Walnut-enriched diet increases the association of LDL from hypercholesterolemic men with human HepG2 cells

      In a randomized, cross-over feeding trial involving 10 men with polygenic hypercholesterolemia, a control, Mediterranean-type cholesterol-lowering diet, and a diet of similar composition in which walnuts replaced ~35% of energy from unsaturated fat, were given for 6 weeks each. Compared with the control diet, the walnut diet reduced serum total and LDL cholesterol by 4.2% (P = 0.176), and 6.0% (P = 0.087), respectively. No changes were observed in HDL cholesterol, triglycerides, and apolipoprotein A-I levels or in the relative proportion of protein, triglycerides, phospholipids, and cholesteryl esters in LDL particles. The apolipoprotein B level declined in parallel with LDL cholesterol (6.0% reduction). Whole LDL, particularly the triglyceride fraction, was enriched in polyunsaturated fatty acids from walnuts (linoleic and α-linolenic acids). In comparison with LDL obtained during the control diet, LDL obtained during the walnut diet showed a 50% increase in association rates to the LDL receptor in human hepatoma HepG2 cells. LDL uptake by HepG2 cells was correlated with α-linolenic acid content of the triglyceride plus cholesteryl ester fractions of LDL particles (r2 = 0.42, P < 0.05). Changes in the quantity and quality of LDL lipid fatty acids after a walnut-enriched diet facilitate receptor-mediated LDL clearance and may contribute to the cholesterol-lowering effect of walnut consumption.
      —Muñoz, S., M. Merlos, D. Zambón, C. Rodríguez, J. Sabaté, E. Ros, and J. C. Laguna. Walnut-enriched diet increases the association of LDL from hypercholesterolemic men with human HepG2 cells. J. Lipid Res. 2001. 42: 2069–2076.

      Supplementary key words

      There is ample evidence that blood cholesterol levels are reduced by dietary substitution of unsaturated fatty acids from vegetable oils and fats and fish products for saturated fatty acids (SFA) from animal fat (
      • Mensink R.P.
      • Katan M.B.
      Effect of dietary fatty acids on serum lipids and lipoproteins: a meta-analisis of 27 trials.
      ), and this effect can be predicted by the amounts of fatty acid classes exchanged (
      • Mensink R.P.
      • Katan M.B.
      Effect of dietary fatty acids on serum lipids and lipoproteins: a meta-analisis of 27 trials.
      ,
      • Hegsted D.M.
      • Ausman L.M.
      • Johnson J.A.
      • Dallal G.E.
      Dietary fat and serum lipids: an evaluation of the experimental data.
      ,
      • Yu S.
      • Derr J.
      • Etherton T.D.
      • Kris-Etherton P.M.
      Plasma cholesterol-predictive equations demonstrate that stearic acid is neutral and monounsaturated fatty acids are hypocholesterolemic.
      ,
      • Clarke R.
      • Frost C.
      • Collins R.
      • Appleby P.
      • Peto R.
      Dietary lipids and blood cholesterol. Quantitative meta-analysis of metabolic ward studies.
      ). Hence, current guidelines for reducing the risk of cardiovascular disease by dietary and other lifestyle practices emphasize replacement of foods with cholesterol-raising fatty acids by nutrients with a high unsaturated fatty acid content in conjunction with an energy intake suitable for maintaining a normal body weight (
      • Willett W.C.
      Diet and health: what should we eat?.
      ,
      • Krauss R.M.
      • Eckel R.H.
      • Howard B.
      • Appel L.J.
      • Daniels S.R.
      • Deckelbaum R.J.
      • Erdman Jr., J.W.
      • Kris-Etherton P.
      • Goldberg I.J.
      • Kotchen T.A.
      • Lichtenstein A.H.
      • Mitch W.E.
      • Mullis R.
      • Robinson K.
      • Wylie-Rosett J.
      • Jeor S.St.
      • Suttie J.
      • Tribble D.L.
      • Bazzarre T.L.
      AHA Dietary Guidelines. Revision 2000: a statement for healthcare professionals from the Nutrition Committee of the American Heart Association.
      ). Among foods with favorable fatty acid profiles, nuts have received particular attention because of the epidemiological association of their frequent intake with protection from coronary heart disease (
      • Fraser G.E.
      • Sabaté J.
      • Beeson W.L.
      • Strahan T.M.
      A possible protective effect of nut consumption on risk of coronary heart disease: the Adventist Health Study.
      ,
      • Kushi L.H.
      • Folsom A.R.
      • Prineas R.J.
      • Mink P.J.
      • Wu Y.
      • Bostick R.M.
      Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women.
      ,
      • Hu F.B.
      • Stampfer M.J.
      • Manson J.E.
      • Rimm E.B.
      • Colditz G.A.
      • Rosner B.A.
      • Speizer F.E.
      • Hennekens C.H.
      • Willet W.C.
      Frequent nut consumption and risk of coronary heart disease in women: prospective cohort study.
      ,
      • Sabaté J.
      Nut consumption, vegetarian diets, ischemic heart disease risk, and all-cause mortality: evidence from epidemiologic studies.
      ). Feeding studies that have incorporated different nuts into test diets have shown a consistent cholesterol-lowering effect of regular nut consumption, with an actual response greater than that predicted from the changes in dietary fatty acids (
      • Kris-Etherton P.M.
      • Yu-Poth S.
      • Sabaté J.
      • Ratcliffe H.E.
      • Zhao G.
      • Etherton T.D.
      Nuts and their bioactive constituents: effects on serum lipids and other factors that affect disease risk.
      ). Thus the mechanisms underlying plasma cholesterol reduction due to nut consumption remain speculative.
      Of the tree nuts, walnuts are unique because they are a rich source of the PUFA 18:2n-6 (linoleic acid) and 18:3n-3 (α-linolenic acid) (

      Exler, J., Weihrauch, J. L., 1986. Provisional Table on the Content of Omega-3 Fatty Acids and Other Fat Components in Selected Foods. U.S. Department of Agriculture Publication No. HNIS/PT-103.

