Lower plasma -carboxyethyl-hydroxychroman after deuterium-labeled -tocopherol supplementation suggests decreased vitamin E metabolism in smo
the Linus Pauling Institute, Oregon State University, Corvallis, OR (RSB, SWL, TMB, and MGT
Roswell Park Cancer Institute, Buffalo, NY (JL)
ABSTRACT
Background: Cigarette smoking increases the fractional disappearance rates of -tocopherol and is associated with increased oxidative stress, but its effects on -tocopherol metabolism are unknown.
Objective: We hypothesized that smokers would have less -tocopherol available and consequently lower plasma -carboxyethyl-hydroxychroman (-CEHC), the -tocopherol metabolite produced by a cytochrome P450–mediated process.
Design: Smokers and nonsmokers (n = 10 per group) were supplemented with deuterium-labeled -tocopheryl acetates (75 mg each d3-RRR--tocopheryl and d6-all-rac--tocopheryl acetate) from day –6 to day –1, and plasma tocopherols and CEHCs were measured (day –6 through day 17).
Results: After 6 d of supplementation, plasma d3- and d6--tocopherol concentrations did not differ significantly between groups. Plasma d3- and d6--CEHCs were detectable only from day –5 to day 5. Before supplementation, unlabeled - and -CEHCs were 60% and 40% lower, respectively, in smokers than in nonsmokers (P 0.05). In addition, d0-, d3-, and d6--CEHC areas under the curves were 50% lower in smokers (P < 0.05), and smokers had lower maximal d3--CEHC (P = 0.004) and d6--CEHC (P = 0.0006) concentrations. Notably, 2.9–4.7 times as much -CEHC was produced from all-rac--tocopherol than from RRR--tocopherol. During supplementation, smokers had about one-half (P < 0.05) the plasma total, d6-, or d3--CEHC concentrations that nonsmokers did given similar -tocopherol concentrations.
Conclusions: Smoking did not increase -tocopherol disappearance through P450-mediated tocopherol metabolism. Therefore, the mechanism of increased -tocopherol disappearance in smokers likely operates through oxidation pathways, which is consistent with -tocopherol’s antioxidant function. Consequently, evaluating the molecular mechanism or mechanisms responsible for tocopherol metabolism under conditions of oxidative stress and the mechanisms that regulate -tocopherol status is warranted.
Key Words: Oxidative stress carboxyethyl-hydroxychroman CEHC smokers tocopherols metabolism cytochrome P450
INTRODUCTION
Oxidative stress, such as cigarette smoke, has been observed during in vitro investigations to deplete -tocopherol from human plasma (1, 2). In humans, direct comparison of plasma -tocopherol concentrations between unsupplemented smokers and nonsmokers has yielded inconsistent results: some investigations reported lower plasma -tocopherol concentrations among smokers (3, 4), whereas other investigations observed no differences in plasma concentrations between these groups (5–7). Recently, we studied the fractional disappearance kinetics of vitamin E in smokers and nonsmokers (8). After 6 days of deuterium-labeled -tocopherol supplementation, plasma labeled -tocopherol concentrations were not significantly different between the groups, but fractional disappearance rates of -tocopherol were 13% faster and -tocopherol half-lives were 10 h shorter in smokers than in nonsmokers (8). Moreover, the fractional disappearance rates of -tocopherol in smokers were inversely related to their plasma vitamin C concentration, which suggests that higher vitamin C status could attenuate -tocopherol disappearance (8) and further supports the role of oxidative stress in vitamin E disappearance.
Because differences in circulating unlabeled -tocopherol concentrations are not consistently observed between control and experimental groups, it has been suggested that additional biomarkers are necessary for the assessment of -tocopherol status (9). Therefore, measurement of the circulating or urinary vitamin E metabolite -carboxyethyl-hydroxychroman (-CEHC) represents a possible means of assessing in vivo -tocopherol status. CEHCs are nonoxidation products of tocochromanols (Figure 1). Their formation is initiated via cytochrome P450–mediated -oxidation, followed by stepwise -oxidation of the phytyl tail (10–13). Subsequently, CEHCs are excreted after gluconuration or sulfation (14).
Metabolism of tocopherols and tocotrienols to their respective CEHCs plays a role in the excretion of unwanted or excess vitamin E forms (15). Urinary recovery of - and -CEHCs from - and -tocotrienol supplementation is only a small fraction of the administered dose (16). As for the major dietary and biological forms of vitamin E, - and -tocopherol (17), -tocopherol appears to be more actively metabolized to -CEHC, as suggested by the >12 times greater plasma concentration of -CEHC than of -CEHC despite significantly higher plasma concentrations of -tocopherol relative to -tocopherol (18). In contrast, -tocopherol appears to be actively metabolized only when a plasma -tocopherol threshold of 30–40 μmol/L is reached or when individuals are supplemented (10, 19, 20). Consistent with the metabolism of excess vitamin E, the -tocopherol transfer protein discriminates between natural (RRR) and synthetic -tocopherols, such that 4 of the 8 stereoisomers in all-rac (2R-, not 2S--tocopherol forms) are maintained in the plasma (21), whereas 3 times more all-rac--tocopherol than RRR--tocopherol is metabolized to -CEHC and excreted in the urine (22).
To date, the role of oxidative stress on vitamin E status and subsequent plasma -CEHC concentrations has not been investigated. Therefore, our aim was to evaluate plasma deuterium-labeled -CEHC concentrations from cigarette smokers and nonsmokers who were supplemented with deuterium-labeled -tocopherol for 6 d (8). In the present investigation, we hypothesized that smokers, as a result of increased oxidative stress (8), would have less -tocopherol available for -CEHC production and consequently lower plasma -CEHC concentrations.
SUBJECTS AND METHODS
Materials
HPLC-grade methanol and glacial acetic acid were obtained from Fisher (Fair Lawn, NJ). Trolox, phosphate-buffered saline, ascorbic acid, and -glucuronidase (type H-1, contains minimum 300 000 U/g -glucuronidase activity and minimum 10 000 U/g sulfatase activity) were from Sigma-Aldrich (St Louis, MO). Diethyl ether was obtained from Mallinckrodt Baker Inc (Phillipsburg, NJ). Standards, including unlabeled (d0), d6-RRR-, and d3-all-rac--tocopheryl acetates and d0--tocopherol, were gifts from James Clark of Cognis Nutrition and Health (LaGrange, IL). all-rac--5,7,8-(CD3)3-tocopheryl acetate (d9-all-rac--tocopheryl acetate) was provided by Carolyn Good of General Mills and was synthesized by Isotec Inc (Miamisburg, OH). The isotopic purity of d9--tocopherol was found to be 88.4% d9 and the remainder d8. 2,5,7,8-Tetramethyl-2-(2-carboxyethyl)-6-hydroxychroman (-CEHC) and 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxychroman (-CEHC, or LLU-) were gifts from WJ Wechter of Loma Linda University (Loma Linda, CA).
