Am J C/in Nutr l991;53:899-907. Printed in USA. 1991 American Society for Clinical Nutrition 899

Effects of diets rich in monounsaturated fatty acids onplasma lipoproteins-the Jerusalem Nutrition Study:high MUFAs vs high PUFAs13Elliot M Berry, Shlomo Eisenberg, Dror Haratz, YechielNathan A Kaufmann, and Yechezkiel Stein

ABSTRACT Twenty-six Yeshiva students were randomlyassigned to a 24-wk crossover study of monounsaturated fattyacid (MUFA) vs polyunsaturated fatty acid (PUFA) diets (50%carbohydrate, 32% fat, 18% protein) fed alternately during two12-wk periods. Total plasma cholesterol (TC) decreased signif-icantly by 10% and 16% on the MUFA and PUFA diets,respectively. Plasma triglyceride response was variable. Low-density-lipoprotein cholesterol (LDL-C) decreased in both groupswith an additional significant effect between periods. Concen-trations of high-density-lipoprotein cholesterol did not changesignificantly. LDL-receptor status in fresh monocytes, affinityofLDL towards the LDL receptor in cultured fibroblasts, zonal-centrifugation profiles, and lipoprotein composition were notsignificantly different between the diets. There was a significantlyhigher tendency toward lipid peroxidation on the PUFA diet,as ascertained by more thiobarbituric acid-reactive-substancesformation on that diet. Dietary PUFA results in somewhat lowerTC and LDL-C concentrations whereas with MUFA the sus-ceptibility of LDL to oxidative stress is lower. Am J C/inNutr 1991;53:899-907.

KEY WORDS Dietary fats, fatty acids, monounsaturatedfatty acids, unsaturated fatty acids, lipoproteins, lipid peroxi-dation

Introduction

The hypothesis that diet can predictably alter plasma lipidconcentrations continues to generate much research and dis-cussion particularly about which type ofdietary fat is preferable.That it still remains a hypothesis after nearly 40 y of investigationis testimony to the difficulties inherent in its proof. One of themajor obstacles is the studying of the effect of natural foods infree-living populations over a long time period while ensuringdietary adherence. We have been able to establish an experi-mental environment that overcomes, at least in part, the prob-lems associated with such studies. Through the cooperation ofthe Har-Etzion Yeshiva, we set up a metabolic kitchen on thecampus where male students participated in two l2-wk diet pe-riods during which they ate diets ofdiffering fatty acid content.Each student acted as his own control in this crossover study.

Oleic acid has been considered neutral with regard to its effecton blood lipid concentrations ( 1 , 2). However, recent work sug-

Friedlander, Yehudit Norman,

gests that there may be an inverse association between dietaryoleic acid and ischemic heart disease (3), which may be relatedto a possible hypolipidemic effect (4). The Jerusalem NutritionStudy plans to investigate in detail the effects of monounsaturatedfatty acids (MUFAs) on lipoprotein structure and function overa 4-y period. Yearly protocols are planned to determine the effectson blood lipids of substituting high or low amounts of MUFAsfor one constituent of the diet while keeping the quantity andquality of other constituents constant. Dietary adherence ismonitored by food-composition analysis and by changes in thefatty acid content of erythrocyte membranes.

We wish to report the first years experiment, which was tocompare effects on lipoprotein structure and function of a dietof high MUFAs and low polyunsaturated fatty acids (PUFAs)with a diet of low MUFAs and high PUFAs, keeping total fat,saturated fatty acids (SFAs), and cholesterol constant.

Subjects and methods

SubjectsThe study was performed on healthy normal male students

ofthe Yeshiva (Talmudic College) Har Etzion outside Jerusalem.A yeshiva is a college in which students study mainly the Talmud(a basic compilation of Jewish law and tradition) in a uniquesystem ofteaching that requires them to attend the college fromearly in the morning to late in the evening. All food is servedby the central kitchen ofthe yeshiva and the opportunity to buyor eat additional food from the outside is very limited. The ye-shiva situation was, therefore, an ideal setting for performing ametabolic study with free-living subjects.

At the beginning of the academic year, all students were as-sembled and the purpose ofthe study, details ofits performance,

I From the Lipid Research Laboratory, Department of Medicine B,Hadassah University Hospital, and the Departments ofSocial Medicineand Nutrition, School of Public Health and Community Medicine, He-brew University-Hadassah Medical School, Jerusalem.

2 Supported by grant HL-39302 from the National Institutes of Health.3 Address reprint requests to NA Kaufmann, Department of Nutrition,

Hebrew University-Hadassah Medical School, POB 1 172, Jerusalem91010, Israel.

Received December 13, 1989.Accepted for publication May 30, 1990.

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and requirements on the part ofthe participants were explainedby the investigators and the directors ofthe yeshiva. Thirty stu-dents volunteered for the study. Each of the volunteers under-went a thorough medical examination and routine biochemicalscreening. Blood lipids were examined twice after an overnightfast at a 3-10-d interval and served as the baseline values forrandomization. Subjects with endocrine or metabolic distur-bances, such as diabetes mellitus, hypothyroidism, obesity, orhigh blood lipid concentrations, or any other chronic diseasewere excluded. Twenty-six subjects were included as participantsof the dietary study and completed the first dietary period, and22 participants remained in the study until the end ofthe secondperiod.

