Objective: To determine whether consumption of five portions of fruit and vegetables per day reduces the enhancement of oxidative stress induced by consumption of fish oil.
Subjects: A total of 18 free-living healthy smoking volunteers, aged 18–63 y, were recruited by posters and e-mail in The University of Reading, and by leaflets in local shops.
Design: A prospective study.
Setting: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK.
Interventions: All subjects consumed a daily supplement of 4 × 1 g fish oil capsules for 9 weeks. After 3 weeks, they consumed an additional five portions of fruits and vegetables per day, and then they returned to their normal diet for the last 3 weeks of the study. Fasting blood samples were taken at the ends of weeks 0, 3, 6 and 9.
Results: The plasma concentrations of ascorbic acid, lutein, β-cryptoxanthin, α-carotene and β-carotene all significantly increased when fruit and vegetable intake was enhanced (P<0.05). Plasma concentrations of α-tocopherol, retinol and uric acid did not change significantly during the period of increased fruit and vegetable consumption. Plasma oxidative stability, assessed by the oxygen radical absorbance capacity (ORAC) assay, also increased from weeks 3–6 (P<0.001) but not in association with increases in measured antioxidants. Lag phase before oxidation of low-density lipoprotein (LDL) significantly decreased in the first 3 weeks of the study, reflecting the incorporation of EPA and DHA into LDL (P<0.0001). Subsequent enhanced fruit and vegetable consumption significantly reduced the susceptibility of LDL to oxidation (P<0.005).
Conclusion: Fish oil reduced the oxidative stability of plasma and LDL, but the effects were partially offset by the increased consumption of fruit and vegetables.
Sponsorship: This study was supported by funding from the Ministry of Agriculture, Fisheries and Food, and from Boots plc.
There is considerable epidemiological evidence that a diet rich in fruit and vegetables may be protective against coronary heart disease (Ness & Powles, 1997; Law & Morris, 1998). There is also increasing experimental evidence that oxidative stress, particularly oxidation of low-density lipoproteins (LDL) plays a significant role in the pathogenic pathway of atherosclerosis, the primary cause of coronary heart disease (Reaven & Witztum, 1996). Fruit and vegetables are a rich source of a wide range of antioxidant compounds such as ascorbic acid, carotenoids and phenolics. These compounds have been shown to effectively scavenge reactive oxygen species and inhibit lipid peroxidation in vitro (Frei, 1991; Lim et al, 1992; Vinson et al, 1995). Increasing the intakes of a range of dietary antioxidants through increased fruit and vegetable consumption may produce effects that are not necessarily seen for individual antioxidants.
The observed protective effect of fruits and vegetables has prompted national bodies to recommend consumption of five portions of fruit and vegetables per day (Department of Health, 1994). However, the beneficial effects of this level of consumption have not been examined. Studies to date have concentrated on dietary enhancement of volunteers with carotenoid-rich vegetables (Hininger et al, 1997; van het Hof et al, 1999; Bub et al, 2000; Chopra et al, 2000) or provided subjects with a highly controlled diet with a very high fruit and vegetable component (9–10 servings per day) (Cao et al, 1998; Miller et al, 1998). Previous studies attempting to increase fruit and vegetable consumption in free-living volunteers demonstrated increases in plasma antioxidants (Zino et al, 1997), but no significant increase in plasma antioxidant capacity (Record et al, 2001). Hence, there is currently a lack of knowledge about the effects of a moderate increase in fruit and vegetable consumption upon plasma markers of antioxidant status in free-living healthy subjects. Since no effects have previously been reported for consumption of five portions of fruits and vegetables per day, subjects who were smokers were selected for this study, and they were supplemented with fish oil. These subjects would represent a group whose level of oxidative stress was at the upper end of the normal range, and it was considered likely that this would make the study more sensitive to the beneficial effects of antioxidant components of fruits and vegetables.
