JACN Did you know that you can get alerts when a new issue is online?
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fuller, C. J.
Right arrow Articles by Martin, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fuller, C. J.
Right arrow Articles by Martin, K. J.
Journal of the American College of Nutrition, Vol. 19, No. 3, 361-369 (2000)
Published by the American College of Nutrition

Original Research

The Effect of Vitamin E and Vitamin C Supplementation on LDL Oxidizability and Neutrophil Respiratory Burst in Young Smokers

Cindy J. Fuller, PhD, RD, Margaret A. May, MSN, MS, RD and Karla J. Martin

Department of Nutrition & Foodservice Systems, The University of North Carolina at Greensboro, Greensboro, North Carolina

Address reprint requests to: Cindy J. Fuller, PhD, RD, Department of Nutrition & Foodservice Systems, The University of North Carolina at Greensboro, P.O. Box 26170, Greensboro, NC 27402-6170.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objective: The purpose of this study was to determine the effect of vitamin E and/or vitamin C supplementation on low-density lipoprotein (LDL) oxidizability and neutrophil (PMN) superoxide anion production in young smokers.

Methods: Thirty smokers with a <5 pack-year history were randomly assigned to take placebo; vitamin C (1 g/day); vitamin E (400 IU/day); or both vitamins in a double-blind fashion. Subjects took the supplements for 8 weeks. At weeks 0 and 8, blood was collected for isolation of LDL and PMN, and for antioxidant vitamin analysis. LDL was oxidized with a copper (Cu) catalyst, and oxidation was measured by formation of conjugated dienes over a 5-hour time course. Lag times and maximum oxidation rates were calculated from the time course data. PMN superoxide anion release was assessed by respiratory burst after stimulation with phorbol ester and opsonized zymosan, and their ability to oxidize autologous LDL following treatment with the above stimuli was measured with the conjugated diene assay.

Results: Subjects who received vitamin E alone had a significant increase in the lag phase of Cu-catalyzed LDL oxidation (week 0, 118 ± 31 min vs. week 8, 193 ± 80 min, mean ± SD, p < 0.05), whereas the vitamin C and placebo groups had no changes in LDL oxidation kinetics. The group receiving both vitamins E and C had a significant reduction in oxidation rate (week 0, 7.4 ± 2.3 vs. week 8, 5.1 ± 2.1, p < 0.05). There were no significant changes for any group in PMN superoxide anion production or PMN LDL oxidation after stimulation with either phorbol ester or opsonized zymosan. Plasma and LDL vitamin E concentrations were significantly increased in both groups that received vitamin E. The subjects who received vitamin C alone had no significant change in plasma vitamin C concentrations; however, when data were pooled from both groups who received vitamin C, the increases were significant.

Conclusion: Vitamin E supplementation of young smokers was effective in reducing Cu-catalyzed LDL oxidizability; however, vitamin E and/or C supplementation showed few significant effects on the more physiologically relevant PMN function. This casts doubt on the ability of antioxidant supplementation to reduce oxidative stress in smokers in vivo. Therefore, smoking cessation remains the only means by which young smokers can prevent premature coronary heart disease.

Key words: vitamin C, vitamin E, low-density lipoprotein, neutrophils, smokers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidation of low-density lipoprotein (LDL) may be a key early step in the pathogenesis of coronary heart disease (CHD). All of the cells of the arterial wall—macrophages, smooth muscle cells, endothelial cells, and neutrophils (polymorphonuclear leukocytes or PMN)—are able to oxidize LDL in vitro [1,2]. Oxidized LDL is taken up via the macrophage scavenger receptor and may contribute to the formation of foam cells [3]. Furthermore, evidence of oxidized LDL has been obtained from arterial walls of animal models of atherosclerosis and of CHD patients [4,5]. Supplementation of humans with antioxidant nutrients has also been shown to decrease the oxidizability of LDL in vitro [6] and reduce the recurrence of myocardial infarctions in CHD patients [7]. Vitamin E has consistently been shown to reduce LDL oxidizability [8–12]. The results of supplementation studies with vitamin C on LDL oxidation are less clear-cut [12–14]. It is conceivable that vitamin C can regenerate vitamin E from the tocopheroxyl radical in the LDL particle [15], which could confer an indirect effect on LDL oxidation. Supplementation with a combination of vitamins E and C showed an additional reduction of LDL oxidizability in only one study to date [13].

Cigarette smokers are at increased risk for CHD. Cigarette smoke contains numerous free radical species that can contribute to the oxidation of LDL in vitro [16–18], although data are conflicting [19]. LDL isolated from smokers may be more susceptible to oxidation than LDL from nonsmokers [20,21]; however, Siekmeier et al. [22] and Marangon et al. [23] did not find any difference in LDL oxidizability between smokers and nonsmokers. Smokers have lower plasma concentrations of vitamin C and ß-carotene [24,25], which may be due to a combination of reduced dietary intakes and the free radicals present in cigarette smoke [26,27]. Earlier investigators have shown that antioxidant nutrient supplementation of long-term smokers (specifically with vitamins E and C) reduced the oxidizability of LDL [9,12,14].

