A randomized controlled trial of vitamin E and selenium on rate of decline in lung function

Background The intake of nutrients with antioxidant properties is hypothesized to augment antioxidant defenses, decrease oxidant damage to tissues, and attenuate age-related rate of decline in lung function. The objective was to determine whether long-term intervention with selenium and/or vitamin E supplements attenuates the annual rate of decline in lung function, particularly in cigarette smokers. Methods The Respiratory Ancillary Study (RAS) tested the single and joint effects of selenium (200 μg/d L-selenomethionine) and vitamin E (400 IU/day all rac-α-tocopheryl acetate) in a randomized double-blind placebo-controlled trial. At the end of the intervention, 1,641 men had repeated pulmonary function tests separated by an average of 3 years. Linear mixed-effects regression models estimated the effect of intervention on annual rate of decline in lung function. Results Compared to placebo, intervention had no main effect on either forced expiratory volume in the first second (FEV1) or forced expiratory flow (FEF25–75). There was no evidence for a smoking by treatment interaction for FEV1, but selenium attenuated rate of decline in FEF25–75 in current smokers (P = 0.0219). For current smokers randomized to selenium, annual rate of decline in FEF25–75 was similar to the annual decline experienced by never smokers randomized to placebo, with consistent effects for selenium alone and combined with vitamin E. Conclusions Among all men, there was no effect of selenium and/or vitamin E supplementation on rate of lung function decline. However, current smokers randomized to selenium had an attenuated rate of decline in FEF25–75, a marker of airflow. Trial registration Clinicaltrials.gov identifier: NCT00241865. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0195-5) contains supplementary material, which is available to authorized users.


Background
Pulmonary function, which is reliably measured by spirometry, is central to the diagnosis and staging of chronic obstructive pulmonary disease (COPD). COPD, the third most common cause of death in the US, led to $29.5B in direct costs and $20B in lost productivity costs in 2010 [1]. The age-related rate of decline in the forced expiratory volume in the first second (FEV 1 ) is a marker of mortality risk in the general population [2] and in healthy smokers [3], the rate of decline is steeper in smokers [4] and in COPD patients, although the latter association varies by disease attributes [5,6]. The forced expiratory flow at the mid-portion of forced vital capacity (FEF  ) reflects the state of small airways, and offers a measure of lung function reflecting airflow rather than volume [7]. Attenuating lung function decline may reduce morbidity and mortality, both in healthy persons and in COPD patients. The identification of factors that affect lung function decline is important to the development of clinical or public health interventions.
Both smoking and aging accelerate the annual rate of decline in FEV 1 [4,8]; the effect of other factors, including diet, is less clear [9]. Observational studies of nutrients (whole foods, micronutrients, dietary patterns, biomarkers) and lung outcomes (COPD, lung function) support the broad hypothesis that nutrients with antioxidant properties improve lung health [10][11][12][13], presumably by altering the oxidant/antioxidant balance in lung tissue. A recent critical review of the evidence for a causal relation between nutrition and lung outcomes [9] concluded there was a "limited/suggestive" role for diet, reflecting that the majority of studies are cross-sectional. However, most existing longitudinal studies report protective associations of antioxidant nutrients and lung outcomes, and there is strong evidence that the beneficial effects of diet may be limited to smokers [14]. Given that observational studies are limited by potential confounding due to lifestyle factors associated with healthful diets, and given that measures of diet based on self-report are subject to bias and have poor precision, experimental studies are needed to fully understand whether nutrition affects lung function and its decline with age, particularly in cigarette smokers.
The few randomized controlled trials considering respiratory endpoints other than lung cancer used post-hoc, secondary analyses [15][16][17] mainly in special populations (all heavy smokers, all with vascular disease), did not study lung function decline, and did not test selenium. Using the infrastructure of the Selenium and Vitamin E Cancer Prevention Trial (SELECT) [18], we conducted an ancillary study nested within SELECT to investigate the a priori hypothesis that supplementation with selenium and/or vitamin E, two nutrients with antioxidant potential, would attenuate the annual decline in lung function; we hypothesized a stronger effect in cigarette smokers given their higher exposure to inhaled oxidants.