The study was undertaken after approval from the Israel Mm-istry of Health and the Hadassah Medical Center Board for theProtection of Human Subjects, and all subjects gave their writtenconsent to participate.

Experimental design

Before the experiment the volunteers were stratified by bloodcholesterol concentrations and divided randomly into twogroups. After randomization, subjects were given all food (mainmeals and snacks) by the study staff. No other food was allowed.During the first 4 wk the participants were given the regular foodofthe yeshiva including in-between meals and snacks. On severaloccasions they were asked to record their 24-h food intake, whichenabled us to calculate the individual energy requirements ofeach participant and plan his personal diet accordingly.

At the end ofthis run-in period, the dietary experiment started.Two diets, MUFA or PUFA, were administered for a 12-wkperiod (period 1) followed by 4 wk of the regular yeshiva diet(not prepared or delivered by us). After this wash-out period,subjects were given diets in reverse order for a second 1 2-wkperiod (period 2). One-halfofthe students (group 1) began withMUFA diet during period 1 and were given PUFA diet duringperiod 2. This order was reversed in the other halfofthe students(group 2). During both experimental periods participants spenttheir time in the yeshiva and, therefore, received all their mealsand food from our staff. Every second or third weekend (half ofFriday and Saturday) the students visited their families. On theseoccasions they were given strict instructions about what to eatat their homes, and they were required to consume the itemsproviding most of the specific fats, such as olive oil, almonds,or walnuts, which were provided in take-away packages. Fastingblood for plasma lipid determination was drawn periodically.At the end ofeach period, blood was also taken for more detailedstudies of lipoprotein structure and functions. These latter cx-aminations were performed in 12 subjects from each group se-lected at random from all the participants. Subjects were weighedevery 2 wk throughout the study.

Diet

The MUFA diet was rich in MUFAs whereas the PUFA dietwas rich in PUFAs. All other components, ie, total fat and sat-urated fatty acids, cholesterol, protein, and carbohydrates, wereequal in both diets. Each diet was planned to provide 2800 kcal/d-96 g fat (33.5%), 100 g protein ( 15.5%), and 325 g carbo-hydrate (5 1%). There was 300 mg cholesterol in each diet.The diets consisted ofnatural and common food items and wereprepared and cooked in customary ways. About 50 food items(the major ingredients ofthe two diets) were analyzed according

to standard methods of the Association of Official AnalyticalChemists (AOAC) (5). Duplicate samples of the different dietswere collected at random on three occasions during each period,the samples were blended, and a 5% portion of each diet waskept frozen. At the end of each period these subsamples werecombined and analyzed. Fatty acid composition was determinedby gas-liquid chromatography (GLC). The composition of theminor food items was derived from standard food tables (6). Onthe basis of these data, special food tables for the study werecompiled. The two experimental diets contained the same basicfood items each day. Thus, the same meat or the same vegetableswere prepared for both the MUFA and PUFA diets. However,in the MUFA diet fat was added in form of olive oil, avocado,and almonds whereas the PUFA diet was supplemented withequivalent amounts of saffioweroil, soy oil, and walnuts. Foreach diet, 12 daily menus of similar composition but of varieditems were prepared and used in rotation. After the food wascooked, portions were weighed and put on individual trays foreach participant. Subjects were asked to eat all food on theirtrays and to report all leftovers. Usually all the food distributedwas consumed. To allow the participants a certain degree of freechoice, several equivalent items were offered. Thus, for breakfastsubjects could choose between various kinds ofbread and crack-cr5 or various kinds of dairy products of equal fat and caloriccontent. Snacks of composition equal to the daily menu wereprepared and subjects could eat them ad libitum provided thatthe total portion was eaten and the number ofsnacks consumedwas reported. In this way the participants could adjust their totalfood intake to their caloric needs without changing the com-position of the diet.

All planning and calculations were done by a qualified dietitianand the food was prepared and cooked under her supervisionin a special kitchen.

Isolation and labeling of lipoproteinsVery-low-density lipoprotein (VLDL) and low-density lipo-

protein (LDL) were isolated, by ultracentrifugation, at d 1.006and 1.019-1 .063 kg/L, respectively, from human plasma con-taming 1 g Na2EDTA/L. The LDL was sterile filtrated, portionedinto plastic tubes, and kept under nitrogen at 4 #{176}C.

Zonal ultracentrifugation of lipoproteins was performed ac-cording to the techniques of Patsch et al (7) as modified by Ei-senberg et al (8). Intermediate-density lipoprotein (IDL) andLDL were separated from 20 mL plasma by zonal centrifugation.The amounts of LDL protein (4-10 mg) were sufficient for fur-ther characterization whereas with IDL it was sometimes nec-essary to combine preparations from two or even three subjectson the same diet. High-density lipoprotein (HDL) was separatedinto HDL2 and HDL3 from lO-mL plasma samples. LDL fromnormolipemic subjects not participating in the study was pm-pared by ultracentrifugation as described above. After dialysisand concentration, a sample of this normal LDL was radioio-dinated to a specific activity of 3.4-10.2 &iJg protein by usingthe iodine monochloride method (9) as modified for lipoproteinlabeling (10).