Smokers also have increased numbers of circulating PMN, the most prevalent leukocyte [28,29]. These phagocytes may be stimulated by compounds in cigarette smoke to increase production of free radicals, such as superoxide anion, that could result in oxidative damage to cells and LDL [30,31]. Kanno et al. [32] reported that vitamin E could reduce PMN superoxide production in vitro. Vitamin C occurs in millimolar levels in PMN [33]. In response to chemical or bacterial stimuli [34,35] vitamin C is further accumulated in PMN by ascorbate recycling, possibly as a defense against endogenous reactive oxygen species. The effect of antioxidant supplementation on PMN function in smokers is unclear.

No one to date has studied the effect of antioxidant nutrient supplementation on younger smokers, i.e., those who have been smoking for fewer than five years. The number of adolescent smokers has increased in recent years [36]. It is vital to give young smokers strong messages and support to cease smoking, as well as reduce the damage smoking takes on them until they are able to quit permanently. Hence, the purpose of this study was to determine the effect of vitamin E and/or vitamin C supplementation on LDL oxidizability and PMN superoxide anion production in young smokers.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and Study Design
This study was approved by the Institutional Review Board of the University of North Carolina at Greensboro, and all subjects gave informed consent. They were also given monetary compensation upon completion of the study. Healthy smokers, both males and females, were recruited from the University of North Carolina at Greensboro student body and from the greater Greensboro community. Subjects had to smoke at least 10 cigarettes/d for <= 5 years (total smoking history of <= 5 pack-years). In addition, they had to meet the following criteria: Age >= 18 years, non-use of vitamins or other supplements, normal fasting plasma glucose, normal liver and kidney function tests, if female, not pregnant. The number of subjects per group was chosen based on results of previous studies involving vitamin E supplementation [10,13].

At baseline, subjects came in after an overnight fast. Blood samples (120–180 mL) were drawn into ethylenediaminetetraacetic acid (EDTA)-containing tubes (1 mg/mL) for isolation of LDL and PMN. Blood for antioxidant measurements was collected in heparinized tubes. The subjects were then randomly assigned to receive either one of the following treatments in a double-blind fashion: Vitamin E (400 IU/d); vitamin C (1000 mg/d), both vitamins E and C, placebo. The vitamin E was in the form of d-{alpha}-tocopheryl acetate and was provided by Eastman Fine Chemical (Kingsport, TN). The vitamin C was provided by Perrigo Corporation (Greenville, SC). The dosages selected have reduced LDL oxidizability in previous studies [10,14]. The combination group was included to determine if there was an additive or synergistic effect of the vitamins on the parameters tested. The subjects were instructed to take their supplements with a meal to reduce any possible gastrointestinal side effects and to increase absorption of vitamin E. Each supplement was in a single tablet or capsule to aid in compliance. The subjects followed their usual diet, activity and smoking habits. At the end of eight weeks, another fasting blood sample was drawn. Compliance was measured by pill counts at the end of the study and biweekly phone calls.

LDL Isolation and Oxidation
Except where noted, all chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO). Low-density lipoprotein was isolated by a two-hour density gradient ultracentrifugation in a Beckman (Palo Alto, CA) Ti-50 fixed-angle rotor in a Beckman model L7-65 ultracentrifuge [19]. The LDL layer from each tube was obtained and pooled, then eluted twice through a BioRad (Hercules, CA) Econo-Pac 10 DG column to remove the salts. Protein was analyzed by the modified Lowry method of Markwell et al. [37]. Bovine serum albumin was used as the standard. The vials containing LDL were flushed with nitrogen, covered with foil, and stored at 4°C.

Each subject’s LDL oxidation experiment was performed on the day of isolation. LDL (200 µg) was oxidized in phosphate-buffered saline, pH 7.4, with 5µmol/L cupric sulfate (Cu) as catalyst. The formation of conjugated dienes was monitored by measuring absorbance at 234 nm every ten minutes for five hours in a Beckman (Fullerton, CA) DU640 spectrophotometer. Data from the LDL oxidation were plotted with the Deltagraph (Delta Point, Inc., Monterey, CA) program and the resulting curve was fitted to a spline function to determine lag phase and rate of oxidation [10].