Study design
The Respiratory Ancillary Study (RAS) was nested within SELECT [18], a phase 3 randomized, placebo-controlled double-blind trial of 35,533 men testing whether selenium (200 μg/d L-selenomethionine) and vitamin E (400 IU/d all rac-α-tocopheryl acetate) alone and/or in combination would prevent prostate cancer. SELECT eligibility included age ≥55 y (≥50 y in African-Americans), serum prostate-specific antigen ≤ 4 ng/mL, and no clinical evidence of prostate cancer. SELECT enrolled men in the United States and Canada between 2001-2004; use of study supplements stopped on 10/23/2008, after an interim analysis determined that there was no effect and that further intervention was unlikely to show significant reduction in prostate cancer incidence [18]. RAS used a post-randomization design, due to rapid enrollment in SELECT relative to the start of RAS; thus, we did not measure pre-randomization lung function, but we captured the rate of decline over the intervention period through repeated measurements of lung function. This design assumed that the intervention effect is reached early in the study, and is stable over time. To test the hypothesis that current smokers benefit more from intervention, RAS enrolled men from the 16 SELECT sites with the greatest number of current cigarette smokers (Additional file 1: Table S1). Based on the predicted effect of intervention on annual FEV 1 decline, assuming a 7-10 year follow-up, the target sample size in RAS was 3000 men.

Participants
Eligibility requirements for registration to RAS included SELECT registration at one of the 16 SELECT sites with a high proportion of smokers and adherence to supplements (either active or placebo) at the time of RAS registration. Each SELECT site invited all eligible current smokers to RAS and, depending on the number of participants at the site, either a random sample or all eligible former and never smokers. Ultimately, men were registered at their first (5%), second (17%), third (38%) or fourth (40%) annual SELECT visit. The RAS was approved by local IRBs at each of the 16 study sites, and by the Cornell University IRB.

End point assessment
The primary endpoint was annual decline in FEV 1 ; the secondary endpoint was annual decline in FEF  . Spirometry was assessed at three out of four annual visits spanning three years. Due to early termination of SELECT, not all RAS participants completed all scheduled pulmonary function tests (PFTs); the endpoint (the third and final PFT) was available for 57% of participants. We assume this is an unbiased sample given the timing of supplementation withdrawal relative to the timing of a participant's annual visit is expected to be random and thus equal across arms.
Pulmonary function testing followed American Thoracic Society guidelines [19] and used the EasyOne handheld, flow-sensing spirometer, which has excellent validity and reliability, and significantly simpler field implementation in comparison to desktop devices [20]. Only PFTs meeting criteria for acceptable start and end of test and for reliability were included in analyses; further details are provided in Additional file 1: Table S2).

Statistical analysis
There were three pre-specified main effect comparisons between each active treatment arm and placebo, with a P-value threshold of 0.018 to account for the three tests with a common placebo group. All analyses were intentto-treat, and effects were estimated using a linear mixedeffects regression model incorporating the repeated measurements of pulmonary function (either FEV 1 or FEF  ) as the outcome. The model included random intercept and slope, and the following fixed effects: time (time between each PFT and baseline), treatment arm and its interaction with time (treatment-by-time), age, height, race and smoking. The treatment-by-time coefficient estimated the effect of treatment on annual rate of decline in lung function. All RAS men with ≥1 (n = 2920) PFT were included in the model and contributed to estimates of effects of age, height, race and smoking, but only men with ≥2 PFTs (n = 1677) were informative for the estimate of the time-by-treatment coefficient. Missing data were assumed to be missing at random, given very low drop-out rates. To test whether treatment effects differed by smoking status, models were extended to include the treatment-by-time-by-smoking interaction terms, and the significance threshold for the interaction effects was a nominal P-value of 0.05.