Cell isolation and culture

Freshly prepared circulating mononuclear cells were isolatedfrom 30-40 mL blood collected under sterile conditions by theprocedures of Boyum (1 1) and Bilheimer et al (12). The cells (6x l09/L) were suspended in RPMI 1640 (Gibco, Paisley, Scot-

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DIETARY MUFA VS PUFA 901

land, UK) medium supplemented with penicillin, streptomycin,and 5 g protein/L human lipoprotein-deficient serum (LPDS;ie, RPMI 1640-5% LPDS). Monocytes were then separated fromlymphocytes by 1-h adherence to plastic (35-mm) Petri dishes.Cell viability was estimated by trypan-blue exclusion.

Human skin fibroblasts were cultured from skin biopsies ofnormal volunteers and used between the 5th and 15th passage.After trypsinization, 3-5 X 10 cells were seeded in 35-mm disheswith 2 mL medium containing 100 mL fetal calfserum/L. Cellswere fed three times weekly with the same medium.

Bovine aortic smooth muscle cells (SMC) were prepared asdescribed previously and subcultured in Dulbecco-Vogt medium(Biological Industries, Kibbutz, Bet Haemeq, Israel) containing100 mL fetal bovine serum/L (13).

LDL receptor-specific degradation in fresh monocytesReceptor-specific degradation of 25I-labeled LDL by mono-

cytes was determined after 8 h incubation with 2 mL mediumcontaining 20 mg/L of 251-LDL from a normal-lipidemic subjectin the RPMI 1640-5% LPDS medium. Degradation productswere determined in culture medium as described ( 1 3). Nonspe-cific degradation was determined in the presence of25-fold excessof unlabeled LDL. No-cell control dishes were processed si-multaneously. The cells were washed in protein-free mediumand were dissolved in 0.5 mol NaOH/L for protein determi-nation. LDL-specific degradation was calculated after substrac-tion of control and nonspecific values.

Competitive displacement of251-labeled control LDLby experimental LDL

The affinity of MUFA- and PUFA-derived LDL towards theLDL receptor was determined by a competition assay in upregu-lated cultured fibroblasts grown for 48 h in 5% LPDS. Ten mi-crograms per milliliter ofcontrol 25I-LDL protein was includedin the incubation medium without or with different concentra-tions (5, 10, 20, and 40 mg/L) of unlabeled LDL preparations,derived from subjects consuming MUFA or PUFA diets. Bind-ing, internalization, and proteolytic degradation of the control25ILDL were determined at the end of 3-h incubation at 37OC by following established procedures (14). The data of theproteolytic degradation were used to construct competitioncurves in which values for incubations without added unlabeledLDL were taken as 100% activity.

Thiobarbituric acid-reactive substances

Conditioning of LDL was carried out by incubation of 50 mgLDL protein/L in Ham F-10 medium (Gibco) without serumfor 24 h in the presence of confluent cultures of bovine aorticSMC and/or 2.5 mol Cu2/L. No-cell control dishes were pro-cessed simultaneously. Thereafter, thiobarbituric acid-reactivesubstances (TBARS) were determined (1 5) by the modified pro-cedure ofLee (16). Briefly, to 1 mL plasma or 50 ig LDL proteinin 1 mL medium, 0.5 mL of 350 g tricarboxylic acid (TCA)/Lwas added; the solution was vortex mixed; 1 mL of 5 g TBA/Lwas added, vortex mixed, and incubated for 90 mm at 60 #{176}Cina shaking waterbath. After incubation, 1 mL of 700 g TCA/Land 2 mL chloroform were added and the tubes were centrifugedfor 20 mm at 1 100 X g. A standard curve was prepared by usingmalondialdehyde, and the intensity of fluorescence was deter-mined at excitation of 5 15 nm and emission of 553 nm as em-ployed by Yagis method (17). The results were expressed as

malondialdehyde equivalent content (imol MDA/L plasma orzmol MDA/g LDL protein).

Analytical methods

All blood samples were collected after a 12-h overnight fast.Plasma cholesterol and triglycerides were determined by an en-zymatic procedure in a batch analyzer (Vitalab, Vital Scientific,Dieren, The Netherlands). HDL-cholesterol (HDL-C) concen-trations were determined after precipitation ofapo B-containinglipoproteins with phosphotungstic acid. LDL-C (including IDL)was obtained by subtraction, using the Friedewald et al equa-tion (18).

To determine the composition of VLDL, LDL, and HDL3,standard procedures were used to measure protein, triglycerides,cholesterol, and phospholipids. Unesterified cholesterol andcholesteryl esters were determined by specific enzymatic methodswith a commercial kit (Boehringer-Mannheim, Mannheim, FRG).

Erythrocytes were separated from EDTA-containing bloodsamples and hemolyzed by the method of Dodge et al (19, 20).Total lipids were extracted with chloroform-methanol andwashed to remove proteins. Fatty acid methyl esters were pre-pared by transesterification with methanolic trimethylammo-nium hydroxide (Meth Prep II, Applied Science, Deerfield, IL)(2 1). During the separation procedures, 2,6-ditert-butyl-9-cresol(BHT; 5 g/l00 L) was used as an antioxidant. The fatty acidmethyl esters were separated and quantified by GLC using aTracor 565 GLC column (1.85 m X 4 mm id, 10% SP-2330 on100/ 120 chromosorb in WAW 1- 185 1 ; Supelco, Bellefonte, PA).Temperature programming was used from 185-220 #{176}C.Peakswere identified by comparing retention times with known stan-dards.

