Mortality in Randomized Trials of Antioxidant Supplements for Primary and Secondary Prevention:  Systematic Review and Meta-analysis FREE

Oxidative stress is implicated in most human diseases.^1- 2 Antioxidants may decrease the oxidative damage and its alleged harmful effects.^3- 6 Many people are taking antioxidant supplements, believing to improve their health and prevent diseases.^7- 10 Whether antioxidant supplements are beneficial or harmful is uncertain.^11- 15 Many primary or secondary prevention trials of antioxidant supplements have been conducted to prevent several diseases.

We found that antioxidant supplements, with the potential exception of selenium, were without significant effects on gastrointestinal cancers and increased all-cause mortality.^14- 15 We did not examine the effect of antioxidant supplements on all-cause mortality in all randomized prevention trials.¹⁶ Our aim with the present systematic review was to analyze the effects of antioxidant supplements (beta carotene, vitamins A and E, vitamin C [ascorbic acid], and selenium) on all-cause mortality of adults included in primary and secondary prevention trials.

The present review follows the Cochrane Collaboration method¹⁷ and is based on the principles of our peer-reviewed protocol and review on antioxidant supplements for gastrointestinal cancer prevention.^{14- 15,18- 19} We included all primary and secondary prevention trials in adults randomized to receive beta carotene, vitamin A, vitamin C, vitamin E, or selenium vs placebo or no intervention. Parallel-group randomized trials and the first period of crossover randomized trials were included.¹⁷ Trials including general or healthy populations were classified as primary prevention. Trials including participants with specific disease were classified as secondary prevention. We excluded tertiary prevention (treatment) trials, like trials on acute, infectious, or malignant diseases except nonmelanoma skin cancer.

We included antioxidant supplements at any dose, duration, and route of administration. We analyzed the antioxidants administered singly, in combination with other antioxidants, or with other vitamins or trace elements. Trials with collateral interventions were included if the interventions were used equally in the trial groups. Subgroup analyses without high-bias risk trials and selenium trials were preconceived. Our outcome measure was all-cause mortality at maximum follow-up.

We searched The Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library (Issue 3, 2005), MEDLINE (1966 to October 2005), EMBASE (1985 to October 2005), and the Science Citation Index Expanded (1945 to October 2005).²⁰ We scanned bibliographies of relevant articles for additional trials.

Two of the 3 authors (G.B. and D.N., and R.G.S.) independently assessed trial eligibility. Excluded trials were listed with the reasons for exclusion. Disagreement was resolved by discussion or in consultation with a third author (C.G.). We contacted authors of the trials for missing information.

From each trial we recorded first author; country of origin, country income category (low, middle, high)²¹; number of participants; characteristics of participants: age range (mean or median) and sex ratio; participation rate; dropout rate; trial design (parallel, factorial, or crossover); type of antioxidant; dose; duration of supplementation; duration of follow-up (ie, treatment duration plus posttreatment follow-up); and cointerventions. We extracted the date, location, sponsor of the trial, and the publication status.

Due to the risk of overestimating intervention effects, analyses were stratified according to the risk of bias (methodological quality).^{14- 15,18- 19,22- 24} Trials with adequate generation of the allocation sequence, adequate allocation concealment, adequate blinding, and adequate follow-up were considered low-bias risk trials (high methodological quality).²⁴ Trials with one or more unclear or inadequate quality components were classified as high-bias risk trials (low methodological quality).²⁴ Generation of the allocation sequence was considered adequate if the allocation sequence was generated by a computer or random-number table, or similar; allocation concealment was considered adequate if concealed up to the point of treatment by central randomization, sealed envelopes, or similar; blinding was considered adequate if the trial was described as double-blind and using identical placebo; follow-up was considered adequate if the numbers and reasons for dropouts and withdrawals in all intervention groups were described or if it was specified that there were no dropouts or withdrawals. Bias risk was assessed without blinding of 2 authors (G.B. and D.N. or R.G.S.). Consensus was reached through discussion or arbitration by a third author (C.G. or L.L.G.) before data entry. We have found high interrater agreement between blinded and unblinded assessments and also between 2 independent assessors.²⁴