Results
Participants RAS enrolled 2,920 men ( Figure 1) at 16 SELECT sites between 7/2/2004 and 4/30/2007. RAS eligibility and enrollment were blind to intervention arm, and indeed the number of participants across the four arms was balanced ( Figure 1). RAS experienced minimal attrition, with only 2-3% of men in each intervention group refusing further RAS and/or SELECT follow-up at some time point after registration. All participants studied herein had at least one acceptable PFT, and 56 to 60% of participants in each arm had repeated PFTs, confirming that repeated measurements for the endpoint assessment were similar by arm. The mean number of PFTs per participant was 2.3 (SD 1.1; median 3) with a mean of 36.1 months (SD 4.2) between first and last PFT (median 35.8 months; range 24 to 52). Spirometry quality control scores ranged from 3.2 to 3.7 out of maximum score of 4 (Additional file 1: Table S2).
RAS participants had similar distributions of age, race/ ethnicity, education, smoking history and height across intervention arms ( Table 1), confirming that the postrandomization design yielded four groups balanced on characteristics. The participants with one PFT were similar on all baseline characteristics to participants with repeated PFTs (data not shown); this is consistent with our expectation because the lack of repeated PFT data was a function of the date the intervention was withdrawn, and was not driven by participant choice or participant characteristics. Thus, we expect the estimate of the effect of treatment on lung function decline to be unbiased. Among participants who completed the final PFT, the mean time from SELECT registration to a participant's last PFT was 60.4 months (SD 10.8; median 59.8), thus results reflect intervention effects of about 5 years duration. Adherence among RAS participants (Table 2), determined using pill count, compares well to all SELECT participants [18]. Across the four arms, 87 to 92% of RAS men were adherent to the selenium supplement (or matching placebo) in year 1, and 80 to 84% were adherent in year 5. Similarly, 87 to 92% of RAS men were adherent to the vitamin E supplement (or matching placebo) in year 1, and 79 to 81% were adherent in year 5. Across all arms, for the full study period, self-supplementation with nonstudy vitamin E and selenium (drop-in rate, assessed by self-reported use of either supplement) was reported by ≤2.3% and ≤1.2% of participants, respectively.

Rate of decline in pulmonary function
Overall, the distribution of rate of decline in FEV 1 was consistent with expectations of decline, and the mean annual change in FEV 1 was −37.5 mL (SD 12.5; Additional file 1: Figure S1). Compared to never smokers, FEV 1 was 363 mL lower in current smokers and annual decline in FEV 1 was 6.9 mL/y steeper. In unadjusted analyses of raw data, compared with the placebo group (Table 3), participants randomized to intervention experienced an attenuation of between 3 and 6 mL/y in rate of change in FEV 1 , but there were no statistically significant differences between arms. Similarly, for rate of decline in FEF 25-75 (mL/ second/y), there were no statistically significant differences between arms (Table 4).
or the registration contact at which men started RAS (reflecting the length of time on study intervention) showed similar results.

Effect modification by cigarette smoking
The hypothesis that smoking modifies the effect of supplementation was pre-specified, and models were extended to estimate intervention effects within categories of cigarette smoking; categories included current, former (quit prior to trial), and never (lifetime never smoker). In the placebo arm, expected differences in rate of decline in FEV 1 were confirmed such that the annual decline in current smokers was 11 to 16 mL/y steeper compared to former and never smokers (Table 3). Similarly, the annual Table 3 Model-based estimated mean annual decline in FEV 1 (mL/year) by treatment group in the full sample, and stratified by cigarette smoking status (Additional file 1: Tables S3 and S4 for model coefficients)  Marginal models combine treatment groups. Any selenium model compares participants on any selenium (selenium alone and in combination with E) to placebo. In a separate model, participants on any vitamin E (vitamin E alone and in combination with selenium) are compared to placebo. rate of decline in FEF  in current smokers on placebo was more than two-fold that in never smokers (P = 0.0189; Table 4). There was no evidence that smoking modified the effect of intervention on rate of decline in FEV 1 , and all P values exceeded the threshold of 0.05. However, for the FEF  outcome, compared to placebo the annual rate of decline in FEF  was attenuated in current cigarette smokers in the selenium arm (P = 0.0219) and in the combined arm (P = 0.0236). Further models testing any selenium (selenium alone and selenium + vitamin E, combined) vs. placebo showed that FEF 25-75 rate of decline was decreased by more than half in current smokers on any selenium compared to current smokers in the placebo group (P = 0.0095).