Statistical analysis

Statistical analysis included t tests for the comparison ofchanges in lipid concentrations in response to dietary treatmentand time period for this basic two-period, crossover design ac-cording to Fleiss (22). For subjects commencing with the MUFAdiet in period 1 and changing to the PUFA diet in the period 2,DMP measures the change in response from period 1 to period2. This value includes a measure of the difference between thetwo treatments regardless of the period, plus the time trend re-gardless ofthe treatments. Similarly, DPM represents the changein response for subjects in group 2 who followed the same pro-cedure in the reverse order. The hypothesis of equal efficacy ofthe two diets was tested by means ofa t test comparing the twoaverages, DMP and DPM (22). The sum of plus DM and itsstandard error was used to test the existence of a period effect.The possible interaction between the dietary treatments and pe-riod was also tested by means ofa t test (22). Values are expressedas mean SD or mean SEM.

Results

The baseline characteristics ofthe students entering the studyare shown in Table 1. During each 12-wk period some studentswere unavailable for blood tests. The experimental crossoverdesign required analysis of those participants on whom therewere data at the beginning and end ofeach period. Such completedata sets were available for 9 students in each group and therefore

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TABLE 1Baseline characteristics of participants in study

Group 1(n=l2)

Group 2(n=l2)

Body mass index (kg/rn2) 23.3 2.4 21.6 2.2Number of smokers 1 1Number of subjects born in Israel 8 9Plasma cholesterol (mmol/L) 3.8 1 0.77 3.8 1 0.78Plasma triglycerides (mmol/L) 1.02 0.22 0.92 0.27Plasma HDL cholesterol (mmol/L) 1.09 0.25 1 . 1 1 0.17

S SD.

plasma lipid concentrations relate to these 18 students. Specialstudies were performed on subgroups of participants at the endof each diet period.

The composition of the planned experimental diets is shownin Table 2. Both diets comprised 2800 kcal with -34% of thecalories from fat. About 17% of the total calories came fromeither MUFAs or PUFAs, the quantities of SFAs, protein andcarbohydrate being nearly identical. During period 1 the amountsofboth MUFAs and PUFAs as percent total calories were slightlyless than planned; during period 2 the discrepancy disappeared.The house diet of the Yeshiva is shown for comparison. Ad-herence to the diet was monitored by comparing changes inerythrocyte membrane fatty acid composition, in particular theratio between 18: 1 (oleic acid) and 18:2 (linoleic acid). In bothperiods on the MUFA diet, the ratio increased (more in period2), and on the PUFA diet the ratio decreased significantly (Table3). During period 1 the average weight change of the studentsfrom both groups was < 0.5 kg; during period 2 the change was< 1.0 kg.

The changes in total plasma cholesterol, triglycerides, HDL-C, and LDL-C at the beginning and end ofthe two study periodsare shown in Table 4. Statistical analysis of the overall effectsof diet and season on plasma lipids is shown in Table 5. Theplasma lipid values reported represent the mean oftwo separatedeterminations before the beginning ofthe experiment (3-10 dapart; baseline) and two determinations at the end (weeks 10and 12). After period 1 for both diets there was a significantdecrease (1 1%) in total cholesterol; after period 2 this decreasewas more prominent on the PUFA diet (20%) and less so onthe MUFA diet (8.5%), but both were significant when comparedwith the baseline values. The data of the crossover study wereanalyzed to distinguish between effects on plasma lipids due tothe diet intervention and those that relate to the timing (season-ality) ofthe experimental period. Our results for cholesterol in-dicated that the overall effect ofthe PUFA diet was significantlygreater than that of the MUFA diet (P < 0.02). There was noseasonality effect. Plasma triglyceride concentrations decreasedsignificantly on both diets in period 1, but after period 2 a slightincrease was observed after the PUFA diet. Statistical analysisshowed a significant effect for the period (seasonality; P < 0.01)but not for the diet manipulation. There were no significantchanges in plasma HDL-C concentrations as a result of the twodiets or the different time periods. LDL-C concentrations de-creased significantly on both diets and over both time periodsof study. The reduction after period 2 on the PUFA diet wassignificantly greater and a decrease of 31% was observed, pro-

BERRY ET AL

TABLE 2Composition of the experimental and house diets during the twodietary periods: planned vs from analysis5

Diet SFAs MUFAs PUFAs Fat Protein CHO

% of kca/

Period 1MUFA

Planned 8.5 17.7 6.3 35.1 15.2 49.7From analysis 8.1 14.9 6.7 31.4 19.2 49.4

PUFAPlanned 8.1 7.1 17.6 35.2 14.7 50.1From analysis 7.2 5.9 14.7 29.6 19.4 51.0

Period 2MUFA

Planned 8.9 16.5 6.3 33.8 16.7 49.5From analysis 7.8 16.9 8.3 34.9 18.4 46.7

PUFAPlanned 8.4 6.4 17.1 33.8 16.8 49.5From analysis 7.0 6.4 17.2 32.3 16.3 51.4

House, from analysis 10.5 1 1.5 15.8 40.6 15.7 43.7

S SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids;PUFAs, polyunsaturated fatty acids; CHO, carbohydrate.

ducing a significant seasonality effect (P < 0.01). The overalleffect of PUFAs on LDL-C concentrations was significantlygreater than that of MUFAs (P < 0.05).

There were no significant differences in lipoprotein compo-sition analysis during either period ofthe diet study or betweenthe two diets (Table 6). The profiles of IDL plus LDL and ofHDL on zonal ultracentrifugation were also similar (Fig 1).

