An olive oil-rich diet results in higher concentrations of LDL cholesterol and a higher number of LDL subfraction particles than rapeseed oil and sunflower oil diets
2000; Elsevier BV; Volume: 41; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)32351-8
ISSN1539-7262
AutoresAnette Tønnes Pedersen, Manfred W. Baumstark, Peter Marckmann, Helena Gylling, Brittmarie Sandström,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoWe investigated the effect of olive oil, rapeseed oil, and sunflower oil on blood lipids and lipoproteins including number and lipid composition of lipoprotein subclasses. Eighteen young, healthy men participated in a double-blinded randomized cross-over study (3-week intervention period) with 50 g of oil per 10 MJ incorporated into a constant diet. Plasma cholesterol, triacylglycerol, apolipoprotein B, and very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) cholesterol concentrations were 10–20% higher after consumption of the olive oil diet compared with the rapeseed oil and sunflower oil diets [analysis of variance (ANOVA), P < 0.05]. The size of IDL, VLDL, and LDL subfractions did not differ between the diets, whereas a significantly higher number (apolipoprotein B concentration) and lipid content of the larger and medium-sized LDL subfractions were observed after the olive oil diet compared with the rapeseed oil and sunflower oil diets (ANOVA, P < 0.05). Total HDL cholesterol concentration did not differ significantly, but HDL2a cholesterol was higher after olive oil and rapeseed oil compared with sunflower oil (ANOVA, P < 0.05). In conclusion, rapeseed oil and sunflower oil had more favorable effects on blood lipids and plasma apolipoproteins as well as on the number and lipid content of LDL subfractions compared with olive oil. Some of the differences may be attributed to differences in the squalene and phytosterol contents of the oils.—Pedersen, A., M. W. Baumstark, P. Marckmann, H. Gylling, and B. Sandström. An olive oil-rich diet results in higher concentrations of LDL cholesterol and a higher number of LDL subfraction particles than rapeseed oil and sunflower oil diets. J. Lipid Res. 2000. 41: 1901–1911. We investigated the effect of olive oil, rapeseed oil, and sunflower oil on blood lipids and lipoproteins including number and lipid composition of lipoprotein subclasses. Eighteen young, healthy men participated in a double-blinded randomized cross-over study (3-week intervention period) with 50 g of oil per 10 MJ incorporated into a constant diet. Plasma cholesterol, triacylglycerol, apolipoprotein B, and very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) cholesterol concentrations were 10–20% higher after consumption of the olive oil diet compared with the rapeseed oil and sunflower oil diets [analysis of variance (ANOVA), P < 0.05]. The size of IDL, VLDL, and LDL subfractions did not differ between the diets, whereas a significantly higher number (apolipoprotein B concentration) and lipid content of the larger and medium-sized LDL subfractions were observed after the olive oil diet compared with the rapeseed oil and sunflower oil diets (ANOVA, P < 0.05). Total HDL cholesterol concentration did not differ significantly, but HDL2a cholesterol was higher after olive oil and rapeseed oil compared with sunflower oil (ANOVA, P < 0.05). In conclusion, rapeseed oil and sunflower oil had more favorable effects on blood lipids and plasma apolipoproteins as well as on the number and lipid content of LDL subfractions compared with olive oil. Some of the differences may be attributed to differences in the squalene and phytosterol contents of the oils.—Pedersen, A., M. W. Baumstark, P. Marckmann, H. Gylling, and B. Sandström. An olive oil-rich diet results in higher concentrations of LDL cholesterol and a higher number of LDL subfraction particles than rapeseed oil and sunflower oil diets. J. Lipid Res. 2000. 41: 1901–1911. Dietary fatty acid composition influences plasma lipids and lipoproteins associated with the development of atherosclerosis and ischemic heart disease (IHD). The effect of dietary fatty acids on plasma total cholesterol, and on low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol, has been subject to many studies and prediction algorithms have been developed (1Keys A. Anderson J.T. Grande F. Serum cholesterol response to changes in the diet. IV. Particular saturated fatty acids in the diet.Metabolism. 