AJCN 19th International Congress of Nutrition
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Upritchard, J. E
Right arrow Articles by Wiseman, S. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Upritchard, J. E
Right arrow Articles by Wiseman, S. A
Agricola
Right arrow Articles by Upritchard, J. E
Right arrow Articles by Wiseman, S. A
American Journal of Clinical Nutrition, Vol. 78, No. 5, 985-992, November 2003
© 2003 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Spread supplemented with moderate doses of vitamin E and carotenoids reduces lipid peroxidation in healthy, nonsmoking adults1,2,3

Jane E Upritchard, Caroelien RWC Schuurman, Anthony Wiersma, Lilian BM Tijburg, Stefan AJ Coolen, Philip J Rijken and Sheila A Wiseman

1 From the Unilever Health Institute, Unilever Research and Development Vlaardingen, Vlaardingen, Netherlands

2 Supported by Unilever Research and Development Vlaardingen.

3 Address reprint requests to JE Upritchard, Unilever Health Institute, Unilever Research and Development Vlaardingen, PO Box 114, 3130 AC Vlaardingen, Netherlands. E-mail: jane.upritchard{at}unilever.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: High doses of vitamin E have been shown to decrease lipid peroxidation in persons under oxidative stress. At present, the data are insufficient to predict whether lower doses offer the same benefit in healthy persons.

Objective: We studied the effect of moderate doses of a combination of vitamin E and carotenoids, incorporated into a food product, on markers of antioxidant status and lipid peroxidation in healthy persons.

Design: One hundred five healthy adults were randomly, evenly assigned in this double-blind, placebo-controlled, parallel, 11-wk intervention study. After a 2-wk stabilization period during which the subjects consumed a commercial unfortified spread, the subjects consumed 25 g/d of spread containing 43 mg {alpha}-tocopherol equivalents ({alpha}-TE; 2-3 fold the US dietary reference intake) and 0.45 mg carotenoids (spread A), 111 mg {alpha}-TE and 1.24 mg carotenoids (spread B), or 1.3 mg RRR-{alpha}-tocopherol without carotenoids (spread C).

Results: In subjects consuming spread A, plasma {alpha}-tocopherol concentrations increased 31% to 32 µmol/L, with small but significant increases in concentrations of {alpha}-carotene and lutein. This resulted in LDL with significantly higher total antioxidant capacity (17%) and an increased resistance to oxidation, as determined by lag time (18%). These improvements were dose dependent: larger increases in these variables were observed in subjects consuming spread B. Furthermore, consumption of spread B significantly reduced concentrations of the plasma lipid peroxidation biomarker F2{alpha}-isoprostane (15%).

Conclusion: The consumption of food products containing moderate amounts of vitamin E and carotenoids can lead to measurable and significant improvements in antioxidant status and biomarkers of oxidative stress in healthy persons.

Key Words: Antioxidant capacity • oxidation resistance • carotenoids • F2{alpha}-isoprostanes • malondialdehyde • spread • oxidized LDL • peroxidation • vitamin E


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is thought to play an important underlying role in the development of several chronic diseases and in age-related physiologic degeneration (1). In the body, vitamin E can modulate some of the adverse effects that reactive oxygen species have on normal physiologic function. High doses of vitamin E have been shown to increase the resistance of LDL to oxidation (2-5), enhance antioxidant capacity, and reduce F2{alpha}-isoprostane concentrations in persons under increased oxidative stress as a result of disease (6-9). In healthy persons, however, little is known about the potential benefits of vitamin E on in vivo markers of lipid oxidation, such as plasma F2{alpha}-isoprostanes. In particular, the effect on lipid peroxidation of lower doses of vitamin E and of vitamin E in combination with other dietary antioxidants is unknown.

The results of cross-sectional epidemiologic studies and experimental investigations have led to the suggestion that plasma {alpha}-tocopherol concentrations > 28 µmol/L may be required to reduce cardiovascular disease risk (10). It is likely that average dietary vitamin E intakes of 15-30 mg/d would be required to achieve these plasma concentrations (11). Achieving dietary intakes of vitamin E in this range can be difficult. A recent dietary intervention reported that < 20% of subjects attained the target of 30 mg/d, despite intensive dietary instruction, provision of vitamin E-rich foods, and high motivation of the subjects (12). Furthermore, vegetable oils and other vitamin E-rich foods tend to also be high in energy, which may be incompatible with dietary advice to maintain or reduce energy intake. Therefore, the objective of the present study was to examine the effect of consuming moderate doses of a combination of vitamin E and selected dietary carotenoids, incorporated into a food product consumed daily, on markers of antioxidant status, antioxidant capacity, and biomarkers of lipid peroxidation in healthy persons.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
 Participants were recruited from the Rotterdam area of the Netherlands through newspaper advertisements. Eligible subjects for the study were aged 35-70 y, had a body mass index (in kg/m2) between 18 and 32, and were assessed to be in good health through a medical history questionnaire and biochemical tests. Exclusion criteria were a plasma vitamin E concentration > 36 µmol/L, smoking, or participation in the month before the start of the study in medical treatments that could interfere with the test results, such as a medically prescribed diet or weight-loss regimen or the consumption of vitamin or mineral supplements. All participants in the study were habitual spread users. The protocol was approved by the Unilever Research and Development Vlaardingen Medical Ethical Committeee, and all subjects gave their written informed consent before participation.