Deuterated -tocopheryl acetates
Capsules containing RRR--5-(CD3)-tocopheryl acetate and all-rac--5,7-(CD3)2-tocopheryl acetate (d3-RRR--tocopheryl acetate and d6-all-rac--tocopheryl acetate, respectively) were a gift from the Natural Source Vitamin E Association and were synthesized by Eastman Kodak, Rochester, NY. The d3-RRR- and d6-all-rac--tocopheryl acetates were encapsulated in a gelatin capsule as nominal 1:1 mixtures in 150-mg quantities. The molar ratio of d3-RRR- to d6-all-rac--tocopherol was determined to be 0.98 (22).
Study participants
The Institutional Review Board at Oregon State University approved the protocol for this investigation, and all participants provided written consent before enrollment. An extensive description of the subjects and protocol was reported previously (8). In brief, healthy, normolipidemic volunteers (n = 10 nonsmokers and 10 smokers) were selected for this study on the basis of age (18–35 y), nonnutritional supplement use (>6 mo), and exercise status (<5 h/wk of aerobic activity). Cotinine, the metabolite of nicotine, was measured by radioimmunoassay (Diagnostics Products Corp, Los Angeles, CA) to verify smoking status. Participants were enrolled in the study only if they had clinical serum chemistry values within normal limits.
Experimental design
On 6 consecutive evenings (day –6 to day –1), the participants ingested the deuterated -tocopheryl acetate supplement containing 75 mg each d3-RRR--tocopheryl acetate and d6-all-rac--tocopheryl acetate immediately after a standard meal (43% carbohydrate, 17% protein, and 41% fat). Blood samples were collected from the antecubital vein into tubes containing 0.05 mL 15% K3 EDTA (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) after the subjects had fasted overnight (10–12 h) on days –6, –5, –4, –3, –2, –1, 0, 1, 2, 3, 4, 5, 6, 8, 10, 13, 15, and 17 (negative days denote the supplementation period). Blood samples were kept on ice for <30 min before plasma isolation. Plasma was separated by centrifugation (500 x g, 15 min, 4 °C; Beckman TJ-6, Paola Alto, CA), aliquoted into cryovials, snap frozen in liquid nitrogen, and then stored at –80 °C until analyzed. Smokers were asked to refrain from smoking for 1 h before blood collection to alleviate transient oxidative stress effects. Last, all participants were instructed to complete a 3-d food record (2 weekdays, 1 weekend day) during the investigation. Nutrient intakes were analyzed by using FOOD PROCESSOR dietary analysis software (version 7.9; ESHA Research Inc, Salem, OR).
Analyses
The chemical structures of labeled and unlabeled tocopherols and CEHCs are illustrated in Figure 1. Plasma labeled and unlabeled - and -CEHCs were extracted (23) and measured by liquid chromatography–mass spectrometry (LC-MS) (24). The linear quantitative range for - and -CEHC analysis was 0.2–20 pmol injected, and the lower limit of detection was 80 fmol injected on the column.
The following were analyzed from plasma and were previously reported (8): labeled and unlabeled tocopherols, ascorbic acid, uric acid, F2-isoprostanes, total cholesterol, and triacylglycerols. All biochemical analyses were conducted such that all samples (day –6 to day 17) from a participant were extracted and measured in the same batch.
Statistical analyses
Statistical analysis was performed by using GraphPad PRISM (version 4.0; GraphPad Software, San Diego, CA). Total -tocopherol or total -CEHC refers to the sum of the corresponding unlabeled (d0-) and labeled (d3-, d6-) compounds. Area under the curve (AUC) was estimated by using the trapezoidal rule. For any undetectable CEHC values, one-half the detectable limit was substituted. An unpaired Student’s t test or two-way analysis of variance with repeated measures was used as appropriate for comparisons between smokers and nonsmokers. Simple linear regression, stratified by smoking status, was used to visualize the associations between -tocopherol and -CEHC and between -tocopherol and -CEHC. To further estimate the main effect and interactive effect of -tocopherol and smoking status on -CEHC concentrations, we conducted multiple linear regression by using SAS (SAS Institute Inc; Cary, NC) GENMOD with the generalized estimating equation to control for within-subject correlation. All data were considered statistically significant when P values were <0.05. All data are reported as means ± SEs unless otherwise noted.
RESULTS
Participant characteristics and -tocopherol biokinetics
Complete details of deuterium-labeled -tocopherol biokinetics were previously reported (8). Before supplementation, there were no significant differences between smokers and nonsmokers with respect to age; body mass index; plasma concentrations of ascorbic acid, uric acid, -tocopherol, and -tocopherol; or dietary vitamin E intakes. Smokers had a greater degree of oxidative stress as marked by elevated F2-isoprostanes (8).
CEHC time course and area under the curve
Plasma - and -CEHC time courses for smokers and nonsmokers are shown in Figure 2 and Figure 3. As expected at baseline (before supplementation; day –6), no plasma d3--CEHCs or d6--CEHCs were detected (Figure 2B, C). However, only 7 of 10 nonsmokers and 8 of 10 smokers had measurable plasma d0--CEHC (Figure 2A), whereas all 20 participants had measurable amounts of d0--CEHC (Figure 3). Baseline plasma d0--CEHC concentrations for the smokers (2.6 ± 0.4 nmol/L) were less than one-half those of the nonsmokers (6.2 ± 2.0 nmol/L; P = 0.04). Similarly, smokers’ plasma d0--CEHC concentrations (62.5 ± 8.8 nmol/L) were strikingly lower than those of the nonsmokers (104.4 ± 16.0 nmol/L; P = 0.017).
After the initial supplement of 75 mg of each d3-RRR- and d6-all-rac--tocopheryl acetate (12 h after the first dose), labeled -CEHCs were found in the plasma of smokers and nonsmokers. The labeled metabolites were detected throughout the supplementation period, but became undetectable in all participants by 5 d postsupplementation. The nonsmokers’ time of maximal concentration (tmax) for labeled and unlabeled plasma -CEHCs occurred at day –2. The smokers’ tmax for labeled -CEHCs peaked later in the time course (day 0), whereas their plasma d0--CEHC remained relatively constant throughout the study period. The smokers’ d3--CEHC maximal concentration (Cmax; 8.6 ± 1.6 nmol/L) was less than one-third the nonsmokers’ Cmax (26.1 ± 5.7; P = 0.004). Similarly, the smokers’ d6--CEHC Cmax (20.2 ± 4.4 nmol/L) was significantly lower than the nonsmokers’ (50.0 ± 9.4 nmol/L; P = 0.007). Moreover, the nonsmokers’ d6--CEHC Cmax was 2.3 times higher than their d3--CEHC Cmax (P = 0.001), whereas this was 3.1 times higher in smokers (P = 0.002).