We used The Cochrane Collaboration software (RevMan Analyses 1.0; www.cochrane.org), STATA 8.2 (STATA Corp, College Station, Tex), Sigma Stat 3.0 (SPSS Inc, Chicago, Ill), and StatsDirect (StatsDirect Ltd, Altrincham, England). We analyzed the data with a random-effects model,²⁵ calculating the relative risk (RR) with 95% confidence intervals (CIs). To account for 0 cells in the 2 × 2 tables, we calculated the RR with 3 different continuity corrections (0.5; 0.1; 0.01).^26- 27 We did not include trials with 0 events in both intervention groups.^27- 28 Because the number of such trials was large, we performed exploratory analysis adding an imagined trial with 1 death and 20 000 participants in each group.

We used the STATA metareg command for the random-effects metaregression to assess which covariates influenced the intervention effect across trials.²⁹ The included covariates were bias risk, type and dose of supplement, single or combined supplement regimen, duration of supplementation, and primary or secondary prevention. Univariate and multivariate analyses including all covariates were performed. Results are presented with regression coefficients and 95% CI.

All analyses followed the intention-to-treat principle. For trials with factorial design, we based our results on at-margins analysis,³⁰ comparing all groups that received antioxidant supplements with groups that did not. To determine the effect of a single antioxidant, we performed inside-the-table analysis³⁰ in which we compared the group taking a single antioxidant with the group taking placebo or receiving no intervention. In trials with more than 2 groups assessing additional therapy, we compared only groups receiving antioxidants, placebo, or no intervention.

We assessed heterogeneity with I² that describes the percentage of total variation across trials due to heterogeneity rather than chance.^17,31 I² can be calculated as I² = 100% × (Qv −df)/Q, where Q is Cochran's heterogeneity statistics and df the degrees of freedom. Negative values of I² are put equal to 0, so I² lies between 0% (no heterogeneity) and 100% (maximal heterogeneity).³¹ We compared the estimated treatment effects in trials with a low- or high-risk of bias with test of interaction.³² We performed adjusted-rank correlation³³ and regression-asymmetry tests³⁴ for detection of bias.

Database searches yielded 16 111 references. Exclusion of duplicates and irrelevant references left 1201 references describing 815 trials. To obtain additional information we wrote to authors of eligible trials. Seventy authors responded. We excluded 816 references (747 trials) due to the following: mortality was 0 in both study groups (n=405 trials, including about 40 000 participants [http://ctu.rh.dk]); did not fulfill inclusion criteria (n=245); was not a randomized trial (n=69); insufficient data (n=24); or still ongoing trial (n=4). We included 385 references describing 68 randomized trials fulfilling our inclusion criteria and able to provide data for our analyses^{35- 6970- 102} (Figure 1 [http://ctu.rh.dk]). This corresponds to a median of 6 references per included trial (range, 1-44). Forty trials used parallel-group design, 26 factorial design (23 trials 2 × 2; 2 trials 2 × 2× 2; 1 trial half replicate of 2 × 2× 2× 2), and 2 crossover design.

A total of 232 606 participants were randomly assigned in the 68 trials. The number of participants in each trial ranged from 24 to 39 876 (Table 1 and Table 2). The mean age was 62 years (range, 18-103 years). The mean proportion of women was 44.5% in the 63 trials reporting sex.

Twenty-one trials were primary prevention trials including 164 439 healthy participants; 47 trials were secondary prevention trials including 68 167 participants with gastrointestinal (n=11), cardiovascular (n=9), neurological (n=6), ocular (n=5), dermatological (n=5), rheumatoid (n=2), renal and cardiovascular (n=1), endocrinological (n=1), or unspecified (n=7) diseases. Main outcome measures in the primary prevention trials were cancer and mortality (cause specific and all cause), and in the secondary prevention trials they were progression of disease and mortality (cause specific and all cause; Table 3 and Table 4).