Subjects and methods
A total of 18 (10 female, 8 male) smoking subjects (mean age 37.8 (range 18–60) y; mean BMI 25.0 (18.5–28.6) kg/m2) were recruited from the Reading area. The sample size was based on a power calculation using LDL lag time increases observed in a previous study with similar protocol (Hininger et al, 1997). A one-tailed t-test confirmed that 16 subjects would give 80% power to detect a 7 min change in LDL lag time at the 5% significance level. Subjects were eligible if they met the following inclusion criteria: (i) apparently healthy as indicated by a general medical questionnaire, (ii) habitual consumption of ≤3 portions of fruit or vegetables per day as assessed by 3-day diet diary; (iii) currently smoking ≥10 cigarettes per day for ≥1 y and no intention to stop during the study; (iv) no use of vitamin, mineral or fish oil supplements or medications; (v) a BMI ≤30 kg/m2; (vi) moderate weekly alcohol consumption (<28 U for men, <21 for women); (vii) less than 3 h of vigorous exercise per week. Those considered suitable attended a briefing session at the Hugh Sinclair Unit of Human Nutrition to have the study fully explained to them. Ethical permission for the study was obtained from the Ethics and Research Committee of the University of Reading. All volunteers provided written consent for their participation and their Medical Practitioners were informed of their participation in the trial.
The study lasted a total of 9 weeks, with three treatment periods. Plans to use a fish oil only supplemented group for 9 weeks as control were considered, but abandoned on ethical grounds because of the risk to health of an extended period of oxidative stress. After a baseline (week 0) blood sample was taken, the subjects were supplemented with four Boots Superstrength fish oil capsules per day. These provided a daily supplement of 1.2 g EPA and 0.83 g DHA in 4 g of fish oil. Subjects continued taking the fish oil supplement throughout the study. After 3 weeks, a second blood sample was taken and subjects increased their fruit and vegetable intake by five portions per day for a further 3 weeks. Another blood sample was then taken and followed by a 3-week wash out period during which volunteers continued to take the fish oil supplement but returned to their original diet. Subjects were asked to complete a 3-day diet diary (2 weekdays and 1 weekend day) in the middle week of each 3-week treatment period (weeks 2, 5 and 8). The importance of not altering their habitual diet in weeks 0–3 and 7–9 was stressed and volunteers were encouraged to eat their habitual diet as far as possible in weeks 4–6.
Supply of study fruit and vegetables
Fruit and vegetables for the study were selected to provide a wide range of antioxidant classes, in a form that could be eaten with minimal disruption to habitual meal preparation. The fruits and vegetables were bought from a local supermarket and individual portions were weighed out before being delivered to volunteers' houses within the same day. Each delivery supplied volunteers with fruit and vegetables to be consumed over a 6-day period and comprised two cycles of the suggested 3-day diet seen in Table 1. Volunteers were advised to eat fruit during the day as snacks or as part of a meal and to supplement their usual evening meal with vegetables and/or salad/soup. Additionally, volunteers were instructed to ensure maximum retention of antioxidants in the frozen vegetables by using microwave cooking or by boiling them for a short time in the minimum volume of water. Addition of frozen vegetables, onion or pepper to habitual meals such as stir fries/curries/pasta sauces was also suggested, as was the inclusion of raw onion/red pepper in salads. Subjects did not have to follow the suggested 3-day diet but were asked to eat all supplied fruit and vegetables before the next delivery. Subjects recorded fruit and vegetables consumed during weeks 4–6 in a diary. Volunteers were asked to identify which fruit and vegetables had been provided for them, and which habitual/additional fruit and vegetables had been eaten.
At baseline (week 0) and at the end of weeks 3, 6 and 9, blood samples were collected from subjects after an overnight fast. For analysis of ascorbic acid and plasma antioxidant capacity, blood was collected in vacutainers containing lithium heparin and centrifuged at 3000 rpm (1560 × g) for 10 min at 4°C. The plasma samples were deproteinised with an equivalent volume of 10% metaphosphoric acid (samples for ascorbic acid analysis) or 0.5 M perchloric acid (samples for determination of plasma antioxidant capacity) and frozen at −80°C within 30 min of collection. Blood for all other analyses was collected in EDTA vacutainers, centrifuged, and frozen, within 1 h of collection (plasma for LDL oxidation analysis was treated with 50% sucrose solution before freezing to give a final concentration of 10% sucrose).
