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Articles

High Linoleic Acid, Low Vegetable, and High Oleic Acid, High Vegetable Diets Affect Platelet Activation Similarly in Healthy Women and Men^{1 ,2}

Division of Nutrition, University of Helsinki, Helsinki, Finland

³To whom correspondence should be addressed. E-mail: marjo.misikangas{at}helsinki.fi.

	ABSTRACT

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Upregulation of protein kinase C (PKC), an important enzyme in platelet activation, could be one step toward platelet hyperactivity. PKC activation can be modulated by dietary components in vitro, but few data are available concerning the in vivo effects. In this strictly controlled human dietary intervention, the influence of dietary unsaturated fatty acids and vegetable compounds on platelet activation was investigated. A high linoleic acid diet (10% of energy) with small amounts of vegetables (no berries or apples) was consumed by 9 women and 4 men (24.1 ± 3.9 y), and was compared with a high oleic acid diet (12% of energy) with considerable amounts of vegetables, berries and apples consumed by 8 women and 4 men (24.2 ± 5.5 y). Subjects were healthy Finnish volunteers. Compliance with the experimental protocol was good, as indicated by changes in plasma fatty acids and concentrations of vitamin C, ß-carotene and -tocopherol. No differences between groups were seen in indices of platelet activation, including platelet aggregation, total PKC activity and distribution of PKC isoenzymes , ß_II and . The results indicate that in apparently healthy and fairly young subjects with adequate vitamin intakes, diets differing markedly in their amounts of linoleic and oleic acids, and vegetables, berries and apples do not differ in platelet activation.

KEY WORDS: • vegetables • unsaturated fatty acids • protein kinase C • platelet activation • humans

	INTRODUCTION

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The role of diet in the etiology of cardiovascular diseases (CVD)⁴ is of constant interest. Epidemiologic data indicate that both polyunsaturated fatty acids (PUFA) and vegetables can protect from CVD, but concern has arisen that the imbalance between dietary PUFA and bioactive vegetable compounds may lead to oxidative stress.

Platelets have an important role in atherosclerosis and thrombosis (1) , and platelet hyperactivity is considered to be a risk factor for thrombosis (2) . Oxygen free radicals modulate platelet activation, and thus oxidative stress may lead to unfavorable platelet activation. There is substantial evidence that diet may affect platelets, and dietary fatty acids, in particular, have been widely studied (3) . Dietary studies have focused mainly on platelet aggregation ex vivo, even though diet can affect the earlier steps of platelet activation, and signaling molecules upstream in the pathway may serve as a marker of activation. The cell-signaling molecules of platelet activation cascades, e.g., protein kinase C (PKC), have been studied in vitro, but their usefulness as a platelet activation marker in dietary studies is unknown.

PKC is an important enzyme in cell-signaling pathways. Activation of PKC is necessary to the platelet activation process, leading to platelet secretion and aggregation (4 ,5) . Upregulation of platelet PKC could be one step resulting in platelet hyperactivity. The translocation of the enzyme from cell cytosol to plasma membrane is considered to be a critical point in the classical PKC activation model (6) .

Cis-unsaturated fatty acids directly activate PKC in vitro (7) , and some evidence indicates that platelet PKC activity may be modulated by dietary fatty acids also in vivo (8) . Oleic acid (9) and (n-6) and (n-3) PUFA (10) can induce PKC translocation to the plasma membrane in vitro. Oxygen free radicals (11) and oxidation products of unsaturated fatty acids (12) stimulate PKC activity in vitro, which could be mediated at least in part by the phosphorylation of PKC (13) .

Some antioxidants and other bioactive molecules from plant sources inhibit PKC activity in vitro. Vitamin E can reduce platelet aggregation through a PKC-dependent mechanism (14) . This action is independent of the antioxidant activity of -tocopherol (15) and seems to be mediated by the ability of -tocopherol to dephosphorylate PKC (16) . -Tocopherol also inhibits redistribution of PKC activity from the cytosol to the membrane compartment (17) . Flavonoids naturally present in plants can inhibit PKC in vitro (18) ; however, information about other possible bioactive compounds in vegetables, fruits and berries is insufficient.

As part of a larger intervention examining the effects of diets differing markedly in their amounts of linoleic or oleic acids, and vegetables, berries and apples, we report here the effects of the two extremes of the experimental diets on platelet activation. The diets studied were a high linoleic acid/low vegetable diet [LA(-)] and a high oleic acid/high vegetable diet [OA(+)]. In addition to platelet aggregation ex vivo, total PKC activity and distribution of PKC isoenzymes were followed as earlier markers of platelet activation. The three isoenzymes chosen, , ß_II and , are modulated by dietary constituents in vitro (9 ,13 ,16) .