All antioxidant supplements were administered orally. The dose and regimen of the antioxidant supplements were: beta carotene 1.2 to 50.0 mg (mean, 17.8 mg) , vitamin A 1333 to 200 000 IU (mean, 20 219 IU), vitamin C 60 to 2000 mg (mean, 488 mg), vitamin E 10 to 5000 IU (mean, 569 IU), and selenium 20 to 200 μg (mean 99 μg) daily or on alternate days for 28 days to 12 years (mean 2.7 years). In one trial⁴⁰ antioxidants were applied in a single dose and participants were followed up for 3 months thereafter. The mean duration of follow-up in all trials was 3.3 years (range, 28 days-14.1 years).

Beta carotene was tested in 25 trials, vitamin A in 16, vitamin C in 34, vitamin E in 55, and selenium in 21. Beta carotene was tested singly in 6 trials, vitamin A in 2, vitamin E in 24, and vitamin C and selenium in 3 trials each. The antioxidant supplements were given in the following combinations: beta carotene and vitamin A; beta carotene and vitamin E; beta carotene and vitamin C; vitamin A and vitamin C; vitamin C and vitamin E; vitamin E and selenium; selenium and zinc; beta carotene, vitamin C, and vitamin E; beta carotene, vitamin C, vitamin E, and selenium; beta carotene, vitamin C, vitamin E, selenium, and zinc; vitamin A, vitamin C, vitamin E, selenium, and zinc; vitamin A, vitamin C, vitamin E, selenium, methionine, and ubiquinone. In 11 trials, participants were supplemented with different mixtures of antioxidants as well as with vitamins and minerals without antioxidant properties.^{39,41- 42,45,51- 52,72,84,91- 92,101}

Sixty-three trials used placebo and 5 trials^{43,48,58,69,82} used no intervention in the control group. In 9 trials^{35,40,46,48,65,81,89,97,99} the active and placebo (control) groups were supplemented with vitamins and minerals (with or without antioxidant properties). In 6 of the trials, the supplementation was with vitamin E 4 IU,^46,89 vitamin A 1000 IU⁴⁰; vitamin C 20 and 50 mg^48,81; riboflavin 10 mg³⁵; or niacin 100 mg.⁶⁵ In the trials with factorial or parallel-group design, the additional interventions tested were multivitamins and minerals (zinc, copper, chromium); ubiquinone; L-methionine; omega-3 polyunsaturated fatty acids; citrus bioflavonoid complex; quercetin, bilberry extract, rutin (bioflavonoids); taurine; N -acetyl cysteine; L-glutathione; aged garlic; deprenyl–selegiline (selective monoamine oxidase B inhibitor); donepezil (acetylcholinesterase inhibitor); riluzole (modulator of glutamatergic neurotransmission); amoxicillin, metronidazole (antibiotics); bismuth subsalicylate; omeprazole (proton-pump inhibitor); aspirin; simvastatin (cholesterol-lowering drug); celecoxib (inhibitor of cyclooxygenase), and ramipril (angiotensin-converting enzyme inhibitor).

In 54 trials (79.4%), the antioxidants were provided at no cost from pharmaceutical companies. In the rest of the trials funding was not reported. The trials were conducted in Europe, North and South America, Asia, and Australia. Six trials came from lower-middle-income countries^{41- 42,47,62,67- 68} and 62 trials from high-income countries.

Forty-seven of the 68 trials (69.1%) had low-bias risk, ie, had adequate generation of the allocation sequence, adequate allocation concealment, blinding, and follow-up.²⁴ The remaining trials had one or more inadequate components.

The pooled effect of all supplements vs placebo or no intervention in all randomized trials was not significant (RR, 1.02; 95% CI, 0.98-1.06). Heterogeneity was not significant (I² = 18.6%, P = .10). Adjusted-rank correlation test (P = .08), but not the regression asymmetry test (P = .26), suggested bias among the trials. Exploratory analysis adding an imagined trial with one death and 20 000 participants in each study group had no noticeable effect on the result.

