The "oxidative hypothesis" of atherosclerosis proposes that oxidative modification of lipids in low-density lipoproteins (LDL) contributes to atherogenesis 12. Antioxidants that are effective against lipid peroxidation should therefore reduce atherosclerosis and hence afford protection from cardiovascular diseases (CVD)1. In contrast to a) epidemiological evidence that antioxidants taken with the diet or as supplements reduce cardiovascular (CV) risk 3, and b) experimental data supporting its anti-atherogenic properties 4, vitamin E failed to show any beneficial effect in recent large intervention studies 5. In two large-scale trials, long-term supplementation with vitamin E (300–400 lU/day) failed to reduce cardiovascular events in post-myocardial infarction patients (GISSI-Prevenzione 6) and in subjects at high CV risk (HOPE 7). In the trial conducted by our group (Primary Prevention Project, PPP 8), vitamin E (300 mg/day) taken over three years also showed no effect on the incidence of cardiovascular events in individuals with one or more major risk factors (see Methods). Therefore, the question whether vitamin E had an effective in vivo antioxidant action in the populations under study in these trials is under debate 9101112.

When these studies were designed, the antioxidant efficacy of vitamin E in humans had not been demonstrated in vivo because of the lack of reliable methods 13. Measurement of urinary or circulating F2-isoprostanes (iPF₂ or F₂-isoP) is now accepted as a reliable tool for evaluating the rate of lipid peroxidation in vivo9141516. Using urinary excretion of 8-epi-PGF_2α (also termed iPF_2α-III or 15-F_2t-isoP) as a biomarker, it has been shown that short-term administration of vitamin E (600 mg/day for 14 days) reduced in vivo lipid peroxidation in some clinical settings where oxidative stress is abnormally high, e.g., diabetes mellitus (-37%), hypercholesterolemia (-58%) and cystic fibrosis (-42%) 171819. In contrast, vitamin E had no antioxidant activity in conditions where lipid peroxidation was normal 20. No data are available on the antioxidant effect of longer-term supplementation with vitamin E in subjects at moderate/high cardiovascular risk.

Using a highly selective and validated mass spectrometric assay for 8-epi-PGF_2α21, we measured in vivo lipid peroxidation in PPP trial participants who had taken vitamin E daily for about three years vs those who did not take vitamin E.

PPP is a large, randomized, controlled 2 × 2 factorial trial on primary prevention of CVD 8. It was designed to test the efficacy of long-term administration of vitamin E (synthetic α-tocopherol, 300 mg/day) and/or aspirin (100 mg/day) in preventing cardiovascular events in subjects of both sexes aged ≥ 50 years, with at least one of the following cardiovascular risk factors: old age (≥ 65 yr); hypertension (systolic blood pressure ≥ 160 mm Hg or diastolic blood pressure ≥ 95 mm Hg on at least three separate occasions); hypercholesterolemia (≥ 250 mg/dL on at least two separate occasions); diabetes mellitus (≥ 140 mg/dL fasting venous plasma glucose, on at least two separate occasions); chronic drug treatment for any of the three latter conditions; obesity (body mass index ≥ 30 kg/m²); and premature myocardial infarction before 55 years of age in at least one parent or sibling. Patients with a history of cardiovascular events or diseases were not included. Table 1 shows the frequency of these CV risk factors in our sample.

Overnight urine was obtained from subjects consecutively presenting at five participating centers for a scheduled follow-up visit after at least one year of randomized treatment. With a sample size of 70 individuals per arm, the study had a 90% power (1-β) to detect, with α = 0.05, a difference of at least 25% in urinary 8-epi-PGF_2α between treated and untreated individuals. Two groups of 72 subjects treated or not treated with vitamin E were studied. Clinical and biochemical variables were reassessed yearly and on the occasion of urine collection.

Urinary 8-epi-PGF_2α was selectively measured as we have described previously 21 using immunoaffinity chromatography for selective extraction/purification and a stable isotope dilution assay with gas chromatography-negative ion chemical ionization mass spectrometry for quantitation, with ²H₄-8-epi-PGF_2α as the internal standard. Urinary excretion of 8-epi-PGF_2α was expressed as pg/mg creatinine. Creatinine was measured highly selectively by stable-isotope dilution HPLC-electrospray-tandem mass spectrometry, using ²H₃-creatinine as the internal standard. Urine was stored at -20°C until analyzed. Analyses were done on blind-coded samples.