One hundred thirty persons volunteered for the study. Twelve persons were not eligible because of plasma vitamin E concentrations > 36 µmol/L (n = 8) or abnormalities in biochemical variables (n = 4). One person failed to attend the screening appointment. From the remaining 117 volunteers, 105 were randomly assigned to 1 of 3 equal intervention groups on the basis of sex and screening plasma vitamin E concentration. Eight subjects withdrew during the study for personal reasons that were unrelated to the intervention regimen.

Experimental design
The study was a randomized, double-blind, placebo-controlled, 11-wk intervention study. After a 2-wk stabilization period during which the subjects consumed a commercial spread (containing 1.3 mg RRR-{alpha}-tocopherol without carotenoids), the subjects consumed 25 g/d of spread containing 43 mg {alpha}-tocopherol equivalents ({alpha}-TE) and 0.45 mg carotenoids (50% lutein, 25% ß-carotene, 15% lycopene, and 10% {alpha}-carotene; spread A), 111 mg {alpha}-TE and 1.24 mg carotenoids (50% lutein, 25% ß-carotene, 15% lycopene, and 10% {alpha}-carotene; spread B), or 1.3 mg RRR-{alpha}-tocopherol without carotenoids (spread C). The daily amounts of {alpha}-TE and carotenoids equaled 3-7 times the recommended daily intake for vitamin E (11) and 5-40% of the usual intake of dietary carotenoids (13). For practical reasons, the study was conducted in 3 cohorts starting 1-2 d apart. Blood samples were collected after the subjects had fasted overnight at the end of the 2-wk run-in phase and during the intervention at weeks 5 and 11.

Intervention regimens
The antioxidant composition of the spreads is presented in Table 1Go. The 4 spreads were prepared from a single low-fat (38% fat) base spread. Spreads A and B contained vitamin E (Roche Products Ltd, Derbyshire, United Kingdom), {alpha}/ß-carotene (palm carotenoids suspended in vegetable oil; CHR Hansen A/S, Hørsholm, Denmark), and encapsulated lutein and lycopene (Lyc-o-Lut beadlets; Hoffmann-La Roche, Basel, Switzerland). These spreads were the color of commercial spread, with the encapsulated lutein and lycopene visible as very small red beads. The spread used during the run-in and for the control group (spread C) did not contain any carotenoids. For masking reasons, spread C was colored orange with synthetic colorants (E102/E110). Participants were instructed to consume 25 g/d of their assigned spread in place of their usual spread. Compliance was evaluated every 2-3 wk by weighing the returned sample containers and by having the subjects complete an interview with a dietitian.


View this table:
[in this window]
[in a new window]
  		TABLE 1. . Measured antioxidant content of the study products1

 
During the 13-wk study, the participants were asked to maintain their usual diet but with the following restrictions: < 15 mg dietary vitamin E/d (excluding the amounts supplied in the test products) and minimal fluctuations in other antioxidant-rich foods, such as vegetables, tea, and fruit juices. Vitamin E intake was monitored by using a dietary checklist, and additional oils were supplied during the study (low-fat spread, deep-frying products, and salad oil). Selected high-carotenoid foods (eg, foods containing >= 10 mg lycopene, 25 mg lutein, or 30 mg ß-carotene per serving) were forbidden during the study.

Clinical data and laboratory analyses
Plasma and serum were prepared within 60 min of collection and were immediately divided into aliquots and stored at -70 °C. LDL was isolated in duplicate from thawed EDTA-treated plasma (stored with 0.6% sucrose) by using an Optima TLX tabletop ultracentrifuge (162 000 x g, 24 h, 10 °C; Beckman Instruments Inc, Palo Alto, CA). Antioxidant and oxidation assays were conducted on LDL immediately after isolation. All biochemical measurements were carried out in duplicate. For each individual, the samples from all of the appointments were measured on the same day.

Antioxidant status of the subjects
Plasma tocopherol and carotenoid concentrations were measured by using HPLC. Extracts were prepared with heptane from deproteinated EDTA-treated plasma (400 µL) containing the internal standards {alpha}-tocopherol acetate (2.6 µmol/L) and retinyl acetate (2.6 µmol/L). For the {alpha}-tocopherol determinations, the extract was dissolved in 100 µL dichloromethane:isopropanol (1: 3, by vol), and 20 µL was injected onto a 50 x 4.6 mm Chromolith SpeedROD RP-18e column (Merck, Darmstadt, Germany) at 20 °C. This was eluted with methanol:water (95:5, by vol) with a flow rate of 3.5 mL/min. {alpha}-Tocopherol was detected by ultraviolet-visible light at wavelengths of 292 nm ({alpha}-tocopherol) and 284 nm ({alpha}-tocopherol acetate) with a CV of 4.1%.