Discussion
This is the first randomized trial of selenium and/or vitamin E intervention that studies the rate of decline in pulmonary function as the endpoint. This study is important because it contributes new information about whether interventions that presumably affect the antioxidant/oxidant balance in lung tissue can ameliorate or attenuate a functional outcome reflecting lung health. Neither supplementation with selenium nor vitamin E had statistically significant main effects on rate of decline in FEV 1 or FEF  . Following our a priori hypothesis that effects are stronger in and/or limited to current cigarette smokers, there was evidence for a differential effect of selenium in current smokers for the flow-related endpoint such that smokers supplemented with any selenium, either alone or in combination with vitamin E, had an attenuated rate of decline in FEF  .
This randomized trial evidence for an effect of selenium on annual rate of decline in lung function in smokers is consistent with prior cross-sectional studies that reported strong positive associations of serum selenium with lung function [14]. An analysis of baseline bloods collected on a subset of SELECT participants [18] found that men were rarely low on serum selenium, where low selenium was defined as ≤ 121.6 ng/mL consistent with prior studies of cancer outcomes [21]. While this suggests the potential-to-benefit from selenium intervention in the overall study may be low, the potential to benefit in smokers is likely to be greater given prior evidence that selenium concentrations are lower in smokers [22], and, indeed, this is supported by our findings.
The pattern of the RAS findings, including the effect of selenium on flow (FEF 25-75 ) but not volume parameters and the magnitude of the effect sizes, are similar to the effects of air pollution on lung function reported in the SAPALDIA study. Based on 11 years of follow-up, SAPALDIA reported mean annual rate of decline in FEV 1 and FEF 25-75 of 35 mL/y (SD 30) and 71 mL/second/y (SD 65), respectively [23]. In the RAS placebo arm, average annual rates of decline were very similar, although the RAS estimates are more variable given the Table 4 Model-based estimated annual decline in FEF  (mL/second/year) by treatment group in the full sample, and stratified by cigarette smoking status (Additional file 1: Tables S3 and S4 for model   Model-based estimated mean decline, mL/second/year and 95% confidence interval; main effects models adjusted age, height, race, smoking status, time and tested time x treatment effect (effect of treatment on slope); smoking interaction models adjusted age, height, race, smoking status, and all two way interactions and tested smoking x time x treatment interaction (effect of treatment on slope within smoking group). a P value from mixed-effects linear regression model, with placebo reference group. b Marginal models combine treatment groups. Any selenium model compares participants on any selenium (selenium alone and in combination with E) to placebo. In a separate model, participants on any vitamin E (vitamin E alone and in combination with selenium) are compared to placebo.
shorter duration of follow-up and more closely spaced PFTs. SAPALDIA reported that reductions in particulate matter ≤ 10 microns in diameter (PM 10 ) were associated with the rate of change in both FEV 1 and FEF  , but the strength of the association and the level of statistical significance were greater for the FEF 25-75 outcome [23], similar to the findings reported herein for supplement effects in RAS. In current smokers the selenium intervention effect size for annual decline in FEV 1 was similar to the effect size for reducing PM 10 exposure by 10 μ/m 3 in SAPALDIA (attenuated FEV 1 decline by 4 mL/y). Greater effect sizes were seen for FEF  in both studies: in SAPALDIA, reducing PM 10 attenuated FEF 25-75 decline by 11 mL/second/y, in RAS selenium supplementation attenuated FEF 25-75 decline by 59 mL/second/y. While FEV 1 is less variable than FEF  in cross-sectional studies [19], longitudinal declines in both endpoints are of interest and FEF  findings may be salient in smokers given that changes in flow rates may signal early changes in small airways function [24]. Although such changes may not be predictive at the individual level, they may be informative in the comparison of treatment groups. In addition, a longitudinal endpoint, which leverages repeated measurements per participant, and uses all available spirometry data on each participant (an approach that is consistent with two prior studies of longitudinal change [5,25]), is less affected by variability in comparison to cross-sectional studies.