Univariate meta-regression analyses revealed significant influences of dose of beta carotene (RR, 1.004; 95% CI, 1.001-1.007; P = .012), dose of vitamin A (RR, 1.000006; 95% CI, 1.000002-1.000009; P = .003), dose of selenium (RR, 0.998; 95% CI, 0.997-0.999; P = .002), and bias-risk (RR, 1.16; 95% CI, 1.05-1.29; P = .004) on mortality. None of the other covariates (dose of vitamin C; dose of vitamin E; single or combined antioxidant regimen; duration of supplementation; and primary or secondary prevention) were significantly associated with mortality.

In multivariate meta-regression analysis including all covariates, dose of selenium was associated with significantly lower mortality (RR, 0.998; 95% CI, 0.997-0.999; P = .005) and low-bias risk trials with significantly higher mortality (RR, 1.16; 1.05-1.29; P = .005). None of the other covariates was significantly associated with mortality.

In trials with low-bias risk mortality was significantly increased in the supplemented group (RR, 1.05; 95% CI, 1.02-1.08) without significant heterogeneity (I² = 7.0%). Exploratory analysis adding an imagined trial with 1 death and 20 000 participants in each study group had no noticeable effect on the result.

In high-bias risk trials (low-methodological quality in ≥1 of the 4 components) mortality was significantly decreased in the supplemented group (RR, 0.91; 95% CI, 0.83-1.00) without significant heterogeneity (I² = 4.5%). The difference between the estimate of antioxidants on mortality in low- and high-bias risk trials was statistically significant by test of interaction (z = 2.88, P = .004; Figure 2 and Figure 3).

Beta carotene used singly significantly increased mortality (Table 5). This effect was not significant when combined with other supplements. After exclusion of high-bias risk and selenium trials, beta carotene singly or combined significantly increased mortality (Table 5).

Vitamin A given singly or in combination with the other supplements did not significantly affect mortality. After exclusion of high-bias risk and selenium trials, vitamin A singly or combined significantly increased mortality (Table 5).

Vitamin E given singly or in combination with the other supplements did not significantly affect mortality (Table 5). Vitamin E given singly in high (≥1000 IU) or low dose (<1000 IU) did not significantly affect mortality (RR, 1.07; 95% CI, 0.91-1.25; I² = 0% and RR, 1.00; 95% CI, 0.94-1.07; I² = 13.0%, respectively). After exclusion of high-bias risk and selenium trials, vitamin E given singly or combined significantly increased mortality (Table 5).

Vitamin C given singly or in combination with the other supplements was without significant influence on mortality, even after the exclusion of high-bias risk trials and selenium trials (Table 5).

Selenium given singly or in combination with other antioxidant supplements had no significant influence on mortality when analyzed separately (Table 5). Selenium given singly or combined significantly decreased mortality when analyzed together. After exclusion of high-bias risk trials, selenium given singly or with other antioxidants had no significant influence on mortality (Table 5).

Our systematic review contains a number of findings. Beta carotene, vitamin A, and vitamin E given singly or combined with other antioxidant supplements significantly increase mortality. There is no evidence that vitamin C may increase longevity. We lack evidence to refute a potential negative effect of vitamin C on survival. Selenium tended to reduce mortality, but we need more research on this question. We confirm that trials with inadequate bias control overestimate intervention effects.^{14- 15,19,22- 24} Our findings support and extend our previous findings regarding antioxidant supplements and increased mortality.^14- 15

Our review offers a number of strengths. It follows a published, peer-reviewed Cochrane protocol,¹⁸ taking into consideration our previous findings in a systematic review on antioxidant supplements for preventing gastrointestinal cancers.^14- 15 Our review represents a comprehensive review of the topic, including 68 randomized trials with almost a quarter of a million participants. This increases the precision and power of our analyses.¹⁷ Previous meta-analyses of preventive trials of antioxidant supplements have included less information (lung cancer, 4 trials with 109 394 participants¹⁰³; cardiovascular diseases, 8 trials with 138 113 participants¹⁰⁴; gastrointestinal cancers, 14 trials with 170 525 participants^14- 15; colorectal adenoma, 8 trials with 17 620 participants¹⁹; cancer or preinvasive lesions, 7 trials with 5112 participants¹⁰⁵; and mortality, 19 trials with 135 967 participants¹⁰⁶).