25I-labeled LDL degradation in fresh monocytes ranged be-tween 80 and 180 LDL protein . g cell protein . h and wasnot changed by the different diets. Mean values were similar onthe MUFA and PUFA diets (137 31 and 133 32 g LDLprotein#{149}gcell protein. 8 h, I SD; n 11; n 8 studiedtwice on each diet). Competition studies in cultured human skinfibroblasts between normal LDL and LDL obtained from sub-jects on either of the diets produced identical curves (Fig 2,Table 7). There was, therefore, no evidence for any change inlipoprotein structure or function in response to the differentdiets.

The effect of dietary MUFAs and PUFAs on the tendencytoward oxidative stress was assessed by TBARS production in

TABLE 3Ratio of oleic acid to linoleic acid in erythrocytes from subjects onMUFA and PUFA diets

Diet Baseline End of study period P5

Period 1MUFA 1.040 0.023t 1 .069 0.027 0.072PUFA 0.980 0.027 0.801 0.026

DIETARY MUFA VS PUFA 903

TABLE 4Plasma lipid concentrations by study period and diet5

Lipid

Period 1 Period 2

Baselinet End of period P Baseline End of period P

mmol/L mmo//L

Total cholesterolMUFA 3.83 0.31 3.42 0.24 0.008 3.96 0.32 3.62 0.26 0.037PUFA 3.89 0.32 3.44 0.27 0.003 4.04 0.32 3.22 0.25

IDL.+ LDL

EC

0c%J4-0

0 100 200 300 400 500Zonal Rotor Effluent (ml)

FIG 1. Zonal ultracentrifugation profile of IDL plus LDL (top) andHDL (bottom) in plasma obtained from a subject at the end of 12 wkof the MUFA (- - -) and PUFA (-) diets.

904 BERRY ET ALTABLE 6Composition oflipoproteins at the end of the dietary periods5

Diet and period Protein TGs FC CE PLs

%

VLDLMUFA, 1 (n = 6) 12.0 0.6 5 1.2 2.4 3.2 0.2 6.8 0.9 26.9 3.0MUFA, 2 (n = 7) 13.2 1.7 40.3 3.4 3.4 0.4 7.5 1.1 26.6 3.7PUFA, 1 (n = 5) 12.0 0.7 54.8 2.2 3.0 0.3 5.8 0.9 24.5 1.9PUFA, 2 (n = 4) 13.2 1.4 48.4 5.3 3.0 0.2 7.8 1.7 27.7 4.5

LDLMUFA, 1 (n = 6) 21.8 2.1 4.6 1.0 8.9 0.5 37.4 2.3 26.4 0.8MUFA, 2 (n = 7) 24.6 1.5 4.5 1.1 8.3 0.9 36.2 3.7 26.3 3.0PUFA, 1 (n = 5) 22.7 2.8 5.1 0.8 9.4 1.0 38.5 2.2 24.4 1.4PUFA, 2 (n = 4) 24.0 1.6 4.0 1.0 8.3 0.5 38.1 1.6 25.6 2.1

HDL3MUFA, 1 (n = 4) 51.0 1.9 3.7 2.0 1.7 0.2 14.8 1.2 28.8 1.4MUFA, 2 (n = 4) 54.1 2.7 3.0 0.3 1.4 0.1 15.4 1.6 26.1 1.1PUFA, 1 (n = 4) 55.7 1.5 4.4 1.8 1.5 0.2 14.5 2.6 23.9 2.0PUFA, 2 (n = 4) 54.8 1.1 3.1 0.7 1.5 0.2 14.5 1.1 26.2 1.7

S SD. TG, triglycerides; FC, free cholesterol; CE, cholesteryl esters; PLs, phospholipids. Values are percent contribution ofindividual componentsto total lipoprotein mass.

was 10.5: 1 1 .5: 15.8 for SFAs:MUFAs:PUFAs. The PUFA-richdiet was accompanied by average changes of -3.4% in SFA,-5.4% in MUFA, and +0.2% in PUFA. The predicted changein plasma cholesterol using Keys equation 2 would be -0.25mmol/L (-9.6 mg/dL) compared with -0.45 and -0.83 mmol/L, observed in periods I and 2, respectively (Table 4). For theMUFA-rich diet the relevant changes are in -2.6% in SFA,+4.4% in MUFA, and -8.3% in PUFA. Accordingly, the pre-dicted change in cholesterol would be an increase in cholesterolof0.10 mmol/L (3.7 mg/dL) compared with -0.41 and -0.34mmol/L, which was found in periods I and 2, respectively. The

obvious question that arises in light of these results and resultsby other workers is why there is such a discrepancy between theobserved and predicted changes in cholesterol concentrationsafter changing dietary MUFAs. There is no clear answer, but apossible explanation may be found in the changes in dietaryhabits that have occurred over the past 30 y since the originalequations were devised from studies on subjects living in theUnited States. Such a change was indeed demonstrated throughthe measurement of adipose-tissue fatty acid composition, oneof the most reliable methods of following the dietary fat intakeofa population (30, 3 1). The percentage oflinoleic acid in adi-pose tissue was < 10% in the late l950s (32). By 1980 it hadrisen to 16% in the United States (33) and to close to 25% inIsrael (34). The comparable figures for oleic acid in adipose tissueare 52% in the 1950s, 46% in 1980(US), and 41% in I 986 (Israel).It may well be that these changes in dietary fatty acid consump-tion over the years may affect cholesterol homeostasis in thebody in a manner that has yet to be clearly defined. In other

