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Mediterranean-inspired diet lowers the ratio of serum phospholipid n–6 to n–3 fatty acids, the number of leukocytes and platelets, and vascular endothelial growth factor in healthy subjects^1,2,3

¹ From the Department of Clinical Physiology (AA, MJ, LMG, and PF), the Lundberg Laboratory for Diabetes Research (MA), and the Department of Paediatrics (BS), The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

² Supported by grants from the Swedish Heart and Lung Foundation and the Sahlgrenska Academy at Göteborg University, Sahlgrenska University Hospital.

³ Reprints not available. Address correspondence to P Friberg, Department of Clinical Physiology, Sahlgrenska University Hospital, SE - 413 45 Göteborg, Sweden. E-mail: peter.friberg{at}mednet.gu.se.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Reduced cardiovascular mortality and morbidity have been shown in persons adhering to Mediterranean-inspired diets (MIDs). Although the underlying mechanisms of this association are poorly understood, the importance of increasing dietary amounts of polyunsaturated fatty acids of the n–3 series has been emphasized.

Objective: We investigated whether a MID provided to healthy subjects would affect 1) the inflammatory process and endothelial indexes such as vasoregulation and vascular endothelial growth factor (VEGF) and 2) serum phospholipid fatty acid composition.

Design: A total of 22 subjects (10 women) received a MID or an ordinary Swedish diet (OSD) for 4 wk in a crossover fashion. Concentrations of lipids and fatty acids, high-sensitivity C-reactive protein, and interleukin 6, both before and after lipopolysaccharide stimulation; the number of leukocytes and platelets; and VEGF and monocyte chemoattractant protein 1 were analyzed.

Results: The plasma ratio of n–6 to n–3 fatty acids was substantially lower after the MID than after the OSD ( ± SEM: 4.72 ± 0.19 and 2.60 ± 0.17, respectively; P < 0.0001). Neither C-reactive protein nor interleukin 6 concentrations changed significantly after the MID compared with the OSD. The total number of leukocytes and platelets was 10% (P < 0.05) and 15% (P < 0.001) lower, respectively, after the MID than after the OSD. Serum VEGF concentrations were lower after the MID than after the OSD (237 ± 30 and 206 ± 25 pg/mL, respectively; P = 0.0014).

Conclusions: A MID reduces the number of platelets and leukocytes and VEGF concentrations in healthy subjects. This may be linked to higher serum concentrations of n–3 fatty acids, which promote a favorable composition of phospholipids.

Key Words: Inflammation • diet • Mediterranean-inspired diet • cholesterol • leukocytes • interleukin 6 • high-sensitivity C-reactive protein

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Diet is of pivotal importance for preventing cardiovascular disease (1), and recent data show reduced mortality in persons adhering to a Mediterranean diet (2). The mechanisms underlying the cardiovascular benefits of Mediterranean-inspired diets (MIDs) are not fully understood. These diets are associated with a lower level of LDL oxidation, which results both from a higher presence of monounsaturated fatty acids (3) and a less proinflammatory state. Elevated concentrations of inflammatory mediators, such as C-reactive protein (CRP) and interleukin 6 (IL-6), may reflect inflammation in the arterial wall, which would allow leukocytes to adhere. Ample evidence suggests that inflammation has an essential role in the development and progression of atherosclerosis (4, 5). Moreover, hypercholesterolemia is one of the major risk factors for the development of atherosclerosis. It is also well established that high blood cholesterol is associated with endothelial dysfunction both in adults and in the young (6). Vascular endothelial growth factor (VEGF), an angiogenic growth factor, is elevated in patients at cardiovascular risk, such as in persons with hypertension and 3 or more clinical risk factors (7), which indicates an increased need for vascular repair.

Dietary interventions, such as a MID or a diet high in plant sterols, soy protein, and fibers, effectively reduce LDL-cholesterol and apolipoprotein (apo) B concentrations in both normocholesterolemic and hypercholesterolemic healthy subjects (8, 9). Jenkins et al (9) further showed that, besides LDL cholesterol, CRP concentrations are also lowered. Sparse information exists as to whether leukocytes and VEGF, the latter also released by neutrophils and platelets (10, 11), are affected by dietary intervention. In the Lyon Diet Heart Study, which was a secondary prevention trial, the number of leukocytes was shown to be linked to cardiovascular disease risk (12, 13).