Previous studies either found no beneficial or harmful effect of the supplements^{19,103- 105,107} or reported a significantly increased mortality.^{14- 15,103- 104,106} We conducted a thorough assessment of trial methodology following the recommendations of the Cochrane Collaboration¹⁷ and findings of methodological studies.^22- 24 More than two thirds of the included trials with more than 180 000 participants fall in the group of low-bias risk trials. This highlights the validity of our results.^22- 24 Antioxidant supplements not only seem to be one of the most researched topics in the world, they also seem to be one of the most adequately researched clinical questions. Only a small proportion of trials use adequate methodologies.^108- 109 Our meta-analyses had little trial heterogeneity. This increases the trustworthiness of our findings. Our analyses were robust to sensitivity analyses involving different imputations of mortality in the 0-event study groups. We gave full account of all 405 identified trials assessing the supplements having 0 events in both study groups. These trials were mostly assessing short-term supplement administration and surrogate outcome measures. Our results were robust to exploratory analyses adding an imagined trial with 20 000 participants and one death in each intervention group. Accordingly, the increased mortality does not seem to be an artifact created by exclusion of trials with 0 events in both study groups.^27- 28 Furthermore, all-cause mortality should generally be connected with unbiased estimates.

A large number of unpublished trials on supplements may exist. Their results are more likely to have been either neutral or negative than to have shown beneficial effects.¹¹⁰ Accordingly, our estimates of increased mortality of about 5% is likely to be conservative.

The choice of statistical model for performing meta-analysis of sparse data are important.^27- 28 Because many methods are based on large sample approximations, they may be unsuitable when events are rare. Bradburn et al²⁸ found that no method gives completely unbiased estimates. At event rates below 1%, the Peto odds-ratio method appears to be the least biased and most powerful method when there is no substantial imbalance in treatment and control group sizes within trials, and treatment effects are not exceptionally large. Bradburn et al²⁸ also demonstrated that the Peto odds ratio works well up to event rates around 10%. The calculation avoids addition of 0.5-event adjustments (or any other adjustment). When we applied Peto odds ratio, we found even stronger support for detrimental effects of the supplements (for all 68 trials: 1.05; 95% CI, 1.02-1.08; for the 47 low-bias risk trials: 1.07; 95% CI, 1.04-1.10; after exclusion of high-bias risk trials and selenium trials, for beta carotene: 1.09; 95% CI, 1.06-1.13; for vitamin A: 1.20; 95% CI, 1.12-1.29; for vitamin C: 1.06; 95% CI, 0.99-1.14; and for vitamin E: 1.06; 95% CI, 1.02-1.10).

Our systematic review has several limitations. As with all systematic reviews, our findings and interpretations are limited by the quality and quantity of available evidence on the effects of specific supplements on mortality. The examined populations varied. The effects of supplements were assessed in the general population or in patients with gastrointestinal, cardiovascular, neurological, skin, ocular, renal, endocrinological, and rheumatoid diseases. These populations mostly came from countries without overt deficiencies of specific supplements. Accordingly, we are unable to assess how antioxidant supplements affect mortality in populations with specific needs.

We have compared antioxidants with different properties, given at different doses and duration, singly or combined. We are aware of the potential risks in assessing the effects of different types of antioxidants together with different mechanisms of action, biotransformation, and bioavailability. There are pros^111- 113 and cons¹¹⁴ in the literature about vitamin A being antioxidant. We fully acknowledge this. Most trials assessed combinations of different supplements, which reflects the way supplements are marketed, sold, and taken by people.^7- 10

The methodological quality of some of the trials was assessed using the published reports, which may not reflect the actual design and bias risk of the trials. Some authors responded to our requests for further information.