      ). Controlled dietary intervention trials also have shown that daily consumption of walnuts has a cholesterol-lowering effect greater than expected from predictive models that include regression coefficients for percentage energy changes in dietary fatty acids (
      • Sabaté J.
      • Fraser G.E.
      • Burke K.
      • Knutsen S.
      • Bennett H.
      • Lindsted K.D.
      Effects of walnuts on serum lipids levels and blood pressure in normal men.
      ,
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). In our randomized cross-over trial in hypercholesterolemic men and women (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ), the replacement of ~35% of energy from unsaturated fatty acids with walnuts, without changing total fat or SFA intake, reduced serum LDL cholesterol by an average of 0.29 mM [95% confidence interval (CI), −0.41 to −0.15 mM] [−11.2 mg/dl (−16.3 to −6.1 mg/dl)] compared with the control diet, whereas the decrease predicted by the equations based on percentage energy changes in dietary fatty acids ranged from 0.13 to 0.18 mM (5 to 7 mg/dl) (
      • Mensink R.P.
      • Katan M.B.
      Effect of dietary fatty acids on serum lipids and lipoproteins: a meta-analisis of 27 trials.
      ,
      • Hegsted D.M.
      • Ausman L.M.
      • Johnson J.A.
      • Dallal G.E.
      Dietary fat and serum lipids: an evaluation of the experimental data.
      ,
      • Yu S.
      • Derr J.
      • Etherton T.D.
      • Kris-Etherton P.M.
      Plasma cholesterol-predictive equations demonstrate that stearic acid is neutral and monounsaturated fatty acids are hypocholesterolemic.
      ,
      • Clarke R.
      • Frost C.
      • Collins R.
      • Appleby P.
      • Peto R.
      Dietary lipids and blood cholesterol. Quantitative meta-analysis of metabolic ward studies.
      ).
      We wondered whether LDL compositional changes produced by the walnut diet, particularly LDL enrichment with PUFA from walnuts, might increase LDL uptake by liver cells, thus reducing the number of circulating LDL particles and contributing to the observed cholesterol-lowering effect. To test this hypothesis, LDL were isolated for association studies at the end of the control diet and the walnut diet periods in a subset of 10 men successfully completing the trial (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). LDL association with cultured human hepatoma cells (HepG2) was used as a functional test to identify differences in the biological properties of LDL from the two dietary periods. The human cell line HepG2 is recognized as a good surrogate for adult hepatocytes, as it is able to reproduce most hepatocytic metabolic functions (
      • Javitt N.B.
      HepG2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids.
      ), in particular the clearance of LDL through functional LDL receptors (
      • Havekes L.
      • van Hinsbergh V.
      • Kempen H.J.
      • Emeis J.
      The metabolism in vitro of human low density lipoprotein by the human hepatoma cell line HepG2.
      ,
      • Dashti N.
      • Wolfbauer G.
      • Koren E.
      • Knowles B.
      • Alaupovic P.
      Catabolism of human low density lipoproteins by human hepatoma cell line HepG2.
      ). LDL association with the LDL receptor has been satisfactorily measured by using the fluorescent probe 3,3′-dioctadecylindocarbocyanine (DiI), which is incorporated into the lipoprotein particle in the phospholipid fraction (
      • Yen C.F.
      • Kalunta C.I.
      • Chen F.S.
      • Kaptein J.S.
      • Lin C.K.E.
      • Lad P.M.
      Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.
      ,
      • Lohne K.
      • Urdal P.
      • Leren T.P.
      • Tonstad S.
      • Ose L.
      Standarization of a flow cytometric method for measurement of low density lipoprotein receptor activity on blood mononuclear cells.
      ). The results suggest that LDL incorporation of PUFA from walnuts increases the rate of association of these particles with the LDL receptor, thus providing a potential explanation for the cholesterol-lowering effect of the walnut diet.

      MATERIALS AND METHODS

       Patients

      Ten men with polygenic hypercholesterolemia, who were enrolled in a dietary intervention trial comparing the lipid effects of a diet enriched with walnuts with those of a control Mediterranean diet, were studied (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). The participants were nonsmokers and had no evidence of alcohol abuse or liver, kidney, or endocrine disorders; any lipid-lowering medication was withdrawn 8 weeks before study. None of them had prior coronary heart disease or were taking other medications known to affect plasma lipid levels. Their ages ranged from 48 to 71 years, the body mass index ranged between 23.1 and 30.0 kg/m2, the systolic blood pressure ranged between 120 and 150 mm Hg, and the diastolic blood pressure ranged between 65 and 90 mm Hg.

       Experimental design

      The study protocol has been described in detail (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). Essentially, it was a randomized, cross-over trial comparing the effects of two cholesterol-lowering diets on blood lipids, LDL fatty acid composition, and LDL resistance to oxidation. Dietary intervention consisted of two consecutive 6-week diet periods after a 4-week preinclusion period. The diet periods were randomly assigned: one group followed the walnut diet during the first period and then switched to the control diet during the second period, while the other group followed the diets in reverse order. The investigators performing laboratory analyses were blinded with respect to the subjects' diet sequence. The study protocol was approved by the institutional review board of the Hospital Clínic of Barcelona, and all subjects gave informed consent.

       Diets

      The composition of the two experimental cholesterol-lowering diets has been detailed previously (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). Participants ate on their own, and prepackaged daily allowances of raw, shelled walnuts were provided for daily consumption in amounts varying from 41 to 56 g (the equivalent of 8 to 11 walnuts) during the walnut diet. The control diet was Mediterranean, composed of natural foodstuffs and with olive oil as the main culinary fat. The walnut diet was similar to the control diet, but walnuts replaced approximately 35% of the total energy provided by olive oil and other sources of monounsaturated fatty acids (MUFA). The composition of the two experimental diets was nearly identical with respect to total fat and SFA, protein, carbohydrate, cholesterol, and fiber content, but their unsaturated fat content was dissimilar. The fatty acid composition of the control diet reflected the high MUFA content of olive oil, whereas that of the walnut diet mirrored the constitution of walnut fat, particularly rich in PUFA (Table 1). Adherence to the study diets was carefully monitored, as described (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ).
      TABLE 1.Composition of prescribed and actually observed study diets
      VariableControl DietWalnut DietPValue between Actual Diets
      PrescribedActual
      Mean ± SD values estimated from one 3-day food record and six 24-h diet recalls during each diet period.
      PrescribedActual
      Mean ± SD values estimated from one 3-day food record and six 24-h diet recalls during each diet period.
      Energy, kcal/day1,600–2,2001,899 ± 1021,600–2,2001,942 ± 178>0.2
      Fat, % energy30.230.9 ± 0.732.731.8 ± 0.60.009
      Saturated fatty acids5.86.0 ± 0.55.05.5 ± 0.60.058
      Monounsaturated fatty acids17.617.5 ± 0.713.212.9 ± 0.5<0.001
      Polyunsaturated fatty acids4.23.9 ± 0.411.811.2 ± 0.3<0.001
      Linoleic (C18:2n-6)3.33.1 ± 0.49.69.3 ± 0.6<0.001
      α-Linolenic (C18:3n-3)0.40.3 ± 0.11.91.8 ± 0.1<0.001
      Protein, % energy18.119.1 ± 1.216.618.8 ± 1.0>0.2
      Carbohydrates, % energy51.748.5 ± 1.450.748.8 ± 1.6>0.2
      Cholesterol, mg/1,000 kcal103.9117.2 ± 22.777.886.1 ± 14.50.002
      Soluble fiber, g/1,000 kcal4.83.7 ± 0.45.03.9 ± 0.4>0.2
      α-Tocopherol, mg/1,000 kcal5.85.2 ± 0.45.14.4 ± 0.4<0.001
      Vitamin C, mg/1,000 kcal76.287.3 ± 14.976.380.7 ± 17.6>0.2
      a Mean ± SD values estimated from one 3-day food record and six 24-h diet recalls during each diet period.

