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American Journal of Clinical Nutrition, Vol. 76, No. 5, 950-960, November 2002
© 2002 American Society for Clinical Nutrition

Original Research Communication

High intakes of vegetables, berries, and apples combined with a high intake of linoleic or oleic acid only slightly affect markers of lipid peroxidation and lipoprotein metabolism in healthy subjects1,2,3

Riitta Freese, Georg Alfthan, Matti Jauhiainen, Samar Basu, Iris Erlund, Irma Salminen, Antti Aro and Marja Mutanen

1 From the Division of Nutrition, University of Helsinki (RF and MM); the Biomarker Laboratory (GA IE, IS, and AA) and the Department of Molecular Medicine (MJ), National Public Health Institute, Helsinki; and the Section of Geriatrics, Faculty of Medicine, Uppsala University, Uppsala, Sweden (SB).

2 Supported by the Ministry of Agriculture and Forestry, the University of Helsinki, the Academy of Finland (project 10141399), the Juho Vainio Foundation, the Finnish Horticultural Products Society, the Geriatrics Research Foundation (Sweden), and Raisio Margarine Ltd. All oils and spreads used were donated by Raisio Margarine Ltd (Raisio, Finland).

3 Reprints not available. Address correspondence to R Freese, University of Helsinki, Department of Applied Chemistry and Microbiology, Division of Nutrition, PO Box 27 (Viikki, Latokartanonkaari 9), University of Helsinki, FIN-00014 Finland. E-mail: riitta.freese{at}helsinki.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: A high consumption of vegetables and fruit is associated with decreased risk of ischemic heart disease and several cancers. The pathophysiology of these diseases involves free radical mechanisms. Diet may either enhance or diminish oxidative stress in the body.

Objective: We studied the effects of high and low intakes of vegetables, berries, and apples on markers of lipid peroxidation and lipoprotein metabolism in subjects consuming diets high in linoleic or oleic acid.

Design: For 6 wk, healthy men and women (n = 77; aged 19–52 y) consumed 1 of 4 controlled isoenergetic diets rich in either linoleic acid (11% of energy) or oleic acid (12% of energy) and containing either 815 or 170 g vegetables, berries, and apples/10 MJ. Nineteen healthy volunteers served as control subjects. Several markers of dietary compliance (plasma fatty acids, vitamin C, carotenoids, and quercetin), lipid peroxidation [ex vivo LDL oxidation, plasma and LDL thiobarbituric acid–reactive substances, paraoxonase (EC 3.1.8.1), and urinary 8-iso-prostaglandin F2{alpha}], and lipoprotein metabolism (plasma lipids, apolipoproteins, and lipid transfer protein activities) were measured from samples collected before and at the end of the experimental period.

Results: Plasma fatty acid composition and antioxidant concentrations showed that compliance with the diets was good. However, there were no significant differences between the diets in the markers of lipid peroxidation and lipoprotein metabolism.

Conclusions: In healthy volunteers with adequate vitamin intakes, 6-wk diets differing markedly in the amounts of linoleic and oleic acid and vegetables, berries, and apples did not differ in their effects on lipid peroxidation or lipoprotein metabolism.

Key Words: Monounsaturated fatty acids • polyunsaturated fatty acids • vegetables • berries • apples • lipid peroxidation • lipoprotein metabolism • antioxidants • human intervention • diet


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diets rich in vegetables and fruit have been associated with decreased risk of cardiovascular diseases in several ecologic and epidemiologic studies (1). The pathogenesis of atherosclerosis most likely involves free radical–mediated processes; thus, the preventive effects of plant products have been largely allocated to their high content of antioxidants, such as vitamin C, tocopherols, carotenoids, and polyphenolic compounds. The results of supplementation trials, however, have not confirmed that antioxidants per se are responsible for the prevention or regression of atherosclerosis or ischemic heart disease (IHD) (2). It seems clear that no single dietary compound acts as the antiatherogenic substance; a combination of different food components is needed, possibly even in the context of the natural food matrix. Furthermore, mechanisms other than antioxidant effects are probably also involved in the protection from atherosclerosis. Vegetables, fruit, and berries contain numerous compounds, some possibly still unknown, that may contribute to their beneficial effects. To track the possible mechanisms, a "food approach" rather than the study of dietary supplements may be useful for elucidating which intermediate markers associated with the development of IHD are affected by vegetables and fruit (3, 4).

Some of the protective effects of vegetables and fruit may be due to their low content of energy and saturated fat; additionally, some studies suggest that fruit and vegetable intake may merely be a marker of an otherwise healthful behavior (5, 6). For this reason, controlled interventions with standardized lifestyle and dietary factors are needed to study the effects of vegetables and fruit on factors associated with the risk of IHD. There are indications that the polyunsaturated fatty acid (PUFA) linoleic acid (18:2n-6) acts as a substrate enhancing lipid peroxidation (7, 8) and oxidative modifications of DNA (9) in humans. Thus, the aim of the present study was to investigate how variables associated with the development of IHD and cancer are affected when a diet high in vegetables is combined with high intakes of either linoleic acid or oleic acid (18:1n-9). In the present article, we report the design of this strictly controlled dietary intervention study and the results from analyses of markers of antioxidant status, lipid peroxidation, and lipoprotein metabolism.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The dietary intervention was carried out at the Division of Nutrition, University of Helsinki. The Ethics Committee of the Faculty of Agriculture and Forestry approved the study protocol.

Subjects
Volunteers were recruited from among the students and employees of the Viikki campus of the University of Helsinki. The candidates’ health status was checked through a questionnaire and screening tests (entailing measurement of body weight and height, blood pressure, urinary glucose, and protein). Eighty apparently healthy men and women were chosen for the study; 4 of them were regular smokers. Oral contraceptives were used by 51% of the women, who were evenly distributed among the groups. The women and men were separately randomly assigned to 4 treatment groups. The subjects gave their informed consent after carefully reading the study protocol. They were, however, free to leave the study any time if they wished. Three women dropped out during the experimental period. The study foods were free to the subjects, who otherwise received no payment. In addition to the study subjects, 19 healthy volunteers who did not intentionally change their dietary habits during the intervention were recruited as control subjects. Descriptive characteristics of the final treatment and control groups are presented in Table 1Go.


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  		TABLE 1 . Characteristics of the subjects at baseline and mean dietary energy intakes during the experimental period1
 
Dietary intervention
Before the experimental period, the habitual diet of all the subjects was checked with 3-d food records, with the use of a picture booklet for estimating portion sizes (10). Dietary composition was calculated from the Fineli database of Finnish foods (National Public Health Institute, Finland; 11). These data were used to check for possible differences in baseline diets between the treatment groups and to evaluate the effect of the experimental diets on the intakes of vegetables, berries, and other fruit (Table 2Go).


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  		TABLE 2 . Habitual dietary intakes of energy, nutrients, and some foods and drinks in the preexperimental period1
 
The dietary intervention was carried out in parallel so that each subject consumed one experimental diet for 6 wk. The subjects lunched every weekday at the Division of Nutrition. After lunch they took food with them for the rest of the day and for the next morning; on Fridays, they took food for the whole weekend. The experimental foods provided 90% of daily energy; the subjects chose the other 10% of energy from a limited list of foods free of fat and cholesterol and low in antioxidants (sweets, sugar, grain products, limited amount of alcoholic beverages excluding red wine and berry wines, soft drinks, and selected vegetables and fruit). These foods were scored for their energy content, and the given sum of scores had to be consumed every day. Alcohol consumption was limited to <= 2% of energy.

The subjects recorded daily all freely chosen foods as well as uneaten foods in their diaries. The subjects also recorded their intake of coffee and tea and any medications and recorded symptoms or illnesses that emerged during the study. Female subjects recorded their intake of oral contraceptives and the dates of menstruation. These data were used to ensure that there were no differences between the menstrual phases of the female subjects in different groups at the beginning of the experimental period. All volunteers were asked to keep their physical activity and their coffee and tea intake at the preexperimental level during the experimental period. The control subjects consumed their habitual diets and kept their physical activity stable throughout the study and gave the same blood and urine samples as did the participating subjects. The stability of the control subjects’ habitual diet was checked by 3-d dietary records before and at the end of the experimental period and by a 2-d food record during the experimental period.

The experimental diets consisted of usual foods. The 3-wk menu was calculated for 10 energy levels (7–16 MJ) and was rotated twice. The initial energy levels were determined by calculating resting metabolic rate (12, 13) and multiplying it by an activity level coefficient estimated on the basis of self-reported physical activity in the recruitment questionnaire. The subjects were weighed twice weekly and energy levels were adjusted accordingly to keep body weight constant during the experimental period.

The diets differed in their fatty acid composition and in the amounts of vegetables, berries, and apples they contained (Table 3Go). Diets P1 and P2 were both high in linoleic acid but were either low in vegetables (P1) or high in vegetables, berries, and apples (P2). Diets M1 and M2 were high in oleic acid and contained a small amount of vegetables (M1) or high amounts of vegetables, berries, and apples (M2). Differences in the fatty acid compositions were achieved by using different oils as spreads and in salad dressings and for cooking and baking. All oils and spreads were generously donated by Raisio Margarine Ltd (Raisio, Finland).


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  		TABLE 3 . Calculated fatty acid compositions and intakes of some antioxidants and certain foods in the experimental diets1
 
The oil used in the P1 and P2 diets was high–linoleic acid sunflower oil, and that used in the M1 and M2 diets was high–oleic acid sunflower oil and refined olive oil. The high–oleic acid sunflower oil was redeodorized to lower the ratio of {alpha}-tocopherol to PUFA in diets M1 and M2 to be closer to that in diets P1 and P2. The analyzed fatty acid distributions [saturated fatty acids, monounsaturated fatty acids (MUFAs), and PUFAs as a percentage of total fatty acids] and {alpha}-tocopherol contents of the oils were as follows: high–linoleic acid sunflower oil, 12%, 20%, and 68% and 63 mg/100 g; high–oleic acid sunflower oil, 11%, 80%, and 9% and 5 mg/100 g; and olive oil, 15%, 76%, and 9% and 14 mg/100 g, respectively.

All berries and apples as well as most vegetables were of Finnish origin and were included in the diet as such or as little processed as possible (vegetables in salads or in lightly cooked side dishes; berries uncooked in berry juice creams or in berry pies). The energy provided by vegetables, berries, and apples in diets P2 and M2 was replaced by foods rich in sugar or starch (eg, sugar, wheat bread, pasta, rice, and potatoes) in diets P1 and M1. Fiber intake was thus not balanced between the diets. The intakes of total fat (33% of energy), saturated fat (10% of energy), n-3 fatty acids (0.4% of energy), protein (13% of energy), total carbohydrates (54% of energy, including fiber), and cholesterol (230–240 mg/10 MJ) were calculated to be similar in all diets. Differences between the diets were designed to be in fatty acid and antioxidant compositions (Table 3Go). Diet calculations were carried out with the FLAMINGO software program (version 1.0; Dipper Software, Helsinki), which uses the Fineli database.

The study was carried out as blinded as possible, so that the subjects and the kitchen personnel were unaware of the identity of the fats. The laboratory personnel were blinded for the treatments. Dietary compliance with the experimental diets was checked from the study diaries and by analyzing biochemical compliance markers.

Blood sampling and urine collections
Blood samples were collected before and at the end of the dietary period. The samples were collected by trained laboratory nurses from the antecubital vein with minimal stasis after the subjects had fasted overnight. Vacuum EDTA and serum tubes were used (Venoject II; Terumo Europe, Leuven, Belgium). Plasma or serum was separated by centrifugation (1000 x g, 10 min, room temperature) between 30 and 60 min after blood sampling. Aliquots were either immediately frozen and stored at -70 °C or were delivered on ice to the National Public Health Institute for analyses. Samples for plasma ascorbic acid analyses were acidified within 1 h after venipuncture by adding 0.5 mL plasma to 4.5 mL 5% metaphosphoric acid and were stored at -70 °C.

All subjects collected three 24-h urine samples at the end of the preexperimental and experimental periods by using urine-collecting aliquot cups (Daisho Co Ltd, Osaka, Japan). With this equipment, a fixed proportion (1/21) of the total urine volume was sampled and the rest was thrown away. The volumes of the collected urine samples were measured and aliquots were stored at -20 °C. Before analyses, the separate 24-h urine samples from the preexperimental and experimental periods were pooled in proportion to total urinary volume.

Plasma fatty acids, antioxidants, and homocysteine
Plasma lipids were extracted and fatty acid gas-liquid chromatography analysis was carried out as described (14). Samples for the analysis of plasma tocopherols and carotenoids were treated as follows. To 0.2 mL plasma, a solution (50% ethanol) containing 1% ascorbic acid and tocol or echinenon as an internal standard for tocopherols and carotenoids, respectively, were added. After mixing the samples by vortex, 4 mL n-hexane was added and extracted, an aliquot was evaporated under vacuum, and the residue was dissolved in 120 µL ethanol and transferred to a vial for separate HPLC analysis of tocopherols (15) and carotenoids (16).

Ascorbic acid was measured with an automated fluorimetric method with orthophenylenediamine and was standardized against daily prepared ascorbic acid in 5% metaphosphoric acid (17). Plasma total homocysteine was measured with an immunofluorimetric IMX-method (Abbott Laboratories, Abbott Park, IL) (18). Plasma folate and vitamin B-12 were analyzed by using the Simultrac-SNB dual radioassay for both folate and vitamin B-12 (Becton Dickinson, Franklin Lakes, NJ). Plasma quercetin aglycone concentrations were analyzed after enzymatic hydrolysis of potential conjugates of quercetin (glucuronic acid, sulfate, and sugar conjugates) as described by Erlund et al (19).

Lipid peroxidation
Fresh plasma samples were delivered on ice to the National Public Health Institute. The density (d) of 3.5 mL plasma was adjusted to 1.21 g/mL with potassium bromide, after which 2.5 mL potassium bromide (d = 1.21 g/mL) was added. Finally, the tubes were filled with potassium bromide solution (d = 1.006 g/mL) to the final volume of 13.5 mL. LDL was isolated by gradient ultracentrifugation (4 °C, 260 000 x g, 3 h) with a vertical rotor (VT 65,1) in a Beckman L-70 ultracentrifuge (Beckman Instruments, Palo Alto, CA). After the top layer was removed, a 2-mL fraction of LDL was removed with a peristaltic pump and was immediately desalted by using a size-exclusion column (PD-10, Sephadex G-25; Amersham Pharmacia Biotech, Uppsala, Sweden) with phosphate-buffered saline (PBS) as the elution buffer. LDL-containing tubes were purged with nitrogen and stored at 4 °C overnight.

The ex vivo susceptibility of LDL to oxidation by copper was monitored for 3.5 h at 234 nm during incubation at 34 °C in a spectrophotometer (Lambda 11; Perkin-Elmer, Überlingen, Germany). To 2 mL LDL with a protein concentration of 0.06 mg/mL, 25 µL of a copper chloride solution was added, giving a final copper concentration of 5 µmol/L. Eight samples were processed in a batch with PBS as the blank. Lag phase, oxidation rate, and maximal diene production were calculated as described by Esterbauer et al (20). Thiobarbituric acid–reactive substances (TBARS) were measured as malondialdehyde from fresh plasma, native LDL, and copper-oxidized LDL according to Wade and van Rij (21). The results are expressed as µmol malondialdehyde/L plasma and µmol malondialdehyde/mg protein for LDL. The protein concentration was determined by the Lowry method (22).

Schiff bases were analyzed by the method of Cominacini et al (23). The fluorescence of LDL solutions diluted to 0.06 mg protein/L PBS was measured immediately after dilution (0.15 mL LDL + 1.35 mL PBS) and after incubation with copper at the excitation and emission wavelengths of 360 nm and 430 nm, respectively (Perkin-Elmer LS-5). The copper solution was added to a final concentration of 5 µmol/L and incubated at 34 °C for 3.5 h. The tubes were cooled on ice and diluted with PBS, and fluorescence was measured. The results are expressed as fluorescence units/mg protein.

The urinary isoprostane 8-iso-prostaglandin F2{alpha} (8-iso-PGF2{alpha}) was analyzed from the 24-h urine samples by radioimmunoassay (24). In brief, an antibody was raised in rabbits by immunization with 8-iso-PGF2{alpha} coupled to bovine serum albumin at the carboxylic acid by the 1,1'-carbonyldiimmidazole method. The cross-reactivity of the antibody with 8-iso-15-keto-13,14-dihydro-PGF2{alpha}, 8-iso-PGF2ß, PGF2{alpha}, 15-keto-PGF2{alpha}, 15-keto-13,14-dihydro-PGF2{alpha}, TXB2, 11ß-PGF2{alpha}, 9ß-PGF2{alpha}, and 8-iso-PGF3{alpha} was 1.7%, 9.8%, 1.1%, 0.01%, 0.01%, 0.1%, 0.03%, 1.8%, and 0.6%, respectively. The detection limit of the assay was {approx}8 pg/mL (23 pmol/L). Unextracted urine samples of 50 µL were used in the assay. The results are expressed as nmol/mmol creatinine. Creatinine was analyzed from the pooled samples with commercial reagents (Mercotest; Merck, Darmstadt, Germany).

Lipoprotein metabolism
Total plasma cholesterol and HDL cholesterol after precipitation of apolipoprotein (apo) B–containing lipoproteins with dextran sulfate and magnesium chloride (25) were measured enzymatically (26). Triacylglycerol concentrations were measured with a fully enzymatic method according to the method of Wahlefeld (27). LDL cholesterol was calculated according to the formula of Friedewald et al (28). Serum apo A-I and apo B-100 were analyzed by using immunoturbidometric assays (Hoffmann-La Roche, Basel, Switzerland, and Orion Diagnostica, Espoo, Finland).

Lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) activity was measured with a radiometric method essentially as described earlier (29), with the freshly prepared proteoliposomes as the substrate. Intra- and interassay deviations were 7% and 5%, respectively. Phospholipid transfer protein activity was measured with a radiometric method essentially as described (30). The intra- and interassay precisions were 9% and 12%, respectively. Cholesterol ester transfer protein activity was measured as the transfer of radiolabeled cholesteryl ester from LDL to HDL by using the method of Groener et al (31). The precision of the method (intra- and interassay) was {approx}10%. Paraoxonase (EC 3.1.8.1) activity was measured from serum by using Paraoxon (diethyl-p-nitrophenyl phosphate; Sigma Chemical Company, St Louis) as a substrate, essentially as reported (32).

Statistical analyses
All statistical analyses were carried out with the SYSTAT statistical software package (version 5.2; SYSTAT Inc, Evanston, IL). Normality of the data was tested by the Lilliefors test. Logarithmic transformations were used in normalizing the data; if the transformed data were not normal, nonparametric tests were used. Differences between preexperimental and experimental values within treatment groups were tested by paired t tests or Wilcoxon’s signed-rank tests. If the control values differed significantly in the same direction as in the treatment groups, a period effect was presumed and the biological significance of possible changes in the treatment groups may thus be questionable. Possible differences in the preexperimental values among the treatment groups were compared by one-way analysis of variance (ANOVA) and post hoc Tukey’s test or with Kruskal-Wallis one-way ANOVA and Mann-Whitney U test with Bonferroni correction.

The treatment effect was calculated as the difference between the experimental and preexperimental values within the groups. If the preexperimental values did not differ among the groups, differences between the treatment effects were tested by one-way ANOVA and post hoc Tukey’s or Kruskal-Wallis one-way ANOVA and Mann-Whitney U test with Bonferroni correction. If there were significant differences among the preexperimental values, differences between the treatment effects were tested with analysis of covariance, taking the preexperimental values as covariates. In all statistical analyses, differences with P values <= 0.05 were considered significant. The group sizes were calculated from previous data (33) to detect a difference of 0.10 nmol/mmol creatinine ({alpha} = 5%, ß = 10%, power = 90%) (34) in urinary 8-iso-PGF2{alpha}, which was considered as the main marker of lipid peroxidation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Seventy-seven subjects completed the study, and none reported any side effects during the experimental period. The 3 dropouts left because of personal reasons. There were no significant differences in habitual diet between the treatment groups before the experimental period (Table 2Go). Comparisons made between the habitual food intake data (Table 2Go) and the composition of the experimental diets (Table 3Go) indicated that the intake of vegetables was nearly doubled and the intake of berries was 17-fold higher during the experimental period in the high-vegetable groups (P2 and M2), whereas the intake of other fruit remained fairly constant. The intakes of vegetables and fruit tended to decrease in the low-vegetable groups (P1 and M1). The dietary records of the control group showed that their diet remained constant during the experimental period (data not shown). Mean energy intakes in the treatment groups did not differ significantly during the experimental period (Table 1Go).

The body weight of the subjects remained constant during the experimental period except in the M2 group, in which the 0.5-kg average drop was significant (Table 4Go). However, the study groups did not differ significantly in body weight changes.


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  		TABLE 4 . Body weight and plasma total fatty acid composition in the preexperimental period (Pre) and changes during the experimental period (experimental - Pre)1
 
Compliance with the experimental diets was evaluated by examining changes in the plasma fatty acid profile and by measuring the concentrations of some vitamins, carotenoids, and quercetin in plasma. There were several significant changes in plasma fatty acids, all of which indicated good compliance with the diets (Table 4Go). Total MUFAs and oleic acid increased in the M1 and M2 groups and decreased in the P1 and P2 groups, and the proportion of total PUFAs, n-6 PUFAs, and linoleic acid increased in the P1 and P2 groups. The treatment effects on total MUFAs, oleic acid, total PUFAs, n-6 PUFAs, and linoleic acid in the high–linoleic acid groups (P1 and P2) differed from those in the high–oleic acid groups (M1 and M2). {alpha}-Linolenic acid decreased in all treatment groups, which was probably the result of a decreased intake of low–erucic acid rapeseed oil, which was widely consumed habitually by the subjects. In the P1 and P2 groups, the proportion of eicosapentaenoic acid also decreased, which may imply that the high–linoleic acid diet decreased the conversion of {alpha}-linolenic acid to eicosapentaenoic acid. Plasma fatty acids did not differ significantly in the control group during the experimental period.

The high intake of vegetables, berries, and apples in the P2 and M2 groups was reflected in increased plasma concentrations of quercetin, vitamin C, and several carotenoids (lutein, cryptoxanthin, {alpha}-carotene, and ß-carotene) relative to baseline (Table 5Go). The treatment effects on plasma quercetin, lutein, cryptoxanthin, {alpha}-carotene, and ß-carotene concentrations differed between the high-vegetable groups (P2 and M2) and the low-vegetable groups (P1 and M1). The effects on plasma vitamin C concentrations differed only between groups M2 and P1. Plasma lycopene concentrations were not sensitive to the dietary modifications. {alpha}-Tocopherol increased in the high–linoleic acid groups (P1 and P2), and the treatment effects differed from those in groups M1 and M2. {gamma}-Tocopherol decreased in all study groups, most likely as a result of decreased rapeseed oil consumption. Plasma folate concentrations tended to increase and total homocysteine concentrations tended to decrease in all treatment groups. There were no significant differences in the control group.


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  		TABLE 5 . Plasma concentrations of quercetin, vitamins, carotenoids, and homocysteine in the preexperimental period (Pre) and the change during the experimental period (experimental - Pre)1
 
The markers of lipid peroxidation were rather insensitive to dietary changes (Table 6Go). Plasma TBARS increased in the P1, P2, and M2 groups but decreased in the control group. Urinary 8-iso-PGF2{alpha} increased in both high-vegetable groups (P2 and M2), but there was no significant difference between the treatment effects. An increase was also seen in the control group, which may indicate a period effect in the 8-iso-PGF2{alpha} results. Markers associated with ex vivo LDL oxidation showed some differences within the groups. There was a tendency toward increased LDL oxidizability in the P2 group (increased oxidation rate and enhanced TBARS and Schiff base production during LDL oxidation), whereas the M2 group showed a decreased susceptibility to oxidation (increased lag time). No significant differences between the treatment groups were seen, although the difference in LDL oxidation velocity between the treatments reached significance (post hoc Tukey’s test: P = 0.0505 between the P2 and M2 groups). Plasma paraoxonase activity decreased in all treatment groups but tended to increase in the control group. There were few significant differences in the markers of lipoprotein metabolism within the treatment groups and no significant differences between the treatments in plasma lipid or apolipoprotein concentrations or lipid transfer protein activities.


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  		TABLE 6 . Markers of lipid peroxidation and LDL oxidizability, serum paraoxonase activity, plasma concentrations of lipids and apoproteins, and lipid transfer enzyme activities in the preexperimental period (Pre) and the change during the experimental period (experimental - Pre)1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We studied whether diets high in vegetables, berries, and apples affect markers of lipid peroxidation and lipoprotein metabolism under strictly controlled conditions. Different types of unsaturated fatty acids, ie, linoleic or oleic acid, were used to change the possible diet-induced oxidative stress in the body. Extremes in this respect were the P1 and M2 diets. Compliance with the diets was good, but our results indicate that none of the experimental diets was superior or inferior with respect to lipid peroxidation or lipoprotein profiles.

The data do not support the hypothesis that high-PUFA diets increase and high-vegetable diets decrease lipid peroxidation in humans. This may imply that vegetables decrease the risk of IHD by other mechanisms. Our results may also imply that the 6-wk experimental periods of high or low intakes of vegetables and fruit were too short to modify the tissue concentrations of antioxidants and affect lipid peroxidation among these healthy volunteers with adequate baseline diets. One possible implication is also that the low-vegetable diets, which contained 170 g vegetables/10 MJ, supplied enough dietary antioxidants and that the high-vegetable diets, which contained 810 g vegetables, berries, and apples/10 MJ, provided no additional protection against lipid peroxidation.

The enhanced plasma carotenoid and quercetin concentrations during the high-vegetable diets was expected because the diets included vegetables, berries, and apples rich in carotenoids and flavonoids. The small increase in plasma vitamin C may have been due to the subjects’ good baseline status and indicates that vitamin C was not a good biomarker for vegetable intake in the present study. Increased intake of mixed vegetables and fruit was shown to increase the concentrations of plasma carotenoids and vitamin C in earlier studies (35–37), but few data are available about plasma quercetin concentrations in dietary interventions with a mixed food approach. Our results clearly show that quercetin was bioavailable from the diets. Also, the urinary excretion of flavonoids reflected differences in fruit and vegetable intake (38).

In the present study, the Finnish mix of vegetables and fruit was not effective on lipid peroxidation or susceptibility to oxidation. Earlier data on the effects of mixed vegetables and fruit are limited. In a well-controlled intervention, serum TBARS, an unspecific marker of lipid peroxidation, was not affected but breath ethane excretion was lower with 9 compared with 4 servings of vegetables and fruit/d (39). In 2 less vigorously controlled studies, increasing the intake of fruit and vegetables from 3 to 7–8 servings/d slightly decreased plasma TBARS (40), and an increase from 6 to 12 servings/d (without a control treatment or group) decreased urinary 8-iso-PGF2{alpha} in female volunteers (41). Additional controlled studies are needed on the effects of mixed vegetables and fruit on markers of lipid peroxidation.

The results of studies that used single or only a few sources of antioxidants are conflicting. Supplementation with a vegetable concentrate (equal to 500 g mixed vegetables/d) and fruit juice did not affect plasma TBARS or F2-isoprostanes in male smokers with low habitual intakes of vegetables and fruit (42). In other studies, plasma TBARS were decreased by 1500 mL black currant and apple juice/d (43) and by tomato juice (330 mL/d) (44) but not by 330 mL carrot juice/d (44). LDL ex vivo oxidizability was decreased by citrus juice (500 mg vitamin C/d) (45) and by tomato juice (330 mL/d) (44) but not by carrot juice (44) or a mixture of orange and carrot juices (145 mg vitamin C/d and 16 mg ß-carotene/d) (46). For flavonoid-rich supplements, red wine extract was shown to decrease LDL oxidation ex vivo (47), whereas grape skin extract did not affect TBARS in plasma or LDL (48), onions and black tea were ineffective on plasma TBARS and F2-isoprostanes (49), and green tea solids did not affect urinary 8-iso-PGF2{alpha} (33).

Our compliance data clearly show that the high-PUFA and high-MUFA diets modified plasma fatty acids differently. However, no significant differences in lipid peroxidation markers were found between the fatty acid intake groups. Our results agree with earlier studies that showed that diets or supplements high in linoleic or oleic acid have similar effects on plasma TBARS (50–52) and 8-iso-PGF2{alpha} in urine (53) and plasma (52). TBARS in LDL were shown to decrease (50) or increase (54) with high-PUFA diets compared with high-MUFA diets.

Despite the borderline significant difference in the ex vivo LDL oxidation velocity between the P2 and M2 diets, no significant differences in LDL susceptibility to oxidation were found between the fat intake groups. Our results disagree with earlier studies that reported increased ex vivo LDL oxidation by PUFAs or linoleic acid compared with diets rich in MUFAs (8, 50, 55, 56). The PUFA intake in the P1 and P2 diets was kept close to the upper range (10% of energy) of the current dietary recommendations (57), and the dietary ratio of vitamin E to PUFAs was kept as constant as possible ({approx}1 mg/g). Thus, linoleic acid intake was lower and the ratio of vitamin E to PUFAs higher than in the high–linoleic acid diets used in earlier studies (50, 56). The larger absolute {alpha}-tocopherol intake in the P1 and P2 groups may have protected from the possible prooxidative effects of linoleic acid in the present study. In a supplementation study, 25 IU all-rac-{alpha}-tocopherol acetate/d (17 mg {alpha}-tocopherol equivalents/d) was enough to increase LDL resistance to oxidation (58).

Plasma {alpha}-tocopherol concentrations did not differ significantly within the fat intake groups (P1 compared with P2 and M1 compared with M2), indicating that the larger intake of other antioxidants in the high-vegetable groups was not associated with vitamin E sparing effects. On the other hand, differences in the intakes of {alpha}-tocopherol (P1 compared with M1 and P2 compared with M2) were not reflected in the plasma data, eg, in plasma vitamin C concentrations, indicating no interaction between these 2 antioxidants in our study. In 2 recent carefully controlled studies, vitamin E sparing effects by vitamin C supplementation (59) or polyphenol-rich grape skin extract (48) were reported.

Paraoxonase is synthesized and secreted by the liver, it is complexed with apo A-I and apo J in a specific HDL subpopulation, and can protect LDL against oxidation, which is accompanied by inactivation of lipoprotein-associated lipid peroxides (60, 61). In the present study, serum paraoxonase activity decreased in the treatment groups, whereas no changes occurred in the control group. One explanation for this unspecific effect may be that the slightly decreased intake of alcohol during the experimental period decreased paraoxonase activity (62). In the preexperimental period, habitual vegetable and fiber intakes were negatively correlated with paraoxonase activity (63), but no further effects were seen when vegetable and berry intakes were increased during the experimental period. More detailed studies are needed to explain the results.

Plasma lipid and apolipoprotein concentrations were not significantly affected in either an antiatherogenic or a proatherogenic direction by our experimental diets, probably because the intake of saturated fat was not markedly modified. The fact that linoleic and oleic acids (or PUFAs compared with MUFAs) did not differ in their effects agrees with the results of several earlier studies (64). Diets high in vegetables and fruit have been speculated to decrease serum lipid concentrations as a result of decreased intake of saturated fat or increased intakes of fiber and vegetable protein (3). However, in the present setting with a controlled intake of saturated fatty acids, high vegetable intake had no significant effect on lipoproteins.

The effect of dietary fatty acids on serum lipoprotein concentrations and composition is mediated by altered lipid transfer protein activities (65) and by changes in LDL receptor expression. In the present study, no significant differences in cholesterol ester transfer protein, phospholipid transfer protein, or LCAT activities were observed between the experimental groups. In one study (66), a low-fat PUFA diet and a high-fat MUFA diet did not differ with respect to cholesterol ester transfer protein activity. LCAT utilizes fatty acids in the sn-2 position of phosphatidylcholine for cholesterol esterification. Because the turnover rate of serum phospholipid fraction is slow (67), our 6-wk dietary period may have been too short to affect LCAT substrate functionality, ie, to increase the proportion of dietary fatty acids in the sn-2 position.

In summary, no measurable effects on lipid peroxidation or lipoprotein metabolism in the fasting state were achieved by markedly increasing the intakes of vegetables, berries, and apples or by altering the intakes of unsaturated fatty acids in healthy volunteers with adequate habitual diets. We conclude that the amount of linoleic acid in the high–linoleic acid diets was not sufficient to enhance oxidative stress in the body, probably because the ratio of vitamin E to PUFAs was {approx}1 mg/g. The lack of difference between the high- and low-vegetable diets is more difficult to explain. The results may have been different in a study population in whom more free radical reactions were taking place in vivo, eg, as a result of intensive smoking or aging. Of special interest in this regard would be subjects with suboptimal fruit and vegetable intakes.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge our volunteers for their invaluable commitment, Irma Nykänen and the students of the Helsinki Polytechnic Degree Programme of Catering Services for their invaluable cooperation, and Jetta Tuokkola, Eva Kammiovirta, Ritva Keva, Pirjo Laaksonen, Kaija Lampi, Päivi Mustonen, and Raija Pahlman for excellent technical assistance.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication May 2, 2001. Accepted for publication December 6, 2001.




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