The AUCs of labeled and unlabeled plasma - and -CEHCs were all significantly lower among the smokers than among the nonsmokers (Figure 4). Specifically, smokers had 50% smaller plasma d0- (P < 0.05), d3- (P < 0.05), and d6- (P < 0.05) -CEHC AUCs and an 40% smaller -CEHC AUC (P < 0.05). In addition, the -CEHC AUC was >3 times the total -CEHC AUC among both nonsmokers (P < 0.01) and smokers (P < 0.01). Taken together, these data suggest that smokers produced fewer CEHCs throughout the investigation.
Plasma ratio of all-rac- to RRR--CEHC
The efficacy or biopotency of all-rac--tocopherol compared with RRR--tocopherol is a subject of much debate (25). The plasma ratio of deuterated all-rac- to RRR--tocopherols was previously reported to be 1:2, which is consistent with the preferential utilization of RRR--tocopherol (8). Therefore, we hypothesized that more all-rac would be available for metabolism and thus measured the ratio of plasma concentrations of d6--CEHC to d3--CEHC (d6:d3--CEHC) to evaluate the relative metabolism of synthetic and natural -tocopherols. The plasma d6:d3--CEHC ratios of all participants are combined, because there were no significant differences between groups. As illustrated in Figure 5, more of the ingested d6-all-rac- than the d3-RRR--tocopheryl acetate was metabolized to -CEHC, as noted by ratios of d6:d3--CEHC that ranged between 2.9 and 4.7 throughout the entire study. Moreover, there was no significant change in the d6:d3--CEHC ratio throughout the study, and the overall mean (±SE) was 3.9 ± 0.2. The greater d6:d3--CEHC in plasma was consistent with the higher d3:d6--tocopherol plasma ratio we previously observed (8). Therefore, all-rac--tocopherol appears to be more actively metabolized than is RRR--tocopherol regardless of smoking status, because a greater concentration of d6--CEHC and a lower concentration of d6--tocopherol was observed in the circulation of all participants.
Effect of -tocopherol supplementation on -CEHC
Previously, we reported that 6 d of supplementation with deuterated -tocopheryl acetate significantly decreases plasma -tocopherol concentrations by 38% by day 0 in smokers and nonsmokers (8), but the effect of -tocopherol supplementation on -CEHC was not explored. From the -CEHC time course (Figure 3), it appeared that the largest increase in -CEHC occurred between day –6 and day –5. Therefore, we performed correlations between plasma total -tocopherol and -CEHC concentrations as well as between the change in total -tocopherol and the change in -CEHC concentrations and observed no significant associations (P > 0.05). Similarly, we performed correlations for day 0 plasma total -tocopherol and -tocopherol but observed no significant associations. Moreover, day 0 plasma -tocopherol and day 0 -CEHC were not significantly correlated (P > 0.05). Therefore, these data suggest that supplementation with -tocopherol did not cause a significant reduction in plasma -tocopherol by increased -tocopherol metabolism to -CEHC.
Plasma -CEHC as a possible marker of in vivo -tocopherol status
Additional biomarkers for assessing -tocopherol status are necessary (9) because plasma -tocopherol concentrations are often similar between unsupplemented smokers and nonsmokers (26, 27). Therefore, we proposed to use plasma -CEHC as a marker of -tocopherol status and visualized the associations between -tocopherol and -CEHC, stratified by smoking status (Figure 6). We observed that d6-, d3-, and total -tocopherol were associated with their respective -CEHCs among smokers and nonsmokers (P < 0.0001). However, we observed no significant correlation for either smokers or nonsmokers between d0--tocopherol and d0--CEHC. To further estimate the main and interactive effects of -tocopherol and smoking status on -CEHC concentrations, we performed multiple linear regression by controlling for within-subject correlation (using SAS GENMOD). We observed that plasma d3-, d6-, and total -tocopherol concentrations were highly associated with respective plasma d3-, d6-, and total -CEHC concentrations among nonsmokers (P < 0.0001). Among smokers, the associations were also highly significant (d3: P < 0.0001; d6: P = 0.0003; total: P = 0.0158). Furthermore, multiple regression analysis indicated that there was a significant interactive effect of labeled -tocopherol and smoking status on labeled -CEHC concentrations (ie, the association between -tocopherol and -CEHC among smokers was different from that among nonsmokers). For example, among nonsmokers, the slope of the linear regression of d6--CEHC on d6--tocopherol was 3.78, whereas the slope among smokers was 1.94 (P = 0.0117). Thus, in the nonsmokers, d6--CEHC increased 3.78 nmol/L for each 1-μmol/L increase in d6--tocopherol, whereas in the smokers, d6--CEHC increased only 1.94 nmol/L for the same plasma d6--tocopherol increment. Similarly, nonsmokers compared with smokers had steeper slopes for d3- (P = 0.0141) and total (P = 0.0170) CEHC. Thus, given similar plasma -tocopherol concentrations, smokers metabolized 50% less -tocopherol to -CEHC than did nonsmokers.
DISCUSSION
In the present investigation, we showed that smokers had lower plasma concentrations of labeled and unlabeled -CEHCs after supplementation with deuterium-labeled -tocopheryl acetate (75 mg each d3-RRR- and d6-all-rac--tocopheryl acetate). The 6-d supplementation period was sufficient for smokers and nonsmokers to achieve plasma labeled and total -tocopherol concentrations that were not significantly different on day 0 (8). However, plasma labeled and unlabeled -CEHCs in smokers were significantly lower than in nonsmokers during the 10 d these labeled materials were detectable (Figure 2).
For this investigation, we used LC-MS to take advantage of the high selectively enhanced analyte specificity and low limit of detection in identifying unlabeled and deuterium-labeled plasma CEHCs. Importantly, the use of LC-MS enabled the determination of baseline unlabeled -CEHCs that were comparable with values previously reported in unsupplemented individuals (14). Likewise, supplementation with deuterium-labeled -tocopheryl acetate resulted in total -CEHC (d0- + d3- + d6--CEHC) concentrations that were comparable with values reported from -tocopherol–supplemented individuals (19). Therefore, LC-MS can be successfully used to evaluate circulating CEHC concentrations. In addition, the use of this technology proved important because plasma CEHCs (nmol/L) were present at substantially lower concentrations than were plasma tocopherols (μmol/L), and LC/MS, unlike traditional HPLC detection techniques, easily enables the discrimination of labeled CEHCs from unlabeled CEHCs.