For the carotenoid determinations, the extract was dissolved in 100 µL eluent solutions A and B (1:1, by vol). Eluent A contained methanol:tertiary butylmethyl ether (TBME):1.5% ammonium acetate in water (830:150:20, by vol), and eluent B contained methanol:TBME:1.0% ammonium acetate in water (80:900:20, by vol). The sample (20 µL) was injected onto a 150 x 4.6 mm YMC C30 column (YMC Inc, Wilmington, DE) at 20 °C and was eluted by a gradient of A and B with a flow rate of 0.8 mL/min. The gradient separation started with A:B of 95:5 (by vol), which was maintained for 9 min; over 24 min, a gradient was run to A:B of 5:95 (by vol), which was maintained for 4 min, and then returned to A:B of 95:5 (by vol) by 44 min, with 5 min between injections. Retinyl acetate was detected by ultraviolet-visible light at wavelengths of 325 and 450 nm for lutein, lycopene, {alpha}-carotene, and ß-carotene. The interassay CVs were as follows: lutein, 9.3%; lycopene, 12.9%; {alpha}-carotene, 10.6%; and ß-carotene, 11.4%.

Plasma vitamin C was measured enzymatically in heparin-treated plasma samples (stored with 4.5% metaphosphoric acid). The analysis was conducted according to Vuilleumier et al (14) and was adapted for use on a Packard Multiprobe II HT analyzer (Packard Instrument Company, Merides, CT) with a CV of 3.7%.

Antioxidant capacity
The ferric-reducing abilities of EDTA-treated plasma and of native LDL were measured according to Benzie and Strain (15) but adapted for use on a Packard Multiprobe II HT analyzer. The CVs for the plasma and LDL assays were 3.7% and 2.7%, respectively.

The susceptibility of native LDL to copper ion-stimulated oxidation was assessed by monitoring conjugated diene formation (16). In brief, LDL (50 µg protein) was added to physiologic phosphate-buffered saline (final volume 1 mL), and oxidation was stimulated with copper chloride (50 µmol/L) at 30 °C with a CV of 10%.

Serum arylesterase activity with the substrate phenylacetate was used to estimate paraoxonase 1 activity (17). Change in absorbance at wavelength 270 nm was monitored for 3 min by using the Spectra-max 190 microplate reader (Molecular Devices, Sunnyvale, CA) at 25 °C with a CV of 3.5%.

Biomarkers of oxidative damage
Plasma concentrations of total F2{alpha}-isoprostanes were measured by using HPLC electron spray ionization mass spectrometry and stable isotope dilution mass spectrometry (18). First, an internal standard (deuterated F2{alpha}-isoprostane, 8-iso-prostaglandin F2{alpha}-d) was mixed with 1000 µL EDTA-treated plasma and 80 µL butylated hydroxytoluene (10 mmol/L):triphenylphosphine (1 mmol/L) in ethanol. Esterified isoprostanes were hydrolyzed in ethanolic potassium hydroxide at 40 °C for 45 min. The sample was loaded onto a solid-phase extraction column (tC18 Sep-pak Vac, 3 mL, 200 mg; Waters Chromatography BV, Etten-Leur, Netherlands) and washed with water followed by heptane and was subsequentially eluted with ethyl acetate:heptane:methanol (50:40:10, by vol). The residue was dissolved in 60 µL acetonitrile:water (60:40, by vol, with 0.05% acetic acid), filtered by using micro spin filter tubes (0.2 µm nylon; Alltech Associates Inc, Deerfield, IL), and centrifuged (13400 x g, 3 min, 25 °C).

The supernatant fluid was analyzed by using HPLC electron spray ionization mass spectrometry, and stable isotope dilution mass spectrometry was used to quantify the 8-isoprostanes in plasma. The sample (30 µL) was injected onto a reversed-phase HPLC C18 Symmetry column (dp 3.5 µm, 150 x 2.0 mm internal diameter; Waters Chromatography BV) at 20 °C attached to a Waters Alliance 2790 liquid chromatographic system. A gradient separation was used starting with water and acetonitrile (60:40, by vol), with a flow rate of 0.2 mL/min. This system was maintained for 4 min. Next, a gradient was run over 1 min to 100% acetonitrile, which was maintained for 8 min before being returned to the original eluent composition. The HPLC system was connected to a Micromass Quatro Ultima Mass Spectrometer (Micromass, Manchester, United Kingdom) with an electrospray interface that was operated in the negative ionization mode. In the multiple reaction mode of the mass spectrometer the dwell time was 400 ms; the pause time was 100 ms. Target ions were selected at a mass-to-charge ratio (m/z) of 353/193 for F2{alpha}-isoprostane and at m/z 357/197 for the deuterium-labeled internal standard. The mass spectrometer was set as follows: capillary voltage, 3 kV; cone voltage, 70 V; collision energy, 25 eV; source temperature, 120 °C; desolvation temperature, 200 °C; cone gas, 175 L/h (nitrogen); and desolvation gas, 525 L/h (nitrogen). The CV was 8.8%.