       Blood sampling and analysis

      Blood samples were obtained after an overnight fast twice during the last 2 weeks of each dietary period. Both serum and 0.1% EDTA plasma were collected. The LDL fraction was isolated by sequential ultracentrifugation immediately after separation of plasma. Serum lipid and lipoprotein lipid analyses were performed as described (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). LDL lipid separation and fatty acid analysis were performed by thin-layer chromatography and subsequent gas chromatography of the fatty acid methyl esters (
      • Vázquez M.
      • Merlos M.
      • Adzet T.
      • Laguna J.C.
      Decreased susceptibility to copper-induced oxidation of rat lipoproteins after fibrate treatment. Influence of fatty acid composition.
      ). Lipoprotein oxidation in the presence of 5 μM CuSO4 ·5H2Owas determined by measuring conjugated diene kinetics, as described previously (
      • Vázquez M.
      • Zambón D.
      • Hernández Y.
      • Adzet T.
      • Merlos M.
      • Ros E.
      • Laguna J.C.
      Lipoprotein composition and oxidative modification during therapy with gemfibrozil and lovastatin in patients with combined hyperlipidaemia.
      ).

       LDL labeling

      The LDL were labeled with the fluorescent probe DiI (Molecular Probes, Eugene, OR) by a modification of the method described by Stephan and Yurachek (
      • Stephan Z.F.
      • Yurachek E.C.
      Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.
      ). Briefly, a stock solution of DiI was prepared by dissolving 30 mg of DiI in 1 ml of dimethyl sulfoxide, and an appropriate volume was added to the LDL solution, adjusted to 1 mg of LDL protein per ml to yield a final concentration of 150 μg of DiI per mg of LDL protein. The mixture was protected from light and incubated at 37 °C for 18 h to provide DiI-labeled LDL (DiI-LDL), which was subsequently layered over with an NaBr solution (density, 1.1 g/ml) and reisolated by ultracentrifugation (100,000 g, 18 h, 20 °C), dialyzed (Spectrapor MWCO 3500; Serva, Heidelberg, Germany) against PBS (18 h, 4 °C), and sterilized by filtration (Nalgene polyether sulfone filter, 0.22 μm; Nalge Nunc International, Naperville, IL). The final protein concentration was determined by the Bradford method (
      • Bradford M.
      A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding.
      ). The DiI-LDL was stored at 4 °C in the dark and used within 7 days.

       DiI and DiI-LDL standard curve

      A standard solution of DiI was prepared in isopropanol at a concentration range of 0 to 200 ng/ml and its fluorescence was measured in a Hitachi F2000 fluorometer with excitation and emission wavelengths set at 522 and 564 nm, respectively. Standard solutions of DiI-LDL samples with a concentration range of 100 to 1,000 ng of protein per ml were prepared from a stock solution of 10 μg of LDL protein per ml in isopropanol and their fluorescence was determined as described above. The specific activity of the DiI-LDL samples was calculated as the amount of DiI (micrograms) incorporated into 1 mg of LDL protein.

       Cell culture and association assay

      Human hepatoma (HepG2) cells were obtained from the European Collection of Cell Culture (Salisbury, Wiltshire, UK). The cells were routinely cultured in DMEM containing 10% fetal bovine serum, penicillin G (100 IU/ml; Sigma, Madrid, Spain), and streptomycin (74.7 IU/ml; Sigma), and maintained at 37 μC in equilibration with 5% CO2-95% air in 10-mm plates. For experiments, HepG2 cells (2.5 × 105 cells/well) were seeded into six-well plates in DMEM supplemented with 10% fetal bovine serum, grown for 72 h, and then switched to DMEM containing 0.5% fatty acid-free bovine serum albumin for an additional 24 h. Afterward, the cells were incubated with 30 μg of DiI-LDL protein per ml for 0, 15, 20, 60, 90, and 120 min at 37 °C. At this temperature, both binding and internalization of DiI-LDL particles were measured (
      • Javitt N.B.
      HepG2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids.
      ). At the end of each incubation period, the cells were extensively washed with PBS, and 2 ml of isopropanol was added to each well, with gentle shaking of the plates for 15 min. The isopropanol extracts were then transferred to 10 × 25 mm glass tubes and centrifuged at 3,500 g for 10 min, and the fluorescence was determined as described above. DiI content in the extracts was calculated from the DiI standard curve. The cells were dissolved in 2 N NaOH and left overnight at 4 °C for protein determination. Taking into account the specific activity for each DiI-LDL sample, the rate of association of the DiI-LDL with the HepG2 cells was obtained from the slope of the adjusted straight line obtained by representing the total amount of DiI-LDL associated with the cells versus time and expressed as micrograms of LDL protein per minute per milligram cellular protein, as described elsewhere (
      • Stephan Z.F.
      • Yurachek E.C.
      Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.
      ). The association rate was linear throughout the interval of time studied (Fig. 1A). Saturation was obtained at concentrations of DiI-LDL above 80 °g of LDL protein per ml of incubation medium (Fig. 1B). Scatchardplots gave a linear representation (bound LDL/free LDL − 0.0096 bound LDL + 0.2132, Bmax = 22 μg of LDL protein per mg of HepG2 cell protein), pointing to a single high affinity binding site for LDL on the surface of HepG2 cells.
      Figure thumbnail gr1
      Fig. 1.A: Time dependence of the rate of association of DiI-LDL with HepG2 cells. LDL concentration was fixed at 30 °g of LDL protein per ml of culture medium. Each point is the mean of three different assays; in each assay, two cell preparations for every time point were determined and averaged. B: Dependence of the rate of association (at 37 °C) and binding (at 4 °C) of DiI-LDL to HepG2 cells with respect to the concentration of LDL protein added to the culture medium. The incubation time was fixed at 2 h. Internalization was calculated as the difference, for each LDL concentration point, of the corresponding mean values for internalization and binding. Each point is the mean of three different assays; in each assay, two cell preparations for every LDL concentration point were determined and averaged.
      To assess the sensitivity of the technique to changes in LDL receptor activity, cells were incubated in DMEM supplemented with 10% fetal bovine serum in the presence of 1 μM lovastatin for 18 h. Cells were then switched to DMEM containing 0.5% fatty acid-free bovine serum albumin for an additional 24 h, again in the presence of 1 μM lovastatin. Lovastatin produced a 19% increase in LDL association (2 h), using pooled LDL (0.064 ± 0.001 vs. 0.054 ± 0.003 μg of LDL protein per min per mg cell protein for lovastatin-treated and control cells, respectively; P < 0.01), which is in accordance with previously published data using this assay (
      • Stephan Z.F.
      • Yurachek E.C.
      Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.
      ).

       Statistical analysis

      For each subject, the results of all measurements made in duplicate at the end of each dietary period were averaged. Descriptivevalues are expressed as means ± SD. Statistical analyses included two-tailed paired t-tests for the comparison of changes in outcome variables in response to dietary treatment. Differences with P < 0.05 were considered significant. Diet period and carry-over effects for the two-period cross-over design were analyzed according to Fleiss (
      • Fleiss J.L.
      ). Correlation analysis between variables was performed by linear regression, using standard software (GraphPad Prism, San Diego, CA).

