opacities. Dr. Ham Rouhiaiaen will act as consultam/co-investigator in issues regarding the assessment of eye Iens opacities. Piivi Ro~en, MD, Ophthalmologist: Planning_ and reporting, measurm~em of eye lens opacities. Dr. Piivi Rouhiainen has been responsible for the ~ of lens opackies in a la~e preventive real, the Kuopio A~lerosis Prevention Study (Salonen et al. 1992d). This is double-masked randomized clinic.a] trial testing the effect of lipid modification on the prOgremion of carotid and femoral a~lerosis, as assessed ulwasonom-aplfically. The methods for the amessment of lens opacities have been developed and tested in this study (see Rouhiainen et al. 1992). Riitta Salon,ca, MD, PhD, Research Investigator: Ultrasonographic assessment of atheros:lerosis Dr. Riitta Salonen has worked earlier at the Rm, earch Institute of Exercise Medicine, as a research associate of the Finnish Academy and lately as a research scientist at the ~ch Institute of Public H.,yalth, University of Kuopio. She has studied and investigated ultrasonogtapkic method to as.tess atherosclerosis for almost 10 years and has published extensively in that field. She has been trained in epidemioloD" by professor Py6rglA. University. of Kuopio. C9 Co LI3 OD BATCo document for Legal Services : Health Canada 30 May 2000
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45 Jorma Takala, MD; Ph.D. Professor of General Practice: Supervision of doctoral work concerning h vpertemion Professor Takala has worked extensively in the area of hypertension. He will provide expertice concerning the recruitment and compliance of the subjects as well as supervise a doctoral studem. Riikka T~nck~nen: Gender differences in risk factors for atherosclerodc progression. Riikka Tanskamn is a medical student who has recently staxted her doctoral work. Aria Tervabauta, Phi), ~h Investigator: Hemochromatosis gen~cs. Dr. Aria Tervahanm is a postdoctoral fellow who has a good previous experience in techniques in molecular biology. Mzrkku TerS.s-dtla, MD, Phl), Acting Professor of Ophmlmology: Planning and re~rfing, eye lens opacities. Dr. T~ has a vas~ experie~ in the treatment of catar, t~. He is supervising Piivi Rouhiainen's doc~ral work and will collaborate in the study in the area of cat.aIagts. Tomi-Pekka Tuomainen, MD, resee.-ch associate: Iron stares, hemochromatosis. Tomi-Pckka Tuomainen is a talented young doctoral student who is working in the area of iron s~ams, hemochromatosis and arhcrosclerosis. Vdi.Pekka Valkonen: The role of free radical sn-css in hypermmion. Veti-Pekka Valkoncn is a medical student who has r~tty started his doctoral work. (..f" BATCo document for Legal Services • Health Canada 30 May 2000
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8. Litera~..ure Cite~ Ames 5N, Shigenaga MK, Park E-M. DNA dama.ce by endoq.enous oxidants as a cause of aging a/:d cancer. In: Davies KJA (ed) Oxidative ,"amage & Repair. Chemical, Biological and Medical Aspects. ~ergamon Press, New York, 1992. A--oqarc -=, Biztolc-Bon G, Cazzola:c G. Presence of a modified low densi',y lipoprc=ein in humans. Ar~erlosclerosis 198 ~ ; 8 : ~.9- 67. Balla G, Jacob MS, Eaton JW era!. He~i,,: e possloie physiological media-or of low density !ipoproKein oxida-,ion a~d er.le~.