Before supplementation, smokers had plasma d0--CEHC and d0--CEHC concentrations that were 60% and 40% lower than those of the nonsmokers, respectively, despite plasma - and -tocopherol concentrations that were not significantly different. About 12 h (day –5) after the first dose of d3-RRR- and d6-all-rac--tocopheryl acetate, all subjects had detectable plasma d3- and d6--CEHCs (Figure 2). However, beginning on day –5, these deuterated -CEHCs were consistently lower among the smokers. This finding is further emphasized by d3- and d6--CEHC AUCs (day –5 to day 5) that were 50% lower in smokers than in nonsmokers (Figure 4). In addition, smokers’ plasma d3- and d6--CEHC Cmax values were 67% and 59% lower than the nonsmokers’, respectively. These findings are surprising in that d3- and d6--tocopherol concentrations were not significantly different between smokers and nonsmokers during the supplementation period (8). Overall, these findings indicate that smokers’ plasma -CEHC responses to deuterated -tocopherol supplementation were depressed in that smokers produced less d3- and d6--CEHC given the same -tocopheryl acetate dose, a similar plasma deuterated -tocopherol response, and similar dietary intakes of -tocopherol (5 mg/d) (8).
To evaluate whether synthetic and naturally occurring -tocopherol were metabolized similarly, we evaluated plasma d6:d3-CEHC ratios. Strikingly, we observed that more of the ingested synthetic d6-all-rac--tocopheryl acetate was metabolized to d6--CEHC (Figure 5). In fact, the d6:d3--CEHC ratio ranged between 2.9 and 4.7 throughout the investigation. Collectively, these findings suggest that synthetic -tocopherol preparations are more actively metabolized, which is consistent with the view that vitamin E forms that are not maintained in plasma are subject to increased catabolism and excretion.
We previously reported (8) that smokers have a faster -tocopherol fractional disappearance rate than do nonsmokers, which is consistent with an in vivo antioxidant function of -tocopherol. Furthermore, at the end of the trial (day 17), smokers had significantly lower plasma d3- and d6--tocopherols and lower urinary d3- and d6--CEHCs. From those observations, we speculated that smokers likely had lower -CEHCs as the result of less -tocopherol available for metabolism. However, during the supplementation period (day –6 to day 0), plasma -tocopherols were not significantly different between the groups; nonetheless, the analysis presented here shows that during the same interval, smokers had lower plasma -CEHC concentrations. Therefore, plasma -tocopherol concentrations are unlikely to be the only factors that regulate the metabolism of -tocopherol to -CEHC, and these data suggest that the liver tocopherol concentration is critical.
Recently, Jeanes et al (28) reported that smokers have lower lymphocyte and platelet -tocopherol concentrations than do nonsmokers despite having plasma -tocopherol concentrations that are not significantly different. Therefore, one possibility for the smokers’ lower plasma - and -CEHCs is that the -tocopheryl acetate supplementation period enabled tocopherol-containing tissues to be repleted. Subsequently, the hepatic -tocopherol transfer protein’s binding capacity for -tocopherol would be exceeded, and excess -tocopherol would accumulate in the liver and thus be available for the production of -CEHC. This view is consistent with that of Schultz et al (10), who suggested that -CEHC excretion is an indicator of optimal -tocopherol status and a saturated binding capacity. However, as an alternative or complementary possibility, it is also feasible that cigarette smoking dysregulated P450-mediated metabolism of tocopherols to CEHCs. Metabolism of tocopherols occurs via a cytochrome P450 of the 3A type (11, 29) or 4F type (13). In support of the CYP3A type, tocopherol was shown to prevent acetaminophen-induced CYP3A4 degradation in HepG2 cells (30). Furthermore, treatment of HepG2/C3A cells with sesamin (a known inhibitor of CYP3A) resulted in lower production of -CEHC (29). Cigarette smoke is known to induce certain cytochrome P450s, such as human CYP1A (31) and mouse CYP2E1 (32), whereas CYP3A protein levels are reportedly lower in smokers than in nonsmokers (33). Therefore, if a CYP3A is involved in -tocopherol metabolism and smoking decreases CYP3A protein levels, then it would be expected that smokers would have a lower rate of -tocopherol metabolism and thus lower plasma - and -CEHC concentrations. Clearly, the molecular pathways involved in -tocopherol metabolism require additional investigations with more invasive models of oxidative stress to discern whether smoking decreases P450-mediated tocopherol metabolism or directly decreases tocopherols such that less tocopherol is available for P450-mediated metabolism.
In the United States, only 8% of men and 2.4% of women consume diets that meet the estimated average requirement for -tocopherol (34). Therefore, establishing a reliable biomarker for -tocopherol status is essential. In this investigation, we attempted to use -CEHC as a marker for -tocopherol status. However, given that all-rac--tocopherol is more actively metabolized and smokers appear to metabolize less -tocopherol than do nonsmokers, it is unclear whether -CEHC concentrations can be validated as a suitable biomarker for -tocopherol status. Moreover, under normal dietary conditions, plasma -CEHC is minimal (in the nmol/L range) or undetectable unless individuals surpass a threshold (yet to be defined) that activates P450-mediated metabolism. Therefore, measurement of -CEHC might actually only represent excess -tocopherol rather than be a suitable marker for those with low or marginally low plasma -tocopherol. This is exemplified by the fact that at baseline, 5 of 20 participants had undetectable d0--CEHC and, thus, may have had suboptimal -tocopherol status.
Additional work is clearly warranted to better understand the molecular mechanisms responsible for tocopherol metabolism and the effect that cigarette smoking or other oxidative stresses have on its regulation. Furthermore, investigations regarding -tocopherol liver trafficking should be considered, because we observed a maximal peak in plasma -CEHCs at day –2 that sharply declined (Figure 2) despite -tocopherol supplementation for 2 additional days. The fact that -CEHC did not remain elevated or continue to rise during the -tocopherol supplementation period suggests that alternative pathways, such as biliary -tocopherol excretion (15, 35, 36), may be activated to dispose of excess hepatic -tocopherol.
ACKNOWLEDGMENTS
We express special thanks to the study participants for their cooperation throughout the investigation.
RSB, TMB, and MGT participated in the study design, data collection, and analyses and wrote the initial draft of the manuscript. JL assisted with the statistical analysis and participated in the editing and review of the manuscript. SWL participated in the sample analysis for CEHCs and contributed to the editing and review of the manuscript. None of the authors had a known conflict of interest.
