Substituting Walnuts for Monounsaturated Fat Improves the Serum Lipid Profile of Hypercholesterolemic Men and Women

Lifestyle modification is the cornerstone of population-based strategies for prevention of coronary heart disease and is the first line of therapy in patients with hypercholesterolemia. Diets low in saturated fatty acids and cholesterol have long been recommended to decrease low-density lipoprotein (LDL) cholesterol levels and reduce cardiovascular risk (1). Ample evidence suggests that polyunsaturated fatty acids and monounsaturated fatty acids have a similar cholesterol-lowering effect when substituted for saturated fatty acids (2-4). However, most studies of fatty acids and blood lipids have been done with fats and oils, rarely with whole fatty foods. Because people usually buy and consume whole food products, it is desirable to know the effects of specific foods on risk factors for coronary heart disease. Recent reports suggest that the regular consumption of nuts might reduce cardiovascular risk (5). Walnuts are particularly rich in polyunsaturated fatty acids (6), and epidemiologic evidence suggests that frequent walnut consumption protects against coronary heart disease (7). In a controlled feeding trial by Sabat and colleagues (8), a diet in which walnuts represented 55% of the energy from fat reduced blood cholesterol levels in normal young men when compared with a standard low-fat diet. However, the results cannot easily be extrapolated to the population at risk for coronary heart disease because women, older age groups, and hypercholesterolemic persons were not studied (8). In addition, because meals were served at a metabolic kitchen, the study did not address the question of whether free-living persons would incorporate substantial quantities of walnuts into their diets. Because oxidized LDL plays a key role in atherogenesis (9) and oxidative damage involves peroxidation of polyunsaturated fatty acids in LDL lipids (10), there is concern that walnut intake may promote LDL oxidation. Therefore, we designed a dietary intervention study in free-living adult men and women with polygenic hypercholesterolemia to compare the effects of a walnut-rich diet with those of a cholesterol-lowering Mediterranean diet on serum lipid levels, lipoprotein levels, and LDL resistance to oxidation. Methods Patients Adult men and women with polygenic hypercholesterolemia attending the Lipid Clinic of the Hospital Clnic of Barcelona were eligible if they had serum LDL cholesterol concentrations greater than 3.36 mmol/L (130 mg/dL) and triglyceride concentrations less than 2.82 mmol/L (250 mg/dL); no evidence of alcohol, tobacco, or drug abuse; absence of diabetes mellitus and liver, kidney, thyroid, or other endocrine diseases, as assessed by medical history, a complete physical examination, and laboratory tests; no intake in the previous 8 weeks of medications known to affect lipid metabolism, including hypolipidemic agents and estrogen compounds in women; infrequent consumption of nuts and no known history of allergy to them; and no use of multivitamin or vitamin E supplements. Because the target population had common (polygenic) hypercholesterolemia, we excluded persons whose elevated blood cholesterol levels had a strong genetic basis (such as heterozygous familial hypercholesterolemia or familial combined hyperlipidemia), as established by standard criteria. On admission to the Lipid Clinic, all patients were advised to follow a Mediterranean-type hypolipidemic diet (11). For a crossover design, statistical power calculations indicated that to detect mean differences of 0.39 mmol/L (15 mg/dL), 34 patients would need to complete the two treatment periods ( statistic, 0.05; power>0.8). From a computerized register of clinical records, 75 hypercholesterolemic patients (35 women and 40 men) who initially met the eligibility criteria were selected for screening and were asked to participate in the study. They were offered free walnuts but no monetary compensation. Study Design A crossover design was used. Patients were randomly assigned to the two diet sequences by using a computer-generated random-number table, with stratification by sex. Because patients followed each diet for 6 weeks and lipoprotein changes due to dietary intervention stabilize in less than 4 weeks (12), we did not incorporate a washout period between diets. In their crossover feeding study with walnuts, Sabat and colleagues (8) did not observe a carryover effect. In the week before the trial began, patients received expert dietary counseling individually and in a group class. Twice during the pretrial week and on weeks 5 and 6 of each one of the two dietary periods, patients came to the clinic for a medical visit, an interview with the dietitian, anthropometric measurements, and blood extraction. The main outcomes of the study were changes in serum levels of total and LDL cholesterol from the control diet period to the walnut diet period. Secondary outcomes were changes in other lipid