Malondialdehyde was measured in EDTA-treated plasma (stored with glutathione and butylated hydroxytoluene) with a CV of 17% (19). Antibody titres specific to oxidatively modified LDL were measured in triplicate in EDTA-treated plasma by use of a commercial enzyme-linked immunosorbent assay (Mercodia AB, Uppsala, Sweden). The intraassay CV was 8.5%.

Other measures
Plasma total cholesterol and triacylglycerols were measured spectrophotometrically by using enzymatic methods with commercially available test kits (CHOD-PAP; Boehringer Mannheim, Mannheim, Germany). HDL cholesterol was measured as described by Lopes-Virella et al (20). The Friedewald equation was used to calculate LDL (21).

Statistical methods
Power calculations performed before the start of the study estimated that 32 persons per group would be required to observe with statistical significance a potential change in plasma total F2{alpha}-isoprostanes of 10%. This number was also judged to be sufficient to detect changes in plasma concentrations of {alpha}-tocopherol and carotenoids, the resistance of LDL to oxidation, and the ferric-reducing ability of plasma and LDL. The data were analyzed by three-way analysis of variance with treatment, sex, and cohort as factors. Change from baseline was calculated and differences in change compared with the control group were established by using Dunnett's multiple-comparison test. Treatment effects are presented as differences between the changes as a result of the intervention with SEMs unless stated otherwise. Data were analyzed with SAS version 8.2 (SAS Institute Inc, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General characteristics of the subjects
The baseline characteristics of the subjects are shown in Table 2Go. Age, BMI, and the distribution of men and women in each group were not significantly different at baseline. All subjects had blood cholesterol concentrations within the normal range; however, at baseline, those assigned to spread A had higher plasma total cholesterol (11%) and LDL (15%) concentrations than did those in group C. Body weights and blood lipid concentrations did not change significantly during the study.


View this table:
[in this window]
[in a new window]
  		TABLE 2. . Clinical characteristics of the subjects1

 
General health of the subjects and compliance with the intervention
A small number of participants reported having influenza, cold symptoms, or headaches during the study, but these persons were excluded only if they were unable to continue the intervention regimen or required medication. The incidence of illness did not differ significantly between the groups, nor were any of these events considered serious as defined by good clinical practice guidelines. All volunteers consumed > 90% of their assigned spread (: 24.3 g/d) and were compliant with the other dietary prescriptions.

Antioxidant status
At baseline, plasma concentrations of vitamin E, vitamin C, and carotenoids were within the range previously reported for the Dutch population (13). Plasma {alpha}-tocopherol concentrations increased significantly in subjects consuming spreads A (31%) and B (73%) but did not change significantly in those consuming spread C (2%; Table 3Go). By 11 wk, all of the subjects consuming spreads A and B had plasma {alpha}-tocopherol concentrations > 28 µmol/L. Lipid-standardized plasma {alpha}-tocopherol concentrations paralleled the increases observed for plasma {alpha}-tocopherol. Subjects assigned to spreads A and B had small but significant increases in plasma carotenoid concentrations, reflecting the changes predicted relative to dietary intakes (Table 3Go). Plasma {alpha}-carotene, lutein, and ß-carotene concentrations also increased in subjects consuming the antioxidant-fortified spreads. Plasma lycopene did not change significantly during the study. Subjects consuming the control spread had stable carotenoid concentrations during the study. Plasma vitamin C increased 18-25% in all groups during the study. It is probable that this increase was due to changes in fruit intake associated with seasonal changes, because the entire increase occurred in the second half of the intervention and the magnitude was not significantly different between the groups.


View this table:
[in this window]
[in a new window]
  		TABLE 3. . Markers of antioxidant status and lipid peroxidation1

 
Antioxidant capacity ex vivo
In line with the increases in plasma concentrations of {alpha}-tocopherol and some carotenoids, the total antioxidant capacity of plasma tended to increase (NS). The ferric-reducing ability of LDL increased significantly in subjects consuming spreads A (11%) and B (24%) compared with a reduction (3%) in subjects consuming spread C. LDL lag time was significantly increased in subjects consuming spreads A (18%) and B (18%) compared with no significant change in subjects assigned to spread C (Figure 1Go).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 1.. Mean (± SEM) lag time of isolated LDL oxidized ex vivo in subjects assigned to spread A (n = 33), B (n = 32), or C (n = 31) at baseline ({square}) and at 5 wk ({blacksquare}). Data were analyzed by three-way ANOVA with sex and cohort as factors. Dunnett's test was used to establish post hoc differences from group C. *Change in lag time significantly different from that for group C, P < 0.001.