The present study was undertaken to explore, in normocholesterolemic, healthy individuals subjected to 1 mo each of a traditional OSD and a MID, whether white blood cell (WBC) count, platelets, red blood cell count, concentrations of VEGF and monocyte chemoattractant protein 1 (MCP-1) as indexes of platelet and WBC activity, and concentrations of high sensitivity (hs)-CRP and IL-6 were lower after the MID, possibly mirroring the lower apo B and LDL-cholesterol concentrations reported earlier (8). Second, we explored whether any association of the inflammatory variables with forearm blood flow could be shown. Furthermore, membrane fatty acid composition adapts dynamically to changes in the dietary fatty acid profile (14). A MID favors an increased intake of polyunsaturated fatty acids (PUFAs) of the n–3 series, particularly, a lowering of the ratio of n–6 to n–3 fatty acids compared with the OSD, thus affecting tissue lipids. To investigate this effect, we measured the concentrations of fatty acids in serum phospholipids at the end of both dietary periods.

	SUBJECTS AND METHODS

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Subjects
Subjects (n = 282) who answered an advertisement in the newspaper of the greater Göteborg area were first interviewed by telephone, and 93 were screened in terms of physical examination, electrocardiogram, and routine biochemistry and lipid profiles. To be involved in the study, the following inclusion criteria had to be fulfilled: total cholesterol between 5 and 7 mmol/L and LDL cholesterol between 3 and 4 mmol/L. These concentrations were within the limits of 95% of the Swedish population (15). Hence, 22 healthy subjects (10 women and 12 men) with normal blood lipid and glucose values and BMIs just at the cutoff for overweight were subsequently included in the study (Table 1). The subjects' mean (±SEM) age was 43 ± 1 y (range: 36–51 y).The subjects were all nonsmokers who were not taking any medication. Written informed consent was obtained from each subject before entry into the study. The study was approved by the local ethics committee of the Sahlgrenska Academy in Göteborg.

The MID consisted of twice the amount of fiber in the OSD, 3–4 times as many antioxidants, almost 3 times the amount of PUFAs and twice the amount of n–3 fatty acids, one-half the amount of saturated fat, one-half the amount of cholesterol, and a 35% reduction in the glycemic index. In addition, sterol esters were included as an ingredient in the margarine (2 g/d) only during the MID. The amount of calories, proteins, carbohydrates, and total of fat was similar between the 2 diets. Sixty percent of the daily caloric need was provided to the subjects once weekly from a stock of food produced and frozen within our research facilities, thus allowing strict control of ingredients. The remaining 40% of daily intake was chosen by the subjects but was restricted to low-fat products. Our intention was to keep the conditions as comparable as possible during the 2 experimental periods in terms of body weight, physical activity, and alcohol intake. To assess the subjects' compliance with consuming the provided foods, uneaten items were to be returned and weighed at each visit. Furthermore, 3 unannounced telephone interviews (24-h recalls) were performed during each period to assess the energy content and macronutrient composition of both the self-selected and supplied food items (ie, total diet).

Before and after the 4-wk diet periods, total cholesterol, LDL cholesterol, and triacylglycerol were measured. Hematologic indexes, including WBC count, differential count, red blood cell count, platelet count, and measurements of hs-CRP, IL-6, VEGF, and MCP-1, were performed at the end of each diet period. Plasma concentrations of phospholipid fatty acids and IL-6 before and 4 h after stimulation with lipopolysaccharide were also measured after each 4-wk diet intervention.

Blood samples
WBCs, red blood cells, and platelets were analyzed by fluorescence-activated cell sorting. Hs-CRP and apo B concentrations were analyzed by immunoturbidimetric assay. Fasting total cholesterol, HDL cholesterol, and triacylglycerol were analyzed by using enzymatic methods (Roche Diagnostics, Mannheim, Germany). LDL-cholesterol concentrations were calculated by using Friedewald's equation.

