The vitamin E isoforms α-tocopherol and γ-tocopherol have opposite associations with spirometric parameters: the CARDIA study

We determined whether there are opposing associations of α-tocopherol and γ-tocopherol with the spirometry parameters FEV₁, FVC, and FEV₁/FVC in humans using the CARDIA study with 20 years participant follow-up and existing data for spirometry and tocopherol isoforms. Participant characteristics are in Table 1. By design for CARDIA, the study had similar numbers of males and females of each race. The mean serum α-tocopherol concentration was lower and the mean γ-tocopherol concentration was higher in black compared to white participants at study year 0 (Table 2). This is consistent with previous reports [15, 16]. Due to racial differences in tocopherol concentrations, and because there are known race-based differences in spirometric parameters [17], we stratified the dataset by race. Also, because females and males are known to differ in spirometric parameters [17], the analysis included adjustments for gender.

The cross-sectional, multivariable linear regressions examined the association of α-tocopherol with spirometric parameters and γ-tocopherol with spirometric parameters. The data are presented as difference in parameter analyzed per 10 μM tocopherol, as previously described for association studies with tocopherol [18]. In the cross-sectional analysis, higher serum γ-tocopherol was significantly associated with lower FEV₁ and FVC in blacks (Table 3). In whites and in analysis of all participants, there was a significant association of higher γ-tocopherol with lower FVC (Table 3).

In contrast, to the inverse association of γ-tocopherol with spirometry, α-tocopherol was positively associated with FVC, suggesting opposing effects of the two tocopherol isoforms in blacks, whites, and all participants (Table 3). There was a positive association between α-tocopherol and FEV₁ in black participants and in all participants and then, in whites, there was a trend for a positive association (Table 3). In whites, the FEV₁/FVC was inversely associated with increasing serum α-tocopherol, although the beta was very small (Table 3). In these analyses for Table 3, the other tocopherol isoform is present at various concentrations with potentially opposing functions. Similar opposing associations of the tocopherol isoforms with spirometry were observed when the data analyses included adjustments for the other tocopherol isoform (Additional file 1: Table S1).

The data in Table 3 suggested that the tocopherol isoforms have opposing associations with spirometry. Furthermore, in our mechanistic studies in vitro and in animals, γ-tocopherol potently increases inflammation and lung hyperresponsiveness when the α-tocopherol tissue concentration is low but this effect of γ-tocopherol is ablated when the α-tocopherol tissue concentration is elevated through supplementation [12‐14]. Therefore, we hypothesized that there would be a significant association of tocopherols with FEV₁ when the concentration of the opposing tocopherol was low, causing the least competing opposing effects. For this analysis, we used quartile analysis rather than α-tocopherol/γ-tocopherol ratios because ratios would be the same when both tocopherol isoforms are low and when both tocopherol isoforms are high and because we previously reported in mechanistic animal studies that there are dose dependent effects of tocopherol isoforms on lung inflammation [12, 13]. Consistent with our hypothesis, in blacks at the lowest quartile of α-tocopherol, there was a significant negative association of FEV₁ with increasing γ-tocopherol (Table 4A). At all quartile concentrations of α-tocopherol with the exception of quartile 2 in blacks for FEV₁, the direction of FEV₁ and FVC was decreasing with increasing units of γ-tocopherol (Table 4). In whites, at the lowest quartile of α-tocopherol, there was a trend for a negative association of FEV₁ with increasing γ-tocopherol (Table 4B). For analysis of all participants (Table 4C), the negative beta for FEV₁ with γ-tocopherol was greatest at the lowest quartile of α-tocopherol and the negative beta for FVC with γ-tocopherol was greatest at the lowest two quartiles of α-tocopherol (Table 4C).

Conversely and consistent with our hypothesis, at the lowest quartile of γ-tocopherol, there was a significant positive association of FEV₁ and a trend for a positive association of FVC with increasing α-tocopherol in blacks (Table 5A). In whites, at the lowest quartile of γ-tocopherol, there was no association for α-tocopherol with FEV₁ and FVC (Table 5B). For analysis of all participants, there was also no association for α-tocopherol with FEV₁ and FVC (Table 5C). These data suggest that, for blacks and whites, associations for α-tocopherol with spirometry may be influenced by environmental or genetic differences that are not known.