	SUBJECTS AND METHODS

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The strictly controlled human dietary intervention was conducted at the Division of Nutrition, University of Helsinki. After giving their informed consent, healthy Finnish volunteers (n = 25; mean age 24.1 y) were divided in two groups (Table 1 ) and consumed one of the experimental mixed diets for 6 wk. The study provided 90% of daily energy (en%) to the subjects; in addition, the subjects had to choose 10 en% from a limited list of foods. The diets were isocaloric but differed in their compositions of unsaturated fatty acids and amounts of vegetables, berries and apples (Table 2 ). Energy intakes were adjusted with sugar and starch. The LA(-) diet contained 11 en% of PUFA [10 en% linoleic acid, 18:2 (n-6)] with a low intake of vegetables, no berries or apples. The OA(+) diet contained 16 en% of monounsaturated fatty acids [MUFA; 12 en% oleic acid, 18:1 (n-9)] with a high intake of vegetables, berries and apples. Control subjects (n = 17) were used to study the time effect. They kept their habitual diets constant as verified by dietary records. The study protocol was approved by the Ethics Committee of the Faculty of Agriculture and Forestry, University of Helsinki.

For PKC analysis, platelets were equilibrated for 30–60 min at 25°C. Equal volumes of ice-cold washing buffer [acid-citrate-dextrose /saline] (19) were added to platelet-rich plasma and platelets were pelleted by centrifugation at 700 x g for 10 min at 4°C. For the separation of platelet cytosolic and membrane fractions, the platelet pellet was resuspended in 5 mL ice-cold homogenization buffer containing 20 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EGTA, 2 mL EDTA, 250 mmol/L sucrose, 1 mmol/L phenylmethylsulfonyl fluoride and 0.5 mg leupeptin. The suspension was sonicated (Labsonic 2000V, B. Braun,Markheidenfelt,Germany) in an ice bath for 30 s. The homogenate was centrifuged at 100,000 x g for 1 h at 4°C (Beckman L8–70M, Palo Alto, CA). The supernatant was collected and used as the PKC cytosolic fraction. The pellet was resuspended in 5 mL of ice-cold homogenization buffer containing 2 mL/L Triton X-100, incubated for 20 min and centrifuged at 100,000 x g for 1 h at 4°C. The resulting supernatant was used as the platelet membrane fraction.

Total PKC activity was measured from the cytosolic and membrane fractions. Membrane fractions were partially purified with DEAE-Sephacel chromatography (Pharmacia, Uppsala, Sweden) and stored at -70°C, whereas cytosolic fractions were stored unpurified. PKC activities in both fractions were analyzed by Biotrak RPN 77 assay (Amersham, Little Chalfont, UK) and standardized by protein concentrations, which were measured using a BioRad protein assay reagent (Hercules, CA) with bovine serum albumin as the standard.

For the analysis of PKC isoenzymes, cytosol and membrane fractions were stored at -70°C until concentrated using Ultrafree-4 concentrators (Millipore, Bedford, MA). Protein contents were measured using the BioRad protein assay reagent with bovine serum albumin as the standard. The Western blotting procedure was the same as that used previously (20) with the exception that 10% Tris-HCl BioRad Ready Gels with 15 sample wells were used. Running conditions were 200 V for 35 min and electroblotting at 100 V for 60 min. Protein content in sample wells was 17 µg and in positive control wells (rat brain homogenate), 5 µg. Each sample was analyzed in duplicate. Primary antibodies used were nPKC(c-20), nPKCß_II(c-18) and nPKC(c-20); the secondary antibody was anti-rabbit immunoglobulin-AP (Santa Cruz Biotechnology, Santa Cruz, CA). Blocking peptides of each PKC isoenzyme (Santa Cruz Biotechnology) were used to ensure that the correct bands from immunoblots were analyzed.

Vitamin C, carotenoids, tocopherols and total fatty acids were measured from EDTA-plasma as markers of dietary compliance. For the ascorbic acid assay, plasma (0.5 mL) was pipetted into 4.5 mL of 5% metaphosphoric acid. The analysis was carried out with an automated fluorimetric method (21) . Plasma tocopherols and carotenoids were analyzed separately by HPLC, tocopherols by fluorescent detection (22) and carotenoids by detection at 450 nm (23) . Plasma total fatty acids were extracted and analyzed by gas-liquid chromatography as described (24) .