PMN Isolation and Superoxide Anion Production
PMN were isolated following the method of Scaccini and Jialal [2]. An aliquot of cells was mixed with Trypan blue, and viable cells were counted in a hemocytometer. The cell density was adjusted to 10 x 106 cells/mL with Gey’s balanced salt solution (GBSS). The PMN experiments were also performed on the day of isolation, as PMN have a short lifespan in vitro. The production of superoxide anion by PMN, as measured by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C, was determined by an adaptation of the respiratory burst assay [38,39]. PMN were plated (2.5 x 105 cells/well) in a 96-well plate with ferricytochrome C (0.98 mg/mL) in the presence or absence of SOD (0.11 mg/mL). They were then stimulated with either opsonized zymosan (OZ; 2.5 mg/mL) or phorbol 12-myristate 13-acetate (PMA; 2 µg/mL). Cells were incubated at 37°C for one hour, then absorbance at 550 nm was measured with the Tecan model 340 ATTC microplate reader (SLT Labinstruments, Grodig/Salzburg, Austria). The superoxide anion production was calculated as nmol O2·-/106 cells.

PMN Oxidation of LDL
The ability of PMN to oxidize autologous LDL after stimulation by PMA and OZ was measured by the method of Scaccini and Jialal [2]. Briefly, PMN (5 x 106 cells/mL) in GBSS were incubated in a 12-well plate with LDL (200 µg/mL) in the presence of either PMA (1.6 µmol/L) or OZ (2 mg/mL) for five hours at 37°C in an atmosphere of 95% air/5% CO2. Cell-free control wells were also run concurrently to assess spontaneous oxidation of LDL under these conditions. Following the incubation, culture supernatants were assayed for conjugated dienes by measuring absorbance at 234 nm. The concentrations of conjugated dienes were calculated with an extinction coefficient of 29,500 M-1 cm-1 [2] and corrected for the cell-free control wells. Data are expressed as nmol conjugated dienes/mg LDL protein.

Antioxidant Analysis
Vitamin E, ß-carotene, and lycopene were extracted from plasma and LDL following the hexane extraction methods of Bieri et al. [40,41]. The extraction and analyses were performed under gold lighting to reduce the risk of photodegradation. All solvents were HPLC grade and obtained from Fisher Scientific (Pittsburgh, PA). Tocol (8 µg/mL) and echinenone (0.2 µg/mL) were used as internal standards for vitamin E and carotenoids, respectively. Extracts were dried down under a gentle stream of nitrogen, then reconstituted in the appropriate mobile phase. The samples were assayed by high-performance liquid chromatography (HPLC), utilizing the Hewlett-Packard (Avondale, PA) HP-1090 chromatograph with UV/Vis detection. The column for both vitamin E and carotenoid analysis was a Microsorb-MV 5 µm, 25 cm x 0.46 cm, C-18 ODS (Rainin, Woburn, MA). The guard column was an Adsorbosphere 5 µm, 0.75 cm x 0.46 cm diameter, c-18 (Alltech, Deerfield, IL). The vitamin E mobile phase was 100% methanol, with a flow rate at 2.5 mL/min and the detector set at 292 nm [40]. The carotenoid mobile phase was 70:20:10 (v:v:v) acetonitrile (with 0.13% triethylamine): methylene chloride:methanol (with 0.01% w/v ammonium acetate), with a flow rate of 1.7 mL/min and the detector set at 450 nm [41].

For vitamin C analysis, duplicate aliquots of plasma were mixed with four volumes of ice-cold 90% methanol/1 mmol/L EDTA in microcentrifuge tubes. The sample was vortexed, placed on ice for ten minutes in the dark, then centrifuged at 12,000 RPM at 4°C for ten minutes. The supernatant was aliquotted into tubes, purged with nitrogen and stored immediately at -70°C [42]. The HPLC method was adapted from Reznick et al. [43]. A Hypersil (Hewlett-Packard, Avondale, PA) BDS 3 µm, 100 x 4 mm, C-18 column was used for the analysis, with a mobile phase of 0.05 mol/L sodium phosphate, 0.05 mol/L sodium acetate, 189 µmol/L dodecyltrimethylammonium chloride and 3.66 µmol/L tetraoctylammonium bromide in 30:70 (v:v) methanol: water, pH 4.8. The detector settings were 0.25 V and positive polarity.

Other Analyses
Serum chemistry and lipid profiles were performed by the Clinical Laboratory at Moses Cone Hospital. For fatty acid analysis, an aliquot of plasma was extracted and transesterified by the method of Lepage and Roy [44]; then, gas chromatography was performed on a Hewlett-Packard (Avondale, PA) model HP5890 Series II gas chromatograph. The fatty acid methyl ester standard and the C17:0 internal standard were obtained from NuChek Prep (Elysian, MN).

Each subject completed a three-day food record before supplementation and at the end of the study to determine customary intakes of vitamins E and C. These were analyzed by the Food Processor software (Esha Research, Salem, OR) package. The week 0 nutrient intakes were compared to a group of age-matched nonsmoking subjects (n = 13).