 
Markers of oxidative stress in vivo
Plasma total F2{alpha}-isoprostane concentrations were significantly reduced in subjects assigned to spread B (Figure 2Go). Total plasma F2{alpha}-isoprostanes decreased by 7% after 5 wk and by 15% after 11 wk. In contrast, there were no significant changes (< 2%) in plasma total F2{alpha}-isoprostane concentrations in the subjects assigned to spread A or C. Subjects who consumed spread B had significantly lower plasma malondialdehyde concentrations at the end of the study than did those in the control group. However, this difference was due to a small (< 10%) increase in plasma malondialdehyde concentrations in the control group rather than to a reduction in the subjects assigned to spread B. Antibody titres to oxidatively modified LDL and serum arylesterase activity did not change significantly during the study (Table 3Go).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2.. Mean (± SEM) plasma concentrations of total F2{alpha}-isoprostanes in subjects assigned to spread A ({circ}; n = 31), B ({blacksquare}; n = 33), or C (•; n = 29) from baseline to week 11. Data were analyzed by three-way ANOVA with sex and cohort as factors. Dunnett's test was used to establish post hoc differences from group C. *Changes in F2{alpha}-isoprostanes in group B after 5 wk (-7%; P < 0.05) and 11 wk (-15%; P < 0.05) significantly different from the changes in group C. There were no significant differences between groups A and C, and there was no significant time-by-diet interaction.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, plasma concentrations of {alpha}-tocopherol and carotenoids increased significantly in the subjects who consumed the antioxidant-fortified spreads. The increase in plasma {alpha}-tocopherol was dose dependent and in line with increases previously observed in healthy persons after supplementation with vitamin E (16). Interestingly, despite the relatively modest concentrations of carotenoids in the antioxidant-fortified spreads, significant dose-dependent increases in {alpha}-carotene, lutein, and ß-carotene were observed. These increases are unlikely to have been a result of background dietary changes because concentrations did not increase in those subjects consuming the control spread. The bioavailability of carotenoids dispersed in oils was previously reported to be better than that from vegetables, and this may account for the high percentage increases in plasma concentrations (22-25). Plasma lycopene concentrations did not increase in subjects consuming the antioxidant-fortified spreads; however, it is likely this 2-4% increase in total dietary intake could not be distinguished from the background diet. Plasma vitamin C concentrations increased in all groups during the study, most likely in response to seasonal variations in dietary intake.

An important finding of the present study was the increase in both plasma {alpha}-tocopherol and carotenoids after consumption of 25 g antioxidant spreads daily. Increasing plasma {alpha}-tocopherol and carotenoid concentrations through diet alone was previously reported to be difficult, with substantial increases in vegetable oils, fruit, and vegetables required to achieve relatively modest increases in plasma concentrations of {alpha}-tocopherol and carotenoids (12, 26, 27). Dietary supplements are effective at increasing plasma concentrations of vitamin E and carotenoids, but compared with food sources, the risk of overconsumption is higher with these compounds (11). Therefore, a low-fat spread fortified with moderate amounts of vitamin E and carotenoids may provide a safe and convenient way to achieve desirable antioxidant status.

Increases in plasma antioxidant concentrations may contribute to enhanced antioxidant defense. This is indicated by the fact that ex vivo measures of LDL antioxidant capacity and resistance to oxidation showed consistent and significant increases after consumption of the antioxidant-fortified spreads. This observation is consistent with published data showing that dosages of 25-200 mg {alpha}-tocopherol/d increased LDL lag time in a dose-dependent manner in healthy persons (16, 28). LDL is vulnerable to oxidative damage, and lag time has been suggested to provide an ex vivo measure of LDL peroxidation resistance. Paraoxonase is associated with HDL, but it can protect HDL and LDL by deactivating certain oxidized fatty acids (17, 29). Preliminary evidence suggests that paraoxonase 1 activity is modulated by oxidative stress (30) and antioxidant status (31). However, in the present intervention study, we did not observe a significant change in paraoxonase 1 activity after consumption of a combination of vitamin E and carotenoids.

LDL that has undergone oxidative modification is immunogenic, allowing antibody titers specific to malondialdehyde on oxidized LDL to be quantified in the plasma (32). To date, most of the published research that used this method was collected from patients with established CVD (32-34), and the effect of antioxidants on this variable is unknown. In the present study, concentrations of oxidized LDL were low at baseline and did not change significantly during the study, despite changes in other variables of lipid peroxidation. More data are required on the sensitivity and specificity of antibodies to oxidized LDL in healthy persons.

Malondialdehyde and F2{alpha}-isoprostanes are produced in the body as a consequence of the peroxidation of PUFAs that contain > 2 double bonds, or in the case of F2{alpha}-isoprostanes, mainly arachidonic acid (1). The validity of the malondialdehyde method has frequently come under scrutiny because malondialdehyde is not exclusively formed via lipid peroxidation (35). Although we observed a reduction in malondialdehyde in subjects assigned to spread B relative to the control group, this was most likely due to differences at baseline.