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Roswell Park Cancer Institute, Buffalo, NY (JL)
ABSTRACT
Background: Cigarette smoking increases the fractional disappearance rates of -tocopherol and is associated with increased oxidative stress, but its effects on -tocopherol metabolism are unknown.
Objective: We hypothesized that smokers would have less -tocopherol available and consequently lower plasma -carboxyethyl-hydroxychroman (-CEHC), the -tocopherol metabolite produced by a cytochrome P450–mediated process.
Design: Smokers and nonsmokers (n = 10 per group) were supplemented with deuterium-labeled -tocopheryl acetates (75 mg each d3-RRR--tocopheryl and d6-all-rac--tocopheryl acetate) from day –6 to day –1, and plasma tocopherols and CEHCs were measured (day –6 through day 17).
Results: After 6 d of supplementation, plasma d3- and d6--tocopherol concentrations did not differ significantly between groups. Plasma d3- and d6--CEHCs were detectable only from day –5 to day 5. Before supplementation, unlabeled - and -CEHCs were 60% and 40% lower, respectively, in smokers than in nonsmokers (P 0.05). In addition, d0-, d3-, and d6--CEHC areas under the curves were 50% lower in smokers (P < 0.05), and smokers had lower maximal d3--CEHC (P = 0.004) and d6--CEHC (P = 0.0006) concentrations. Notably, 2.9–4.7 times as much -CEHC was produced from all-rac--tocopherol than from RRR--tocopherol. During supplementation, smokers had about one-half (P < 0.05) the plasma total, d6-, or d3--CEHC concentrations that nonsmokers did given similar -tocopherol concentrations.
Conclusions: Smoking did not increase -tocopherol disappearance through P450-mediated tocopherol metabolism. Therefore, the mechanism of increased -tocopherol disappearance in smokers likely operates through oxidation pathways, which is consistent with -tocopherol’s antioxidant function. Consequently, evaluating the molecular mechanism or mechanisms responsible for tocopherol metabolism under conditions of oxidative stress and the mechanisms that regulate -tocopherol status is warranted.
Key Words: Oxidative stress carboxyethyl-hydroxychroman CEHC smokers tocopherols metabolism cytochrome P450
INTRODUCTION
Oxidative stress, such as cigarette smoke, has been observed during in vitro investigations to deplete -tocopherol from human plasma (1, 2). In humans, direct comparison of plasma -tocopherol concentrations between unsupplemented smokers and nonsmokers has yielded inconsistent results: some investigations reported lower plasma -tocopherol concentrations among smokers (3, 4), whereas other investigations observed no differences in plasma concentrations between these groups (5–7). Recently, we studied the fractional disappearance kinetics of vitamin E in smokers and nonsmokers (8). After 6 days of deuterium-labeled -tocopherol supplementation, plasma labeled -tocopherol concentrations were not significantly different between the groups, but fractional disappearance rates of -tocopherol were 13% faster and -tocopherol half-lives were 10 h shorter in smokers than in nonsmokers (8). Moreover, the fractional disappearance rates of -tocopherol in smokers were inversely related to their plasma vitamin C concentration, which suggests that higher vitamin C status could attenuate -tocopherol disappearance (8) and further supports the role of oxidative stress in vitamin E disappearance.
Because differences in circulating unlabeled -tocopherol concentrations are not consistently observed between control and experimental groups, it has been suggested that additional biomarkers are necessary for the assessment of -tocopherol status (9). Therefore, measurement of the circulating or urinary vitamin E metabolite -carboxyethyl-hydroxychroman (-CEHC) represents a possible means of assessing in vivo -tocopherol status. CEHCs are nonoxidation products of tocochromanols (Figure 1). Their formation is initiated via cytochrome P450–mediated -oxidation, followed by stepwise -oxidation of the phytyl tail (10–13). Subsequently, CEHCs are excreted after gluconuration or sulfation (14).
Metabolism of tocopherols and tocotrienols to their respective CEHCs plays a role in the excretion of unwanted or excess vitamin E forms (15). Urinary recovery of - and -CEHCs from - and -tocotrienol supplementation is only a small fraction of the administered dose (16). As for the major dietary and biological forms of vitamin E, - and -tocopherol (17), -tocopherol appears to be more actively metabolized to -CEHC, as suggested by the >12 times greater plasma concentration of -CEHC than of -CEHC despite significantly higher plasma concentrations of -tocopherol relative to -tocopherol (18). In contrast, -tocopherol appears to be actively metabolized only when a plasma -tocopherol threshold of 30–40 μmol/L is reached or when individuals are supplemented (10, 19, 20). Consistent with the metabolism of excess vitamin E, the -tocopherol transfer protein discriminates between natural (RRR) and synthetic -tocopherols, such that 4 of the 8 stereoisomers in all-rac (2R-, not 2S--tocopherol forms) are maintained in the plasma (21), whereas 3 times more all-rac--tocopherol than RRR--tocopherol is metabolized to -CEHC and excreted in the urine (22).
To date, the role of oxidative stress on vitamin E status and subsequent plasma -CEHC concentrations has not been investigated. Therefore, our aim was to evaluate plasma deuterium-labeled -CEHC concentrations from cigarette smokers and nonsmokers who were supplemented with deuterium-labeled -tocopherol for 6 d (8). In the present investigation, we hypothesized that smokers, as a result of increased oxidative stress (8), would have less -tocopherol available for -CEHC production and consequently lower plasma -CEHC concentrations.
SUBJECTS AND METHODS
Materials
HPLC-grade methanol and glacial acetic acid were obtained from Fisher (Fair Lawn, NJ). Trolox, phosphate-buffered saline, ascorbic acid, and -glucuronidase (type H-1, contains minimum 300 000 U/g -glucuronidase activity and minimum 10 000 U/g sulfatase activity) were from Sigma-Aldrich (St Louis, MO). Diethyl ether was obtained from Mallinckrodt Baker Inc (Phillipsburg, NJ). Standards, including unlabeled (d0), d6-RRR-, and d3-all-rac--tocopheryl acetates and d0--tocopherol, were gifts from James Clark of Cognis Nutrition and Health (LaGrange, IL). all-rac--5,7,8-(CD3)3-tocopheryl acetate (d9-all-rac--tocopheryl acetate) was provided by Carolyn Good of General Mills and was synthesized by Isotec Inc (Miamisburg, OH). The isotopic purity of d9--tocopherol was found to be 88.4% d9 and the remainder d8. 2,5,7,8-Tetramethyl-2-(2-carboxyethyl)-6-hydroxychroman (-CEHC) and 2,7,8-trimethyl-2-(-carboxyethyl)-6-hydroxychroman (-CEHC, or LLU-) were gifts from WJ Wechter of Loma Linda University (Loma Linda, CA).