For the analysis of biomarkers of oxidative status, plasma aliquots were frozen immediately (-40°C) or delivered fresh to the Department of Biochemistry, National Public Health Institute where LDL were isolated by ultrasentrifugation at 260,000 x g for 3 h at 4°C (vertical rotor VT 65, 1 in a Beckman L-70 ultracentrifuge) after adjusting the density of plasma to 1.21 kg/L with potassium bromide. The LDL-fraction was desalted (PD-10, Sephadex, Pharmacia) and eluted with PBS. The test tubes were filled with nitrogen and stored at 4°C overnight. The lag-time of Cu²⁺-induced LDL oxidation in vitro was analyzed as described (25) . Malondialdehyde (MDA) was measured as thiobarbituric acid-reactive substances (26) .

Statistical analysis of the data was performed with the Systat 5.2 program (Systat, Evanston, IL). Normality of the data was tested by Lilliefors test. Depending on the normality of the data, parametric or nonparametric tests were used. Possible differences between the preexperimental period (PRE) levels in the different groups were analyzed by ANOVA and the post-hoc Tukey’s test. Differences from PRE to the experimental period (EXP) within the treatment groups were analyzed with paired t test or the Wilcoxon Signed Ranks test. Difference between the treatment effects ( EXP-PRE within treatment groups) was analyzed with independent samples t test or the Mann-Whitney U test. Correlation analyses were performed using Pearson’s correlation or the Spearman rank-order correlation. Statistical significance of difference was assumed at P < 0.05.

	RESULTS

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Experimental groups did not differ before the study (Tables 1 , 3 and 4 ). Intended differences between the experimental diets were achieved, and the diets were reflected in the dietary compliance markers, i.e., proportions of LA and OA in plasma fatty acids showed a clear difference between the LA(-) and OA(+) groups. Furthermore, the experimental groups differed in plasma vitamin C, ß-carotene and -tocopherol concentrations (Table 3) .

View this table: [in this window] [in a new window]   Table 4. Platelet aggregation, LDL-oxidation in vitro, plasma malondialdehyde (MDA), total protein kinase (PKC) activity and distribution of isoenzymes (PKC, PKCßII, PKC) between platelet cytosol and membrane in the beginning (PRE) and at the end of experiment (EXP) in subjects fed the LA(-) diet or the OA(+) diet for 6 wk or in the control group1 ,2

Biomarkers of oxidative status, LDL-oxidation in vitro and plasma MDA, did not differ between groups (Table 4) . These oxidative markers, plasma fatty acids, antioxidants, flavonoids and lipoprotein fractions did not correlate with platelet PKC activity or the distribution of isoenzymes (data not shown).

When data from all groups were combined, correlations between PKC total activity and PKC cytosolic isoenzymes (r = 0.47, P = 0.003), ß_II (r = 0.44, P = 0.004) and (r = 0.50, P = 0.001) were found at the end of the experiment. Correlations between PKC activity and PKC membrane isoenzymes (r = 0.45, P = 0.004), ß_II (r = 0.50, P = 0.001) and (r = 0.35, P = 0.032) followed the same pattern. No correlations were found before the experiment when the subjects consumed their habitual diets.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The aim of this study was to compare platelet function during controlled isocaloric diets, which differed in unsaturated fatty acid composition (PUFA vs. MUFA) and amounts of vegetables, berries and apples. Compliance with the experimental diets was good, as indicated by changes in plasma fatty acids and concentrations of vitamin C, ß-carotene and -tocopherol. In addition to the biomarkers shown here, the plasma flavonoid quercetin, as well as the excretion of several flavonoids in urine, were greater in the OA(+) group compared with the LA(-) group (data not shown). Although the fatty acid and antioxidant status changed differently in the experimental groups, no differences between the groups were seen in platelet aggregation, PKC activity or isoenzyme localization. Our results are consistent with one earlier study in humans in which dietary linoleic acid and oleic acid diets did not differently affect PKC activity (8) .