TABLE 7Competition of 251-labeled LDL degradation in cultured humanfibroblasts by LDL isolated from subjects on the MUFA and PUFAdiets at the end ofthe dietary period5

Concentration of unlabeled LDL protein

Diet 5 mg/L 10 mg/L 20 mg/L 40 mg/L

%

Control (n = 5) 67 I l.1 S 1 10.8 42 9.1 26 8.1MUFA (n = 12) 66 10.5 51 9.0 39 8.6 22 5.7PUFA (n = 13) 66 8.6 51 8.4 37 8.5 25 8.1

S SD. Results are expressed as percent ofdegradation in the pres-ence oflabeled LDL only. Eleven subjects were studied twice at the endof each diet period. One subject was studied only on the MFA and twowere studied only on the PFA diet.

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DIETARY MUFA VS PUFA 905

S

-I

I

Unlabeled LDL (pg/mI)FIG 2. Competition of 25I-Iabeled LDL metabolism in cultured human skin fibroblasts with unlabeled LDL from

control subjects (healthy normal humans) and with LDL obtained at the end of 12 wk of the MUFA or PUFA diets.The 25I-labeled LDL was prepared from control subjects.

words, it may be necessary to repeat and redefine the Keys equa-tion for the nutritional state ofthe population in the l990s. Oneofthe major aims ofthis ongoing study on the biological effectsofMUFAs is to collect data sufficient for addressing this problem.

The principal differences between this nutritional study andother investigations are that it was carried out in a free-livingpopulation eating normal food (not formula) over two 12-wkperiods. The population studied was homogenous, was non-smoking, and consumed negligible amounts of alcohol. Thiscrossover study shows that diets with similar amounts of eitherPUFAs or MUFAs produced a significant decrease of plasmacholesterol concentrations (- 16% and 10% on the PUFA andMUFA diets, respectively, during the two periods) withoutchanging levels of HDL-C. These results are in general similarto those of Mensink and Katan (27), who also carried out theirinvestigations in a free-living population. The fact that we founda somewhat larger decrease of total and LDL-C on the PUFAthan on the MUFA diet whereas Mensink and Katan (27) didnot may be due to differences in the design of the diets and theduration of the experiment.

The percent decrease in total cholesterol we observed compareswith the 13% observed by Mattson and Grundy (4), who useddiets of between 27% and 33% MUFAs, and with the - 12.8%found by Mensink and Katan (27), who used a diet of 15.1%MUFAs. The concentration of MUFAs in our diets was 17%and although the control diets in these experiments were notsimilar, interesting questions regarding the dose response ofMUFAs on plasma cholesterol arise, to be studied in the future.The amount of MUFAs in the diet is of major importance inlight ofrecent findings in experimental animals that high-MUFAdiets may cause accumulation of cholesterol in the liver (35).On the other hand, a study from Crete (36), where the diet con-tains an average 27% MUFAs, failed to show a more favorableserum lipoprotein pattern in young boys when compared withboys from a more westernized society.

The response of plasma triglycerides was variable with a sea-sonality effect between the two experimental periods. The effectsofseasonality in the concentrations oftriglycerides are not clearlyunderstood. The baseline values at the start of period 2 (Table4) were considerably lower than in period 1 . This may be dueto confounding dietary factors related to the 1-mo wash-out pe-riod which included the Passover holidays. We (37) investigated

the effect ofseasonality on plasma lipid concentrations in Israelin a previous study. In middle-aged men a decrease of0.07 mmoltriglycerides/L was found between November and March. Con-centrations in April (corresponding to the baseline values forperiod 2 in our study) were lowest and thereafter increased by0.24 mmol/L until July. The effects of season on plasma cho-lesterol and LDL-C concentrations in this previous study weremuch smaller than the differences found in the present experi-ment.

Adherence to diet was monitored by changes in erythrocytemembrane composition. Although it has been recognized for along time that dietary manipulation with essential fatty acids(such as linoleic acid) will alter erythrocyte membrane compo-sition (38), it was not clear whether diet enrichment with MUFAswould also be detected. Our findings (Table 3) suggest thaterythrocyte membrane composition may be altered by MUFAs(mainly oleic acid). In both groups the high-MUFA diet led toan increase in 18: 1-1 8:2 ratio whereas the high-PUFA diet pro-duced converse changes. Oleic acid is the most abundant fattyacid of stored adipose-tissue triglyceride (30). It is derived bothfrom dietary sources or by desaturation of stearic acid. Inter-estingly, stearic acid is unique among SFAs in that it does notappear to elevate plasma cholesterol concentrations (28, 39).

TABLE 8Thiobarbituric acid-reactive substances in native and conditionedLDL from subjects on MUFA and PUFA diets at the end of thesecond dietary period5

Smooth-Smooth-

muscle cellsDiet Native muscle cells + Cu2 Cu2

nmo/ MDA/mg LDL protein

MUFA(n = 5) 1.05 0.03 15.70 4.00 47.00 2.00 7.20 2.00

PUFA(n = 6) 1.16 0.04t 24.40 2.00 49.80 2.60 18.60 l.40t

S SEM. MDA, malondialdehyde equivalentstt Significantly different from MUFA (one-tail I test): tP < 0.05, jP

906 BERRY ET AL

These indices of diet adherence suggest that the major meth-odological aim of the study was achieved and strengthens thevalidity ofresults for a free-living population. Whether the resultsare applicable to other populations and also to females in lightofthe findings of differing responses between the sexes (27, 40)requires further investigation. Another point concerns the issueofwhether the lipid concentrations would be maintained or driftback to the initial values over a longer time period.