      RESULTS

      The nutrient content of the self-reported diets was well matched to that of the planned diets (Table 1). As expected, there were significant differences in unsaturated fatty acid intake between the two diets, reflecting the different fatty acid composition of the main fatty foods exchanged (olive oil and walnuts). Other nutrients had differences that were small but statistically significant. This can be explained by the use of calorie-adjusted nutrient values and the participant's close adherence to the prescribed diets, resulting in small standard deviations. Body weight was stable throughout the two dietary periods: 74.4 ± 7.0 kg after the control diet and 74.5 ± 7.4 kg after the walnut diet (P > 0.2). Serum lipid, lipoprotein cholesterol, and apolipoprotein B values while the subjects were on the control diet or the walnut diet are shown in Table 2. Total cholesterol, LDL cholesterol, and apolipoprotein B levels decreased in parallel in all but two participants (patients 3 and 10 in Table 2) during the walnut diet, whereas no changes in HDL cholesterol and VLDL cholesterol levels were observed. There was no evidence of a carryover effect. The resistance of LDL to oxidation, measured as the lag time of conjugated diene production to an in vitro 5 μM Cu2+ challenge, was similar during the control diet and the walnut diet (39.7 ± 6.2 vs. 36.7 ± 5.0 min, respectively, P > 0.2; Table 3). Essentially, lipoprotein changes and LDL oxidizability reported here do not differ from those observed in the main study (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ).
      TABLE 2.Serum lipid and lipoprotein levels at the end of each diet period
      CD, control diet; WD, walnut diet. Values are expressed as mM, except for apoB (mg/dl). To convert values for total, LDL, VLDL, and HDL cholesterol to mg/dl, multiply by 38.67. To convert values for triglycerides to mg/dl, multiply by 88.57.
      PatientAgeTotal CholesterolTriglyceridesLDL CholesterolHDL CholesterolVLDL CholesterolApoB
      CDWDCDWDCDWDCDWDCDWDCDWD
      1526.935.971.671.564.854.121.321.120.710.62169153
      2568.157.751.941.905.975.501.281.011.051.29209203
      3715.605.951.521.593.693.981.231.230.840.89140135
      4638.167.891.862.036.145.841.161.121.281.02218187
      5486.235.611.311.324.273.851.361.160.590.57148118
      6726.436.141.481.104.193.981.561.640.610.44150156
      7646.715.401.050.674.673.481.561.620.480.17150121
      8566.796.871.160.914.914.691.341.760.580.45156148
      9556.716.581.501.464.944.891.091.020.630.67168178
      10545.596.341.271.474.034.470.971.190.560.62148156
      Mean59.16.736.451.481.404.774.481.291.290.730.67165.3155.3
      SD8.00.880.840.290.420.800.750.190.280.250.3226.927.6
      Difference, CD vs. WD (%)−4.2−5.1−6.00−8.0−6.0
      P value
      Paired t-test, two tailed.
      0.176>0.20.087>0.2>0.20.075
      a CD, control diet; WD, walnut diet. Values are expressed as mM, except for apoB (mg/dl). To convert values for total, LDL, VLDL, and HDL cholesterol to mg/dl, multiply by 38.67. To convert values for triglycerides to mg/dl, multiply by 88.57.
      b Paired t-test, two tailed.
      TABLE 3.LDL oxidation-related analytes (n = 6)
      CDWD% Change WD vs. CDP
      A0 (absorbance units)0.375 ± 0.0300.333 ± 0.031−10.3 ± 12.90.106
      Cmax (nmol CDS/mg LDL protein)731.9 ± 63.3776.2 ± 38.36.5 ± 7.50.099
      Vmax (nmol CDS/min per mg LDL protein)29.7 ± 3.440.7 ± 6.338.5 ± 25.40.012
      Lag time (min)39.7 ± 6.236.7 ± 5.0−6.5 ± 14.20.247
      CD, control diet; WD, walnut diet; CDS, conjugated dienes.
      The relative proportions of the different constituents of LDL particles did not differ significantly with the two diets, although there was a trend to a decrease in phospholipid content following the walnut diet (data not shown). Notably, the cholesterol:protein ratio in LDL was essentially the same with the two diets (2.20 ± 0.24 vs. 2.25 ± 0.54 for the control and walnut diet, respectively). The analysis of fatty acids from LDL lipids during the two periods confirmed that participants had closely adhered to the prescribed diets. As expected from the high PUFA content of walnuts, the walnut diet resulted in an enrichment in linoleic acid and α-linolenic acid, with reciprocal changes in oleic acid in the whole particle (Table 4 and Table 5) and in each of the three lipidfractions (triglycerides, phospholipids, and cholesteryl esters) present in LDL (Table 6). Therefore, the relative proportion of PUFA increased whereas that of MUFA was reduced after the walnut diet, thus increasing the unsaturation index of the whole particle measured as PUFA/SFA + MUFA. The incorporation of fatty acids from walnuts into the different lipid fractions of LDL was not homogeneous. The triglyceride fraction was more enriched in linoleic acid and, especially, α-linolenic acid than the cholesteryl ester and phospholipid fractions. Interestingly, the walnut diet produced a significant decrease (14%, P < 0.01) in the unsaturation index of LDL surface components (phospholipids) in relation to the unsaturation index of LDL core lipids (triglycerides and cholesteryl esters) (Table 6).
      TABLE 4.Quantitative fatty acid composition in whole LDL
      Values are expressed as micrograms of fatty acid per milligram of LDL protein.
      PatientPalmitic (16:0)Stearic (18:0)Oleic (18:1n-9)Linoleic (18:2n-6)γ-Linolenic (18:3n-3)α-Linolenic (18:3n-3)Arachidonic (20:4n-6)Eicosapentaenoic (20:5n-3)Docosahexaenoic (22:6n-3)
      CDWDCDWDCDWDCDWDCDWDCDWDCDWDCDWDCDWD
      1273.5176.890.654.4294.3156.5430.0347.177.6109.97.279.05136.568.223.911.146.316.0
      2235.4172.763.783.7203.9118.4393.2350.471.175.25.819.32103.287.38.97.828.328.9
      3209.6183.659.878.3254.7178.7383.6437.5100.788.55.069.1469.858.59.66.520.822.2
      4265.1166.069.242.7293.8193.0329.2365.569.141.94.7011.1985.164.432.012.430.616.6
      5298.6244.373.3103.9336.4186.8422.1491.9116.481.23.966.4092.093.114.212.320.834.9
      6157.0210.854.165.9173.8168.7252.1440.072.973.32.565.5765.580.312.410.920.919.8
      7261.7149.365.852.9236.9101.9470.3295.498.586.25.134.78123.566.121.57.817.416.3
      8156.9239.954.877.1149.0201.0343.6559.363.9149.41.6310.9766.458.716.722.727.021.3
      9158.5240.850.883.5147.3204.3240.0509.668.2110.22.639.1967.589.318.811.326.926.6
      10204.2246.562.596.1188.9172.5372.9413.9124.4104.94.149.1175.8109.211.915.120.331.4
      Mean222.1203.164.573.8227.9168.2363.7421.086.392.14.298.4788.577.517.011.825.923.4
      SD52.837.511.519.665.934.174.483.021.928.71.692.1725.317.07.24.68.46.7
      Difference, CD vs. WD (%)−9.011.4−26.215.810.797.5−12.0−31.0−10.0
      Pvalue
      Paired t-test, two tailed.
      >0.2>0.20.020>0.2>0.2<0.001>0.20.072>0.2
      a Values are expressed as micrograms of fatty acid per milligram of LDL protein.
      b Paired t-test, two tailed.
      TABLE 5.Quantitative fatty acid composition in whole LDL
      Values are expressed as micrograms of fatty acid per milligram LDL protein.
      PatientSFAMUFAPUFAn-6n-3
      CDWDCDWDCDWDCDWDCDWD
      1373.4235.1327.8174.7759.0587.4670.6542.083.939.1
      2304.0261.6225.2129.7643.7582.2591.2528.950.650.7
      3273.2266.5274.8192.1621.2647.1583.3604.637.740.4
      4342.4215.6323.9211.8595.7532.9517.5488.272.842.3
      5381.2355.4373.8204.1721.9744.6676.6684.743.558.9
      6216.7282.8190.5193.9459.6655.0413.7613.539.941.5
      7333.7205.0262.0112.7770.8498.7720.0465.448.331.7
      8213.2320.1158.8216.5540.1849.5490.2791.949.357.6
      9215.1331.2165.8222.0445.6797.7390.5738.353.656.2
      10271.5348.2207.4191.1633.2718.2593.0655.239.160.9
      Mean292.4282.2251.0184.9619.1661.3564.7611.351.947.9
      SD64.654.673.836.5113.7115.0110.9107.615.210.2
      Difference, CD vs. WD (%)−3.5−26.36.88.3−7.6
      Pvalue
      Paired t-test, two tailed.
      >0.20.037>0.2>0.2>0.2
      SFA, saturated fatty acids (14:0 + 16:0 + 18:0 + 20:0); MUFA, monounsaturated fatty acids (16:1 + 18:1 + 20:1); PUFA, polyunsaturated fatty acids (18:2n-6 + 18:3n-6 + 18:3n-3 + 20:3n-6 + 20:3n-9 + 20:4n-6 + 20:5n-3 + 22:4n-6 + 22:5n-3 + 22:6n-3).
      a Values are expressed as micrograms of fatty acid per milligram LDL protein.
      b Paired t-test, two tailed.
      TABLE 6.Changes in percent molar fatty acid composition and unsaturation index of triglycerides, phospholipids, and cholesteryl esters in LDL particles
      Values are means (SD) of 10 patients. Unsaturation index obtained from the ratio PUFA/MUFA + SFA; un-saturation index surface:unsaturation index core ratio obtained from the ratio unsaturation index phospholipids: unsaturation index triglycerides + cholesteryl esters.
      Fatty AcidLipid FractionControl DietWalnut DietPercent ChangeP
      Paired t test, two tailed.
      Oleic acid, C18:1n-9TG35.3 (4.1)28.8 (3.8)−18<0.001
      PL11.7 (1.6)9.4 (1.0)−19<0.001
      CE19.1 (2.2)14.5 (2.2)−24<0.001
      Linoleic acid, C18:2n-6TG15.5 (4.4)23.4 (2.9)51<0.001
      PL19.6 (2.0)24.5 (1.7)25<0.001
      CE43.9 (3.7)50.8 (2.7)16<0.001
      α-Linolenic acid, C18:3n-3TG0.69 (0.26)1.93 (0.76)181<0.001
      PL0.20 (0.11)0.27 (0.09)34>0.2
      CE0.40 (0.13)0.79 (0.24)96<0.001
      Unsaturation indexTG0.45 (0.10)0.68 (0.14)49<0.001
      PL0.71 (0.06)0.84 (0.08)19<0.001
      CE1.74 (0.26)2.31 (0.27)33<0.001
      Whole LDL1.18 (0.20)1.43 (011)21<0.001
      Unsaturation index surface: unsaturation index core0.33 (0.04)0.28 (0.05)−14<0.01
      a Values are means (SD) of 10 patients. Unsaturation index obtained from the ratio PUFA/MUFA + SFA; un-saturation index surface:unsaturation index core ratio obtained from the ratio unsaturation index phospholipids: unsaturation index triglycerides + cholesteryl esters.
      b Paired t test, two tailed.
      Cellular assays demonstrated a significant (P < 0.05) 50% increase in the binding to HepG2 cell receptors of LDL obtained during the walnut diet compared with LDL harvested from the same patients during the control diet (Fig. 2). Considering only the eight individual patients who showed a reduction in LDL cholesterol during the walnut diet, the increase in the association rate was even higher, reaching 72% (0.023 ± 0.009 vs. 0.037 ± 0.014 μg of LDL protein per min per mg cell protein for the control diet and the walnut diet, respectively).
      Figure thumbnail gr2
      Fig. 2.Rate of association of DiI-labeled LDL obtained during the control and walnut diet periods to cultured HepG2 cells (n = 10). Experimental conditions are described in Materials and Methods. Horizontal lines correspond to the mean association rate for each dietary period: control, 0.024 ± 0.009 μg of LDL protein per min per mg cell protein; walnut, 0.036 ± 0.013 μg of LDL protein per min per mg cell protein (P < 0.05). Individual values for the rate of association of DiI-labeled LDL (expressed as micrograms of LDL protein per minute per milligram of cell protein) are shown in the inset.
      Correlation analyses among the different variables showed that part of the decrease in LDL cholesterol levels with the walnut diet could be attributed to the increase in LDL binding to receptors (r 2 = 0.31; P = 0.097). Moreover, LDL binding was correlated to α-linolenic acid enrichmentin LDL core lipids (triglycerides plus cholesteryl esters) (r 2 = 0.41; P < 0.05).

      DISCUSSION

      In a subgroup of 10 hypercholesterolemic men participating in a larger dietary intervention trial, the inclusionof walnuts (about 50 g/day) in a Mediterranean-type hypocholesterolemic diet produced an additional decrease in serum cholesterol and LDL cholesterol. Although in this subset of patients the decrease in LDL cholesterol with the walnut diet in comparison with the control diet did not reach statistical significance (−6%, P = 0.09), the effect was similar in magnitude to that observed in the main study involving 49 patients (−5.9%; P <0.001). This is attributable to the low statistical power of the sample in the present study. The decrease in plasma lipids was associated with a proportional decrease in serum apolipoprotein B, suggesting that the observed effect was due to a reduction of apolipoprotein B-rich circulating particles.
      A reduction of serum and LDL cholesterol has been consistently found in studies using diets supplemented with nuts (
      • Kris-Etherton P.M.
      • Yu-Poth S.
      • Sabaté J.
      • Ratcliffe H.E.
      • Zhao G.
      • Etherton T.D.
      Nuts and their bioactive constituents: effects on serum lipids and other factors that affect disease risk.
      ). In the present study we observed that the decrease in LDL cholesterol levels after the walnut diet was accompanied by an increased association of LDL with the receptors present in human hepatoma cells (HepG2). The increase in LDL association explained approximately 30% of the LDL cholesterol decrease, suggesting the presence of other factors accounting for the hypocholesterolemic effects of the walnut diet. Nevertheless, to our knowledge this is the first time that the relative contribution of plasma LDL association with hepatic LDL receptors has been quantified and correlated with changes in the composition of LDL fatty acids.
      It is well known that the quality of ingested fats modifies the composition and physicochemical properties of cellular membranes, leading to changes in the microenvironment of proteins, such as receptors or enzymes, penetrating the asymmetric lipid bilayer (
      • Murphy M.G.
      Dietary fatty acids and membrane protein function.
      ). The ingestion of SFA increases blood cholesterol by reducing the activity of hepatic LDL receptors (
      • Srivastava R.A.
      • Ito H.
      • Srivastava N.
      • Schonfeld G.
      Regulation of low density lipoprotein receptor gene expression in HepG2 and Caco2 cells by palmitate, oleate, and 25-hydroxycholesterol.
      ,
      • Mustad V.A.
      • Etherton T.D.
      • Cooper A.D.
      • Mastro A.M.
      • Pearson T.A.
      • Jonnalagadda S.S.
      • Kris-Etherton P.M.
      Reducing saturated fat intake is associated with increased levels of LDL receptors on mononuclear cells in healthy men and women.
      ). Conversely, the hypocholesterolemic activity of n-6 PUFA such as linoleic acid has been attributed to enhanced LDL uptake and degradation due to facilitated LDL receptor activity (
      • Kuo P.C.
      • Neinfeld M.
      • Loscalzo J.
      Effect of membrane fatty acyl composition on LDL metabolism in HepG2 hepatocytes.
      ,
      • Bucci C.
      • Serù R.
      • Annella T.
      • Vitelli R.
      • Lattero D.
      • Bifulco M.
      • Mondola P.
      • Dietschy J.M.
      Free fatty acids modulate LDL receptor activity in BHK-21 cells.
      ), and to increased activity of the hepatic scavenger receptor BI or HDL receptor (
      • Spady D.K.
      • Kearney D.M.
      • Hobbs H.H.
      Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-B1) expression and HDL cholestryl ester uptake in the hamster.
      ). Increased cellular degradation of LDL, however, may be due not only to increased affinity of the receptor for plasma LDL because PUFA in surrounding membrane phospholipids facilitate expression of receptor-binding sites, but also to changes in the affinity of circulating LDL particles for the receptor. Thus, it has been reported that LDL uptake by HepG2 cells is increased in triglyceride-rich LDL particles (
      • McKeone B.J.
      • Patsch J.R.
      • Pownall H.J.
      Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells.
      ). Conversely, triglyceride depletion of LDL by fish oil treatment in hypertriglyceridemic patients results in lower binding to fibroblasts (
      • Hsu H-C.
      • Lee T-T.
      • Chen M-F.
      Effect of n-3 fatty acids on the composition and binding properties of lipoprotein in hypertriglyceridemic patients.
      ). The lipid composition data in our study and the relative constancy of lipid-to-protein ratios during the two dietary periods indicate that the increased binding to HepG2 cells of LDL formed during the walnut diet probably does not result from an increase in particle size. However, important differences were apparent in the fatty acid composition of the esterified lipid components (Tables 4 and 5). The overall results from compositional analyses of LDL obtained during the two dietary periods suggest that enrichment of the particles with the fatty acids predominating in each diet was an important determinant of their affinity for LDL receptors in HepG2 cells. The short incubation time (2 h) used in the association experiments is insufficient to modify substantially the fatty acid composition of membrane phospholipids present in HepG2 cells (
      • Lindsey S.
      • Pronczuk A.
      • Hayes C.
      Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells.
      ). Thus, under our experimental conditions, changes in the affinity of LDL particles for the LDL receptor may be ascribed to changes in LDL composition rather than to modifications of the receptor microenvironment.
      Fatty acids incorporated into LDL triglycerides were previously identified as the most sensitive marker of participant's adherence to the prescribed diets (
      • Zambón D.
      • Sabatwé J.
      • Muñoz S.
      • Campero B.
      • Casals E.
      • Merlos M.
      • Laguna J.C.
      • Ros E.
      Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
      ). The enrichment in PUFA, particularly α-linolenic acid, was localized mainly in the triglyceride-rich core of the LDL particle, whereas the phospholipid-rich surface was much less affected. Interestingly, there was a significant correlation (r 2 = 0.42, P < 0.05) between the increase in α-linolenic acid in the core (triglycerides plus cholesteryl esters) of LDL isolated from patients during the walnut diet, related to the LDL obtained from the same patients during the control diet, and the increase in the rate of association of the corresponding LDL to HepG2 cells. Thus, LDL-core enrichment in this quantitatively minor fatty acid, by changing the shape of the lipoprotein, may be in part responsible for increased LDL clearance and reduction of circulating LDL particles during the walnut diet. Of note, the conformation of apolipoprotein B in LDL can be altered depending on the curvature radius of the particle (
      • Kinoshita M.
      • Krul E.S.
      • Schonfeld G.
      Modification of the core lipids of low density lipoproteins produces selective alterations in the expression of apo B-100 epitopes.
      ,
      • Galeano M.F.
      • Milne R.
      • Marcel Y.L.
      • Walsh M.T.
      • Levy E.
      • Ngu'yen T.D.
      • Gleeson A.
      • Arad Y.
      • Witte L.
      • Al-Haideri M.
      • Rumsey S.C.
      • Deckelbaum R.J.
      Apo-B structure and receptor recognition of triglyceride-rich low density lipoproteins is modified in small LDL but not in triglyceride-rich LDL of normal size.
      ), thus affecting the exposure of epitopes responsible for recognition by the LDL receptor. High dietary intake of α-linolenic acid has been associated with reduced risk of coronary artery disease in both epidemiological studies (
      • Dolecek T.A.
      Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial.
      ,
      • Ascherio A.
      • Rimm E.B.
      • Giovannucci E.L.
      • Spiegelman D.
      • Stampfer M.J.
      • Willett W.C.
      Dietary fat and risk of coronary heart disease in men: cohort follow-up study in the United States.
      ,
      • Hu F.B.
      • Stampfer M.J.
      • Manson J.E.
      • Rimm E.B.
      • Wolk A.
      • Colditz G.A.
      • Hennekens C.H.
      • Willett W.C.
      Dietary intake of α-linolenic acid and risk of fatal ischemic heart disease among women.
      ) and a landmark clinical trial, the Lyon Diet Heart Study (
      • De Lorgeril M.
      • Renaud S.
      • Mamelle N.
      • Salen P.
      • Martin J.L.
      • Monjaud I.
      • Guidollet J.
      • Touboul P.
      • Delaye J.
      Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease.
      ,
      • De Lorgeril M.
      • Salen P.
      • Martin J.L.
      • Monjaud I.
      • Delaye J.
      • Mamelle N.
      Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction.
      ). Reduced coronary risk could be attributed to improvement of the serum lipid profile, but the recognized antiarrhythmic and antithrombotic properties of α-linolenic acid may also contribute to this beneficial effect (
      • Kang J.X.
      • Leaf A.
      Antiarrhythmic effects of polyunsaturated fatty acids: recent studies.
      ).
      Epidemiological and clinical studies suggest that frequent nut consumption may be protective against coronary artery disease (
      • Fraser G.E.
      • Sabaté J.
      • Beeson W.L.
      • Strahan T.M.
      A possible protective effect of nut consumption on risk of coronary heart disease: the Adventist Health Study.
      ,
      • Kushi L.H.
      • Folsom A.R.
      • Prineas R.J.
      • Mink P.J.
      • Wu Y.
      • Bostick R.M.
      Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women.
      ,
      • Hu F.B.
      • Stampfer M.J.
      • Manson J.E.
      • Rimm E.B.
      • Colditz G.A.
      • Rosner B.A.
      • Speizer F.E.
      • Hennekens C.H.
      • Willet W.C.
      Frequent nut consumption and risk of coronary heart disease in women: prospective cohort study.
      ,
      • Sabaté J.
      Nut consumption, vegetarian diets, ischemic heart disease risk, and all-cause mortality: evidence from epidemiologic studies.
      ), a beneficial effect that may be attributed, at least in part, to improvement of the serum lipid profile. In clinical trials, the cholesterol-lowering effect of regular nut consumption is greater than that predicted from changes in common dietary fatty acids in the tested diets (
      • Kris-Etherton P.M.
      • Yu-Poth S.
      • Sabaté J.
      • Ratcliffe H.E.
      • Zhao G.
      • Etherton T.D.
      Nuts and their bioactive constituents: effects on serum lipids and other factors that affect disease risk.
      ). In the present study we describe the changes in lipoprotein composition as well as the heterogeneous incorporation of the PUFA provided by walnuts into the different lipid fractions present in LDL following a controlled hypocholesterolemic diet enriched with walnuts. Moreover, we put forward the importance of changes in LDL fatty acid composition in the clearance process by the LDL receptor, suggesting an additional mechanism that may contribute to the reduction of LDL cholesterol levels. Nevertheless, it should be mentioned that other events, including changes in plasma membrane fatty acid composition, impairment of LDL synthesis, and hypolipidemic effects derived from nonlipid constituents present in nuts, may also occur and contribute to the overall cholesterol-lowering effect of regular walnut consumption.

      Acknowledgments

      This work was supported by grants from the California Walnut Commission, USA, and Fondo de Investigaciones Sanitarias (00/1124), Comisión Interministerial de Ciencia y Tecnología (SAF 97/0265 and OLI96-2132), Generalitat de Catalunya (2000SGR00045), and Fundació Privada Catalana de Nutrició i Lípids, Spain.

      REFERENCES

        • Mensink R.P.
        • Katan M.B.
        Effect of dietary fatty acids on serum lipids and lipoproteins: a meta-analisis of 27 trials.
        Arterioscler. Thromb. 1992; 12: 911-919
        • Hegsted D.M.
        • Ausman L.M.
        • Johnson J.A.
        • Dallal G.E.
        Dietary fat and serum lipids: an evaluation of the experimental data.
        Am. J. Clin. Nutr. 1993; 57: 875-883
        • Yu S.
        • Derr J.
        • Etherton T.D.
        • Kris-Etherton P.M.
        Plasma cholesterol-predictive equations demonstrate that stearic acid is neutral and monounsaturated fatty acids are hypocholesterolemic.
        Am. J. Clin. Nutr. 1995; 61: 1129-1139
        • Clarke R.
        • Frost C.
        • Collins R.
        • Appleby P.
        • Peto R.
        Dietary lipids and blood cholesterol. Quantitative meta-analysis of metabolic ward studies.
        Br. Med. J. 1997; 314: 112-117
        • Willett W.C.
        Diet and health: what should we eat?.
        Science. 1994; 264: 532-537
        • Krauss R.M.
        • Eckel R.H.
        • Howard B.
        • Appel L.J.
        • Daniels S.R.
        • Deckelbaum R.J.
        • Erdman Jr., J.W.
        • Kris-Etherton P.
        • Goldberg I.J.
        • Kotchen T.A.
        • Lichtenstein A.H.
        • Mitch W.E.
        • Mullis R.
        • Robinson K.
        • Wylie-Rosett J.
        • Jeor S.St.
        • Suttie J.
        • Tribble D.L.
        • Bazzarre T.L.
        AHA Dietary Guidelines. Revision 2000: a statement for healthcare professionals from the Nutrition Committee of the American Heart Association.
        Circulation. 2000; 102: 2296-2311
        • Fraser G.E.
        • Sabaté J.
        • Beeson W.L.
        • Strahan T.M.
        A possible protective effect of nut consumption on risk of coronary heart disease: the Adventist Health Study.
        Arch. Intern. Med. 1992; 152: 1416-1424
        • Kushi L.H.
        • Folsom A.R.
        • Prineas R.J.
        • Mink P.J.
        • Wu Y.
        • Bostick R.M.
        Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women.
        N. Engl. J. Med. 1996; 334: 1156-1162
        • Hu F.B.
        • Stampfer M.J.
        • Manson J.E.
        • Rimm E.B.
        • Colditz G.A.
        • Rosner B.A.
        • Speizer F.E.
        • Hennekens C.H.
        • Willet W.C.
        Frequent nut consumption and risk of coronary heart disease in women: prospective cohort study.
        Br. Med. J. 1998; 317: 1341-1345
        • Sabaté J.
        Nut consumption, vegetarian diets, ischemic heart disease risk, and all-cause mortality: evidence from epidemiologic studies.
        Am. J. Clin. Nutr. 1999; 70: 500S-503S
        • Kris-Etherton P.M.
        • Yu-Poth S.
        • Sabaté J.
        • Ratcliffe H.E.
        • Zhao G.
        • Etherton T.D.
        Nuts and their bioactive constituents: effects on serum lipids and other factors that affect disease risk.
        Am. J. Clin. Nutr. 1999; 70: 504S-511S
      1. Exler, J., Weihrauch, J. L., 1986. Provisional Table on the Content of Omega-3 Fatty Acids and Other Fat Components in Selected Foods. U.S. Department of Agriculture Publication No. HNIS/PT-103.