Deuterated -tocopheryl acetates
Capsules containing RRR--5-(CD3)-tocopheryl acetate and all-rac--5,7-(CD3)2-tocopheryl acetate (d3-RRR--tocopheryl acetate and d6-all-rac--tocopheryl acetate, respectively) were a gift from the Natural Source Vitamin E Association and were synthesized by Eastman Kodak, Rochester, NY. The d3-RRR- and d6-all-rac--tocopheryl acetates were encapsulated in a gelatin capsule as nominal 1:1 mixtures in 150-mg quantities. The molar ratio of d3-RRR- to d6-all-rac--tocopherol was determined to be 0.98 (22).
Study participants
The Institutional Review Board at Oregon State University approved the protocol for this investigation, and all participants provided written consent before enrollment. An extensive description of the subjects and protocol was reported previously (8). In brief, healthy, normolipidemic volunteers (n = 10 nonsmokers and 10 smokers) were selected for this study on the basis of age (18–35 y), nonnutritional supplement use (>6 mo), and exercise status (<5 h/wk of aerobic activity). Cotinine, the metabolite of nicotine, was measured by radioimmunoassay (Diagnostics Products Corp, Los Angeles, CA) to verify smoking status. Participants were enrolled in the study only if they had clinical serum chemistry values within normal limits.
Experimental design
On 6 consecutive evenings (day –6 to day –1), the participants ingested the deuterated -tocopheryl acetate supplement containing 75 mg each d3-RRR--tocopheryl acetate and d6-all-rac--tocopheryl acetate immediately after a standard meal (43% carbohydrate, 17% protein, and 41% fat). Blood samples were collected from the antecubital vein into tubes containing 0.05 mL 15% K3 EDTA (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) after the subjects had fasted overnight (10–12 h) on days –6, –5, –4, –3, –2, –1, 0, 1, 2, 3, 4, 5, 6, 8, 10, 13, 15, and 17 (negative days denote the supplementation period). Blood samples were kept on ice for <30 min before plasma isolation. Plasma was separated by centrifugation (500 x g, 15 min, 4 °C; Beckman TJ-6, Paola Alto, CA), aliquoted into cryovials, snap frozen in liquid nitrogen, and then stored at –80 °C until analyzed. Smokers were asked to refrain from smoking for 1 h before blood collection to alleviate transient oxidative stress effects. Last, all participants were instructed to complete a 3-d food record (2 weekdays, 1 weekend day) during the investigation. Nutrient intakes were analyzed by using FOOD PROCESSOR dietary analysis software (version 7.9; ESHA Research Inc, Salem, OR).
Analyses
The chemical structures of labeled and unlabeled tocopherols and CEHCs are illustrated in Figure 1. Plasma labeled and unlabeled - and -CEHCs were extracted (23) and measured by liquid chromatography–mass spectrometry (LC-MS) (24). The linear quantitative range for - and -CEHC analysis was 0.2–20 pmol injected, and the lower limit of detection was 80 fmol injected on the column.
The following were analyzed from plasma and were previously reported (8): labeled and unlabeled tocopherols, ascorbic acid, uric acid, F2-isoprostanes, total cholesterol, and triacylglycerols. All biochemical analyses were conducted such that all samples (day –6 to day 17) from a participant were extracted and measured in the same batch.
Statistical analyses
Statistical analysis was performed by using GraphPad PRISM (version 4.0; GraphPad Software, San Diego, CA). Total -tocopherol or total -CEHC refers to the sum of the corresponding unlabeled (d0-) and labeled (d3-, d6-) compounds. Area under the curve (AUC) was estimated by using the trapezoidal rule. For any undetectable CEHC values, one-half the detectable limit was substituted. An unpaired Student’s t test or two-way analysis of variance with repeated measures was used as appropriate for comparisons between smokers and nonsmokers. Simple linear regression, stratified by smoking status, was used to visualize the associations between -tocopherol and -CEHC and between -tocopherol and -CEHC. To further estimate the main effect and interactive effect of -tocopherol and smoking status on -CEHC concentrations, we conducted multiple linear regression by using SAS (SAS Institute Inc; Cary, NC) GENMOD with the generalized estimating equation to control for within-subject correlation. All data were considered statistically significant when P values were <0.05. All data are reported as means ± SEs unless otherwise noted.
RESULTS
Participant characteristics and -tocopherol biokinetics
Complete details of deuterium-labeled -tocopherol biokinetics were previously reported (8). Before supplementation, there were no significant differences between smokers and nonsmokers with respect to age; body mass index; plasma concentrations of ascorbic acid, uric acid, -tocopherol, and -tocopherol; or dietary vitamin E intakes. Smokers had a greater degree of oxidative stress as marked by elevated F2-isoprostanes (8).
CEHC time course and area under the curve
Plasma - and -CEHC time courses for smokers and nonsmokers are shown in Figure 2 and Figure 3. As expected at baseline (before supplementation; day –6), no plasma d3--CEHCs or d6--CEHCs were detected (Figure 2B, C). However, only 7 of 10 nonsmokers and 8 of 10 smokers had measurable plasma d0--CEHC (Figure 2A), whereas all 20 participants had measurable amounts of d0--CEHC (Figure 3). Baseline plasma d0--CEHC concentrations for the smokers (2.6 ± 0.4 nmol/L) were less than one-half those of the nonsmokers (6.2 ± 2.0 nmol/L; P = 0.04). Similarly, smokers’ plasma d0--CEHC concentrations (62.5 ± 8.8 nmol/L) were strikingly lower than those of the nonsmokers (104.4 ± 16.0 nmol/L; P = 0.017).
After the initial supplement of 75 mg of each d3-RRR- and d6-all-rac--tocopheryl acetate (12 h after the first dose), labeled -CEHCs were found in the plasma of smokers and nonsmokers. The labeled metabolites were detected throughout the supplementation period, but became undetectable in all participants by 5 d postsupplementation. The nonsmokers’ time of maximal concentration (tmax) for labeled and unlabeled plasma -CEHCs occurred at day –2. The smokers’ tmax for labeled -CEHCs peaked later in the time course (day 0), whereas their plasma d0--CEHC remained relatively constant throughout the study period. The smokers’ d3--CEHC maximal concentration (Cmax; 8.6 ± 1.6 nmol/L) was less than one-third the nonsmokers’ Cmax (26.1 ± 5.7; P = 0.004). Similarly, the smokers’ d6--CEHC Cmax (20.2 ± 4.4 nmol/L) was significantly lower than the nonsmokers’ (50.0 ± 9.4 nmol/L; P = 0.007). Moreover, the nonsmokers’ d6--CEHC Cmax was 2.3 times higher than their d3--CEHC Cmax (P = 0.001), whereas this was 3.1 times higher in smokers (P = 0.002).