Dietary nutrient intakes for each of the three treatment periods were estimated from the 3-day diet diaries using the FOODBASE (v 1.3; Institute of Brain Chemistry, London) nutrient database. Plasma total cholesterol, HDL cholesterol and triglyceride concentrations were measured using an IL Monarch Automatic Analyser (Instrumentation Laboratories, Warrington, Cheshire, UK) and enzymatic colorimetric kits (Instrumentation Laboratories Ltd). Plasma LDL cholesterol was calculated using the Friedewald formula (Friedewald et al, 1972), modified for molar concentrations.
Fatty acids in plasma phospholipids were measured as methyl esters using gas chromatography with flame ionisation detection (Leigh-Firbank, 2000). Plasma ascorbic acid and uric acid concentrations were measured by HPLC using the method described by Liau et al (1993) with slight modifications. The equipment used comprised a Dionex P580 pumping system, ASI-100T automated sample injector and PDA-100 photodiode array detector (Sunnyvale, CA, USA). The sample (20 μl) was injected onto a 5 μm Nucleosil (250 mm × 4.6 mm ID) C18 analytical column protected by a C18 guard column (Hichrom, Reading). The column was in a Dionex STH 585 column thermostat set at 15°C and samples within the autosampler were kept at 4°C to prevent degradation. Wavelengths of 245 and 285 nm were used to quantify ascorbic acid and uric acid, respectively (Ross, 1994).
Plasma α-tocopherol, retinol and carotenoid concentrations were quantified using the HPLC equipment described above and a modified version of a method described previously (Aebischer et al, 1999). Lipid-soluble antioxidant concentrations were then expressed relative to the total plasma lipid content, using the sum of cholesterol and triglyceride as an estimate of total lipid (Thurnham et al, 1986).
Plasma antioxidant capacity was measured using the manual version of the ORAC assay as described by Cao & Prior (1999). One run of the ORAC assay comprised one blank, one standard and eight plasma samples. Phosphate buffer (2625 μl) and β-phycoerythrin (150 μl) were added to each of 10 fluorimeter cuvettes. The cuvettes were covered to prevent evaporation and preincubated for 15 min in a water bath set at 37°C. A volume of 150 μl of buffer (blank), 20 μM trolox (standard) or diluted plasma (sample) was then added and the cuvette was inverted five times. The fluorescence was measured before the addition of AAPH, using a Perkin-Elmer 3000 fluorescence spectrophotometer (emission 565 nm, excitation 540 nm). This fluorescence was taken as the initial fluorescence. The reaction was then started by the addition of 75 μl of AAPH solution and the cuvette was inverted a further five times. Cuvettes were stored in the water bath at 37°C between measurements and the fluorescence was recorded every 5 min until the fluorescence was less than 5% of the initial value. Analysis of four pooled plasma samples gave an intra-assay coefficient of variation of 1.2%.
LDL was isolated from plasma using ultracentrifugation (Leigh-Firbank, 2000). The susceptibility of LDL to copper-induced oxidation was measured as the lag phase before oxidation by monitoring the increase in conjugated dienes at 234 nm (Esterbauer et al, 1989). A volume of isolated LDL, corresponding to 100 μg of protein, was pipetted into a quartz cuvette and diluted to 1900 μl with Dulbeco's phosphate buffer solution (PBS). Copper sulfate (0.1 mM in distilled water, 100 μl) was added to each sample cuvette and mixed by gentle inversion. The concentrations of LDL and copper in the cuvette were 50 μg protein/ml and 5 μM, respectively. The conjugated diene formation was then measured at 37°C, against matched cuvettes containing a blank of 100 μl distilled water and 1900 μl Dulbeco's PBS solution. Absorbance at 234 nm was measured every 2 min for 180 min using a Perkin-Elmer Lamda bio 20 UV/Vis Spectrometer. This was connected to an IBM compatible personal computer running UVWinlab software and had the capacity to measure eight samples simultaneously. Data were then transferred to Microsoft Excel and the lag phase and maximum peroxidation rate were calculated.
All four LDL samples corresponding to a particular volunteer were isolated and oxidised within the same sample batch. Isolation and oxidation of six pooled plasma samples gave an intra-assay variability of 4.9%. The CV was <5% for the intra-assay variation for all analyses.