There were no differences in lipoprotein structure as shownby compositional analysis or profiles on zonal ultracentrifuga-tion. LDL-receptor activity of monocytes also remained un-changed as was the ability ofdiet LDL to displace control LDLat the fibroblast receptor. However, when the tendency towardoxidative stress was measured, LDL from the PUFA diet pro-moted significantly more TBARS formation than did LDL iso-lated after the MUFA-rich diet. Recent work showed that unes-terified fatty acids may inhibit iron-dependent lipid peroxidation(41). The effect of oleic acid was significantly greater than thatoflinoleic acid. It may also be that oleic acid is a poorer substratethan linoleic acid for peroxidation. However, the possibility thatthe MUFA diet contained more antioxidants, such as vitaminE, cannot be excluded. Thus, there may be a theoretical advan-tage to the MUFA diet. The possible adverse effects ofthe PUFAdiet on the tendency to peroxide formation is of major interestbecause free radicals and modification of LDL are consideredto be important pathoetiological factors in the development ofatherosclerosis (42). In conclusion, our observations lend supportto the concept that MUFAs may serve as a substitute for SFAsin optimizing plasma lipid concentrations and, in addition, anMUFA-rich diet may reduce the susceptibility of LDL to oxi-dative stress. U

We thank the heads and teachers ofthe Har Etzion Yeshiva for theirenthusiastic help, the administrative staffofthe Yeshiva, and especiallythe personnel involved in preparation ofthe special diets. We thank andappreciate the student volunteers for their devoted cooperation.

References

1. Keys A, Anderson JT, Grande F. Prediction of serum-cholesterolresponses of man to changes in fats in the diet. Lancet 1957;l:959-66.

2. Hegsted DM, McGandy RB, Myers ML, Stare FJ. Quantitative effectsofdietary fat on serum cholesterol in man. Am J Can Nutr 1965; 17:281-95.

3. Keys A, Menotti A, Karvonen MJ, et al. The diet and 15-year deathrate in the seven countries study. Am J Epidemiol 1986;124:903-15.

4. Mattson FH, Grundy SM. Comparison ofeffects ofdietary saturated,mono-unsaturated and polyunsaturated fatty acids on plasma lipidsand lipoproteins in man. J Lipid Res l985;26:194-.202.

5. Horwitz W, Senzel A, Reynolds H, Park DL, eds. Official methodsof analysis. 12th ed. Washington DC: Association of Official Ana-lyticalChemists, 1975.

6. Guggenheim KJ, Kaufmann NA, Reshef A. Tables of food corn-position 6th ed. Jerusalem: College of Nutrition and Home Eco-nomics, 1980.

7. Patsch JR. Sailer 5, Kostner G, et al. Separation of the main lipo-protein density classes from human plasma by rate-zonal centrifu-gation. J Lipid Rca l974;15:356-66.

8. Eisenberg S. Gavish D, Oschry Y, Fainaru M, Deckelbaum Ri. Ab-normalities in very low, low and high density lipoproteins in hy-pertriglyceridemia. Reversal toward normal with bezafibrate treat-ment. J Clin Invest 1984;74:470-82.

9. MacFarlane AS. Efficient trace labeling ofproteins with iodine. Na-ture l958;l82:53.

10. Bilheimer DW, Eisenberg 5, Levy RI. The metabolism of very lowdensity lipoprotein proteins. I. Preliminary in vitro and in vivo oh-servations. Biochim Biophys Acta l972;260:2 12-21.

1 1. Boyum A. Isolation of leucocytes from human blood: further oh-servations. Scand J Gin Lab Invest [Suppl) 1968;2l:3l-S0.

12. Bilheimer DW, Ho YK, Brown MS. Anderson RG, Goldstein JC.Genetics of low density lipoprotein receptor. diminished receptoractivity in lymphocytes from heterozygotes with familial hypercho-lesterolemia. J Clin Invest 1978;6 1:678-96.

13. Bierman EL, Stein 0, Stein Y. Lipoprotein uptake and metabolismby rat aortic smooth muscle cells in tissue culture. Circ Res 1974;34:136-SO.

14. Pitas RE, Innerarity KS, Arnold KS, Mahley RW. Rate and equi-librium constants for binding of apo-E-containing lipoproteins ascompared with low density lipoproteins to human fibroblasts. Evi-dence for multiple receptor binding ofapo-E HDL. Proc NatI AcadSci USA 1979;76:231l-S.

15. Fong K-L, McCay PB, Poyer JL, Keele BB, Misra H. Evidence thatperoxidation oflysosomal membranes is initiated by hydroxyl freeradicals produced during flavin enzyme activity. J Biol Cheml973;248:7792-7.