Means were compared by the non-parametric Mann-Whitney U test to avoid assumptions about the distribution of the variables. Levels of 8-epi-PGF_2α were expressed as median (range) values. Multiple regression analysis was used to 1) evaluate the effect of vitamin E on urinary excretion of 8-epi-PGF_2α, taking into account the following potential confounding variables (age, sex, aspirin treatment, smoking, systolic and diastolic blood pressure, blood glucose, blood cholesterol and obesity), and 2) assess whether any of these variables was independently associated with lipid peroxidation. Linear correlation analysis was also used. Probability values of p ≤ 0.05 (two tails) were considered to be statistically significant.

Long-term supplementation with vitamin E-at a dose largely exceeding that of a vitamin E-rich diet (300 mg/day)-did not substantially reduce lipid peroxidation in people with one or more major cardiovascular risk factors. This may help explain why vitamin E was not effective for CVD prevention in the PPP study. As opposed to other studies showing an antioxidant effect of vitamin E in small, uncontrolled subgroups of patients, our observations were obtained in a sample of subjects that more realistically represent a population with cardiovascular risk factors. In fact, as usually occurs in clinical practice, most candidates for cardiovascular prevention are treated-although not necessarily controlled-for their modifiable risk factors. The lack of antioxidant effect in our sample may be explained by the rather surprising finding that lipid peroxidation was normal in a large proportion of these subjects.

We investigated whether oxidative stress was increased in high-risk subjects, given that this category could, in principle, be more sensitive to antioxidant therapy. However, we found no evidence of a correlation between CV risk score and urinary excretion of 8-epi-PGF_2α in untreated subjects, suggesting that lipid peroxidation was not associated with their global CV risk. Although, on average, lipid peroxidation was normal in our population, it was clearly increased in relation to extreme conditions reportedly associated with oxidative stress. Lipid peroxidation was, in fact, significantly higher in smokers, in agreement with consistent evidence of elevated oxidative stress in cigarette smokers, mostly obtained using 8-epi-PGF_2α and/or other F₂-isoprostanes as biomarkers 212227. We also confirmed previous observations that excessive lipid peroxidation in smokers cannot be reduced by vitamin E 2223.

A secondary, but original finding of this study is the direct relationship between urinary excretion of an F₂-isoprostane and systolic blood pressure in treated hypertensive patients with different degrees of blood pressure control. Whether this relationship also exists in untreated hypertensive patients should be investigated. An association between oxidative stress and arterial hypertension has been suggested by several clinical and experimental studies 2829303132. The hypothesis that free radical-mediated mechanisms may play a role in the pathophysiology of hypertension has recently gained support from observations that antioxidants lower blood pressure in hypertensive patients 3033343536. However, vitamin E in particular does not seem effective 37, possibly because it was not antioxidant in these circumstances.

As to other factors reportedly related to in vivo lipid peroxidation, our findings agree with previous evidence. In particular, we confirmed that enhanced urinary excretion of 8-epi-PGF_2α is associated with impaired glycemic control in diabetic patients 17. We did not find a correlation between urinary excretion of 8-epi-PGF_2α and blood cholesterol, which, on average, was slightly elevated in our sample. Such a correlation was, in fact, found in subjects with homozygote familial hypercholesterolemia and in subjects with very high blood cholesterol, but not in those with normal cholesterol levels 24.

Prolonged supplementation with 300 lU/day vitamin E did not reduce lipid peroxidation in subjects with one or more major cardiovascular risk factors. On average, however, lipid peroxidation was near-normal in this population. These data may help explain the overall lack of benefit of vitamin E in recent cardiovascular prevention trials 678. They also suggest the need to reassess whether lipid peroxidation is indeed an epidemiologically relevant determinant of cardiovascular diseases and, consequently, to reconsider the utility of antioxidants as a general preventive measure.

None declared.

Participating physicians: Marina Bosisio Pioltelli (general practitioner, Monza), Alberto Capra (Ospedale Civile, Voghera), Mario Cristofari (Ospedale di Desio), Gaetana Palumbo (Ospedale S. Carlo Borromeo, Milano) and Susanna Rossi (Ospedale di Rovereto)

Coordinating group: Chiara Chiabrando, Fausto Avanzini, Roberto Fanelli and Maria Carla Roncaglioni (Istituto Mario Negri, Milano)