Given the difference in γ-tocopherol concentrations in Americans versus Western Europeans and Asians (Additional file 1: Table S1) [1, 4] and the lung hyperreponsiveness promoting effect of a 5-fold increase in γ-tocopherol in our animal models [12, 13], we determined whether a 5-fold higher γ-tocopherol associates with lower spirometry parameters in the CARDIA participants at ages 21–55 (Figure 1). For the analysis, we defined a priori a γ-tocopherol group to model the average Western European plasma γ-tocopherol levels and two γ-tocopherol groups to model the average USA plasma γ-tocopherol levels. The average Western European γ-tocopherol levels are 1–2 μM γ-tocopherol (Additional file 2: Table S2) [1] and therefore the group that models the average Western European plasma γ-tocopherol is 1–2 μM γ-tocopherol. The average USA γ-tocopherol levels in Table 2 and in previous reports (Additional file 2: Table S2) are 4–7 μM γ-tocopherol [19]. To model the average USA γ-tocopherol levels, two groups were defined a priori as moderate (3–4.8 μM, the average published for NHANES in the U. S.A. [19]) and moderate-high (4.9-10 μM, [1‐4]). In addition, because a 5-fold higher γ-tocopherol in our animal studies reduced lung responsiveness, another group at >10 μM γ-tocopherol was defined a priori to model 5-fold higher γ-tocopherol than the average European plasma γ-tocopherol group (Figure 1).

Analysis was performed separately for non-asthmatics and for the participants with self-reported ever having asthma, because in Table 1, 20% of the participants reported ever having asthma and because our previous studies showed that γ-tocopherol reduced lung function of mice with experimental asthma [4, 12, 13]. In this analysis for participants at ages 21–55, we used spirometry from study years 5, 10, 20 and 25 and the tocopherol concentrations from study years 0, 7 and 15. We addressed the data limitation of lack of concordant spirometry and tocopherol data after year 0 of CARDIA by analyzing with GEE and averaging the CARDIA year 0, 7 and year 15 serum γ-tocopherol concentrations for each individual study participant because individuals tended to maintain their tocopherol relative positions over 15 years as analyzed by covariance of slope and intercept parameters (described in detail in the Manuscript Additional file 3: Methods). Also, the average of the three tocopherol measures reflects the long term exposure of the tocopherol. To limit the potential effects of opposing functions of very high α-tocopherol on spirometric parameters [13], and because it was determined in the cross-sectional analysis at CARDIA study year 0 that there were opposing effects of α-tocopherol and γ-tocopherol, it was determined a priori to include participants with a serum α-tocopherol level less than 37.1 μM because it is two standard deviations above the mean concentration of serum α-tocopherol in the 20–39 year age group from the U.S. National Health and Nutrition Examination Survey (NHANES) [19]. In addition, the average plasma α-tocopherol concentrations are about the same among several countries (Additional file 2: Table S2) [1, 4]. After exclusions primarily for individuals who were missing data for tocopherol, spirometry or covariate at follow-up years (n = 623) and exclusion of a few individuals with very high serum α-tocopherol levels (over 37.1 μM) (n = 146), there were 3757 participants from years 5, 10, 20 and 25 of CARDIA for analysis. Because our analysis of this CARDIA cohort at year 0 revealed similar associations of α-tocopherol and γ-tocopherol in both races, we did not stratify the data by race in this analysis, but instead we adjusted for race to maintain the sample size of the highest and lowest γ-tocopherol groups. A GEE analysis was also performed with percent predicted values for FEV₁ and the statistical outcomes were the same (data not shown).