Data are presented in the tables as mean±s.e.m. Statistical analysis was performed using SPSS (version 10.0). The normality of each variable distribution at each time point was checked using the Shapiro-Wilk's W-Test. Normally distributed data were tested using repeated measure ANOVA to assess if there were any differences within the biological measurements with time. A P-value of ≤0.05 signified significant differences identified using matched paired, two-sided t-tests. Friedman tests and Wilcoxon signed ranks tests were used for non-normally distributed variables. Again a P-value of ≤0.05 was accepted as statistically significant. Spearman correlation coefficients (and P-values) were calculated to assess statistical associations between changes in the outcome parameters.
The mean estimated daily intake of fruit and vegetables and nutrients during the three treatment periods are shown in Table 2. Analysis of the daily fruit and vegetable diary in weeks 4–6 showed good compliance with 92% of supplied portions of fruit and vegetables being consumed. Dietary intervention with five portions of fruit and vegetables per day in weeks 4–6 also produced a 4.9% decrease in the dietary energy obtained from fat (P=0.028) with 2.9% of this decrease because of a reduction in the energy obtained from saturated fat (P=0.0002). There was a significant decrease in dietary cholesterol when fruit and vegetable intake was increased (P=0.026). In contrast, estimated dietary fibre (P=0.022) and folic acid (P=0.043) intakes increased significantly when fruit and vegetable consumption was enhanced. As expected, intakes of vitamin C (P=0.0004) and carotenoids (P<0.0001) increased significantly when volunteers increased their fruit and vegetable intake. Vitamin E intakes remained unchanged throughout the 9 weeks of the study.
Concentrations of plasma lipids and the percentage of EPA and DHA in plasma phospholipids are shown in Table 3. The tendency of LDL cholesterol to increase during fish oil supplementation meant that fasting plasma LDL levels at week 9 were significantly higher than at week 0 (P=0.042). Fasting triglyceride levels were significantly reduced from 1.43 to 1.03 mmol/l between weeks 0 and 3 (P=0.0095) and then remained unchanged between weeks 3 and 9. The percentage of n-3 fish oil fatty acids, EPA and DHA in plasma phospholipids increased significantly between weeks 0 and 3 (P<0.006) and then remained stable for the remainder of the study.
Levels of antioxidants analysed in plasma are given in Table 4. Fasting plasma concentrations of ascorbic acid significantly increased when subjects increased their fruit and vegetable intake from weeks 3 to 6 (P=0.012) and decreased again (P=0.0004) when subjects returned to their habitual diet. Similar patterns were observed for plasma concentrations of carotenoids. The small increase observed for lycopene did not reach a level of statistical significance (P=0.10) but highly significant increases in plasma concentrations of lutein (P=0.007), β-cryptoxanthin (P<0.001), α-carotene (P=0.006) and β-carotene (P=0.047) were observed between weeks 3 and 6. Plasma concentrations of β-cryptoxanthin and α-carotene at week 9 also remained significantly higher than week 3 (P<0.011). Fasting plasma concentrations of uric acid and α-tocopherol showed no significant changes between weeks 0 and 9. Retinol was significantly higher at weeks 3, 6, and 9 than at week 0 (P=0.022). The fish oil used as a dietary supplement did not contain significant concentrations of α-tocopherol or retinol.
Fasting plasma antioxidant activities and lag phases of isolated LDL are shown in Table 5. Plasma ORAC levels increased significantly when fruit and vegetable intake was increased between weeks 3 and 6 (P<0.001) and then significantly decreased (P=0.002) when subjects returned to their habitual diet in weeks 6–9. Plasma ORAC values at week 9 were significantly higher than at week 0 (P=0.006) but not significantly higher than those at week 3 (P=0.078). Supplementation with fish oil only between weeks 0 to 3 produced a significant decrease in LDL lag phase (P<0.001). When fruit and vegetable intakes were increased between weeks 3 and 6 there was a significant increase in LDL lag phase (P=0.003). However, the lag phase at week 6 was still lower than the lag phase at week 0, although the difference just failed to reach significance (P = 0.051). A decrease in lag phase was observed between weeks 6 and 9 when subjects returned to their habitual diets. However, this did not reach a level of statistical significance (P=0.16) and LDL lag phases at week 9 were still significantly higher than at week 0 (P=0.002).