The AUCs of labeled and unlabeled plasma - and -CEHCs were all significantly lower among the smokers than among the nonsmokers (Figure 4). Specifically, smokers had 50% smaller plasma d0- (P < 0.05), d3- (P < 0.05), and d6- (P < 0.05) -CEHC AUCs and an 40% smaller -CEHC AUC (P < 0.05). In addition, the -CEHC AUC was >3 times the total -CEHC AUC among both nonsmokers (P < 0.01) and smokers (P < 0.01). Taken together, these data suggest that smokers produced fewer CEHCs throughout the investigation.
Plasma ratio of all-rac- to RRR--CEHC
The efficacy or biopotency of all-rac--tocopherol compared with RRR--tocopherol is a subject of much debate (25). The plasma ratio of deuterated all-rac- to RRR--tocopherols was previously reported to be 1:2, which is consistent with the preferential utilization of RRR--tocopherol (8). Therefore, we hypothesized that more all-rac would be available for metabolism and thus measured the ratio of plasma concentrations of d6--CEHC to d3--CEHC (d6:d3--CEHC) to evaluate the relative metabolism of synthetic and natural -tocopherols. The plasma d6:d3--CEHC ratios of all participants are combined, because there were no significant differences between groups. As illustrated in Figure 5, more of the ingested d6-all-rac- than the d3-RRR--tocopheryl acetate was metabolized to -CEHC, as noted by ratios of d6:d3--CEHC that ranged between 2.9 and 4.7 throughout the entire study. Moreover, there was no significant change in the d6:d3--CEHC ratio throughout the study, and the overall mean (±SE) was 3.9 ± 0.2. The greater d6:d3--CEHC in plasma was consistent with the higher d3:d6--tocopherol plasma ratio we previously observed (8). Therefore, all-rac--tocopherol appears to be more actively metabolized than is RRR--tocopherol regardless of smoking status, because a greater concentration of d6--CEHC and a lower concentration of d6--tocopherol was observed in the circulation of all participants.
Effect of -tocopherol supplementation on -CEHC
Previously, we reported that 6 d of supplementation with deuterated -tocopheryl acetate significantly decreases plasma -tocopherol concentrations by 38% by day 0 in smokers and nonsmokers (8), but the effect of -tocopherol supplementation on -CEHC was not explored. From the -CEHC time course (Figure 3), it appeared that the largest increase in -CEHC occurred between day –6 and day –5. Therefore, we performed correlations between plasma total -tocopherol and -CEHC concentrations as well as between the change in total -tocopherol and the change in -CEHC concentrations and observed no significant associations (P > 0.05). Similarly, we performed correlations for day 0 plasma total -tocopherol and -tocopherol but observed no significant associations. Moreover, day 0 plasma -tocopherol and day 0 -CEHC were not significantly correlated (P > 0.05). Therefore, these data suggest that supplementation with -tocopherol did not cause a significant reduction in plasma -tocopherol by increased -tocopherol metabolism to -CEHC.
Plasma -CEHC as a possible marker of in vivo -tocopherol status
Additional biomarkers for assessing -tocopherol status are necessary (9) because plasma -tocopherol concentrations are often similar between unsupplemented smokers and nonsmokers (26, 27). Therefore, we proposed to use plasma -CEHC as a marker of -tocopherol status and visualized the associations between -tocopherol and -CEHC, stratified by smoking status (Figure 6). We observed that d6-, d3-, and total -tocopherol were associated with their respective -CEHCs among smokers and nonsmokers (P < 0.0001). However, we observed no significant correlation for either smokers or nonsmokers between d0--tocopherol and d0--CEHC. To further estimate the main and interactive effects of -tocopherol and smoking status on -CEHC concentrations, we performed multiple linear regression by controlling for within-subject correlation (using SAS GENMOD). We observed that plasma d3-, d6-, and total -tocopherol concentrations were highly associated with respective plasma d3-, d6-, and total -CEHC concentrations among nonsmokers (P < 0.0001). Among smokers, the associations were also highly significant (d3: P < 0.0001; d6: P = 0.0003; total: P = 0.0158). Furthermore, multiple regression analysis indicated that there was a significant interactive effect of labeled -tocopherol and smoking status on labeled -CEHC concentrations (ie, the association between -tocopherol and -CEHC among smokers was different from that among nonsmokers). For example, among nonsmokers, the slope of the linear regression of d6--CEHC on d6--tocopherol was 3.78, whereas the slope among smokers was 1.94 (P = 0.0117). Thus, in the nonsmokers, d6--CEHC increased 3.78 nmol/L for each 1-μmol/L increase in d6--tocopherol, whereas in the smokers, d6--CEHC increased only 1.94 nmol/L for the same plasma d6--tocopherol increment. Similarly, nonsmokers compared with smokers had steeper slopes for d3- (P = 0.0141) and total (P = 0.0170) CEHC. Thus, given similar plasma -tocopherol concentrations, smokers metabolized 50% less -tocopherol to -CEHC than did nonsmokers.
DISCUSSION
In the present investigation, we showed that smokers had lower plasma concentrations of labeled and unlabeled -CEHCs after supplementation with deuterium-labeled -tocopheryl acetate (75 mg each d3-RRR- and d6-all-rac--tocopheryl acetate). The 6-d supplementation period was sufficient for smokers and nonsmokers to achieve plasma labeled and total -tocopherol concentrations that were not significantly different on day 0 (8). However, plasma labeled and unlabeled -CEHCs in smokers were significantly lower than in nonsmokers during the 10 d these labeled materials were detectable (Figure 2).
For this investigation, we used LC-MS to take advantage of the high selectively enhanced analyte specificity and low limit of detection in identifying unlabeled and deuterium-labeled plasma CEHCs. Importantly, the use of LC-MS enabled the determination of baseline unlabeled -CEHCs that were comparable with values previously reported in unsupplemented individuals (14). Likewise, supplementation with deuterium-labeled -tocopheryl acetate resulted in total -CEHC (d0- + d3- + d6--CEHC) concentrations that were comparable with values reported from -tocopherol–supplemented individuals (19). Therefore, LC-MS can be successfully used to evaluate circulating CEHC concentrations. In addition, the use of this technology proved important because plasma CEHCs (nmol/L) were present at substantially lower concentrations than were plasma tocopherols (μmol/L), and LC/MS, unlike traditional HPLC detection techniques, easily enables the discrimination of labeled CEHCs from unlabeled CEHCs.
Before supplementation, smokers had plasma d0--CEHC and d0--CEHC concentrations that were 60% and 40% lower than those of the nonsmokers, respectively, despite plasma - and -tocopherol concentrations that were not significantly different. About 12 h (day –5) after the first dose of d3-RRR- and d6-all-rac--tocopheryl acetate, all subjects had detectable plasma d3- and d6--CEHCs (Figure 2). However, beginning on day –5, these deuterated -CEHCs were consistently lower among the smokers. This finding is further emphasized by d3- and d6--CEHC AUCs (day –5 to day 5) that were 50% lower in smokers than in nonsmokers (Figure 4). In addition, smokers’ plasma d3- and d6--CEHC Cmax values were 67% and 59% lower than the nonsmokers’, respectively. These findings are surprising in that d3- and d6--tocopherol concentrations were not significantly different between smokers and nonsmokers during the supplementation period (8). Overall, these findings indicate that smokers’ plasma -CEHC responses to deuterated -tocopherol supplementation were depressed in that smokers produced less d3- and d6--CEHC given the same -tocopheryl acetate dose, a similar plasma deuterated -tocopherol response, and similar dietary intakes of -tocopherol (5 mg/d) (8).
To evaluate whether synthetic and naturally occurring -tocopherol were metabolized similarly, we evaluated plasma d6:d3-CEHC ratios. Strikingly, we observed that more of the ingested synthetic d6-all-rac--tocopheryl acetate was metabolized to d6--CEHC (Figure 5). In fact, the d6:d3--CEHC ratio ranged between 2.9 and 4.7 throughout the investigation. Collectively, these findings suggest that synthetic -tocopherol preparations are more actively metabolized, which is consistent with the view that vitamin E forms that are not maintained in plasma are subject to increased catabolism and excretion.
We previously reported (8) that smokers have a faster -tocopherol fractional disappearance rate than do nonsmokers, which is consistent with an in vivo antioxidant function of -tocopherol. Furthermore, at the end of the trial (day 17), smokers had significantly lower plasma d3- and d6--tocopherols and lower urinary d3- and d6--CEHCs. From those observations, we speculated that smokers likely had lower -CEHCs as the result of less -tocopherol available for metabolism. However, during the supplementation period (day –6 to day 0), plasma -tocopherols were not significantly different between the groups; nonetheless, the analysis presented here shows that during the same interval, smokers had lower plasma -CEHC concentrations. Therefore, plasma -tocopherol concentrations are unlikely to be the only factors that regulate the metabolism of -tocopherol to -CEHC, and these data suggest that the liver tocopherol concentration is critical.
Recently, Jeanes et al (28) reported that smokers have lower lymphocyte and platelet -tocopherol concentrations than do nonsmokers despite having plasma -tocopherol concentrations that are not significantly different. Therefore, one possibility for the smokers’ lower plasma - and -CEHCs is that the -tocopheryl acetate supplementation period enabled tocopherol-containing tissues to be repleted. Subsequently, the hepatic -tocopherol transfer protein’s binding capacity for -tocopherol would be exceeded, and excess -tocopherol would accumulate in the liver and thus be available for the production of -CEHC. This view is consistent with that of Schultz et al (10), who suggested that -CEHC excretion is an indicator of optimal -tocopherol status and a saturated binding capacity. However, as an alternative or complementary possibility, it is also feasible that cigarette smoking dysregulated P450-mediated metabolism of tocopherols to CEHCs. Metabolism of tocopherols occurs via a cytochrome P450 of the 3A type (11, 29) or 4F type (13). In support of the CYP3A type, tocopherol was shown to prevent acetaminophen-induced CYP3A4 degradation in HepG2 cells (30). Furthermore, treatment of HepG2/C3A cells with sesamin (a known inhibitor of CYP3A) resulted in lower production of -CEHC (29). Cigarette smoke is known to induce certain cytochrome P450s, such as human CYP1A (31) and mouse CYP2E1 (32), whereas CYP3A protein levels are reportedly lower in smokers than in nonsmokers (33). Therefore, if a CYP3A is involved in -tocopherol metabolism and smoking decreases CYP3A protein levels, then it would be expected that smokers would have a lower rate of -tocopherol metabolism and thus lower plasma - and -CEHC concentrations. Clearly, the molecular pathways involved in -tocopherol metabolism require additional investigations with more invasive models of oxidative stress to discern whether smoking decreases P450-mediated tocopherol metabolism or directly decreases tocopherols such that less tocopherol is available for P450-mediated metabolism.
In the United States, only 8% of men and 2.4% of women consume diets that meet the estimated average requirement for -tocopherol (34). Therefore, establishing a reliable biomarker for -tocopherol status is essential. In this investigation, we attempted to use -CEHC as a marker for -tocopherol status. However, given that all-rac--tocopherol is more actively metabolized and smokers appear to metabolize less -tocopherol than do nonsmokers, it is unclear whether -CEHC concentrations can be validated as a suitable biomarker for -tocopherol status. Moreover, under normal dietary conditions, plasma -CEHC is minimal (in the nmol/L range) or undetectable unless individuals surpass a threshold (yet to be defined) that activates P450-mediated metabolism. Therefore, measurement of -CEHC might actually only represent excess -tocopherol rather than be a suitable marker for those with low or marginally low plasma -tocopherol. This is exemplified by the fact that at baseline, 5 of 20 participants had undetectable d0--CEHC and, thus, may have had suboptimal -tocopherol status.
Additional work is clearly warranted to better understand the molecular mechanisms responsible for tocopherol metabolism and the effect that cigarette smoking or other oxidative stresses have on its regulation. Furthermore, investigations regarding -tocopherol liver trafficking should be considered, because we observed a maximal peak in plasma -CEHCs at day –2 that sharply declined (Figure 2) despite -tocopherol supplementation for 2 additional days. The fact that -CEHC did not remain elevated or continue to rise during the -tocopherol supplementation period suggests that alternative pathways, such as biliary -tocopherol excretion (15, 35, 36), may be activated to dispose of excess hepatic -tocopherol.
ACKNOWLEDGMENTS
We express special thanks to the study participants for their cooperation throughout the investigation.
RSB, TMB, and MGT participated in the study design, data collection, and analyses and wrote the initial draft of the manuscript. JL assisted with the statistical analysis and participated in the editing and review of the manuscript. SWL participated in the sample analysis for CEHCs and contributed to the editing and review of the manuscript. None of the authors had a known conflict of interest.
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