Concentrations of IL-6 in plasma and supernatant fluid were determined by bioassay assessment. The effect of test samples on the proliferation of the IL-6–dependent cell line B13.29 (17) was assessed after 4 h of stimulation with lipopolysaccharide (Escherichia coli 055:B5; Sigma Chemical Co, St Louis, MO). The results were analyzed by incorporation of [³H]thymidine (Radiochemical Centre, Amersham, United Kingdom). Proliferation in the presence of test samples was compared with that induced by standard dilutions of recombinant IL-6 (Genzyme, Cambridge, MA). Supernatant fluid was stored at –70 °C until analyzed. VEGF and MCP-1 were measured by using a Randox Evidence fully automated immunoanalyzer (Randox Laboratories Ltd, Crumlin, United Kingdom). Assays were performed on the surface of a dedicated biochip, which is transported to various treatment stations within the analyzer. The assay endpoint is the generation of light from a chemiluminescent reaction that is measured by using a charge-coupled-device camera. Imaging technology is used to quantify the light output from discrete test regions (reactions site) on the biochip surface.

Sera were kept frozen (–70 °C) until the fatty acid composition of phospholipids was analyzed. Lipids were extracted and fractionated as previously reported (18). Fatty acid methyl derivatives were separated by capillary gas-liquid chromatography in a Hewlett-Packard 6890 gas chromatograph with the use of helium as a carrier gas. The separation was recorded with HP GC CHEM STATION software (HP GC, Wilmington, DE). The fatty acid 21:1 was used as an internal standard, and the fatty acid fractions were identified by comparison with retention times of pure reference substances (Sigma Aldrich Sweden AB, Stockholm, Sweden). The interassay CV was 0.6% for linoleic acid (18:2n–6) and 0.5% for arachidonic acid (20:4 n–6) (n = 15).

Statistical analyses
STATVIEW for WINDOWS (version 5.0.1; SAS Institute Inc, Cary, NC) was used, and the results are expressed as means ± SEMs. Student's t test for paired observations was used to identify statistically significant differences. The statistical significance of variables also obtained as starting values was calculated as the difference between the starting values and the values at the end of each 4-wk diet period, and then these differences were tested by using paired t tests. The relation between 2 variables was assessed from bivariate scatter plots and calculation of Pearson correlation coefficients. Significance testing for correlations as well as the difference between correlations was assessed by permutation test. Statistical significance was defined as a two-sided P < 0.05.

	RESULTS

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In general, compliance with the diets was excellent, and all subjects tolerated the dietary changes well, as assessed by 24-h recalls and return of uneaten food items. Energy intake, as determined from 24-h recalls, was slightly lower during the MID period than during the OSD period, being 1869 ± 80 and 2090 ± 87 kcal (P < 0.05), respectively. A small but statistically significant lowering of BMI (difference between diet periods: 0.209 ± 0.082) occurred after 4 wk of the MID versus 4 wk of the OSD (Table 1).

As further indicated in Table 1, each diet period caused a lowering of glucose, insulin, and cholesterol fractions, which made comparisons between the ends of each diet period essential. For all variables in Table 1, the value before starting the OSD was not significantly different from that before starting the MID. Total and LDL cholesterol and triacylglycerol were lower by 17% (P < 0.001), 23% (P < 0.001), and 15% (P = 0.07) after the MID than after the OSD (Table 1). Apo B concentrations were 16% lower after the MID than after the OSD (P < 0.001; Table 1). HDL cholesterol was slightly but significantly lower after the MID than after the OSD (P < 0.05; Table 1).

Mean total serum concentrations of saturated fatty acids were lower and n–3 PUFAs were higher after 4 wk of the MID versus the OSD (Table 2). Concentrations of linoleic acid were unchanged between diets, and concentrations of arachidonic acid were lower after the MID, probably reflecting competition between the elongase and desaturase activities of the n–3 fatty acids. This was further supported by the lower -linolenic acid (18:3n–3) concentrations, whereas eicosapentaenoic acid (20:5n–3) and docosahexaenoic acid (22:6n–3) were both higher after the MID (Table 2). Hence, a low ratio between arachidonic acid and docosahexaenoic acid prevailed in response to the MID (Figure 1, top panel). As further indicated in Table 2, total mean concentrations of n–3 fatty acids were higher and those of n–6 fatty acids were lower, resulting in a lower ratio of n–6 to n–3 fatty acids during the MID period than during the OSD (Figure 1, lower panel). A statistically significant correlation was found between the ratio of n–6 to n–3 fatty acids and apo B concentrations considering both diet periods (r = 0.38, P = 0.04).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Individual values for the ratio of arachidonic acid (AA; 20:4n-6) to docosahexaenoic acid (DHA; 22:6n–3) and the ratio of total amounts of n–6 to n–3 polyunsaturated fatty acids (PUFAs) in subjects changing from the ordinary Swedish diet (OSD) to the Mediterranean-inspired diet (MID) in a crossover design. The mean (±SEM) value for each diet is also indicated. n = 22. #Significantly different from the OSD, P < 0.0001 (paired t test).

Table 3

Figure 2

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Individual changes in vascular endothelial growth factor (VEGF) in response to change in dietary regimen. Note the variation in absolute values. There was a marked consistency in the response pattern, ie, VEGF was lower during the 4-wk Mediterranean-inspired diet (MID) period than during the ordinary Swedish diet (OSD) period. The mean (±SEM) value for each diet is also indicated. n = 22. Significantly different from the OSD, P = 0.0014 (paired t test).

Significant correlations were also found between the change in LDL cholesterol and the change in VEGF comparing the diets (r = 0.54, P = 0.0078), between the change in LDL cholesterol and the change in platelets (r = 0.76, P = 0.0002), and between the change in platelets and the change in VEGF (r = 0.66, P = 0.0032). The number of lymphocytes correlated inversely with docosahexaenoic acid concentrations considering both diet periods together (r = –0.54, P = 0.006; Figure 3). No associations were detected between inflammatory markers, fatty acid composition, or VEGF and maximal endothelial-dependent vasodilatation; the latter variable is reported elsewhere (8) and is presented here for correlation purposes only.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Number of lymphocytes in relation to increasing concentrations of docosahexaenoic acid (DHA) after the ordinary Swedish diet (•; n = 19) and the Mediterranean-inspired diet (; n = 21). The significance of the Pearson correlation coefficient (r = –0.54, P = 0.006) was assessed by permutation.

	DISCUSSION

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Although the present study showed clearcut changes in lipid, fatty acid, and hematology profiles, the lack of significant difference in oxidative stress measurements could be related to the rather low number of subjects used, which resulted in limited statistical power. Another limitation is that not all major outcome variables were measured at baseline before each diet arm, although glucose and lipid concentrations were, which suggests that the risk of carryover effects between the diet periods was rather low and that 1 mo of washout was enough to reestablish baseline concentrations.

Leukocyte count was shown to be a marker of increased risk of coronary heart disease mortality (12, 19). However, most of these results were in subjects at high risk of cardiovascular disease. Little information is available in low-risk groups. The phospholipid fatty acids are an integral component of membrane constituents that influence the physical properties of membrane function. Fatty acids and their metabolites also interfere with many steps of inflammation, such as vascular contraction, chemotaxis, cell adhesion and diapedesis, and cell activation. Fatty acids can directly or indirectly, via the eicosanoids, modulate leukocyte function and hence control the proliferation and production of cytokines and adhesion molecules (20). Persons living in countries with a high intake of saturated fats have higher concentrations of plasma LDL cholesterol and a higher incidence of coronary artery disease, whereas persons living in Mediterranean countries, who commonly consume diets rich in monounsaturated fatty acids, n–3 essential fatty acids, and antioxidants, have a lower incidence of coronary artery disease (21). Part of the beneficial effect of the MID is related to reduced atherosclerosis, and convincing evidence suggests that inflammation plays an essential role in the development and progression of atherosclerosis (4, 5). It has also been recognized that both monounsaturated fatty acids and PUFAs of the n–3 series have antiinflammatory actions that may contribute to their beneficial effects on LDL fatty acid composition (22) and endothelial function (23, 24).