%='.ial l'_.jur/. Ar~erioscler Thromb 1991;11:1700- 1711. Bendich A, Cohen M. Ascorbic acid safe~,/: ~u~a!ysis of factors affecting iron absorption. Toxicol L~tt 1990;-=i :189-201. Benclich A, Mac.~-!~u- LJ. Safe=y of oral intake cf vitaE.in E. Am J Clin Nu-.r 1988;48:612- 619. Berg A-M, Pel~oniemi J. Puska ~. Health behaviuur --~ng Finnish adult population. Spring 1991. Publications of the Na',!ona! ~hlic ~.ealP, h Institute, Helsinki B ~/1991. Be.--ry EM. The effec',s of nutrients o.~. lipcprotein susceptibility to oxidation. Curt Opin/on Lipidoi 1992 ; 3 : 5-11. BjSrkhem 7, He.uriksson-Freyschi'.uss A, Breuer Cet al. The an~ioxida~ buzylaUed hydrox~o!uene pro~ects against auherosclerosis. Arterioscler Thromb 1991;11: 15-22. BlazLkmz~hor= DH, Se!zer RH, Crawford-DW etal. Be.ueficial effects of Colestipol-Niaci~ therapy on the common carotid artery: two- and £~-year reduction of in~ima-media th/ckmess measured by ultrasound. Cir,~ula-.ion 1993;88:20-28. Bruckdorfer KR. Free rad/cals, lipid peroxidation and atherasclerosis. C~urrent Opinion Lipido! 1990; 1 : 5~9°535. Caz~w TE, Schwenke DO, S~e~r~ D. A-~-ia'.herogenic effec~ of prohucol 'u~Irelated to i~s hypocholes~erolemic effect: Evidence uha~. an~icxidan~s in vivo can selectively inh/bi~ low density lipoprouein d egradatio-- in ~cro~hage-rich fatty streaks slowing the progression of auherosclerosis ~ ~he ~'~[L rabbi:. Proc Nail Acad Sci USA 1987 ;84:7725-7729. Carland D. Role of si~e-specific, metal-catalyzed oxidation in lens aging a=d cataract. A h.v'po~hesis. Exp Eye Res 1990;$0:677-682. Cazzolato G, Avogaro P, Bit~olo-Bon G. Characterization of a m~re elec~ronega~ively cha~ed LDL subfraction by ion exchange ~LC. Free Rad Biol Meal 1991;11:247-253. Cerami A, Crabbe JC. Recent advances in co&far cataract research. TIPS, July 1986. Elsevier Science Publishers, Amsterdam 19B6. Cai~ A. Methods of assessing lipid and lipopr~tein oxidation. Curt Opinion Lipido! 1992;3:389-394. Chazov El, Orekhov AN, Teruov VV en al. A=herogenicity of blood plasma from patie_nus wi~h coronary a-,herosclerosis and its correction. Atherosclerosis Roy 1988 ;17 : 9-20. Chazov E~, Ter',ov W, Orekhov AN etal. A~her~enici~y of blood serum from patients with coronary heart disease. Lancet 1986;2:595-598. Che_ng JT, Hsieh-Chen SO, Tsai C-~. L-Asccrbic acid prduces hypoglycaem/a and hyp~rinsulinae.~.--~a in anaesthatized rats. J Pharm Pharmaool 1989;41:345-546. Chow OK. Vitamin E and oxidative stress. Free .Rad Biol Med 1991;11:215232. Christen WG, Ma=son JE, Seddon HM, Giynn RJ, Buxing JE, Roster B, Hennekens OH. A Prospec~.ive S~u~y of Cigarette Smoking am.d Risk of Cataract in Men. JAM;, 1992; 268 : 989-~9"... CD L~, BATCo document for Legal Services - Health Canada 30 May 2000