Statistical data for Figure 1 is as follows: For 21–27 year old CARDIA participants, the FEV₁ for the highly-elevated γ-tocopherol group is lower than the low, moderate, and moderate-high γ-tocopherol groups by: A) 317 mL (p = 0.006), 284 mL (p = 0.0003), and 263 mL (p = 0.0008), respectively, for all CARDIA participants (non-asthmatics and those who self-reported that they ever had asthma); B) 255 mL (p = 0.04), 213 mL (p = 0.01) and 175 mL (p = 0.03), respectively, for non-asthmatics; and C) 460 mL (p = 0.08), 475 mL (p < 0.009), and 517 mL (p = 0.004), respectively, for self-reported ever asthmatics. For 21–27 year old CARDIA participants, the FVC for the highly-elevated γ-tocopherol group is lower than the low, moderate, and moderate-high γ-tocopherol groups by: D) 322 mL (p = 0.01), 263 mL (p = 0.004), and 254 mL (p = 0.005), respectively, for all participants; E) 263 mL (p = 0.05), 160 mL (p = 0.08) and 156 mL (p = 0.08), respectively, for non-asthmatics; and F) 357 mL (p = 0.22), 568 mL (p = 0.009) and 545 mL (p = 0.01), respectively, for self-reporting ever asthmatics. For FEV_1, the tests for interaction of the continuous age variable for the highly-elevated γ-tocopherol group compared to the low, moderate and moderate-high γ-tocopherol groups were as follows: A) p = 0.07, p = 0.12, and p = 0.11, respectively, in all participants; B) p = 0.23, p = 0.45 and p = 0.58, respectively, in the non-asthmatics; and C) p = 0.21, p = 0.10 and p = 0.05, respectively, in those with “ever asthma”. For FVC_, the tests for interaction of the continuous age variable for the highly-elevated γ-tocopherol group compared to the low, moderate and moderate-high γ-tocopherol groups were as follows: D) p = 0.60, p = 0.89, and p = 0.63, respectively, for all participants; E) p = 0.98, p = 0.25 and p = 0.65, respectively, for non-asthmatics; and F) p = 0.53, p = 0.31 and p = 0.22, respectively, for those reporting “ever asthma”.

Figure 1 presents data for all CARDIA participants and also stratifies results by asthma status. For the 21–27 year olds, FEV₁ for the highly-elevated γ-tocopherol group was significantly lower than the low, moderate, or moderate-high γ-tocopherol groups that modeled the average European/Asian and USA plasma γ-tocopherol levels (Figure 1A-C). Similarly, in the 21–27 year olds, FVC of the highly-elevated γ-tocopherol group was significantly lower than the other γ-tocopherol groups (Figure 1D-F). For ages 21–55, there was no difference in slopes of FEV1 and FVC (NS test for interaction with age). Therefore, because FEV₁ and FVC for the highly-elevated γ-tocopherol group was significantly lower than the other γ-tocopherol groups at the age 21–27 and then there was no difference among the slopes of the lines to age 55, FEV₁ and FVC of the highly-elevated γ-tocopherol group was lower than the other γ-tocopherol groups (Figure 1A-F). The analysis of FEV₁/FVC resulted in no significant differences among the γ-tocopherol groups (Figure 1G-I).

CARDIA is a multi-center longitudinal cohort study designed to examine the risk factors for cardiovascular disease in young adults enrolled at 18–30 years [29‐31]. By design, the study enrolled 5,114 participants in equal subgroups of race (black or white), and gender. Participants were assessed at year 0 (1985–1986) and years 2, 5, 7, 10, 15, and 20. Young Adult Longitudinal Trends in Antioxidants (YALTA), an ancillary CARDIA study, measured serum tocopherols from blood samples of fasting participants in years 0, 7, and 15 of the study [32, 33]. After exclusions in Figure 2, there were 4,526 participants.

The spirometry measurements FEV₁ and FVC were at years 0, 2, 5, 10, and 20 as described in the Additional file 3. American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines were followed to ensure quality control and testing procedures [34].

Tocopherols at years 0, 7, and 15 of the study were extracted from the serum and analyzed by high pressure liquid chromatography (HPLC) [35] as described in the Additional file 3.

Details of each statistical analysis are in the Manuscript Additional file 3. Briefly, multivariable linear regressions were performed at study year 0 to determine the association between tocopherol concentrations and spirometry. Due to significant differences in tocopherol concentrations (p < 0.0001) by race, data were either stratified by race or adjusted for race as indicated in the tables and figures. To analyze associations of opposing effects of tocopherol isoforms, the association of each isoform was analyzed by strata based on quartiles of the opposing tocopherol using the SAS (Cary, NC, USA) GLM procedure.

To examine the association of γ-tocopherol with spirometry as a function of participant age, we used generalized estimating equations (GEE) as described in the Additional file 3 and figure legend. We used GEE analysis for FEV₁, FVC and FEV₁/FVC as a function of the age groups for presentation in the figure and also did analysis treating age as a continuous variable as described in the Additional file 3. The GEE analysis was also performed with percent predicted values for FEV₁ and the statistical outcomes were the same (data not shown).