        • Sabaté J.
        • Fraser G.E.
        • Burke K.
        • Knutsen S.
        • Bennett H.
        • Lindsted K.D.
        Effects of walnuts on serum lipids levels and blood pressure in normal men.
        N. Engl. J. Med. 1993; 328: 603-607
        • Zambón D.
        • Sabatwé J.
        • Muñoz S.
        • Campero B.
        • Casals E.
        • Merlos M.
        • Laguna J.C.
        • Ros E.
        Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women.
        Ann. Intern. Med. 2000; 132: 538-546
        • Javitt N.B.
        HepG2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids.
        FASEB J. 1990; 4: 161-168
        • Havekes L.
        • van Hinsbergh V.
        • Kempen H.J.
        • Emeis J.
        The metabolism in vitro of human low density lipoprotein by the human hepatoma cell line HepG2.
        Biochem. J. 1983; 214: 951-958
        • Dashti N.
        • Wolfbauer G.
        • Koren E.
        • Knowles B.
        • Alaupovic P.
        Catabolism of human low density lipoproteins by human hepatoma cell line HepG2.
        Biochim. Biophys. Acta. 1984; 794: 373-384
        • Yen C.F.
        • Kalunta C.I.
        • Chen F.S.
        • Kaptein J.S.
        • Lin C.K.E.
        • Lad P.M.
        Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.
        J. Immunol. Methods. 1994; 177: 55-67
        • Lohne K.
        • Urdal P.
        • Leren T.P.
        • Tonstad S.
        • Ose L.
        Standarization of a flow cytometric method for measurement of low density lipoprotein receptor activity on blood mononuclear cells.
        Cytometry. 1995; 20: 290-295
        • Vázquez M.
        • Merlos M.
        • Adzet T.
        • Laguna J.C.
        Decreased susceptibility to copper-induced oxidation of rat lipoproteins after fibrate treatment. Influence of fatty acid composition.
        Br. J. Pharmacol. 1996; 117: 1155-1162
        • Vázquez M.
        • Zambón D.
        • Hernández Y.
        • Adzet T.
        • Merlos M.
        • Ros E.
        • Laguna J.C.
        Lipoprotein composition and oxidative modification during therapy with gemfibrozil and lovastatin in patients with combined hyperlipidaemia.
        Br. J. Clin. Pharmacol. 1998; 45: 265-269
        • Stephan Z.F.
        • Yurachek E.C.
        Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.
        J. Lipid Res. 1993; 34: 325-330
        • Bradford M.
        A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding.
        Anal. Biochem. 1976; 72: 248-254
        • Fleiss J.L.
        The Design and Analysis of Clinical Experiments. John Wiley & Sons, New York1986
        • Murphy M.G.
        Dietary fatty acids and membrane protein function.
        J. Nutr. Biochem. 1990; 1: 68-79
        • Srivastava R.A.
        • Ito H.
        • Srivastava N.
        • Schonfeld G.
        Regulation of low density lipoprotein receptor gene expression in HepG2 and Caco2 cells by palmitate, oleate, and 25-hydroxycholesterol.
        J. Lipid Res. 1995; 36: 1434-1446
        • Mustad V.A.
        • Etherton T.D.
        • Cooper A.D.
        • Mastro A.M.
        • Pearson T.A.
        • Jonnalagadda S.S.
        • Kris-Etherton P.M.
        Reducing saturated fat intake is associated with increased levels of LDL receptors on mononuclear cells in healthy men and women.
        J. Lipid Res. 1997; 38: 459-468
        • Kuo P.C.
        • Neinfeld M.
        • Loscalzo J.
        Effect of membrane fatty acyl composition on LDL metabolism in HepG2 hepatocytes.
        Biochemistry. 1990; 29: 6626-6632
        • Bucci C.
        • Serù R.
        • Annella T.
        • Vitelli R.
        • Lattero D.
        • Bifulco M.
        • Mondola P.
        • Dietschy J.M.
        Free fatty acids modulate LDL receptor activity in BHK-21 cells.
        Atherosclerosis. 1998; 137: 329-340
        • Spady D.K.
        • Kearney D.M.
        • Hobbs H.H.
        Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-B1) expression and HDL cholestryl ester uptake in the hamster.
        J. Lipid Res. 1999; 40: 1384-1394
        • McKeone B.J.
        • Patsch J.R.
        • Pownall H.J.
        Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells.
        J. Clin. Invest. 1993; 91: 1926-1933
        • Hsu H-C.
        • Lee T-T.
        • Chen M-F.
        Effect of n-3 fatty acids on the composition and binding properties of lipoprotein in hypertriglyceridemic patients.
        Am. J. Clin. Nutr. 2000; 71: 28-35
        • Lindsey S.
        • Pronczuk A.
        • Hayes C.
        Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells.
        J. Lipid Res. 1992; 33: 647-658
        • Kinoshita M.
        • Krul E.S.
        • Schonfeld G.
        Modification of the core lipids of low density lipoproteins produces selective alterations in the expression of apo B-100 epitopes.
        J. Lipid Res. 1990; 31: 701-708
        • Galeano M.F.
        • Milne R.
        • Marcel Y.L.
        • Walsh M.T.
        • Levy E.
        • Ngu'yen T.D.
        • Gleeson A.
        • Arad Y.
        • Witte L.
        • Al-Haideri M.
        • Rumsey S.C.
        • Deckelbaum R.J.
        Apo-B structure and receptor recognition of triglyceride-rich low density lipoproteins is modified in small LDL but not in triglyceride-rich LDL of normal size.
        J. Biol. Chem. 1994; 269: 511-519
        • Dolecek T.A.
        Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial.
        Proc. Soc. Exp. Biol. Med. 1992; 200: 177-182
        • Ascherio A.
        • Rimm E.B.
        • Giovannucci E.L.
        • Spiegelman D.
        • Stampfer M.J.
        • Willett W.C.
        Dietary fat and risk of coronary heart disease in men: cohort follow-up study in the United States.
        Br. Med. J. 1996; 313: 84-90
        • Hu F.B.
        • Stampfer M.J.
        • Manson J.E.
        • Rimm E.B.
        • Wolk A.
        • Colditz G.A.
        • Hennekens C.H.
        • Willett W.C.
        Dietary intake of α-linolenic acid and risk of fatal ischemic heart disease among women.
        Am. J. Clin. Nutr. 1999; 69: 890-897
        • De Lorgeril M.
        • Renaud S.
        • Mamelle N.
        • Salen P.
        • Martin J.L.
        • Monjaud I.
        • Guidollet J.
        • Touboul P.
        • Delaye J.
        Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease.
        Lancet. 1994; 343: 1454-1459
        • De Lorgeril M.
        • Salen P.
        • Martin J.L.
        • Monjaud I.
        • Delaye J.
        • Mamelle N.
        Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction.
        Circulation. 1999; 99: 779-785
        • Kang J.X.
        • Leaf A.
        Antiarrhythmic effects of polyunsaturated fatty acids: recent studies.
        Circulation. 1996; 94: 1774-1780