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Chy!ack LT Jr, Leske HC, HcCar',h.v D et el. Lens Opacities Classification System II. Arch Oph~ha!mcl 1989;107:99!-997. Chylack LT Jr. Leske MC, Sperdu:o R e'. el. Lens Opacities Classifica~_icn system. Arch Oph=halmoll988 ; 106:330-334. Chylack LT Jr, Leske MC, Sperdu'.o R0 McCart.~y D. Profess in expanding the Lens Opacities Classificatio.~ System. LOCS If. Lens Res 1988;5:109-115. Clauss A. Geringua=sphysic!cgische $c~,-nellme~.ode zur Bestimmung des Fibrinogens. Acta Haematol 1957;17:2~7-46. Costagliola C, luiiano G, Rina!di E et al. l~.vivo measttrement of huma~ le~s aging ~a.~i~g ~he Lens Opacity Meter. Ophthalmolog-'ca 1989.199:158-161. Cutler RG. Aging and Ox-y~en Radicals. In: Taylor A2, Matalo¢1 S, Ward PA (eds). Physiology of O~en Radicals. Clinical Physiology Series. American Physiological Soci-'ty. Waverly Press Inc., Baltimore, Maryland, 1986. De Natale R, Flammer J, Zulauf M, Bebie T. Influence of age on the transparency of the lexLs in no,--me!s: a population study wit/~ help ofthe Lens Opacity Meter 701. Oph~.halmologica 1988 ; 197 : 14-18. Dieber-Rot.heneder M, Puhl H, Waeg Get el. Effect of oral supplementation with d- alpha-tocophero! e= the vitamin E conte.ut of human low density lipoproteims and its oxidation resistazce. J Lipid Res 1991;8:1325-1332. Eichholzer M, Stahe!in HB, Gey KF. Inverse correlation between essential antioxidants in plasma and subsequent risk to develop cancer, ischemIc heart: disease and stroke respectively: 12-year follow-up of ~he Basel Study. EXS 1992;62:398-410° Ena~rom JE, Kanim LE, Klein MA. Vitamin C intake aitd mmz-cality among a sample of ~he United States population. Epide.~.iology. 1982; 3:194-202.. Erden F, Bacisalihog.lu A, Kocer Z, Simsek B, Nebioglu S. Effects of vitamin c intake on whole blood plasma, leucocy~..e and urine ascorbic acid and urine oxalic acid levels. Acta Vitaminol Enzymol 1985;7:123-130. Esterbauer H, J~Irgeas, G, Quehember~er O, Koller E. Autoxidation of human low density lipopronein: Loss of polyunsaturated fatty acids and vitam/n E and generation of aldehydes. J Lipid Res 1987;28:495-509. Esuerb~uer H, Die.ber-Rothen~er M, WeeS G et el. Bioch~nical, s~ruc~ttrKl, a~Id functional prope~ies of oxidized low=density lipoprotein, them Res Toxicol 1990;3:83-84. Esterhauer If, Dieber-Rot.heneder M, Striegl G, Waeg G. Rmle of rite,nit E in preventing the oxidation of low-denslty lipopronein. Am J C!in Nuur 1991;53:-~14S-321S. Es=erbauer H, Ge.bicki J, Puhi-E, G~nther J. The role of lipid peruxida'.ion and a~Itioxidants in oxidative modification of LDL. Free Red Meal Biol 1992;13:341-390. Esterbauer H, Puh! H, Dieber-Rctheneder Met el. Effect of antioxidants on oxidative modification of LDL. Axulals of Medicine 1991;23:573-S81. Esterbauer H, Striegl G0 Pub! H, Rotheneder M. Continuou~ ~monit.oring olin vicro oxidation of human low density lipoprotelzl. FreeoRad.Res Co~m~s 1989;6:67-75. Flammer J, Behie H. Lens Opacity Meter: a new inst,-nmmen~ uo quantify lens opacity. Ophthalmologica 1987; 195: 69- 72. Frei B. Ascorbic acid protects lipids in human plasma a~d low-density lipoprotein against oxidative damage. Am J Clln Nu=r 1991; 54: II13S-II18S. Frei B, England L, Ames BN. Ascorbate is an ou'.standing antioxidant in huma~ blood plasma. Proc Nail Acad So--" - USA 1989; 86: 6377-6381. huma,= blood plasma. Adv Exp. Hod @io! !990; 264: i~5-163. xD Cx BATCo document for Legal Services • Health Canada 30 May 2000