F2{alpha}-isoprostanes in plasma and associated urinary metabolites are recognized as valid markers of oxidative stress in vivo and are the preferred biomarker of lipid peroxidation (36). Persons consuming the spread that provided 111 mg {alpha}-tocopherol and 1.24 mg carotenoids/d had a 15% reduction in plasma total F2{alpha}-isoprostane concentrations during the 11-wk study. This finding shows that in healthy persons with normal concentrations of basal F2{alpha}-isoprostanes, improvements in the antioxidant defense system can result in measurable reductions in lipid oxidation.

The present study had several strengths, including a randomized, placebo-controlled design and a sample size sufficient to detect small changes with statistical confidence. Furthermore, the use of HPLC with tandem mass spectrometry to measure plasma F2{alpha}-isoprostane concentrations allowed the less volatile components to be measured without derivatization, resulting in lower CVs without compromising specificity and sensitivity (37). Our findings agree with the results of several vitamin E intervention studies that showed reductions in F2{alpha}-isoprostane metabolites in the urine (34-58%) after supplementation with 67-900 mg {alpha}-tocopherol/d (6-9, 38). However, not all intervention studies have recorded a benefit in F2{alpha}-isoprostane concentrations with comparable doses of vitamin E or a combination of dietary tocopherols and carotenoids (39-45). The findings of these studies may vary because they were conducted in subjects with differences in health (high cholesterol, diabetes, cystic fibrosis, or prothrombotic disorder) or smoking status or used different forms of vitamin E (2R-{alpha}-tocopherol, RRR-{alpha}-tocopherol, tocopherols, and tocotrienols), different dosages (7-2000 mg/d), or different time periods (5-60 d).

In conclusion, using a controlled, 11-wk intervention study with sufficient statistical power, we showed that the consumption of food products containing moderate amounts of vitamin E and selected dietary carotenoids can lead to measurable and significant improvements in antioxidant status and biomarkers of oxidative stress, such as F2{alpha}-isoprostane concentrations and resistance of LDL to oxidation ex vivo.


   ACKNOWLEDGMENTS
 
We thank LA Akerboom-Voogd, SY Gielen, M Jäkel, A Porcu, PC Remmerswaal, VIO Ringelberg-van Eerdenburg, M Slotboom, C van Tuijl, and RLC Weterings for recruitment and clinical procedures and our colleagues who produced, packed, and analyzed the spreads. We are indebted to J Gerrits, L van Buren, M van der Ham, and WGL van Nielen for their careful analytic work and to JNJJ Mathot for the quality control and coordination of the laboratory analyzes. We acknowledge the expert help of FHM van de Put in automating several key assays and JJ Schilt and A van Unnik for data management. Last, we thank the volunteers who participated in the study.