Statistical Analysis
Results are expressed as mean ± standard deviation (SD). One-way analysis of variance was used to detect group differences at week 0. Differences within each group (week 8–week 0) were then tested with paired-t tests. In some cases, data from the vitamin E and vitamins E&C groups, or vitamin C and vitamins E&C groups were pooled for analysis. The dietary intake data for smokers and the nonsmoking controls were analyzed by Student’s t tests for unpaired samples. Pearson’s correlation coefficients were calculated to determine the relationship between plasma and LDL antioxidant concentrations, LDL oxidizability and PMN function. The threshold for statistical significance was set at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subjects and Plasma Analyses
Thirty-three subjects were initially enrolled in the trial. A total of 30 subjects completed the study. One subject quit smoking on her own during the study, one subject left school, and a third began to smoke clove cigarettes during the study. Since the effects of this type of cigarettes on LDL oxidizability and PMN function are unknown, it was decided to drop this subject. The demographics of the four groups are shown in Table 1. The proportion of males was higher in the vitamin C group than in the others. Recruitment of males who had a <= 5 pack-year history was difficult in this population; hence, all of the other groups were predominantly female. Ages were similar across all study groups. Smoking history in the vitamin E group was significantly lower than in the vitamin C group (p < 0.01). None of the subjects reported any side effects of the supplementation. Compliance with the supplement regimen was > 80%, as assessed by pill counts. Dietary intakes of vitamins E and C did not change over the eight weeks of the study (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Subject Characteristics (mean ± SD)

 
Plasma lipid profiles for the groups before and after supplementation are shown in Table 2. No differences were seen for total cholesterol, triglycerides, LDL or HDL cholesterol between or within the groups. Plasma fatty acids are shown in Table 3. Again, there were no statistically significant differences within or between the groups for the levels of polyunsaturated fatty acids in plasma, or in LDL (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 2. Plasma Lipid Profiles over Course of Study, mmol/L (Mean ± SD)

 

View this table:
[in this window]
[in a new window]
  		Table 3. Plasma Fatty Acid Composition, µmol/L (Mean ± SD)

 
Plasma and LDL antioxidant concentrations are shown in Table 4. Plasma and LDL {alpha}-tocopherol increased in both groups that received vitamin E (plasma p < 0.01 for E group, p < 0.005 for E&C group; LDL p < 0.01 for E and E&C groups). The mean percent increases were 109 ± 58 and 89 ± 66% for plasma and LDL {alpha}-tocopherol in the vitamin E group, respectively; and 111 ± 82 and 160 ± 250% for plasma and LDL {alpha}-tocopherol in the vitamins E and C group, respectively. Plasma vitamin C increased in the subjects that received vitamin C alone or combined with vitamin E; however, these increases were not statistically significant. When data from both groups that received vitamin C were combined, the difference between weeks 0 and 8 was statistically significant (p < 0.05). Plasma ß-carotene and LDL ß-carotene and lycopene were significantly reduced in the vitamin E group at 8 weeks relative to 0 weeks (plasma ß-carotene p < 0.01, LDL ß-carotene p < 0.05, LDL lycopene p < 0.05); however, this difference was not seen in the vitamins E&C group nor when data from the vitamin E and E&C groups were pooled. There were no other statistically significant changes in plasma or LDL ß-carotene or lycopene within the groups.


View this table:
[in this window]
[in a new window]
  		Table 4. Plasma and LDL Antioxidant Concentrations (Mean ± SD)

 
Cu-Catalyzed LDL Oxidizability
The results of the Cu-catalyzed LDL oxidation assays are shown in Table 5. The group receiving vitamin E had a significant increase in the lag phase of oxidation (p < 0.05), but no change in oxidation rate. The group receiving vitamin C showed no change in lag time or oxidation rate. The E&C group had no change in lag phase, but a 31% decrease in oxidation rate, which was statistically significant at p < 0.05. The difference between week 0 and week 8 oxidation rates in the pooled vitamin C and E&C groups was highly significant (p < 0.001). The placebo-supplemented group had no change in either lag time or oxidation rate.


View this table:
[in this window]
[in a new window]
  		Table 5. Cu-Catalyzed LDL Oxidation Kinetics (Mean ± SD)

 
PMN Superoxide Production and Oxidation of LDL
The production of superoxide anion by subjects’ PMN after stimulation with PMA and OZ are shown in Fig. 1. Use of the stimulant PMA induced the PMN to produce twice as much superoxide anion as did OZ. Analysis of variance revealed no differences by group or week.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Neutrophil (2.5 x 105 cells/well) superoxide anion production after stimulation with phorbol 12-myristate-13 acetate (PMA, top) and opsonized zymosan (OZ, bottom). Bars depict mean ± SD. There were no significant differences within each group between week 0 and week 8.

 
The ability of PMN to oxidize autologous LDL in response to PMA and OZ stimulation, as measured by the production of conjugated dienes, is shown in Fig. 2. Analysis of variance revealed no differences between the groups at week 0. There were no significant differences found for either stimulant within any of the groups between weeks 0 and 8.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Conjugated diene formation by PMN (5 x 106 cells) of autologous LDL (200 µg/mL) after stimulation with phorbol 12-myristate-13 acetate (PMA, top) and opsonized zymosan (OZ, bottom). Bars depict mean ± SD. No changes were seen in any group between weeks 0 and 8.