Besides our findings of lower LDL-cholesterol and apo B plasma concentrations, the MID resulted in a reduction in saturated fatty acid intake in combination with a more than tripled amount of monounsaturated fatty acids and PUFAs compared with the OSD according to our previously reported 24-h recalls (8). This is in good agreement with results showing lower total serum saturated fatty acid concentrations, lower arachidonic acid concentrations, and substantially higher n–3 PUFA concentrations, which yielded a much lower ratio of n–6 to n–3 PUFAs after the MID than after the OSD.

Fatty fish and flax seed, which contain n–3 fatty acids, were used during the MID period. This group of fatty acids is known to generate a series of actions important for the inflammatory process through cell mediators such as prostaglandins and leukotrienes. It has been shown that fatty acids and mediators derived from them can also regulate the expression of adhesion proteins in both leukocytes and endothelial cells (25). A cross-sectional study of habitual dietary intake of fatty acids (n–3 and n–6) and the relation to inflammatory markers confirmed these beneficial effects (26). Taken together, our findings of a lower leukocyte count during the MID period corroborate these previously reported findings pointing toward a lower inflammatory state. Notably, the present study showed a reduction in the number of platelets during the MID compared with the OSD, which may have influenced the activity of the platelet pool, possibly conferring a protective effect against atherogenesis even in healthy subjects.

Ridker et al (27) suggested that circulating hs-CRP represents one of the strongest independent predictors of vascular death. We did not detect the hypothesized decrease in hs-CRP concentrations after the MID, although hs-CRP values showed a rather strong correlation with the WBC count after the OSD; no such correlation was evident after the MID. This pattern may represent an early indication of beneficial inflammatory change, detected even before a change in baseline and lipopolysaccharide-stimulated IL-6 concentrations, given that no such changes were observed in the present healthy subjects. The increase in IL-6 concentrations resulting from lipopolysaccharide stimulation, when expressed either in plasma or related to the respective WBC type, with special emphasis on monocytes, was not significantly different between the diet periods. In addition, we did not detect any change in MCP-1. This may be because the degree of vascular inflammation was still low in these healthy subjects and was not yet affecting measurable cytokine production, compared with, for example, the situation in patients with metabolic syndrome, as elegantly shown by Esposito et al (28). In that study, hs-CRP concentrations were 2.5 times those in the present study, which supports the contention of our subjects being without apparent cardiovascular disease risk.

Although we did not observe any relation between inflammatory markers and acetylcholine-induced forearm vasodilatation, several other variables suggest a beneficial reduction induced by the MID. Interestingly, we found a negative correlation between the number of lymphocytes and the concentrations of docosahexaenoic acid after both diet periods, which may also reflect a change in inflammatory characteristics, although the mechanism needs to be unraveled.

In summary, the MID reduced the number of platelets and leukocytes, and possibly as a consequence, lowered VEGF concentrations. Taken together, these observations are indicative of a beneficial influence of the MID, which may be linked to the marked reduction in the ratio of n–6 to n–3 fatty acids.

	ACKNOWLEDGMENTS

 
We thank Gun Bodehed-Berg, Tom Elfverson, Berit Holmberg, Magdalena Laffrenzen, and Staffan Nilsson for providing excellent help.

AA participated in the planning of the study, recruited the subjects, performed the experiments, and contributed to the writing of the manuscript. MJ participated in designing the study, recruited and examined the subjects, performed the experiment, and helped with writing the manuscript. MA took part in designing the study and planned the diet regimens, supervised the subjects while they participated in the study, and calculated energy intake. LG helped with blood sample analysis and manuscript writing. BS was responsible for the fatty acid analysis and manuscript writing. PF took part in designing and planning the study, recruiting the subjects, and analyzing the data and was the principal investigator responsible for the study and for drafting and finishing the manuscript. None of the authors had a conflict of interest to disclose.

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