The 7% increase in plasma LDL concentration and 28% decrease in plasma triglyceride concentration agree well with the 5–10% increase in LDL and 20–30% decrease in triglycerides observed in a recent review of EPA/DHA supplementation studies (Harris, 1997). These observations, together with the observed increases in plasma phospholipid EPA and DHA concentrations, indicate that subjects complied with the fish oil supplementation protocol. Fish oil supplementation also produced a 15% reduction in LDL lag phase between weeks 0 and 3. This potentially adverse effect is a measure of the increased susceptibility of LDL to oxidation because of the incorporation of highly unsaturated n-3 PUFA into the phospholipids of the LDL particles, as observed in previous studies (Wander et al, 1996; Sorensen et al, 1998; Foulon et al, 1999). One of the effects of the increased consumption of fruits and vegetables was a 9.7% reduction in caloric intake, with a significant reduction in percent energy from fat from 34.9 to 30.0%. There is some evidence that a high-fat diet induces endothelial dysfunction, which is associated with oxidative stress (Cuevas et al, 2000). However, it is unlikely that the change in energy intake from dietary fat had a significant effect on oxidative stability of LDL or plasma ex vivo. The effects of dietary antioxidants on these parameters are likely to be much more significant.
The baseline plasma antioxidant concentrations were generally lower than those reported by a multicentre study quantifying plasma antioxidant concentrations in healthy nonsmoking subjects (Olmedilla et al, 2001). Plasma ascorbic acid, β-carotene and β-cryptoxanthin concentrations were decreased compared to the literature values for nonsmoking subjects. However, lower plasma concentrations of antioxidants have previously been observed in smoking subjects (Lykkesfeldt et al, 2000; Ma et al, 2000). The observed increase in plasma retinol from weeks 0 to 3 was unexpected, since the fish oil was not a liver extract and did not contain a significant concentration of retinol. The fish oil may have caused some mobilisation of hepatic retinol. There were no significant changes because of the initial 3 weeks of fish oil consumption in any of the other plasma antioxidant concentrations compared to baseline measurements, suggesting that, with the exception of retinol, the increases in dietary n-3 PUFA did not lead to changes in circulating concentrations of antioxidants.
Increasing the fruit and vegetable intake of the subjects resulted in a significant 7.4 min increase in LDL lag phase, indicating an increased resistance to copper-induced oxidation ex vivo. A similar increase was observed by Hininger et al (1997), who supplemented smokers with a variety of carotenoid-rich vegetables. In our study, these LDL changes coincided with significant increases in the plasma concentrations of lutein, β-cryptoxanthin, α-carotene and β-carotene. Plasma concentrations of lycopene also increased but this increase did not reach a level of statistical significance. Additional lycopene would mainly come from the tomato soup, which would provide about 1.2 mg lycopene per day, based on the tomato content (O'Neill et al, 2001). The lack of change in plasma levels of retinol and α-tocopherol when fruit and vegetable intake was increased agrees with previous intervention studies. α-Tocopherol was only present at low levels in the fruit and vegetables used in the study, and only about 1.5 mg/day α-tocopherol would be consumed from this source. Displacement of other foods in the diet would reduce the net change in α-tocopherol intake compared to the run-in period. The plasma level of α-tocopherol decreased slightly, but not significantly, during the fruit and vegetable intervention period. Reduction in plasma α-tocopherol would not necessarily lead to a reduction in lag phase for LDL oxidation since α-tocopherol may have a pro-oxidant effect by reducing copper (II) to copper (I) (Kontush et al, 1996). Metabolism of carotenoids did not increase the retinol level significantly. Previous studies have observed that the increases in plasma carotenoid levels reflect the changes seen in LDL concentrations (Chopra et al, 2000). However, in our study there were no significant changes in LDL concentration from week 3 to 6. Hence, it seems reasonable to suggest that the increases in LDL lag phase may well be because of increases in concentrations of the antioxidants within LDL. However, changes in plasma concentrations of β-carotene were actually negatively correlated with the changes in lag phases between weeks 3 and 6 (R2=−0.525, P=0.025). No other correlations between changes in individual or total plasma carotenoid levels and the changes in lag phase LDL were observed between weeks 3 and 6. However, recent carotenoid supplementation studies have failed to show any significant decreases in susceptibility to oxidation (Carroll et al, 2000; Hininger et al, 2001). Hence, the increases in LDL lag phases seen in carotenoid-rich vegetable supplementation studies are probably due to other antioxidant components within the vegetables. Carotenoids may simply be markers of fruit and vegetable consumption and hence markers for the true protective agents (Halliwell, 1999).
The only parameter to correlate significantly with the increases in LDL lag phases between weeks 3 and 6 was the corresponding increase in plasma ORAC values (P=0.007). However, the increases in plasma ORAC values were not correlated with corresponding increases in ascorbic acid. This may be explained by the small contribution of ascorbic acid to total plasma ORAC capacity. It has been estimated that approximately 50% of the total plasma ORAC value is from compounds that are as yet unidentified (Rice-Evans, 2000). Phenolic compounds such as flavonoids in fruit and vegetables may also have contributed to the observed increase in plasma ORAC values after increased fruit and vegetable consumption. However, determining the extent of this contribution is impossible at present, as very little is known about the absorption and metabolism of flavonoids. Phenolic components in the fruits and vegetables consumed in this study include flavonol conjugates and glycosides, flavanones, anthocyanin conjugates and glycosides, and hydroxycinnamate derivatives (Proteggente et al, 2002). It is likely that water-soluble antioxidant components that contribute to the oxidative stability of plasma bind to the LDL and contribute to its stability. Dietary phenolic compounds incubated in plasma for 1 h have been shown to inhibit oxidation of subsequently isolated LDL with the extent of inhibition being strongly correlated with the protein binding affinity of the phenolic compound (Wang & Goodman, 1999).
The study was performed in the summer months (May–July). Consumption of salads and summer fruits in the background diet at this time of year would be higher than in the winter months. However, the mean dietary consumption of fruits and vegetables was 1.58 ± 0.24 portions per day in the initial period, and it increased to 5.68 ± 0.49 during the supplementation period. Although the diet diaries only covered a 3-day period, and therefore only provided a rough picture, analysis of the diaries indicated that marked changes in nutrient intake can occur when high levels of fruit and vegetables are incorporated into the diets of free-living volunteers. Indeed, the diet in weeks 3–6 was significantly lower in saturated fat and cholesterol, high intakes of which have been linked with coronary heart disease. There was also a significant increase in dietary fibre intake when fruit and vegetable consumption was increased by five portions per day. Dietary fibre from fruit and vegetable sources has been shown to have hypocholesterolemic action in humans (Lampe, 1999). Mean estimated folic acid intake also significantly increased in weeks 3–6. Deficiency of this nutrient has been linked to hyperhomocysteinaemia, an independent risk factor for coronary heart disease (Broekmans et al, 2000). Increased intakes of fruit and vegetables have been shown to decrease plasma homocysteine concentration although this was not measured in the present study.
Fish oil causes a reduction in the oxidative stability of LDL ex vivo, but the present study has shown that the effect of 4 g/day fish oil is partially offset in smokers by dietary supplementation with five portions of fruit and vegetables per day. Plasma stability ex vivo as assessed by the ORAC method was not reduced by fish oil, but fruit and vegetables caused a significant increase in the plasma stability. The improvement in the oxidative stability of the plasma and LDL after 3 weeks of enhanced fruit and vegetable consumption could not be related to plasma carotenoids or vitamin A, C or E concentrations. The antioxidants responsible were not identified, but dietary phenolics such as flavonoids or their metabolites are likely to be at least partially responsible.
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Thanks are due to the volunteers for their time and patience, and to Julie Lovegrove, Anne-Marie Minihane and Kim Jackson for phlebotomy, and to Jan Luff for help on clinic days.
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Roberts, W., Gordon, M. & Walker, A. Effects of enhanced consumption of fruit and vegetables on plasma antioxidant status and oxidative resistance of LDL in smokers supplemented with fish oil. Eur J Clin Nutr 57, 1303–1310 (2003). https://doi.org/10.1038/sj.ejcn.1601692
- LDL oxidation
- fruit and vegetables
- fish oil supplementation
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