All available nonenzymatic antioxidants work differently in the human body, and most of them exert effects that are nonantioxidant. We are not able to point to the specific biochemical mechanisms behind the detrimental effects. We found that trials examining the individual supplements singly were rare. It has been suggested that antioxidant supplements may show interdependency and may have effects only if given in combination.¹¹⁵

Most trials investigated the effects of supplements administered at higher doses than those commonly found in a balanced diet, and some of the trials used doses well above the recommended daily allowances and even above the tolerable upper intake levels.^116- 117 Our meta-regression analyses revealed significant effects of dose of beta carotene, vitamin A, and selenium on mortality. The duration of supplementation and follow-up differed among the trials. However, we found no significant effect of treatment duration on our results.

We only assessed all-cause mortality. We are not able to determine the cause of the increased mortality. It is likely that increased cancer and cardiovascular mortality are the main reasons for the increased all-cause mortality.^103- 104 Further study of causes of mortality is needed. We fear that its assessment may be difficult due to varying definitions in the included trials. Our results extend previous reviews^{14- 15,19,103- 107} and guidelines,^118- 120 suggesting that antioxidant supplements may not be beneficial.

Beta carotene, administered singly or in combination with other antioxidants, significantly increased all-cause mortality. Recent studies have suggested that beta carotene may act as a cocarcinogen.^121- 122 Vitamin A combined with other antioxidants significantly increased mortality. We found that vitamin E given singly or combined with 4 other antioxidants did not significantly influence mortality. After exclusion of high-bias risk trials, however, vitamin E given singly or combined significantly increased mortality. This is in agreement with a recent meta-analysis.¹⁰⁶ Dose of vitamin E was without significant effect on mortality in our analysis. The chance that vitamin E may benefit seems low.^123- 125

The trials in which vitamin C was applied singly or in different combinations with beta carotene, vitamin A, vitamin E, and selenium found no significant effect on mortality. According to the CIs, small beneficial or large harmful effects cannot be excluded. We calculated the proportion of participants who died in the trials in which participants took vitamin C alone. In the control group it was 0.019 and in the vitamin C group it was 0.017. With α set to .05 and power to .90, the required sample size would be 186 000 participants. We are still far from having examined a sufficient sample. Studies have demonstrated that vitamin C may act as both a pro-oxidant and as an antioxidant in vivo,^126- 127 and trials should be monitored closely for harm.

Selenium given singly or in combination with other supplements seemed to significantly decrease mortality, but after exclusion of high-bias risk trials, the effect disappeared. Results of ongoing randomized trials with selenium will likely increase our understanding of the effects of selenium.¹²⁸

Our findings contradict the findings of observational studies, claiming that antioxidants improve health.^129- 132 Considering that 10% to 20% of the adult population (80-160 million people) in North America and Europe may consume the assessed supplements,^7- 10 the public health consequences may be substantial. We are exposed to intense marketing with a contrary statement, which is also reflected by the high number of publications per included randomized trial found in the present review.

There are several possible explanations for the negative effect of antioxidant supplements on mortality. Although oxidative stress has a hypothesized role in the pathogenesis of many chronic diseases, it may be the consequence of pathological conditions.¹³³ By eliminating free radicals from our organism, we interfere with some essential defensive mechanisms like apoptosis, phagocytosis, and detoxification.^134- 136 Antioxidant supplements are synthetic and not subjected to the same rigorous toxicity studies as other pharmaceutical agents.¹³⁷ Better understanding of mechanisms and actions of antioxidants in relation to a potential disease is needed.¹³⁸

Because we examined only the influence of synthetic antioxidants, our findings should not be translated to potential effects of fruits and vegetables.

We did not find convincing evidence that antioxidant supplements have beneficial effects on mortality. Even more, beta carotene, vitamin A, and vitamin E seem to increase the risk of death. Further randomized trials are needed to establish the effects of vitamin C and selenium.