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Garland D. Role of site-specific, me~al-cazalvzed oxidation in lens agln~ and cauarac'.: A h'/pouhesis. Exp Eye Res 1990;5C:~77-682. Gerster H, Moser U. Is high-dose vi~ami,". C ~n:ake associated with sys:e-ic con~i~icnlng? Nu=r Res 1988 ; 8 : 1327-1332. Ge.v E2. Crr. ~ha a/~ioxidan~ hypothesis wi~h re~ard ~o arteriosclerosis, Bibl Nutr ,~ieza 1966;37:53-91. Gey F, P.'ska P, Jor~n P, Moser D-~. inverse correlation between plasma vitamin E and ,.-~rua!i',v from ischemic heart disease in cross-cultural epidemiology. AT~ J Clin Nutr 1991;53 :~265-345. Gree~ LC, Wa~ner DA, Glogowski J, Fkipper-PL, WisP~nok JS, Tannenbaum $K. Analysis of Ni~.ra~e, Nitrite, and [t~] Nitrate in Biolcqical Fluids. Analytical Biochem/stry 1982 ; 126:31-138. Griffi~h OW. Glu~a~hione and glunathione disulphide. Zn: Me~hods of R.~..-ymatic Aua!ysis. Be,'~Jmeyer HU, Ber~mayer J, Grassl M (eds), VCH Verlagsgesel!schafU, we~-_~heim, FRG, 1985. Habe.-la~d M~, Fogelman AM. The role of alue---.d !ipoproneins in the pa:hogenesis of a-,h~ercsc!erosis. Am Heart J 1987;113:573-577. Rag!or L, He_--man RH. Oxalaue metabolism. I- Am J Cli~ Nutr 1973;26:882-889. Hall ED. Tntensive an~i-oxidant preureaume~-, reKards motor nerve dogeS_oration. Brain Res 1987;413 : 175-178. Hal!iwel! B, Gutteridge JMC. Free rad/cals i~ biology and medicine. Clarendon Press, Oxford 1989. F~liliw~!l B, G~ueridge JMC. Role of f_T~o--radic~-is.~r~,ca~aly~ic me~al ions in human disease: an overview. In: Packer L, Glazer ~ (eds.~. Me~hods in Enz~!ogy, Vo! 186. Sat Diego: Academic Press 1990, pp. 1-85. ~Kamkinson SE, Willet RC, Coldi~z GA, $eddon JM, Roster B, Speizer FE, St~fer MJ. A Pl-ospec~ive Study of Cigarette Smoking and Risk of Cataract Surgery in Women. JAMA 199~;268 : 994 -998. Ka=a~s D, Be_u-Naim M, Dabach Y, Hollander G, Havivi E, Stein Y. Effec'. of Vitamin C and E S%qoplemen~a~i~n of Susceptibility of ~lasma LipoproueizLs uo Percxidauion 7~=ed hn~ Acute S~k~g. Atherosclerosis 1990 ; 85 : 47-S4. Har;~ing JJ, van Heyningen R. Epidem/ology a~d risk factors for catarao'.. Eye 1987;I :537-541. ~a.--aan D. Free radical ~heory of aging: History. In: Emerit I, Chance B (eds.) . Free radicals and aging, pp. i-I0. BirkhAuser Verlag: Basel, Switzerland 1992. B~l~ H. Vitamin C and lowering blood pressure: need for in~erven~io= ~rials. J Bype=uens 1991 ; 9 : 1076-1078. Holler A. Ascorbic acid and kidney stones. Car, Med Assoc J 1985;132:320. Eoruig DH, Moser U. The safety of high vitamin C intake in man. In: Vitamin C Ascorbic Acid. JN Counsell and DH Nor~/g (eds) . Applied Science Publishers, E~glewood, NJ, 1981, pp. 225-248. ~hamainen M, Salonen R, Sepp~nen R eta!. Nutrition data collection i= the Kuopio Is-Cuaemic Heart Disease Risk Factor Study: nuurien~ intake of middle-aged Eastern F~ish me--. Nu~r Res 1989;9:597-604. Jacob RA, Skala YS, Omaye ST, Turnlund JR. Effect of varying ascorbic acid in~akes on cc~-per absorption and cerulopls~tin levels of young men. J Nutr 1987;117:2109-2115. _ja~-ues PF, Chylack LT Jr. Epidemic!o~ic ~vidmnce of a role for the antioxidant vitamins and carc:encids in ca~arac~ prever.~ion. Am J olin Nutr 1991;'S.'.'-3"~251"3555. 0 0 Co BATCo document for Legal Services : Health Canada 30 May 2000
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