 
Intakes of vitamins C and E did not differ between the smokers and the nonsmoking controls. The mean consumption of vitamin C over three days was 107 ± 79 mg for the smokers and 122 ± 54 mg for the nonsmokers; for vitamin E, the mean intakes were 4.3 ± 3.2 mg for the smokers and 3.9 ± 2.0 mg for the nonsmokers.

Correlations
No statistically significant correlations were seen between plasma vitamin C or E or LDL vitamin E concentrations and Cu-catalyzed LDL oxidation kinetics, whether expressed as absolute values or percent changes from weeks 0 to 8.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cigarette smoking is a major risk factor for coronary heart disease. One possible mechanism is the increased oxidative stress produced by cigarette smoke, either directly or by secondary activation of PMN. This study is the first to examine the effect of antioxidant supplementation on both direct (Cu-catalyzed LDL oxidizability) and indirect (PMN superoxide anion production and LDL oxidation) pro-atherogenic actions of cigarette smoke. Our results indicate that the effects of supplementation are mixed. While vitamin E increased the lag phase of copper-catalyzed LDL oxidizability, consistent with other studies [9–13], vitamin C had no effect on the duration of the lag phase. This contradicts earlier research [12–14], but a similar conclusion was reached by Wen, Cooke and Feely [45], who found that 1 g/d vitamin C did not affect LDL oxidizability in healthy nonsmokers. In addition, Aghdassi et al. [46] reported that supplementation of smokers with 500 mg vitamin C per day did not affect breath pentane output or plasma malondialdehyde concentrations.

The lag phase of LDL oxidation reflects the depletion of endogenous antioxidants. In whole plasma oxidation systems, vitamin C is depleted before the lipophilic tocopherols and carotenoids [47]. Esterbauer et al. [48] reported that addition of vitamin C to the LDL oxidation system lengthened the lag phase of oxidation. However, Stait and Leake [49] found that vitamin C actually increased the oxidation of minimally-modified LDL by macrophages. It is possible that the LDL fatty acids of the subjects had been modified by cigarette smoke, which may have accounted for the lack of effect of vitamin C on LDL oxidizability. The rate of LDL oxidation is a reflection of both antioxidant and polyunsaturated fatty acid content of the LDL particle [50].

Several reasons may account for the discrepant results for vitamin C supplementation and LDL oxidation with smokers. Our study population was unique in that the maximum smoking history was five years. One previous study [14] utilized smokers with longer smoking histories (mean 17.2 pack-years for supplemented group; 19.4 pack-years for placebo group) and higher daily cigarette consumption (>=20/day); other investigators did not provide this information on their subjects. It is possible that younger smokers may still have some of their adaptive mechanisms for dealing with oxidative stress intact. Hulea et al. [51] reported that plasma lipid peroxides, total antioxidant capacity and chemiluminescence of leukocytes stimulated with OZ was not different between smokers and nonsmokers aged 18 to 25; however, older smokers showed more evidence of oxidative stress than their nonsmoking counterparts. Our subjects also consumed their usual diets. In a previous study by Fuller et al. [14], subjects followed a low vitamin C diet (<= 30 mg/day) for the duration of the six-week trial. The supplements in the present study were given as single doses to maximize subject compliance. Fractional absorption of vitamin C decreases with increasing dosages [52]; this could explain the reduced response to supplementation in the present study. The subjects in the present study, on average, consumed greater than the 1989 RDA of vitamin C for smokers of 100 mg/day [53]; this was not different from an age-matched group of nonsmokers. Their week 0 plasma levels were, with the exception of the placebo group, within the reference range of 34–79 µmol/L [54]. Thus, the baseline vitamin C status of smokers in the present study may have been better than smokers who participated in previous trials. Perhaps the most plausible explanation for our discrepant findings in the vitamin C and vitamins C&E groups were the small sample sizes, which afforded us less statistical power to detect differences after supplementation.

There were no effects of antioxidant supplementation on either PMN superoxide anion production or PMN LDL oxidation. Kanno et al. [32] showed that vitamin E added in vitro reduced rat and guinea pig PMN superoxide anion production via the respiratory burst. In addition, Herbaczynska-Cedro et al. reported that a combination of vitamins C and E (600 mg of each/day) reduced reactive oxygen species production by PMN in both healthy subjects [55] and in patients with acute myocardial infarction [56]. Their assessment of reactive oxygen species was by lucigenin-amplified chemiluminescence after stimulation with arachidonic acid. This method primarily measures superoxide anion output. To date, no one has examined PMN LDL oxidation with the subjects’ autologous LDL, which is the closest approximation to in vivo conditions. Since both PMN and LDL would be affected by vitamin E and C supplementation, the respiratory burst assay and Cu-catalyzed LDL oxidation were used to demonstrate the separate effects of these nutrients on PMN and LDL, respectively. Another approach to this question would have been to utilize an artificial LDL of consistent composition for PMN to oxidize, as Devaraj et al. [57] did in their study of the effects of vitamin E supplementation on monocyte function. They reported that vitamin E (1200 IU/day for eight weeks) supplementation of healthy non-smokers significantly decreased monocyte superoxide anion release and lipid peroxidation in response to the stimulant lipopolysaccharide.