The study was conceived and designed by SAW, LBMT, JEU, and PJR. CRWCS coordinated the trial and JEU supervised the analytic aspects. SAJC gave significant advice on the methods used in the study. AW performed all statistical testing. JEU wrote the manuscript, and all authors were involved in interpreting the results and in critical revision of the paper. No authors had any advisory board affiliations.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Halliwell B, Gutteridge JM. Oxidative stress and disease. Free radicals in biology and medicine. Oxford, United Kingdom: Oxford University Press, 2000:617-783.
  2. Jialal I, Fuller CJ, Huet BA. The effect of alpha-tocopherol supplementation on LDL oxidation. A dose-response study. Arterioscler Thromb Vasc Biol 1995;15:190-8.[Abstract/Free Full Text]
  3. Fuller CJ, Chandalia M, Garg A, Grundy SM, Jialal I. RRR-{alpha}-Tocopheryl acetate supplementation at pharmacologic doses decreases low-density-lipoprotein oxidative susceptibility but not protein glycation in patients with diabetes mellitus. Am J Clin Nutr 1996;63:753-9.[Abstract/Free Full Text]
  4. Reaven PD, Herold DA, Barnett J, Edelman S. Effects of vitamin E on susceptibility of low-density lipoprotein and low-density lipoprotein subfractions to oxidation and on protein glycation in NIDDM. Diabetes Care 1995;18:807-16.[Abstract]
  5. Upritchard JE, Sutherland WH, Mann JI. Effect of supplementation with tomato juice, vitamin E, and vitamin C on LDL oxidation and products of inflammatory activity in type 2 diabetes. Diabetes Care2000; 23:733-8.[Abstract/Free Full Text]
  6. Ciabattoni G, Davi G, Collura M, et al. In vivo lipid peroxidation and platelet activation in cystic fibrosis. Am J Respir Crit Care Med2000; 162:1195-201.[Abstract/Free Full Text]
  7. Davi G, Ciabattoni G, Consoli A, et al. In vivo formation of 8-iso-prostaglandin F2{alpha} and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation 1999;99:224-9.[Abstract/Free Full Text]
  8. Davi G, Alessandrini P, Mezzetti A, et al. In vivo formation of 8-epi-prostaglandin F2{alpha} is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol 1997;17:3230-5.[Abstract/Free Full Text]
  9. Pratico D, Ferro D, Iuliano L, et al. Ongoing prothrombotic state in patients with antiphospholipid antibodies: a role for increased lipid peroxidation. Blood 1999;93:3401-7.[Abstract/Free Full Text]
  10. Gey KF. Vitamins E plus C and interacting conutrients required for optimal health. A critical and constructive review of epidemiology and supplementation data regarding cardiovascular disease and cancer.Biofactors 1998;7:113-74.[Medline]
  11. Committee for Dietary Reference Intakes. Vitamin E. Dietary reference intakes for vitamin C, vitamin E, selenium and carotenoids. Washington, DC: National Academy Press, 2000:186-324.
  12. McGavin JK, Mann JI, Skeaff CM, Chisholm A. Comparison of a vitamin E-rich diet and supplemental vitamin E on measures of vitamin E status and lipoprotein profile. Eur J Clin Nutr 2001;55:555-61.[Medline]
  13. Olmedilla B, Granado F, Southon S, et al. Serum concentrations of carotenoids and vitamins A, E, and C in control subjects from five European countries. Br J Nutr 2001;85:227-38.[Medline]
  14. Vuilleumier JP, Keck EJ. Fluorimetric assay of vitamin C in biological materials using a centrifugal analyser with fluorescence attachment.Micronutr Anal 1989;5:25-34.
  15. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem1996; 239:70-6.[Medline]
  16. Princen HM, van Duyvenvoorde W, Buytenhek R, et al. Supplementation with low doses of vitamin E protects LDL from lipid peroxidation in men and women. Arterioscler Thromb Vasc Biol 1995;15:325-33.[Abstract/Free Full Text]
  17. Watson AD, Berliner JA, Hama SY, et al. Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin Invest1995; 96:2882-91.
  18. Coolen SAJ, van der Ham M, Wiseman SA, Verhagen H. Fast and sensitive determination of free and esterified 15-F2t-isoprostanes in plasma with high performance liquid chromatography tandem mass spectrometry. Free Radic Res 2002;36:120 (abstr).
  19. Wong SH, Knight JA, Hopfer SM, Zaharia O, Leach CN Jr, Sunderman FW Jr. Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin Chem 1987;33:214-20.[Abstract/Free Full Text]
  20. Lopes-Virella MF, Stone P, Ellis S, Colwell JA. Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem 1977;23:882-4.[Abstract/Free Full Text]
  21. Friedewald WT, Levy RI, Fredrickson DS. Estimation of low-density lipoprotein cholesterol in plasma, without the use of preparative ultrcentrifuge. Clin Chem 1972;18:499-502.[Abstract]
  22. Castenmiller JJ, West CE, Linssen JP, het Hof KH, Voragen AG. The food matrix of spinach is a limiting factor in determining the bioavailability of beta-carotene and to a lesser extent of lutein in humans. J Nutr 1999;129:349-55.[Abstract/Free Full Text]
  23. Chug-Ahuja JK, Holden JM, Forman MR, Mangels AR, Beecher GR, Lanza E. The development and application of a carotenoid database for fruits, vegetables, and selected multicomponent foods. J Am Diet Assoc 1993;93:318-23.[Medline]
  24. Micozzi MS, Brown ED, Edwards BK, et al. Plasma carotenoid response to chronic intake of selected foods and beta-carotene supplements in men. Am J Clin Nutr 1992;55:1120-5.[Abstract/Free Full Text]
  25. van het Hof KH, Gartner C, Wiersma A, Tijburg LBM, Weststrate JA. Comparison of the bioavailability of natural palm oil carotenoids and synthetic beta-carotene in humans. J Argric Food Chem 1999;47:1582-6.
  26. Maskarinec G, Chan CL, Meng L, Franke AA, Cooney RV. Exploring the feasibility and effects of a high-fruit and -vegetable diet in healthy women. Cancer Epidemiol Biomarkers Prev 1999;8:919-24.[Abstract/Free Full Text]
  27. Zino S, Skeaff M, Williams S, Mann J. Randomized controlled trial of effect of fruit and vegetable consumption on plasma concentrations of lipids and antioxidants. BMJ 1997;314:1787-91.[Abstract/Free Full Text]
  28. van het Hof KH, Tijburg LBM, de Boer HS, Wiseman SA, Weststrate JA. Antioxidant fortified spread increases the antioxidant status. Eur J Clin Nutr 1998;52:292-9.[Medline]
  29. Durrington PN, Mackness B, Mackness MI. Paraoxonase and atherosclerosis. Arterioscler Thromb Vasc Biol 2001;21:473-80.[Abstract/Free Full Text]
  30. Sutherland WH, Walker RJ, de Jong SA, van Rij AM, Phillips V, Walker HL. Reduced postprandial serum paraoxonase activity after a meal rich in used cooking fat. Arterioscler Thromb Vasc Biol 1999;19:1340-7.[Abstract/Free Full Text]
  31. Aviram M, Rosenblat M, Billecke S, et al. Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants. Free Radic Biol Med 1999;26:892-904.[Medline]
  32. Holvoet P, Vanhaecke J, Janssens S, Van de WF, Collen D. Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation 1998;98:1487-94.[Abstract/Free Full Text]
  33. Holvoet P, Mertens A, Verhamme P, et al. Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease.Arterioscler Thromb Vasc Biol 2001;21:844-8.[Abstract/Free Full Text]
  34. Holvoet P, Van Cleemput J, Collen D, Vanhaecke J. Oxidized low density lipoprotein is a prognostic marker of transplant-associated coronary artery disease. Arterioscler Thromb Vasc Biol 2000;20:698-702.[Abstract/Free Full Text]
  35. Moore K, Roberts LJ. Measurement of lipid peroxidation. Free Radic Res 1998;28:659-71.[Medline]
  36. Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. Drug Metab Rev 2000;32:377-85.[Medline]
  37. Griffiths HD, Møller L, Bartosz G, et al. Biomarkers. Mol Aspects Med 2002;23:101-208.[Medline]
  38. Marangon K, Devaraj S, Tirosh O, Packer L, Jialal I. Comparison of the effect of alpha-lipoic acid and alpha-tocopherol supplementation on measures of oxidative stress. Free Radic Biol Med 1999;27:1114-21.[Medline]
  39. Dietrich M, Block G, Hudes M, et al. Antioxidant supplementation decreases lipid peroxidation biomarker F(2)-isoprostanes in plasma of smokers. Cancer Epidemiol Biomarkers Prev 2002;11:7-13.[Abstract/Free Full Text]
  40. Meagher EA, Barry OP, Lawson JA, Rokach J, FitzGerald GA. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA 2001;285:1178-82.[Abstract/Free Full Text]
  41. Patrignani P, Panara MR, Tacconelli S, et al. Effects of vitamin E supplementation on F(2)-isoprostane and thromboxane biosynthesis in healthy cigarette smokers. Circulation 2000;102:539-45.[Abstract/Free Full Text]
  42. Reilly M, Delanty N, Lawson JA, FitzGerald GA. Modulation ofoxidant stress in vivo in chronic cigarette smokers. Circulation 1996;94:19-25.[Abstract/Free Full Text]
  43. Simons LA, von Konigsmark M, Simons J, Stocker R, Celermajer DS. Vitamin E ingestion does not improve arterial endothelial dysfunction in older adults. Atherosclerosis 1999;143:193-9.[Medline]
  44. van den BR, van Vliet T, Broekmans WM, et al. A vegetable/fruit concentrate with high antioxidant capacity has no effect on biomarkers of antioxidant status in male smokers. J Nutr 2001;131:1714-22.[Abstract/Free Full Text]
  45. Weinberg RB, VanderWerken BS, Anderson RA, Stegner JE, Thomas MJ. Pro-oxidant effect of vitamin E in cigarette smokers consuming a high polyunsaturated fat diet. Arterioscler Thromb Vasc Biol 2001;21:1029-33.[Abstract/Free Full Text]
Received for publication July 2, 2002. Accepted for publication April 29, 2003.




