CLINICAL INVESTIGATION Effects of Vitamin E on Lipid Peroxidation in Healthy Persons Emma A. Meagher, MD Context Oxidative stress may play a role in the development or exacerbation of many Orla P. Barry, PhD common diseases. However, results of prospective controlled trials of the effects of antioxidants such as vitamin E are contradictory. John A. Lawson, BS Objective To assess the effects of supplemental vitamin E on lipid peroxidation in vivo Joshua Rokach, PhD in healthy adults. Garret A. FitzGerald, MD Design Randomized, double-blind, placebo-controlled trial conducted March 1999 to June 2000. XIDATIVE STRESS APPEARS TO Setting A general clinical research center in a tertiary referral academic medical center. be of fundamental rel- Participants Thirty healthy men and women aged 18 to 60 years. evance to diseases as di- Interventions Participants were randomly assigned to receive placebo or a-tocoph- verse as atherosclerosis, erol dosages of 200, 400, 800, 1200, or 2000 IU/d for 8 weeks (n=5 in each group), Ocancer, and Alzheimer disease.1-3 How- followed by an 8-week washout period. ever, prospective, controlled clinical tri- Main Outcome Measures Three indices of lipid peroxidation, urinary 4-hydroxynon- als of antioxidants present a confused pic- enal (4-HNE) and 2 isoprostanes, iPF2a-III and iPF2a-VI, measured by gas chromatography/ ture. For example, while administration mass spectrometry and compared among the 6 groups at baseline, 2, 4, 6, and 8 weeks, of vitamin E appeared to benefit pa- and 1, 3, and 8 weeks after discontinuation. tients with coronary disease in the 4 Results Circulating vitamin E levels increased in a dose-dependent manner during the CHAOS study, the HOPE and GISSI Pre- study. No significant effect of vitamin E on levels of urinary 4-HNE or either isoprostane venzione studies failed to detect such a was observed. Mean (SEM) baseline vs week 8 levels of iPF2a-III were 154 (20.1) vs 168 benefit.5,6 Similarly, while dietary flavo- (22.3) pg/mg of creatinine for subjects taking placebo; 165 (19.6) vs 234 (30.1) pg/mg noids appear to reduce cardiovascular for those taking 200 IU/d of vitamin E; and 195 (26.7) vs 213 (40.6) pg/mg for subjects mortality, supplemental b-carotene and taking 2000 IU/d. Corresponding iPF2a-VI levels were 1.43 (0.6) vs 1.62 (0.4) ng/mg of vitamin A appear to increase the risk of creatinine for subjects taking placebo; 1.64 (0.3) vs 1.24 (0.8) ng/mg for those taking death from lung cancer and heart dis- 200 IU/d of vitamin E; and 1.83 (0.3) vs 1.94 (0.9) ng/mg for those taking 2000 IU/d. ease in smokers and workers exposed to Baseline vs week 8 levels of 4-HNE were 0.5 (0.04) vs 0.4 (0.05) ng/mg of creatinine for 7,8 subjects taking placebo; 0.4 (0.06) vs 0.5 (0.02) ng/mg with 200 IU/d of vitamin E; and asbestos. Various possibilities have 0.2 (0.02) vs 0.2 (0.1) ng/mg with 2000 IU/d. been advanced to explain this discrep- ancy, including differences in patient Conclusions Our results question the rationale for vitamin E supplementation in healthy individuals. Specific quantitative indices of oxidative stress in vivo should be considered characteristics, the antioxidant content as entry criteria and for dose selection in clinical trials of antioxidant drugs and vitamins of their diets, dose selection, and ran- in human disease. dom distribution of outcomes about the JAMA. 2001;285:1178-1182 www.jama.com mean. However, a striking feature of these and other trials of antioxidants is vitro.15,16 Despite these observations, However, this assay bears an uncertain the absence of a biochemical basis for pa- nothing is known about the suscepti- relationship to actual oxidation of LDL tient inclusion or dose selection. bility of clinical trial patients to supple- in vivo, and its relationship to oxida- Elaborate and diversified antioxi- mentation with antioxidants or whether dant mechanisms protect tissues from the doses selected exhibit antioxidant ef- Author Affiliations: Center for Experimental Thera- oxidative damage in humans and other fects. What little is known of the dose- peutics, University of Pennsylvania, Philadelphia (Drs 9-11 Meagher, Barry, and FitzGerald and Mr Lawson); and organisms, and susceptibility to ben- response relationships of antioxidants in the Claude Pepper Institute and Department of Chem- efit from exogenous antioxidants in vitro humans is based on an ex vivo assay of istry, Florida Institute of Technology, Melbourne (Dr Rokach). is conditioned by the degree of their the oxidizability of low-density lipopro- Corresponding Author and Reprints: Garret A. FitzGer- depletion.12-14 Additionally, certain vi- tein (LDL) cholesterol. Small studies us- ald, MD, Center for Experimental Therapeutics, 811 Bio- medical Research Bldg II/III, University of Pennsylva- tamins with antioxidant properties can ing this approach report variable ef- nia, 421 Curie Blvd, Philadelphia, PA 19104-6160 function as prooxidants, at least in fects of vitamin E supplementation.17,18 (e-mail: [email protected]). 1178 JAMA, March 7, 2001—Vol 285, No. 9 (Reprinted) ©2001 American Medical Association. All rights reserved. Downloaded From: by a Florida Institute of Technology User on 11/13/2017 VITAMIN E AND LIPID PEROXIDATION tion of other lipid substances and to dis- subjects were available for measure- was used for gas chromatography/mass ease pathogenesis is unclear. ment at all study points. Compliance was spectrometry analysis. The mass spec- Reliable, quantitative indices of free monitored by regular capsule counts and trometer was operated in the negative- radical–induced modification of lipids, serum vitamin E levels. Twenty-four- ion, electron capture ionization mode, proteins, and DNA in vivo have begun hour urine collections for isoprostanes using ammonia as the moderating gas. to emerge only recently.2,19 Most infor- and serum vitamin E, vitamin C, and se- Ions monitored were of mass-to-mass mation acquired in humans relates to lenium levels were measured at base- charge ratio 283 and 286 for 4-HNE and lipid peroxidation. F2 isoprostanes are line and at 2, 4, 6, and 8 weeks of dos- d3-HNE, respectively. free radical–catalyzed prostaglandin F2 ing and 1, 3, and 8 weeks after dosing. Serum levels of vitamin E and plasma isomers.20,21 They are chemically stable concentrations of vitamin C were mea- and can be measured noninvasively in Isoprostane Measurements sured by high-performance liquid chro- 28,29 urine with precision and sensitivity us- Urinary iPF2a-III and iPF2a-VI were as- matography. Serum selenium con- ing homologous internal standards and sayed by stable-isotope dilution gas chro- centrations were measured by atomic mass spectrometry.22-25 We used this ap- matography/mass spectrometry as pre- absorption spectrometry.30 proach to investigate the dose-response viously described.22,23 Urinary creatinine relationships of vitamin E with lipid per- was determined using a standard auto- Statistical Analysis oxidation in healthy volunteers. mated colorimetric assay (Beckman Syn- Tests of statistical hypothesis for main chron CX System, Beckman Instru- effects were based on a fixed type I er- METHODS ments, Arlington Heights, Ill). ror rate of 5%. The study was powered Participants and Design to detect a 10% change in urinary iso- To investigate the dose-response rela- Analysis of 4-Hydroxynonenal prostane and 4-HNE measurements. tionship of vitamin E with lipid peroxi- The method for urinary measurement of Data were initially subjected to analy- dation in healthy volunteers, we per- 4-hydroxynonenal (4-HNE)26 was sis of variance with subsequent pair- formed a randomized, double-blind, adapted from one previously devel- wise analysis using a 2-tailed t test, as placebo-controlled study from March oped27 to measure 4-hydroxyalkenals in appropriate. Data are expressed as mean 1999 to June 2000. The study was ap- oxidized LDL. Briefly, 5-mL urine (SEM). To account for potential differ- proved by the institutional review board samples were spiked with 5 ng of ences among group baseline values, all of the University of Pennsylvania and the d3-HNE, mixed well, and allowed to successive time values were adjusted for General Clinical Research Center Ad- equilibrate for 15 minutes at room tem- each subject’s baseline measure: D(i,k) visory Committee and all participants perature. Two milligrams of (2,3,4,5,6- =value(i,k)−value(i,0), for each sub- provided informed consent. Thirty vol- pentafluorobenzyl) hydroxylamine ject i at k=2, 4, 8, 9, 12, and 16 weeks. unteers, 15 men and 15 women, be- hydrochloride was added to each sample, These adjusted measurements were sub- tween the ages of 18 and 60 years (mean and they were allowed to stand for 30 jected to an analysis of variance appro- [SD], 38 [12.5] years) were random- minutes at room temperature. 4-Hy- priate for a 2-factor experiment design ized to 1 of 6 dosing groups: placebo or droxynonenal was extracted using with 1 repeated measure (time) and 1 vitamin E at 200, 400, 800, 1200, or reverse-phase solid-extraction car- nonrepeated measure (dose). 2000 IU/d for 8 weeks, followed by an tridges (C18 EC, 500 mg; International 8-week washout period. There were 5 Sorbent Technology Ltd, Mid Glamor- RESULTS subjects in each group. All volunteers gan, Wales) under the following condi- Prior to dosing, all measurements of en- were nonsmokers. In addition, they were tions. The cartridge was conditioned dogenous antioxidants were within nor- all less than 120% of ideal body weight with 5 mL of ethanol and washed with mal limits.