1965; 14: 776-787Google Scholar, 2Hegsted D.M. McGandy R.B. Myers M.L. Stare F.J. Quantitative effects of dietary fat on serum cholesterol in man.Am. J. Clin. Nutr. 1965; 17: 281-295Google Scholar, 3Mensink R.P. Katan M.B. 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Influence of n-3 fatty acids from fish oils on concentration of high- and low-density lipoprotein subfractions and their lipid and apolipoprotein composition.Clin. Biochem. 1992; 25: 338-340Google Scholar) and an increased LDL particle size (30Tinker L.F. Parks E.J. Behr S.R. Schneeman B.O. Davis P.A. (n-3) fatty acid supplementation in moderately hypertriglyceridemic adults changes postprandial lipid and apolipoprotein B responses to a standardized test meal.J. Nutr. 1999; 129: 1126-1134Google Scholar) compared with baseline. Thus, the limited available evidence suggests that the amount and type of dietary fat influence the LDL subfraction profile, but it is not clear whether common edible vegetable oils leading to varying blood lipid levels also differ with respect to lipoprotein subclass profiles. In the present study, we therefore compared the effects of three experimental diets rich in different vegetable oils (olive oil [OO], rapeseed oil [RO], and sunflower oil [SO]) on blood lipids and on the number, size, and composition of LDL and HDL subfractions. It was hypothesized that RO, because of its lower content of saturated fatty acids (SFA) compared with OO and its higher content of an n–3 fatty acid, would be associated with the most favorable lipoprotein subclass profile. Furthermore, we expected that SO, because of its higher content of polyunsaturated fatty acids (PUFA), would lead to lower blood lipid concentrations, but higher, less favorable total and LDL:HDL cholesterol ratios compared with the OO and RO diets. The effects on blood coagulation factor VII (31Larsen L.F. Jespersen J. Marckmann P. Are olive oil diets antithrombotic? Diets enriched with olive, rapeseed, or sunflower oil affect postprandial factor VII differently.Am. J. Clin. Nutr. 1999; 70: 976-982Google Scholar) and the oxidation resistance of VLDL and LDL particles (N. S. Nielsen, P. Marckmann, and C-E. Høy, unpublished observations) were also studied, but are presented separately. Eighteen male students were recruited for the study by local advertisement. The subjects were aged 20–28 years (mean, 24 years), weighed from 62 to 99 kg (mean, 79 kg), and had body mass indexes from 18 to 27 kg/m2 (mean, 23 kg/m2). All subjects were nonsmokers and did not use any medication. They were apparently healthy, did not exercise excessively, and had no family history of atherosclerotic disease or hypertension. Mean fasting lipid concentrations at inclusion were as follows: plasma total cholesterol, 4.74 mM (range, 3.09 to 6.25 mM); HDL-cholesterol, 1.10 mM (range, 0.84 to 1.50 mM); and plasma total TAG, 1.2 mM (range, 0.41 to 3.29 mM). The aim of the stud y was explained orally to each subject and written information was given, before the subjects gave their written consent. The research protocol was approved by the Scientific Ethics Committee of the municipalities of Copenhagen and Frederiksberg (01-272/95). The study was performed as a double-blinded, randomized, cross-over experiment with three periods of 3 weeks separated by wash-out periods of 5–12 weeks. Six groups of three subjects consumed the diets in the following order: OO, RO, SO/OO, SO, RO/RO, OO, SO/RO, SO, OO/SO, OO, RO/SO, RO, OO, respectively. Subjects were carefully instructed not to change habitual diets during the wash-out periods and to keep the same physical activity level during the study. Subjects received all foods from our department and were not allowed to consume other foods in the study periods, except for water, plain coffee, and tea (coffee and tea in small amounts). Subjects were allocated to fixed energy levels according to body weight, age, and physical activity indexes. Body weight was measured every second day in each period, and a weight variation of more than 1 kg resulted in either the allocation to a different energy level or to consumption of extra rolls (same nutrient composition as the total diet). The mean energy intake during the study was 15.4 MJ/day (range, 13–18 MJ). Consumption of extra rolls was noted in a diary, as were daily records of physical activity, any sign of illness, medication, coffee and tea intake, and any deviation from the diet. On weekdays lunch was consumed in the department under supervision, whereas beverages, dinner, snack, and breakfast for the next morning were provided daily as a package with guidelines for its preparation. Food and beverages for the weekend were provided on Fridays. Leftovers were brought back to the department to be registered. The amounts of returned foods were small and did not affect the average fat intake and fat composition for any of the subjects. Most food for each intervention period was prepared in one batch in the metabolic kitchen, weighed, coded in color according to the oil used, and frozen until use. Duplicate portions of each diet were collected during 2 weeks in each period of the study. The portions were pooled per diet and analyzed for energy content and nutritional composition. The three diets were identical except for the quality of fat. Nineteen percent of the total dietary energy intake, that is, 50 g/10 MJ, derived from either extra virgin OO (Navarino, Danton Trading, Århus, Denmark), physically refined RO (in a pilot scale) (Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark), or chemically refined SO (Solex W, Århus Olie, Århus, Denmark). All other food items were held constant and identical in the three experimental diet periods. Menus were repeated every week and consisted of common prepared and cooked foods. The test oils were included in bread, rolls, cakes, dressing, and dinner dishes consisting of vegetables, meat, pasta, rice, or potatoes. Duplicate portions of each of the three 3-week diets were collected, homogenized, lyophilized, and chemically analyzed. Nitrogen was determined according to the principle of Dumas (32Kirsten W.J. Hesselius G.U. Rapid, automatic, high capacity Dumas determination of nitrogen.Microchem. J. 1983; 28: 529-547Google Scholar) on an automatic nitrogen analyzer (NA 1500; Carlo Erba Strumentazione, Milan, Italy). Fat content was measured after extraction according to the procedure by Folch, Lees, and Stanley (33Folch J. Lees M. Stanley G.H.S. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Google Scholar), and the content of dietary fiber was determined enzymatically (34Asp N.G. Johansson C.G. Hallmer H. Siljeström M. Rapid enzymatic assay of insoluble and soluble dietary fiber.J. Agric. Food Chem. 1983; 31: 476-482Google Scholar). The diet macronutrient composition is shown in Table 1.TABLE 1.Analyzed macronutrient composition (% of total energy) and fiber and cholesterol content (g/10 MJ) of olive oil, rapeseed oil, and sunflower oil dietsOO DietRO DietSO DietFat353535Saturated fatty acids11910Monounsaturated fatty acids21189Polyunsaturated fatty acids3715Carbohydrates535253Protein121312Fiber (g/10 MJ)252224Cholesterol (mg/10 MJ)aThe cholesterol content of the experimental diet without added oil was calculated by the use of a national database (Dankost 2.0, Danish Catering Center A/S, Herlev, Denmark) and the analyzed cholesterol content of the oils was added.257259257Abbreviations: OO, olive oil; RO, rapeseed oil; SO, sunflower oil.a The cholesterol content of the experimental diet without added oil was calculated by the use of a national database (Dankost 2.0, Danish Catering Center A/S, Herlev, Denmark) and the analyzed cholesterol content of the oils was added. Open table in a new tab Abbreviations: OO, olive oil; RO, rapeseed oil; SO, sunflower oil. The fatty acid composition of the 3-week diets was analyzed by gas chromatography (8420; Perkin-Elmer, Birkerød, Denmark) after extraction with diethyl ether-petroleum ether and subsequent methylation with methanolic BF3 (35Morrison W.R. Smith L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J. Lipid Res. 1964; 5: 600-608Google Scholar) by the National Food Administration in Denmark (Table 2). The squalene and sterol contents of the oils (Table 3) were determined from non-saponifiable material by gas-liquid chromatography on a 50-m-long capillary column (Ultra 1R; Hewlett-Packard, Palo Alto, CA) as trimethylsilyl derivatives, using 5α-cholestane as internal standard (36Miettinen T.A. Cholesterol metabolism during ketoconazole treatment in man.J. Lipid Res. 1988; 29: 43-51Google Scholar), at the University of Helsinki (Helsinki, Finland).TABLE 2.Analyzed fatty acid composition (mol%) of the three experimental diets containing 50 g/10 MJ of olive oil, rapeseed oil, and sunflower oilFatty AcidsOO DietRO DietSO DietSaturated fatty acids, total312729Lauric acid (C12:0)111Myristic acid (C14:0)343Palmitic acid (C16:0)191516Stearic acid (C18:0)546Monounsaturated fatty acids, total605027Oleic acid (C18:1, n–9)554524Polyunsaturated fatty acids, total82142Linoleic acid (C18:2, n–6)71541α-Linolenic acid (C18:3, n–3)0.860.8Abbreviations: OO, olive oil; RO, rapeseed oil; SO, sunflower oil. Open table in a new tab TABLE 3.Sterol and squalene concentrations of the test oils (mg/kg)Olive OilRapeseed OilSunflower OilCholesterol10277Campesterol552,753543Sitosterol9583,8922,417Squalene3,65110134 Open table in a new tab Abbreviations: OO, olive oil; RO, rapeseed oil; SO, sunflower oil. Fasting blood samples were taken after 10 min of supine rest before the study and on days 21 and 22 in each diet period. Blood samples were collected in tubes without additives for the analysis of serum C-reactive protein (CRP), and in ethylenediaminetetraacetic acid tubes for the analysis of plasma insulin, NEFA, cholesterol, TAG, apolipoproteins, squalene, sterols, fatty acid composition of plasma cholesteryl esters, and lipoprotein fractionation. All plasma samples were immediately placed on ice, and centrifuged at 3,000 g for 15 min at 4°C. Samples for serum CRP were kept at –20°C, samples for plasma insulin, NEFA, squalene, sterols, and fatty acid composition of plasma cholesterol esters at –80°C, and samples for lipids, apolipoproteins, and lipoprotein fractionation were shipped the same day (to Freiburg University, Freiburg, Germany) and kept cooled (4°C) until analysis. Elevated CRP concentration in one subject on one occasion and technical problems with samples from one subject on one occasion resulted in the exclusion of these observations. Serum CRP was assessed by a commercial immuno-turbidimetric method (Roche, Basel, Switzerland), plasma insulin by an enzyme-linked immunosorbent assay method (Dako, Glostrup, Denmark), and plasma NEFA by a commercial enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany). The fatty acid composition of plasma cholesteryl esters was determined after separation by thin-layer chromatography. Plasma cholesteryl esters were methylated with BF3 (35Morrison W.R. Smith L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J. Lipid Res. 1964; 5: 600-608Google Scholar) and the methyl esters were analyzed by gas chromatography (HP 6890; Hewlett-Packard) at the Technical University of Denmark. Plasma squalene and noncholesterol sterols were determined as described in the food analysis section. VLDL (d < 1.0063 g/ml), IDL (1.0063 g/ml < d < 1.019 g/ml), LDL (1.019 g/ml < d < 1.063 g/ml), and HDL (1.063 g/ml < d < 1.21 g/ml) were prepared by sequential flotation according to Lindgren et al. (37Lindgren F.T. Jensen L.L. Hatch F.T. The isolation and quantitative analysis of serum lipoproteins.in: Nelson G.J. Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. Wiley-Interscience, New York1972: 181-274Google Scholar, 38Lindgren F.T. Preparative ultracentrifugal laboratory procedures and suggestions for lipoprotein analysis.in: Perkins E.G. Analysis of Lipids and Lipoproteins. American Oil Chemists' Society, Champaign, IL1975: 204-224Google Scholar). HDL subfractions (HDL 2b, 1.063 g/ml < d < 1.100 g/ml; HDL2a, 1.100 g/ml < d < 1.125 g/ml; HDL3, 1.125 g/ml < d < 1.21 g/ml) were prepared by equilibrium density gradient centrifugation according to Anderson et al. (39Anderson D.W. Nichols A.V. Forte T.M. Lindgren F.T. Particle distribution of human serum high density lipoproteins.Biochim. Biophys. Acta. 1977; 493: 55-68Google Scholar, 40Anderson D.W. Nichols A.V. Pan S.S. Lindgren F.T. High density lipoprotein distribution. Resolution and determination of three major components in a normal population sample.Atherosclerosis. 1978; 29: 161-179Google Scholar) with minor modifications. Total LDL was fractionated into six density classes by equilibrium density gradient centrifugation as described previously (41Baumstark M.W. Kreutz W. Berg A. Frey I. Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering.Biochim. Biophys. Acta. 1990; 1037: 48-57Google Scholar). The density ranges of the subfractions as determined by precision refractometr y (38Lindgren F.T. Preparative ultracentrifugal laboratory procedures and suggestions for lipoprotein analysis.in: Perkins E.G. Analysis of Lipids and Lipoproteins. American Oil Chemists' Society, Champaign, IL1975: 204-224Google Scholar) of blank gradients were as follows: LDL-1, d < 1.031; LDL-2, 1.031 < d < 1.034; LDL-3, 1.034 < d < 1.037; LDL-4, 1.037 < d < 1.040; LDL-5, 1.040 < d < 1.044; LDL-6, d > 1.044 g/ml. All centrifugation steps were performed at a temperature of 18°C using partially filled (6 ml) polycarbonate bottles in a 50 Ti rotor. Phospholipid (PL), free cholesterol (FC), total cholesterol (TC), and TAG were measured by automated (EPOS; Eppendorf, Hamburg, Germany) enzymatic methods. Reagent kits for TC and TAG were obtained from Boehringer Mannheim (Mannheim, Germany), kits for FC were purchased from Wako Chemicals, and kits for PL were from bioMérieux (Nürtingen, Germany). Apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) were measured by end-point nephelometr y (Behring, Marburg, Germany). ApoB was standardized according to the Centers for Disease Control (CDC, Atlanta, GA) standard. This standard was confirmed by amino acid analysis in an LDL-3/LDL-4 pool (41Baumstark M.W. Kreutz W. Berg A. Frey I. Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering.Biochim. Biophys. Acta. 1990; 1037: 48-57Google Scholar). Particle composition of apoB-containing particles was assessed by calculating the molar ratio of lipid per apoB, using previously published molecular weights (41Baumstark M.W. Kreutz W. Berg A. Frey I. Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering.Biochim. Biophys. Acta. 1990; 1037: 48-57Google Scholar). The average particle volume of each subfraction was calculated from the above-described number of lipid molecules per particle and the volume of the lipid molecules and the estimated volume of one apoB molecule (41Baumstark M.W. Kreutz W. Berg A. Frey I. Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering.Biochim. Biophys. Acta. 1990; 1037: 48-57Google Scholar). A radius corresponding to this volume was calculated, assuming a spherical lipoprotein particle. Radii calculated in this way are in good agreement with radii measured by X-ray small-angle scattering (41Baumstark M.W. Kreutz W. Berg A. Frey I. Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering.Biochim. Biophys. Acta. 1990; 1037: 48-57Google Scholar). The described method for the analysis of cholesterol and TAG in lipoproteins resulted in a mean recovery for cholesterol of 0.85 (range, 0.79–0.92) and for TAG of 0.91 (range, 0.84–1.03). The discrepancy may be due to the fact that concentrations in plasma were measured in samples other than those used to determine concentrations in lipoproteins. HDL cholesterol was also measured by a classic polyethylene glycol precipitation method (Quantolip; Immuno AG, Vienna, Austria) and resulted in slightly higher HDL cholesterol levels than those presented in this article. However, neither of the methods revealed significant differences in HDL cholesterol between the diets. In addition, total, chylomicron, VLDL, and LDL plus HDL cholesterol concentrations were measured in a different laborator y (Research Department of Human Nutrition, Frederiksberg, Denmark) (after separation by sequential ultracentrifugation) with kits from Boehringer Mannheim and the results showed lower cholesterol concentrations in total plasma and higher concentrations in the individual lipoproteins, resulting in better recover y for these data. However, the statistical analyses of the two sets of data resulted in similar results. Therefore, it was decided that the data including measurements of subfractions should be presented despite a risk of minor compositional bias due to the incomplete recovery. The intra-assay c
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