This article has been cited by other articles:


Home page
 J. Nutr.Home page
K. M. Tomey, M. R. Sowers, X. Li, D. S. McConnell, S. Crawford, E. B. Gold, B. Lasley, and J. F. Randolph Jr
Dietary Fat Subgroups, Zinc, and Vegetable Components Are Related to Urine F2a-Isoprostane Concentration, a Measure of Oxidative Stress, in Midlife Women
J. Nutr., November 1, 2007; 137(11): 2412 - 2419.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
D. Sircar and P. V. Subbaiah
Isoprostane Measurement in Plasma and Urine by Liquid Chromatography-Mass Spectrometry with One-Step Sample Preparation
Clin. Chem., February 1, 2007; 53(2): 251 - 258.
[Abstract] [Full Text] [PDF]

Home page
 Arch Intern MedHome page
M. Cesari, S. B. Kritchevsky, B. J. Nicklas, B. W. H. J. Penninx, P. Holvoet, P. Koh-Banerjee, S. R. Cummings, T. B. Harris, A. B. Newman, and M. Pahor
Lipoprotein Peroxidation and Mobility Limitation: Results From the Health, Aging, and Body Composition Study
Arch Intern Med, October 10, 2005; 165(18): 2148 - 2154.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
S. Vega-Lopez, K.-J. Yeum, J. L Lecker, L. M Ausman, E. J Johnson, S. Devaraj, I. Jialal, and A. H Lichtenstein
Plasma antioxidant capacity in response to diets high in soy or animal protein with or without isoflavones
Am. J. Clinical Nutrition, January 1, 2005; 81(1): 43 - 49.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Upritchard, J. E
Right arrow Articles by Wiseman, S. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Upritchard, J. E
Right arrow Articles by Wiseman, S. A
Agricola
Right arrow Articles by Upritchard, J. E
Right arrow Articles by Wiseman, S. A

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS