Ascorbate is depleted by smoking and repleted by moderate supplementation: a study in male smokers and nonsmokers with matched dietary antioxidant intakes

¹ From the Department of Molecular and Cell Biology, University of California, Berkeley, the Western Human Nutrition Research Center, US Department of Agriculture, Davis, CA; and the Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Copenhagen; and University of Berne, Institute of Medical Microbiology (Infectious Diseases), Berne, Switzerland.

² Reference to a company or product name does not imply approval or recommendation of the product by the US Department of Agriculture to the exclusion of others that may be suitable.

³ Supported in part by Tobacco Related Diseases Research Program grant 7RT-0178, National Cancer Institute Outstanding Investigator grant CA39910 and NIEHS Center grant ESO1896 (to BNA), Danish Natural Science Research Council grant SNF9502434 (to JL), and a fellowship from the Swiss Foundation for Medical-Biological Stipends (to SC). The Pharmavite Corporation (Mission Hills, CA) provided the vitamin supplement and placebo.

⁴ Address reprint requests to BN Ames, University of California, Department of Molecular and Cell Biology, 401 Barker Hall, Berkeley, CA. E-mail: bnames{at}uclink4.berkeley.edu.

ABSTRACT  
Background: Lack of reliable dietary data has hampered the ability to effectively distinguish between effects of smoking and diet on plasma antioxidant status. As confirmed by analyses of comprehensive food-frequency questionnaires, the total dietary intakes of fruit and vegetables and of dietary antioxidants were not significantly different between the study groups in the present study, thereby enabling isolation of the effect of smoking.

Objective: Our objective was to investigate the effect of smoking on plasma antioxidant status by measuring ascorbic acid, -tocopherol, -tocopherol, ß-carotene, and lycopene, and subsequently, to test the effect of a 3-mo dietary supplementation with a moderate-dose vitamin cocktail.

Design: In a double-blind, placebo-controlled design, the effect of a vitamin cocktail containing 272 mg vitamin C, 31 mg all-rac--tocopheryl acetate, and 400 µg folic acid on plasma antioxidants was determined in a population of smokers (n = 37) and nonsmokers (n = 38). The population was selected for a low intake of fruit and vegetables and recruited from the San Francisco Bay area.

Results: Only ascorbic acid was significantly depleted by smoking per se (P < 0.01). After the 3-mo supplementation period, ascorbic acid was efficiently repleted in smokers (P < 0.001). Plasma -tocopherol and the ratio of - to -tocopherol increased significantly in both supplemented groups (P < 0.05).

Conclusions: Our data suggest that previous reports of lower concentrations of plasma vitamin E and carotenoids in smokers than in nonsmokers may primarily have been caused by differences in dietary habits between study groups. Plasma ascorbic acid was depleted by smoking and repleted by moderate supplementation.

Key Words: Ascorbic acid • -tocopherol • -tocopherol • ß-carotene • lycopene • folic acid • smoking • plasma antioxidants • population study • dietary supplementation • poor diet • San Francisco Bay area

INTRODUCTION  
Long-term smoking is known to be associated with an increased risk of developing cancer, cardiovascular disease, and many other chronic diseases (1, 2). Tobacco smoke contains a large number of toxic chemicals such as NO_x and other oxidizing radicals (3, 4) that cause damage to cellular functions (5–7). The high oxidant content of smoke explains the low antioxidant status and increased oxidative stress and damage that is consistently observed in smokers (8, 9). On this basis, it has been suggested that smokers in particular would benefit from increasing their dietary intake of antioxidants (10, 11).

Dietary guidelines currently recommend 5–9 servings/d of fruit and vegetables (12). Recent studies indicate that only a relatively small proportion of the US population routinely has an intake in the recommended range (13–15). Like smoking, a low daily intake of fruit and vegetables has been associated with an increased risk of developing chronic diseases such as cancer (16–18) and atherosclerosis (19). Smokers have been found to have poorer diets than nonsmokers (20–25). This difference in dietary habits has so far made it difficult to distinguish between the effects of diet and smoking per se on plasma antioxidants in population studies.

In the present study, we investigated antioxidant status in a cohort of smokers and nonsmokers by measuring total ascorbic acid (TAA; ascorbic acid + dehydroascorbic acid) and the lipid-soluble antioxidants -tocopherol, -tocopherol, ß-carotene, and lycopene in blood plasma. The subjects were selected for an equally low daily intake of fruit and vegetables. A consequence of selecting for similar dietary habits was that vitamin C and E intakes from the normal diet did not differ significantly between smokers and nonsmokers. This enabled the isolation of the effect of smoking itself. Dietary intakes of vitamins, fruit, and vegetables were measured by comprehensive food-frequency questionnaires and analyzed as described by Block et al (26, 27). Subsequently, the effect of a moderate dietary supplement of a vitamin cocktail consisting of 272 mg vitamin C, 31 mg all-rac--tocopheryl acetate, and 400 µg folic acid was examined over a 3-mo period in a double-blind, placebo-controlled fashion. The amounts used in the daily supplementation regimen were chosen with reference to intakes that could be obtained through a more balanced diet. At the end of the supplementation period, TAA and the tocopherols, -tocopherol and -tocopherol, were measured again and compared with the baseline data. Data concerning folic acid will be reported elsewhere.

The aims of the study were to establish 1) which of the plasma antioxidants are depleted by smoking per se, 2) whether moderate supplementation can restore the depleted antioxidants to normal concentrations, and 3) whether smokers in particular respond to supplementation.

SUBJECTS AND METHODS  
Subjects and study design
Men from the San Francisco Bay area aged between 20 and 50 y and within 90–130% of ideal body weight were recruited by newspaper advertisements and flyers. One thousand eighty-nine men completed telephone prescreening, which included questions about dietary supplement use and consumption of fortified foods within the previous 6 mo. Fruit and vegetable intake was crudely assessed by using a 7-question questionnaire developed for us by Gladys Block, based on a version published by her previously (28). The cutoff point for low fruit and vegetable intake was 3 servings/d. Eighty-two nonsmokers and 149 smokers (10 cigarettes/d) with low self-reported fruit and vegetable intake who did not use vitamin supplements or fortified food completed a full in-house screening at the US Department of Agriculture Western Human Nutrition Research Center. Medical history, height, weight, pulse, and blood pressure were measured. Standardized tests for health and infectious disease markers were performed on blood and urine samples. The participants completed the 1995 Block revision of the National Cancer Institute Health Habits and History Questionnaire (HHHQ) (26, 27), which was used to verify low fruit and vegetable intake and to evaluate their dietary habits. Subjects were excluded from further study if they were in poor health (had evidence of cerebrovascular, liver, or kidney disease; cancer; anemia; or type 1 diabetes), tested positive for infectious disease markers, or consumed >3 servings/d of fruit and vegetables. Although no restrictions regarding ethnicity were made, the study population was primarily white. This study was approved by the University of California Berkeley Committee for Protection of Human Subjects and by the US Department of Agriculture/Agricultural Research Service Human Studies Review Committee.

Thirty-nine nonsmokers and 38 smokers willing to participate in the intervention study came to the center for a second visit to provide baseline samples. Blood was taken for the analysis of TAA, lipid-soluble antioxidants, and markers of the subjects' general health status. The men were grouped by smoking status and were then randomly assigned by our statistician in a double-blind, restricted fashion into either the supplement or the placebo group with stratification for age, body mass index, and alcohol consumption.

The supplement was generously donated to us by Pharmavite Corp (Mission Hills, CA) and contained by analysis 272 mg vitamin C, 31 mg all-rac--tocopheryl acetate, and 400 µg folic acid. The placebo tablets, which were also supplied by the Pharmavite Corp, contained the vehicle used to prepare the supplement (nondigestible carbohydrate and <2% stearic acid by wt) and laboratory analysis confirmed the absence of the supplemented vitamins. The design of the supplement was based on Food and Nutrition Board estimates of intakes of these nutrients that would be provided by a diet with 5 combined servings/d of fruit and vegetables (29). The subjects were asked to take the supplement or corresponding placebo daily for 90 d and to continue with their usual dietary and smoking habits. Our nursing coordinator monitored compliance by conducting monthly telephone consultations. The subjects were also asked to keep a daily log in which they recorded supplement consumption and food intake. At the end of the 90-d intervention period the participants returned to the center to give a second set of blood samples. Information supplied by their log was reviewed to ascertain that the men complied with the study requirements. Additionally, the men were asked to return their pill bottles and any remaining pills were counted. Overall study compliance was 95% and was not significantly different between the treatment groups. The participants filled out a second HHHQ to examine whether there were any changes in dietary habits during the study.

Sample collection and laboratory methods
Blood
Overnight fasting blood samples were taken by venipuncture into evacuated glass tubes (Becton Dickinson, Franklin Lakes, NJ) and were immediately placed on ice along with fasting urine samples. Some of the unprocessed blood and urine was used for a complete blood count (SmithKline Beecham, Dublin, CA), for standard chemistry panels, and to screen for infectious disease markers. The remaining blood was centrifuged at 3000 x g for 10 min (4°C) within 15 min after collection, and the plasma was removed and stored in amber vials for the analysis of lipid-soluble antioxidants.

Ascorbic acid
Plasma ascorbic acid was stabilized immediately with an equal amount of 10% (wt:vol) meta-phosphoric acid. The precipitate was removed by centrifugation at 3000 x g for 20 min (4°C) and the supernate was stored at -80°C in amber vials for <6 mo until analyzed. We showed previously that ascorbic acid is stable under these conditions (30). The concentration of TAA in EDTA-anticoagulated blood plasma was measured after reduction with dithiothreitol using reversed-phase HPLC with coulometric detection as described previously (30). All standards were from Fluka (Milwaukee).

Lipid-soluble antioxidants
Lipid-soluble antioxidants (-tocopherol, -tocopherol, ß-carotene, and lycopene) were measured in hexane extracts of EDTA-anticoagulated blood plasma as described previously (31). The conditions for the reversed-phase HPLC assay with electrochemical detection were specifically optimized for the separation of -tocopherol and -tocopherol (32). The concentrations reported for -tocopherol also include those for ß-tocopherol, which does not separate from the former under reversed-phase conditions but represents <10% of the 2 forms in blood plasma (33). Because of the nature of the separation conditions used, only the all-trans-forms of lycopene and ß-carotene are reported, which account for 65% and 95% of the total amounts of all isomers, respectively, in comparable subjects (34). Tocopherol standards were from Fluka; lycopene and ß-carotene were generously provided by Hoffmann–La Roche (Nutley, NJ). The data from 2 subjects were excluded from this study because the measured ratio of -tocopherol to -tocopherol was >20, which exceeds by far the range for normal, unsupplemented subjects (39–41) and indicates noncompliance (33, 42). Removal of those 2 subjects did not change the outcome for any measures other than those concerning vitamin E.

Lipids
Plasma total cholesterol and triacylglycerol were measured enzymatically with a clinical analyzer (SmithKline Beecham). Plasma total cholesterol was also determined by HPLC of the hexane extracts (prepared for the analysis of lipid-soluble antioxidants) by individually measuring unesterified cholesterol and the quantitatively major cholesterol esters (31). Mean values for the 2 types of determination before and after treatments were not significantly different, indicating that the extraction procedure was quantitative. Lipid-soluble antioxidants are reported both as absolute concentrations and as a ratio to total neutral lipids (ie, total cholesterol + triacylglycerol determined enzymatically) to adjust for possible differences in lipid status (35–38).

Dietary data
Dietary intake data from the HHHQ were analyzed commercially (Block Dietary Systems, Berkeley, CA). Nutrient intakes were estimated from each individual subject's reported frequency of consumption and portion sizes on the HHHQ. The data obtained were evaluated for reporting errors by determining whether energy intakes seemed unusually high (>5000 kcal, or 20.9 kJ) or if too few (15% of the foods selected) or too many foods per day were selected. If these indexes appeared unreasonable, the data were recalculated by adjusting the portion sizes initially selected by the subject. If recalculated energy intakes were still unusually high or low, the assumption was made that the subject's reporting was inaccurate and the data were excluded from the database. On average, 20% of the dietary intake data were excluded from statistical analyses because of inaccurate baseline data, end of study data, or both. Both under- and overreporting occurred with similar frequencies and were equally spread across all 4 treatment groups. Two-way analysis of variance (ANOVA) showed no significant differences between the complete data set and the reduced data set.

Statistical analysis
Data were analyzed by using STATISTICA 5.1 (StatSoft, Tulsa, OK). Homogeneity of variances was verified by Levene's test. Differences between smokers and nonsmokers were analyzed by using one-way ANOVA (Table 1). Differences between data from before and after supplementation were analyzed by using paired t tests (Tables 2 and 3). Changes in dietary intakes and plasma antioxidants were also tested by using two-way ANOVA with smoking and supplementation as factors. A two-tailed P value <0.05 was considered statistically significant. Values are reported as means ± SDs.

View this table:
TABLE 1.. Baseline measurements of the study population¹  

View this table:
TABLE 2.. Dietary intakes of vitamin C, vitamin E, vegetables, and fruit at the beginning and end of the 3-mo supplementation period¹  

View this table:
TABLE 3.. Effect on plasma antioxidants of a 3-mo multivitamin supplementation regimen in smokers and nonsmokers¹  

RESULTS  
The baseline statistics for the study population are summarized in Table 1. Although smokers had a slightly, albeit significantly, lower fruit intake (P < 0.01) than the nonsmokers, total fruit and vegetable intake was not significantly different between the groups. Self-reported dietary intakes of vitamin C, vitamin E (-tocopherol equivalents), ß-carotene, and lycopene were not significantly different between groups, indicating an effective matching of the study population—a prerequisite to effectively isolating the effect of smoking per se on plasma antioxidant concentrations. To correct for subject-dependent variances in lipid status, which codetermine the concentration of lipid-soluble antioxidants in blood plasma (39–42), their concentrations are also reported as a ratio to total neutral lipids (ie, the sum of total cholesterol and triacylglycerol). None of the lipid-soluble antioxidants (-tocopherol, -tocopherol, ß-carotene, and lycopene), either corrected or uncorrected, was significantly lower in smokers than in nonsmokers (Table 1). To our surprise, however, lipid-adjusted -tocopherol was significantly higher in smokers (P < 0.05), in contrast with the results of a smaller study that suggested that smoking preferentially depletes plasma -tocopherol (43). Total neutral lipids did not differ significantly between smokers and nonsmokers (5.96 ± 1.65 compared with 5.98 ± 1.68 mmol/L; P = 0.97). In contrast with the lipid-soluble antioxidants, the plasma concentration of TAA was 34% lower in smokers than in nonsmokers (Table 1).

After the collection of the initial baseline sample, smokers and nonsmokers were divided into subgroups receiving either the daily supplement or placebo for 3 mo. The daily dietary intakes of vitamin C, vitamin E, and servings of vegetables and fruit for all groups as measured by the HHHQ before and after the 3-mo period are summarized in Table 2. There were no significant changes in dietary intakes except for a significant increase in vitamin C during the 3-mo period among nonsmokers who received the supplement. As is often seen in studies like this, a nonsignificant trend toward a better diet could be observed by slight overall increases in fruit and vegetable intake, even though subjects were asked to maintain their usual diet and smoking habits during the supplementation period. Two-way ANOVA with the changes in dietary intakes as the response and smoking and supplementation as factors showed no effects of either smoking or supplementation for any of the variables.

The effect of the 3-mo supplementation regimen on plasma TAA, -tocopherol, and -tocopherol in smokers and nonsmokers is summarized in Table 3. Compared with baseline, a 43% increase in TAA was observed for supplemented nonsmokers whereas the supplemented smokers showed a 194% increase during the same period. Plasma -tocopherol, both corrected and uncorrected for total neutral lipids, increased significantly both in nonsmokers and smokers compared with the baseline. Conversely, supplementation caused a significant decrease in uncorrected plasma -tocopherol concentrations compared with baseline, an effect that was ascribed to the presence of -tocopherol in the supplement (33, 38). As a consequence, the ratio of -tocopherol to -tocopherol increased significantly in both nonsmokers and smokers (Table 3). Lipid-adjusted -tocopherol did not decrease significantly.

DISCUSSION  
The National Research Council recommends 5–9 servings/d of fruit and vegetables. A substantial proportion of the US population does not meet this dietary recommendation (13–15). This constitutes a potentially severe health problem because low fruit and vegetable intake has been associated with an increased risk of several diseases such as atherosclerosis (19) and cancer (16–18). To investigate the consequences of inadequate micronutrient intake, the study population was selected for a low intake of fruit and vegetables. We assumed that by selecting for low fruit and vegetable intakes, we would isolate a cohort with inadequate vitamin C, ß-carotene, lycopene, and possibly, vitamin E intakes. As summarized in Table 1, the compiled food-frequency questionnaire data showed an average of only 2.7 servings/d of fruit and vegetables for the present cohort of smokers and nonsmokers.

The most common fruit choices were orange juice and bananas, whereas the most common vegetables were fried potatoes and tomato products. In a report of the second National Health and Nutrition Examination Survey (NHANES II), vitamin C intake for persons consuming 2 combined servings/d of fruit and vegetables was 96 mg/d (44). Thus, our finding of 77 mg/d for all study subjects eating 2.7 servings/d of fruit and vegetables provides a lower estimate. Furthermore, our finding of vitamin C intakes slightly above the recommended dietary allowance (RDA) in nonsmokers, when they were consuming amounts of fruit and vegetables well below those recommended for disease prevention, shows the inconsistency between the vitamin C RDA and the fruit and vegetable intakes recommended by the National Research Council, and thereby indirectly supports arguments to increase the RDA (45). The intake of vitamin C in smokers was only about three-quarters of the smokers' RDA (10).

The mean vitamin E intake for all study subjects was very similar to population estimates for similarly aged men (46) and did not differ significantly by smoking status. Mean intake was somewhat higher than that reported in a New Zealand study of 87 smoking and nonsmoking men and women who habitually consumed 3 servings/d of fruit and vegetables (47). This discrepancy may be due to differences in fat intake or, simply, to differences in the national food supplies.

The low fruit and vegetable intakes of the men were reflected by a mean ß-carotene intake for all study subjects approximately one-third lower than reported national mean intakes for men (48). Mean lycopene intakes were not significantly different by smoking status and were actually 3 times higher than estimates reported in a study that used the same food-frequency questionnaire in an older, mixed population of men and women (both smokers and nonsmokers) who were not selected for any particular dietary habits (49). Lycopene is the major carotenoid found in tomato products, and because of their high consumption, tomato products provide > 80% of the lycopene consumed in the United States (50). The men's few vegetable choices (highly concentrated tomato products such as ketchup and pizza sauce) may have contributed to their unexpectedly high lycopene intakes.

In general, smokers eat more fat and less fiber, fruit, and vegetables than do nonsmokers (20–24), a fact that should be taken into consideration when the effects of smoking on the plasma concentrations of dietary antioxidants are studied. In this study, total intake of fruit and vegetables and dietary intake of the major plasma antioxidants from food sources were not significantly different between the study groups. This enabled us to isolate the effect of smoking in the depletion of plasma antioxidants. We found ascorbic acid to be the only plasma antioxidant depleted by smoking per se. That depletion of ascorbic acid in smokers is due primarily to the toxicity of the smoke and not only to dietary differences agrees with our previous work (51). The mean plasma TAA concentration for our full study population agrees well with estimates from NHANES II data in a population of smoking and nonsmoking men consuming 50–96 mg vitamin C/d (52). The difference between the smokers' and nonsmokers' plasma TAA concentrations was 14.6 µmol/L, which agrees with NHANES II data (53).

For vitamin E (Table 1), our data are consistent with many previous reports that plasma or serum concentrations of vitamin E (ie, -tocopherol) are not affected by smoking (41, 54), although lower concentrations of -tocopherol in smokers have also been reported (7) even when dietary intakes were the same as those in the corresponding nonsmokers (55).

The mean plasma concentration of ß-carotene for all study subjects compared favorably with that of other studies (47, 48). In agreement with the mean concentrations we report for smokers and nonsmokers, Pamuk et al (56) found no significant differences in ß-carotene concentrations between female African-American smokers and nonsmokers after adjustment for various factors, including diet. Lycopene concentrations were also similar to reported population concentrations (50) and were not significantly different by smoking status. Our results suggest that the reported depletion of tocopherols and carotenoids in smokers is probably primarily the effect of differences in diet rather than of smoking—at least in men with low fruit and vegetable intakes. In addition, we provide new reference values for antioxidant intakes and plasma concentrations in male nonsmokers and smokers consuming few fruits and vegetables.

To investigate whether beneficial effects could be achieved exclusively from supplementation with a few of the major antioxidant vitamins, smokers and nonsmokers were randomly assigned to subgroups receiving either a simple vitamin supplement containing moderate amounts of vitamin C and -tocopherol (as acetate) together with folic acid or a placebo for 3 mo. In the present paper, we only report on the changes in plasma concentrations of ascorbic acid and the 2 main forms of tocopherol. The amounts provided were chosen with reference to what could be obtained from a diet richer in fruit and vegetables. The presence of folic acid in the supplement was based on its proposed specific role as a cofactor in uracil and homocysteine methylation reactions rather than on its potency as an antioxidant. The folate results will be reported elsewhere.

The food-frequency-questionnaire data before and after the 3-mo supplementation period are shown. The data show that the subjects, as requested, were able to keep their dietary habits more or less unchanged during the study. Thus, no significant changes in total fruit and vegetable intake were observed. However, we did find significant increases in the dietary intake of vitamins C and E among supplemented nonsmokers and unsupplemented nonsmokers, respectively. Overall, a nonsignificant trend toward a better diet over the 3-mo supplementation period was observed for the cohort, as is frequently found in studies in which diet is not strictly controlled. Two-way ANOVA showed no effect of either smoking or supplementation on any of the dietary variables.

The blood plasma concentrations of TAA, -tocopherol, and -tocopherol before and after the supplementation period are shown in Table 3. TAA increased markedly in the supplemented groups. However, because dietary vitamin C intakes increased during the study period in nonsmokers receiving the vitamin supplement, the effect of the supplementation itself cannot be properly evaluated in this group. Among the smokers, no difference was observed in dietary vitamin C intakes increased whereas the plasma concentration showed a significant 3-fold increase after supplementation. Most likely, at least part of the better response among smokers could be attributed to their lower TAA concentration at baseline. Thus, smokers in general seem to benefit more from vitamin C supplementation than do nonsmokers. Additionally, perhaps smokers have a higher homeostatic requirement, thereby contributing to a higher degree of supplement bioavailability in this group. No significant changes in dietary or plasma TAA were observed in the placebo groups over the course of the study.

Plasma concentrations of -tocopherol increased significantly in the supplemented groups, whereas their dietary intakes remained unchanged. For -tocopherol, the effect of -tocopherol supplementation was clearly negative. With the exception of lipid-adjusted -tocopherol in smokers, -tocopherol decreased significantly in both supplemented groups, whereas no significant changes were observed in the placebo groups. The nonsignificant decrease of lipid-adjusted -tocopherol in supplemented smokers, together with the significantly higher lipid-adjusted -tocopherol concentration in smokers at baseline, is unexpected in light of the recent findings that smoking cessation results in an increase in plasma -tocopherol but not in -tocopherol (43). The significant increase in the ratio of -tocopherol to -tocopherol in both smokers and nonsmokers receiving the supplement in our study also indicated that the participants complied with the study protocol and took the supplement as requested (33, 38).

On the basis of the results of this study, we conclude that 1) of the major plasma antioxidants measured, ascorbic acid is the only plasma antioxidant depleted by smoking per se; 2) depleted plasma TAA concentrations can be normalized by moderate supplementation in smokers; and 3) smokers in particular seem to respond to this supplementation. Of course, the conclusions can only legitimately be based on the original selection criteria of the study because the cohort differs from the average population. Extending this work to populations that have other dietary habits but differ only in smoking status is therefore desirable. However, it was shown recently that only 10–14% of similarly aged men consume the minimum recommended 2 servings of fruit and 3 servings of vegetables per day (13). Consequently, our study cohort may represent a large segment of the US population.

ACKNOWLEDGMENTS  
We thank Gladys Block for her help in providing the customized prescreening fruit and vegetable intake questionnaire.

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