A missing piece to the results of this study is the effect of supplementation on PMN levels of vitamins E and C. Due to technical problems, these analyses were unable to be performed. Devaraj et al. [57] reported that 1200 IU/day vitamin E supplementation increased monocyte vitamin E concentrations by 186%. In their study of vitamin C requirements in males, Levine et al. [52] found that PMN vitamin C plateaued at a dose of 100 mg per day at 1.3 mmol/L.

In conclusion, supplementation of young smokers with vitamin E showed a reduction in the lag phase of Cu-catalyzed LDL oxidation, but no effect on oxidation rate. Vitamin C supplementation showed no effects on Cu-catalyzed LDL oxidation, but showed a trend toward reduction in the superoxide anion output of PMN when stimulated with phorbol ester. The combination of vitamins E and C did not confer any additional benefit to the subjects. Vitamin E supplementation alone reduced LDL concentrations of both lycopene and ß-carotene. Based on the results of this small study, the only way for young smokers to reduce oxidative damage to the vasculature by tobacco is to quit smoking rather than to take antioxidant vitamin supplements.


   ACKNOWLEDGMENTS
 
Funding for this study came from the North Carolina Agricultural Research Service and the University of North Carolina at Greensboro. The authors thank Dr. Andreas Pappas at Eastman Fine Chemicals of Kingsport, TN, for providing vitamin E and placebo capsules and Perrigo Chemical of Greenville, SC, for providing the vitamin C and placebo tablets. The authors are also grateful to Beth Alexander and Zhixin Huang for technical assistance and Dawn Koschnitzki for the nutrient analysis.