Although the vitamin E effect sizes were clinically meaningful and consistent in effect direction (attenuated rate of decline) across two pulmonary function measures, with stronger effects of intervention in current smokers, the effects did not meet pre-set criteria for statistical significance. These findings reflect either a true lack of effect of vitamin E on rate of decline in lung function, the possibility that baseline vitamin E levels were high (or at minimum, not deficient) and thus there was limited potential to benefit from supplementation, or the possibility that attenuation in decline might occur only with a longer period of supplementation. While several past observational studies reported that associations of vitamin E were limited to smokers [26], such studies are more likely to be affected by confounding than the randomized trial findings reported herein.
The Respiratory Ancillary Study (RAS) to SELECT used a post-randomization design, and participants were registered to the RAS after active supplementation began. The primary endpoint was rate of decline in lung function; absolute differences in lung function due to the supplements over a fixed period of time were not calculated because first measurements were obtained after the participants started taking their supplements. The design assumes that effects are achieved quickly and are stable over the supplemented period, which is reasonable given the hypothesis of support for antioxidant function provided by the supplements.
This study measured pre-bronchodilator spirometry, but the lack of post-bronchodilator spirometry is not a serious weakness given the primary outcome is rate of decline, which relies on within-person repeated measurements. A recent study shows similar associations of pre-and post-bronchodilator spirometry with mortality [3], which is welcome news given the added participant burden of conducting post-bronchodilator spirometry in healthy population studies.
The strengths of RAS include the enrichment of the study sample with current cigarette smokers, which was part of the a priori intent to test effect modification, and the inclusion of a diverse sample (24% African Americans), which supports inference to broader population groups. An additional strength was the extensive infrastructure provided by SELECT, which allowed RAS to be conducted with efficiencies of cost and effort. SELECT infrastructure included an online data collection tool, which allowed incorporation of web-based uploading of spirometry data on a weekly basis, and bi-annual meetings of study personnel, which allowed for optimal training and refresher courses on spirometry methodology.
A few limitations are worth noting. In the postrandomization design, we cannot directly estimate whether the intervention increased FEV 1 early in the supplementation period in smokers on the active study supplements. This question is important given that prior studies among individuals with COPD show that some clinical treatments increase FEV 1 , but have no effect on FEV 1 rate of decline [27], and in light of the Lung Health Study, which showed that smoking cessation led to a small but significant initial rebound in FEV 1 , followed by an attenuation in the rate of decline [28]. Our study was conducted in male participants in the SELECT prostate cancer prevention trial, thus whether findings apply to women requires further study. In addition, the design of SELECT did not vary dose and/or formulation, and did not consider whether genetic variation might influence nutrient requirements for optimal health. Finally, the premature termination of supplements meant that final pulmonary function test on some participants was collected well after supplements had been discontinued, and thus analyses were based on fewer participants than originally planned.

Conclusions
While smoking cessation is the key public health intervention to prevent smoking-related health effects, about 20% of the population continues to smoke [1]. This study investigated the role of nutritional supplementation in lung function decline to identify possible intervention strategies to mitigate lung effects in continuing smokers. This randomized controlled trial found statistically significant protective effects of selenium, specifically 200 μg/d L-selenomethionine, on rate of decline in FEF  in current cigarette smokers. Supplementation with selenium attenuated the annual rate of decline in FEF  in current cigarette smokers, but neither vitamin E nor selenium had effects on rate of decline in FEV 1 . Further studies are needed to understand whether intervention effects are modified by baseline selenium nutriture and/or selenium-related genetic variation [29].