16. Lee DM. Malondialdehyde formation in stored plasma. BiochemBiophys Res Commun 1980;95: 1663-72.

17. Yagi K. A simple fluorimetric assay for lipoperoxide in blood plasma.Biochem Med l976;lS:212-6.

18. Friedewald W, Levy RI, Fredrickson DS. Estimation ofthe concen-tration oflow-density lipoprotein cholesterol in plasma, without useof the preparative ultracentrifuge. Gin Chem 1972;l8:499-502.

19. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemicalcharacteristics of hemoglobin-free ghosts of human erythrocytes.Arch Biochem Biophys l963;lOO:l 19-27.

20. Dodge JT, Phillips GB. Composition ofphospholipids and of phos-pholipid fatty acids and aldehydes in human red cells. J Lipid Resl967;8:667-77.

21. MacGee J, Allen KG. Preparation ofmethyl esters from the sapon-ifiable fatty acids in small biological specimens for gas-liquid chro-matographic analysis. J Chrornatogr 1974;lOO:35-42.

22. fleiss J. The design and analysis ofclinical experiments. New York:Wiley, 1986.

23. Baggio G, Pagnan A, Muraca M, et al. Olive oil enriched diet: effecton serum lipoprotein levels and biliary cholesterol saturation. AmJ Clin Nutr 1988;42:960-4.

24. Grundy SM. Comparison of mono-unsaturated fatty acids and car-bohydrates for lowering plasma cholesterol. N EngI J Med I986;314:745-8.

25. Grundy SM, Florentin L, Nix D, Whelan MI. Comparison ofmonounsaturated fatty acids and carbohydrates for reducing raisedlevels of plasma cholesterol in man. Am J Gin Nutr 1988;47:965-9.

26. Mensink RP, de-Groot MJ, Van den Broeke LT, Scverijnen-NobelsAP, Demacker PH, Katan MB. The effects ofmonounsaturated fattyacids vs complex carbohydrates on serum lipoproteins and apopro-teins in healthy men and women. Metabolism 1989;38:172-8.

27. Mensink RP, Katan MB. Effect of a diet enriched with monoun-saturated or polyunsaturated fatty acids on levels oflow-density andhigh-density lipoprotein cholesterol in healthy women and men. NEngl J Med l989;321:436-4l.

28. Keys A, Anderson iT, Grande F. Serum cholesterol response tochanges in the diet IV: particular saturated fatty acids in the diet.Metabolism 1965; 14:776-87.

29. Keys A. Diet and blood cholesterol in population surveys-lessonsfrom analysis of the data from a major survey in Israel. Am J ClinNutr l988;48:l 161-S.

by guest on March 25, 2015

ajcn.nutrition.orgD

ownloaded from

DIETARY MUFA VS PUFA

30. Hirsch J, Farquhar JM, Ahrens EG, Jr. Peterson ML, Stoffel W.Studies of adipose tissue in man. A micro-technique for samplingand analysis. Am J Clin Nutr 1960;8:499-S1 1.

31. Van Staveren WA, Duerenberg P. Katan MB, et al. Validity of thefatty acid composition of subcutaneous fat tissue microbiopsies asan estimate of the long-term average fatty acid composition of thediet of separate individuals. Am J Epidemiol 1986;123:4S5-63.

32. Insull W Jr, Bartsch GE. Fatty acid composition of human adiposetissue related to age, sex and race. Am J Gin Nutr 1967;20:l3-23.

33. Berry EM, Hirsch J, Most J, McNamara DJ, Thornton J. The re-lationship of dietary fat to plasma lipid levels as studied by factoranalysis ofadipose tissue fatty acid composition in a free-living pop-ulation of middle-aged American men. Am J Clin Nutr 1986;44:220-31.

34. Berry EM, Zimmerman J, Peser M, Ligumsky M. Dietary fat, adiposetissue composition, and the development ofcarcinoma ofthe colon.JNCI l986;77:93-7.

35. Beynen AC. Mono-unsaturated fatty acids and liver cholesterol. Ar-tery l988;lS:l70-S.

907

36. Aravanis C, Mensink RP, Karalias N, Chistodoulou B, Kafatos A,Katan MB. Serum lipids, apoproteins and nutrient intake in ruralCretan boys consuming high olive oil diets. J Gin Epidemiol l988;41:1117-23.

37. Harlap 5, Kark JD, Barns M, Eisenberg 5, Stein Y. Seasonal changesin plasma lipid and lipoprotein levels in Jerusalem. Isr J Med Sci1982;18:l 158-65.

38. Farquhar JW, Ahrens EJ Jr. Effects ofdietary fats on human eryth-rocyte fatty acid patterns. J Clin Invest l963;42:675-80.

39. Bonanome A, Grundy SM. Effect ofdietary stearic acid on plasmacholesterol and lipoprotein levels. N Engl J Med l988;3 18:1244-8.

40. Mensink RP, Katan MB. Effect of mono-unsaturated fatty acidsversus complex carbohydrates on high-density lipoproteins in healthymen and women. Lancet 1987;1:l22-S.

41. Balasubramanian KA, Nalini 5, Cheeseman KH, Slater TF. Non-esterified fatty acids inhibit iron-dependent lipid peroxidation.Biochim Biophys Acts 1989;l003:232-7.

42. Steinberg D, Parthasarathy 5, Carew TE, Khoo JC, Witztum JLBeyond cholesterol: modifications of low-density lipoprotein thatincrease its atherogenicity. N Engl J Med l989;320:9l5-24.

by guest on March 25, 2015

ajcn.nutrition.orgD

ownloaded from