Received December 1, 1999. Revised February 1, 2000. Accepted February 1, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Müller K, Carpenter KLH, Mitchinson MJ: Cell-mediated oxidation of LDL: Comparison of different cell types of the atherosclerotic lesion. Free Rad Res 29: 207–220, 1998.[Medline]
  2. Scaccini C, Jialal I: LDL modification by activated polymorphonuclear leukocytes: A cellular model of mild oxidative stress. Free Radical Biol Med 16: 49–55, 1994.[Medline]
  3. Brown MS, Goldstein JL: Lipoprotein metabolism in the macrophage. Ann Rev Biochem 52: 233–261, 1983.
  4. Haberland ME, Fong D, Cheng L: Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science 241: 215–218, 1988.[Abstract/Free Full Text]
  5. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84: 1086–1095, 1989.
  6. Fuller CJ, Jialal I: Antioxidants and LDL oxidation. In Garewal HS (ed): "Antioxidants and Disease Prevention." Boca Raton, FL: CRC Press, pp 115–130, 1997.
  7. Stephens SG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ, Brown MJ: Randomized controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 347: 781, 1996.[Medline]
  8. Jialal I, Grundy, SM: Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein. J Lipid Res 33: 899–906, 1992.[Abstract]
  9. Princen HMG, van Poppel G, Vogelezang C, Buytenhek R, Kok FJ: Supplementation with vitamin E but not ß-carotene in vivo protects low density lipoprotein from lipid peroxidation in vitro: Effect of cigarette smoking. Arterioscler Thromb 12: 554, 1992.[Abstract/Free Full Text]
  10. Jialal I, Fuller CJ, Huet BA: The effect of {alpha}-tocopherol supplementation on LDL oxidation: A dose-response study. Arterioscler Thromb Vasc Biol 15: 190, 1995.[Abstract/Free Full Text]
  11. Simons LA, Von Konigsmark M, Balasubramaniam S: What dose of vitamin E is required to reduce susceptibility of LDL to oxidation? Aust NZ J Med 26: 496–503, 1996.[Medline]
  12. Harats D, Ben-Naim M, Dabach Y, Hollander G, Havivi E, Stein O, Stein Y: Effect of vitamin C and E supplementation on susceptibility of plasma lipoproteins to peroxidation induced by acute smoking. Atheroscler 85: 47, 1990.
  13. Rifici VA, Khachadurian AK: Dietary supplementation with vitamins C and E inhibits in vitro oxidation of lipoproteins. J Am Coll Nutr 12: 631–637, 1993.[Abstract]
  14. Fuller CJ, Grundy SM, Norkus EP, Jialal I: Effect of ascorbate supplementation on low density lipoprotein oxidation in smokers. Atherosclerosis 119: 139–150, 1996.[Medline]
  15. Packer JR, Slater TF, Willson RL: Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278: 737–738, 1979.[Medline]
  16. Church DF, Pryor WA: Free-radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspect 64: 111–126, 1985.[Medline]
  17. Yokode M, Kita T, Arai H, Kawai C, Narumiya S, Fujiwara M: Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract. Proc Natl Acad Sci USA 85: 2344–2348, 1988.[Abstract/Free Full Text]
  18. Santanam N, Sanchez R, Hendler S, Parthasarathy S: Aqueous extracts of cigarette smoke promote the oxidation of low density lipoprotein by peroxidases. FEBS Lett 414: 549–551, 1997.[Medline]
  19. Chen C, Loo G: Cigarette smoke extract inhibits oxidative modification of low density lipoprotein. Atherosclerosis 112: 177–185, 1995.[Medline]
  20. Scheffler E, Huber L, Frühbis J, Schulz I, Ziegler R, Dresel HA: Alteration of plasma low density lipoprotein from smokers. Atherosclerosis 82: 261–265, 1990.[Medline]
  21. Gouazé V, Dousset N, Dousset J-C, Valdiguié P: Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers. Clin Chim Acta 277: 25–37, 1998.[Medline]
  22. Siekmeier R, Wülfroth P, Wieland H, Groß W, März W: Low-density lipoprotein susceptibility to in vitro oxidation in healthy smokers and nonsmokers. Clin Chem 42: 524–530, 1996.[Abstract/Free Full Text]
  23. Marangon K, Herbeth B, Artur Y, Esterbauer H, Siest G: Low and very low density lipoprotein composition and resistance to copper-induced oxidation are not notably modified in smokers. Clin Chim Acta 265: 1–12, 1997.[Medline]
  24. Schectman G, Byrd JC, Gruchow HW: The influence of smoking on vitamin C status in adults. Am J Public Health 79: 158–162, 1989.[Abstract/Free Full Text]
  25. Zondervan KT, Ocké MC, Smit HA, Seidell JC: Do dietary and supplementary intakes of antioxidants differ with smoking status? Int J Epidemiol 25: 70–79, 1996.[Abstract/Free Full Text]
  26. Bridges RB, Chow CK, Rehm, SR: Micronutrient status and immune function in smokers. Ann NY Acad Sci 587: 218–231, 1990.[Medline]
  27. Handelman GJ, Packer L, Cross CE: Destruction of tocopherols, carotenoids, and retinol in human plasma by cigarette smoke. Am J Clin Nutr 63: 559–565, 1996.[Abstract/Free Full Text]
  28. Bridges RB, Wyatt RJ, Rehm SR: Effect of smoking on peripheral blood leukocytes and serum antiproteases. Eur J Respir Dis 66 (Suppl): 24–33, 1985.
  29. Van Antwerpen VL, Theron AJ, Richards GA, van der Merwe CA, Viljoen E, van der Walt R, Anderson R: Plasma levels of beta-carotene are inversely correlated with circulating neutrophil counts in young male cigarette smokers. Inflammation 19: 405–414, 1995.[Medline]
  30. Bridges RB, Fu MC, Rehm, SR: Increased neutrophil myeloperoxidase activity associated with cigarette smoking. Eur J Resp Dis 67: 84–93, 1985.[Medline]
  31. Pitzer JF, Del Zoppo GJ, Schmid-Schönbein GW: Neutrophil activation in smokers. Biorheology 33: 45–58, 1996.[Medline]
  32. Kanno T, Utsumi T, Takehara Y, Ide A, Akiyama J, Yoshioka T, Horton AA, Utsumi K: Inhibition of neutrophil-superoxide generation by {alpha}-tocopherol and coenzyme Q. Free Rad Res 24: 281–289, 1996.[Medline]