variables and oxidizability of LDL particles. The study protocol was approved by the institutional review board of the Hospital Clnic of Barcelona, and all patients gave informed consent. Diets The experimental diets were individually prescribed and were based on estimated energy requirements. Because participants ate on their own, detailed dietary information was provided to them and, if appropriate, to their partners. Diets were calculated in increments of 200 kcal to cover the range from 1600 to 2200 kcal. The control diet was Mediterranean and was composed of natural foodstuffs. Red meat and eggs were limited, vegetable products and fish were emphasized, olive oil was indicated for culinary use, and no nuts were allowed. The walnut diet was similar to the control diet, but walnuts partially replaced olive oil and other fatty foods. Prepackaged daily allowances of raw, shelled walnuts were provided daily in amounts varying from 41 g to 56 g (the equivalent of 8 to 11 walnuts), according to the participants' total energy intake. Walnuts were consumed as snacks or with meals in desserts or salads. In the walnut diet, walnuts contributed approximately 18% of the total energy and 35% of the total fat. To improve compliance, each family unit was given 1000-g packs of walnuts at the beginning of the walnut diet period. Adherence to the study diets was carefully monitored. Unannounced 24-hour diet recalls were performed weekly by telephone during the two dietary periods, for a total of 12 recalls per patient. This method allows reliable estimations of food intake (13). The nutrient composition of the diets was calculated with Food Processor Plus software, version 5.0 (ESHA Research, Salem, Oregon), which was adapted to nutrient databases of specific Mediterranean foods when appropriate. We defined noncompliance as at least 20% deviation from dietary instructions regarding walnut or nutrient intake. Compliance during the walnut diet was also assessed at each clinic visit by a count of the empty walnut packages. The fatty acid content of LDL lipids was analyzed as a biological measure of adherence to the prescribed diets. Laboratory Measurements Blood samples were obtained after an overnight fast, and serum and EDTA plasma were collected and processed immediately. Serum lipid and apolipoprotein levels were determined as described elsewhere (11). In brief, serum cholesterol and triglyceride levels were measured by using enzymatic reagents (Trinder, Bayer Diagnostics, Tarrytown, New York) adapted to a Cobas Mira automated analyzer (Hoffmann-LaRoche, Basel, Switzerland). High-density lipoprotein (HDL) cholesterol was quantified after precipitation with phosphotungstic acid and magnesium chloride. Levels of apolipoprotein A-I and apolipoprotein B were determined by an immunoturbidimetric method (Unimate 3, Roche Diagnostic Systems, Basel, Switzerland). Measurements of the cholesterol content of very-low-density lipoprotein particles and LDL particles were based on preparative ultracentrifugation (11). Lipoprotein(a) levels were measured by using an immunoturbidimetric method (Lipoprotein[a] SPQ II Test Kit, DiaSorin, Stillwater, Minnesota). To obtain the LDL fraction for fatty acid and oxidizability studies, lipoproteins were fractionated by sequential centrifugation adjusting with NaBr to separate very-low-density lipoprotein particles (d=1.006 g/mL) and to obtain LDL particles (d=1.063 g/mL), as described elsewhere (14). Low-density lipoprotein fatty acids were measured by capillary gas chromatography in the cholesteryl ester, phospholipid, and triglyceride lipid fractions (14). Low-density lipoprotein susceptibility to oxidation was determined by measuring the -tocopherol content of LDL particles and conjugated diene kinetics after incubation of 50 g of LDL protein with 5 mol of copper sulfate at 37 C (15, 16). Statistical Analysis The two measurements obtained at baseline and at the end of each dietary period were averaged. Means and SDs are presented for each measurement. With methods described by Fleiss (17), two-tailed t-tests were used to compare changes in outcome variables in response to dietary treatment and diet period and carryover effects for the two-period crossover design. Differences between the walnut and control diets were also tested by analysis of covariance using general linear models; baseline values or sex were used as covariates. Analyses were performed by using SAS software (SAS, Inc., Cary, North Carolina) (18). Role of the Funding Sources The California Walnut Commission provided funding and walnuts. Research grants were also obtained from national and local nonprofit agencies. The funding sources were not involved in the design of the study and had no role in the collection, analysis, or interpretation of the data or in the decision to submit the manuscript for publication. None of the authors has any financial interest in the nut food industry. Results Patient Characteristics Of the 75 eligible patients, 20 left the study before randomization for va