The possibility of harmful fatty acid oxidation is suggested to increase as the unsaturation of the fatty acids increases. Because PUFA may be potential sources of oxidative stress in the body, a large difference in PUFA intake was used in this study in an attempt to change the oxidative status of the study groups. Another strategy to modify the oxidative status was the use of vegetables, berries and apples, which contain antioxidants and other bioactive molecules that may prevent oxidative stress. The intake of PUFA in the LA(-) group was very close to the maximal possible intake from a normal mixed diet, whereas the intake of vegetable compounds was kept very low. In the OA(+) group, intake of PUFA was considerably lower and the main dietary fatty acids were MUFA. The portions of vegetables, berries and unpeeled apples in the OA(+) group were approximately fourfold higher than in the normal Finnish diet (27) . It is almost impossible to increase amounts of vegetables, fruits and berries (and thus the possible bioactive plant molecules) from normal mixed dietary sources more than was done in this study without supplementation because portions would be too large to consume. In supplementation trials, it is possible to use pharmacologic doses but their relevance to normal physiology is questionable. Lipid peroxidation or LDL oxidation did not change differently in the present study, indicating that the LA(-) diet did not increase or the OA(+) diet decrease oxidative stress in our healthy subjects.

Oxidative compounds may cause platelet hyperactivation and this can be seen as a risk factor for thrombosis. Unwanted platelet activation could proceed through PKC enzyme activation. Cis-unsaturated fatty acids and oxidation products of unsaturated fatty acids can activate PKC in vitro (7 ,12) and also induce PKC translocation to the plasma membrane (9 ,10) . These mechanisms seemed not to be involved here, i.e., the changes found in the experimental groups in platelet aggregation or PKC did not differ from those found in the control group.

Plasma concentrations of vitamin C, carotenoids and quercetin were higher in the OA(+) group, whereas -tocopherol was higher in the LA(-) group. In vitro studies have shown that these and other bioactive molecules from vegetables, berries and apples inhibit platelet activation, and this may be mediated at least in part by PKC inhibition (18 ,28) . In this study, -tocopherol or vegetable compounds did not inhibit platelet aggregation or PKC compared with the controls. Studies that have found -tocopherol to inhibit PKC activity have used pharmacologic doses of supplementation, thus comparisons with our results are questionable (14) .

Because fatty acids activate platelet PKC in vitro, we wanted to determine whether there were correlations between fatty acids and PKC. Plasma total fatty acid composition was measured in this study, but not platelet fatty acid composition. No correlations between plasma total fatty acids and platelet PKC total activity or isoenzymes were found. One reason may be that because only certain phospholipid classes seem to be involved in PKC activation (29) , plasma total fatty acids may be far too crude a marker to reflect associations between platelet phospholipid fatty acid composition and PKC activation.

It is difficult to explain why PKC activity and all three isoenzymes studied correlated at the end of the experiment but not at the beginning. It is possible that the experimental diets had some effect on platelet PKC, after all. The dietary changes may have favored participation of PKC isoenzymes , ß_II and in PKC activity compared with consumption of habitual diets.

The use of PKC in dietary interventions has been quite rare. Our study indicates that there might be some difficulties using PKC in in vivo interventions. Because intraindividual variation in PKC activity and isoenzyme localization seems to be quite large, the study populations should be much larger than that used here to be able to detect small changes in these variables. This study also showed the importance of the control group in dietary interventions, i.e., a small time trend in PKC activity and isoenzyme translocation could have led to incorrect conclusions without the control group.

Earlier studies have shown that even a 4-wk period is long enough to induce differences in fatty acid profiles and physiologic responses during different dietary treatments (8 ,19) . In this study, 6 wk was enough to induce changes in dietary compliance markers. Nevertheless, in our apparently healthy and fairly young subjects with adequate vitamin intakes, modification of dietary unsaturated fatty acids and vegetable compounds did not change the balance in the body, and platelet activation remained unchanged, as monitored by platelet aggregation and a specific enzyme of platelet activation, PKC.

	FOOTNOTES

 
¹ Presented in poster form at the XVth Annual European Symposium on Blood Platelets, October 19–21, 2000, Bischenberg, Alsace, France (Misikangas, M., Freese, R., Turpeinen, A. M. & Mutanen, M. Similar effects of low and high intakes of natural antioxidants when combined with high linoleic or oleic acid diets on human platelet PKC activation or isoenzyme , ß_II, translocation in healthy subjects).

² Supported by the Academy of Finland (project number 101 41399), the Ministry of Agriculture and Forestry, the University of Helsinki.

⁴ Abbreviations used: CVD, cardiovascular diseases; EXP, experimental period; LA(-), diet rich in PUFA (mainly linoleic acid) and low in vegetables, no berries or apples; MDA, malondialdehyde; MUFA, monounsaturated fatty acids; en%, percentage of daily energy intake; OA(+), diet rich in MUFA (mainly oleic acid) and high in vegetables, berries and apples; PKC, protein kinase C; PRE, preexperimental period; PUFA, polyunsaturated fatty acids.

Manuscript received September 5, 2000. Initial review completed November 1, 2000. Revision accepted March 2, 2001.

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