  33. Washko P, Rotrosen D, Levine M: Ascorbic acid transport and accumulation in human neutrophils. J Biol Chem 264: 18990–19002, 1989.
  34. Washko PW, Wang YN, Levine M.: Ascorbic acid recycling in human neutrophils. J Biol Chem 268: 15531–15535, 1993.[Abstract/Free Full Text]
  35. Wang Y, Russo TA, Kwon O, Chanock S, Rumsey SC, Levine M: Ascorbate recycling in human neutrophils: induction by bacteria. Proc Natl Acad Sci USA 94: 13816–13819, 1997.[Abstract/Free Full Text]
  36. Centers for Disease Control and Prevention: Tobacco use and usual source of cigarettes among high school students—United States, 1995. J Am Med Assoc 276: 184–185, 1996.
  37. Markwell MAK, Haas SM, Bieber, Tolbert NE: A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206–209, 1978.[Medline]
  38. Cerasoli F, Gee M, Ishihara Y, Albertine K, Tahamont M, Gottlieb J, Peters S: Biochemical analysis of the activation of adherent neutrophils in vitro. Tissue & Cell 20: 505–517, 1988.[Medline]
  39. Carey L, Schuhl R, Gee M: Microplate reader assay for measurement of opsonized zymosan-stimulated superoxide production by neutrophils. Biotechniques 19: 824–829, 1995.[Medline]
  40. Bieri JG, Tolliver TJ, Catignani GL: Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr 32: 2143–2149, 1979.[Abstract/Free Full Text]
  41. Bieri JG, Brown ED, Smith Jr JC: Determination of individual carotenoids in human plasma by high performance liquid chromatography. J Liq Chromatog 8: 473–484, 1985.
  42. Washko PW, Hartzell WO, Levine M: Ascorbic acid analysis using high performance liquid chromatography with coulometric electrochemical detection. Anal Biochem 181: 276–282, 1989.[Medline]
  43. Reznick A, Cross CE, Hu ML, Suzuki YJ, Khwaja S, Safadi A, Motchnik PA, Packer L, Halliwell B: Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation. Biochem J 286: 607–611, 1992.
  44. Lepage G, Roy CC: Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27: 114–120, 1986.[Abstract]
  45. Wen Y, Cooke T, Feely J: The effect of pharmacological supplementation with vitamin C on low-density lipoprotein oxidation. Br J Clin Pharmacol 44: 94–97, 1997.[Medline]
  46. Aghdassi E, Royall D, Allard JP: Oxidative stress in smokers supplemented with vitamin C. Internat J Vit Nutr Res 69: 45–51, 1999.
  47. Frei B, England L, Ames BN: Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci USA 86: 6377–6381, 1989.[Abstract/Free Full Text]
  48. Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G: Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr 53: 314S–321S, 1991.[Abstract/Free Full Text]
  49. Stait SE, Leake DS: Ascorbic acid can either increase or decrease low density lipoprotein modification. FEBS Letters 341: 263–267, 1994.[Medline]
  50. Croft KD, Williams P, Dimmitt S, Abu-Amsha R, Beilin LJ: Oxidation of low-density lipoproteins: effect of antioxidant content, fatty acid composition and intrinsic phospholipase activity on susceptibility to metal ion-induced oxidation. Biochim Biophys Acta 1254: 250–256, 1995.[Medline]
  51. Hulea SA, Olinescu R, Nitã S, Crocnan D, Kummerow FA: Cigarette smoking causes biochemical changes in blood that are suggestive of oxidative stress: A case-control study. J Environ Pathol Toxicol Oncol 14: 173–180, 1995.[Medline]
  52. Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal, KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR: Vitamin C pharmacokinetics in healthy volunteers: Evidence for a recommended dietary allowance. Proc Natl Acad Sci USA 93: 3704–3709, 1996.[Abstract/Free Full Text]
  53. Food and Nutrition Board: "Recommended Dietary Allowances", 10th ed. Washington, DC: National Academy Press, 1989.
  54. Beers MH, Berkow R (eds): "The Merck Manual of Diagnosis and Therapy", 17th ed. West Point, PA: Merck & Co., 1999.
  55. Herbaczynska-Cedro K, Wartanowicz M, Panczenko-Kresowska B, Cedro K, Klosiewicz-Wasek B, Wasek W: Inhibitory effect of vitamins C and E on the oxygen free radical production in human polymorphonuclear leukocytes. Eur J Clin Invest 24: 316–319, 1994.[Medline]
  56. Herbaczynska-Cedro K, Klosiewicz-Wasek B, Cedro K, Wasek W, Panczenko-Kresowska B, Wartanowicz M: Supplementation with vitamins C and E suppresses leukocyte oxygen free radical production in patients with myocardial infarction. Eur Heart J 16: 1044–1049, 1995.[Abstract/Free Full Text]
  57. Devaraj S, Li D, Jialal I: The effects of alpha tocopherol supplementation on monocyte function: Decreased lipid oxidation, interleukin 1ß secretion, and monocyte adhesion to endothelium. J Clin Invest 98: 756–763, 1996.[Medline]



This article has been cited by other articles:


Home page
 ChestHome page
D. G. Yanbaeva, M. A. Dentener, E. C. Creutzberg, G. Wesseling, and E. F. M. Wouters
Systemic Effects of Smoking
Chest, May 1, 2007; 131(5): 1557 - 1566.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
J. N Hathcock, A. Azzi, J. Blumberg, T. Bray, A. Dickinson, B. Frei, I. Jialal, C. S Johnston, F. J Kelly, K. Kraemer, et al.
Vitamins E and C are safe across a broad range of intakes
Am. J. Clinical Nutrition, April 1, 2005; 81(4): 736 - 745.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
R. A. Jacob, G. M. Aiello, C. B. Stephensen, J. B. Blumberg, P. E. Milbury, L. M. Wallock, and B. N. Ames
Moderate Antioxidant Supplementation Has No Effect on Biomarkers of Oxidant Damage in Healthy Men with Low Fruit and Vegetable Intakes
J. Nutr., March 1, 2003; 133(3): 740 - 743.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fuller, C. J.
Right arrow Articles by Martin, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fuller, C. J.
Right arrow Articles by Martin, K. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS