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Influence of a Diet High in Unsaturated Fatupon Composition of Arterial Tissue and

Atheromata in ManBy SEYMOUR DAYTON, M.D., SAM HASHIMOTO, M.S.,

AND MORTON LEE PEARCE, M.D.

A NUMBER of modifications of the usualAmerican-European diet have been found

effective in depressing serum cholesterol levelsin man, leading to hope that such modificationsmight retard the development of atherosclero-sis and its complications. Clinical recommen-dations based on these considerations have forthe most part emphasized either reduction intotal fat intake or rigorous substitution of high-ly unsaturated for saturated fat.A clinical trial of the latter type of diet

was begun in this Center in 1959, with theprimary objective of evaluating its possibleeffectiveness in preventing complications ofatherosclerosis. The experimental diet resultedin an early drop in serum cholesterol levels,'which have remained depressed throughoutthe study. Over-all difference between serumcholesterol concentrations of the experimentalsubjects and those of control subjects has beenabout 14 per cent of the baseline level through-out the period of observation. Mean bodyweight has fallen about 2 per cent in thecontrol group and risen about 2 per cent inthe experimental group.

In the course of the study, which is still inprogress, a number of subjects of experimentaland control groups have come to autopsy.

From the Medical Services of Wadsworth Hospitaland Domiciliary, and the Research Service, VeteransAdministration Center, and the Department of Medi-cine, University of California at Los Angeles, Schoolof Medicine, Los Angeles, California.

Supported in part by grants from the Arthur DoddFuller Foundation, the Los Angeles County HeartAssociation (241), and the U. S. Public Health Ser-vice (HE-03734 and HE-04900).A preliminary report was presented at the 1964

meeting of the American Institute of Nutrition (Fed.Proc. 23: 144, 1964).Circulation, Volume XXXII, December 1965

Following evaluation of the extent of athero-sclerosis by gross inspection, arterial tissue hasbeen analyzed in some detail with regard tocomponent lipids. Results of the chemicalanalyses are described herein and related totype of diet. Relationships between atheromalipids and antemortem serum lipids of thesame subjects have also been studied.

Methods

SubjectsThe participants in the study are elderly men

living in a Veterans Administration Domiciliaryunit. Upon entering the study each man was as-signed, by use of a table of random numbers,either to the control group or to the experimentalgroup. The control group was placed on a con-ventional diet containing 40 per cent fat calories.This diet differs only in minor respects from theregular institutional diet, which all subjects hadbeen eating for 4 months or more prior to thestudy. The experimental group was placed on adiet also containing 40 per cent fat calories, butembodying major -substitution of vegetable oilfor the saturated fats of the natural foods. Thesubjects and experimental conditions are de-scribed in greater detail in earlier publications." 2

Since men living in the Domiciliary are free toleave during the day, it is not possible to enforcetotal adherence to the study diets. Presence orabsence of each participant is therefore recordedat every meal.* The present report includes ma-terial from men with varying adherence rates. The15 subjects of the control group had adherencerates from 43 to 93 per cent (mean, 76 per cent);

*Independent evidence that this gives a realisticappraisal of adherence is provided by data on com-position of subcutaneous fat. By the end of 5 years,mean linoleic acid content had risen to 32 per centof adipose tissue fatty acid in men of the experimentalgroup with a mean adherence rate of 92 -per cent,from a starting value of 11 per cent. The linoleic acidcontent of dietary fat (fig. 1) is 38 per cent.

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DAYTON ET AL.

MEAN COMPOSITION OF DIETARY FAT

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Abundance of fatty acids in fat of experimental andcontrol diets, determined by analysis of extracted fat.The fatty acids are identified by two numbers, desig-nating chain length and the number of double bonds.Results are means of seven analyses, each representinga 4- to 9-week pool, weighted as to length of collec-tion period. Reprinted with permission of the NewEngland Journal of Medicine.1

the range for the 11 experimental subjects was

53 to 99 per cent, with a mean of 75 per cent.*Time on the study diet ranged from 279 to 1,536days (mean, 891 days) for the control group, and495 to 1,351 days (mean, 964 days) for the ex-

perimental group. Mean age on entering thestudy, for subjects of the present report, was 67years in the control group and 69 in the experi-mental group.

Diets

Diets and methods of diet analysis have beendescribed previously.1 2 Details of diet composi-tion are summarized in table 1 and figure 1.

Tissues

Because it has been the routine practice in thisinstitution to embalm bodies prior to autopsy,most of the tissues analyzed had been in contactwith embalming fluid, generally for less than 1week, prior to extraction. It was therefore neces-

sary to evaluate the effects of embalming fluidand storage on the compounds being assayed. Todo this, unembalmed atheromatous aortic tissuewas obtained within 24 hours after death. Thetissue was reduced to a fine mince, which was

thoroughly mixed. Part of the mince was ana-

lyzed fresh and the remainder stored as a suspen-

Table 1Composition of Diets

Control Experimental

Total daily calories* 2,400 2,425Protein, Gm./day* 91 92Fat, Gm./day* 108 106Fat calories, % of total* 41 39Iodine value of fat* 53 101Cholesterol, mg./dayt 721 365a-sitosterol, mg./dayt 0 33i3-sitosterol, mg./dayt 16 187a-tocopherol, mg./day: 2.4 22.6

*Mean of 216 1-week pooled collections.tMean of seven analyses, each representing a 4- to

9-week pool, weighted as to length of collectionperiod.tMean of three 1-week pools.

sion in embalming fluidt at -20 C. for varyingperiods prior to analysis, which was carried outby the procedures described below. Table 2shows results of silicic acid fractionation as wellas the fatty acid content of the sterol ester frac-tion. Apart from some small fluctuations in analyt-ical values, attributable to the difficulties ofsampling minced tissue, there were no significantchanges for 8 weeks. Fatty acids of triglyceridesand phosphatides, not shown in the table, weresimilarly stable. Since tissue concentrations ofembalming compounds in aortas from embalmedbodies must have been a great deal lower than inthis experiment, one may be quite confident thatembalming and subsequent storage of tissues didnot produce changes affecting these analyses.Because not all bodies were embaLmed and be-cause embalming might have been incomplete,a similar experiment was done with minced tissuesuspended in saline. Here, too, there was nochange during storage for 8 weeks.

Degree of hydrolysis of lipids during storagewas evaluated in a separate experiment, in whichaliquots of diced aortic tissue were extractedwith acidified isopropanol-heptane, and the ex-tracted free fatty acid (FFA) was titrated withsodium hydroxide. Samples extracted immediate-ly contained 3.28 ,uEq. of FFA per gram of fat-free tissue. After 1 week in embalming fluid at-20 C. the FFA content had increased by 58 percent, and after 1 week in saline at -20 C. it hadincreased by 74 per cent. The FFA fractions thusinclude significant contributions from hydrolysis,

t Composition of the embalming fluid is sodiumcarbonate monohydrate 16 Gm., sodiurn borate 53Gm., formaldehyde (37 per cent) 200 ml., diethyleneglycol 118 ml., eosin Y 0.16 Gm., Aquarome Special5.3 ml., Igepon 1.7 ml., water to make 1 litr.

Czrculation, Volume XXXII, December 1 65

*The -numbers- of cases in this report bear norelationship t-o- the relative mortality rates of the twogroups.

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including an uncertain amount of hydrolysis priorto autopsy. However, FFA of atheroma samplesaveraged only 2 per cent of total lipid, indicatingthat hydrolysis would have had no importantinfluence on the data for major esterified lipidfractions.

Coronary atheroma was freed from underlyingmedia and adventitia. The coronary atheromasample for each subject consisted of a pool of allatheromatous material that could be dissectedfrom all the coronary arteries in this manner. Aor-tas were stripped free from adherent fat andadventitia, then divided lengthwise into two partsof roughly equal size. One half of each samplewas used, without further dissection, for quantita-tion of major lipids and of calcium in wholeaorta. The other half was used as a source ofisolated atheromata, which were analyzed sepa-rately. Aortic atheromata were dissected througha plane just beneath the lesions and then trimmedcarefully to remove all surrounding uninvolvedtissue. Aortic lesions were sorted into two poolsof tissue, one consisting of uncomplicated andthe other of complicated atheromata. The latterincluded all lesions displaying gross calcification,ulceration, thrombosis, or aneurysm formation.Tissues were stored at -20 C. until extracted.Serum samples from fasting blood, obtained

from all subjects at 4-month intervals, were placedinto ampoules, gassed with pre-purified nitrogen,and sealed. Foaming during gassing was controlledby addition of Polyglycol (Dow Chemical Co.).Sera were stored at -20 C.

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Chemical ProceduresTissues were dried to constant weight in vacuo

at 60 C. and reduced to a fine mince for lipidextraction. Serum and tissue lipids were extractedby the Folch procedure.3 Cholesterol,4 triglyc-eride,5 and lipid phosphorus6 were determinedcolorimetrically on the extract of each undis-sected half-aorta. Lipids of serum samples andatheromata were fractionated by column chroma-tography on silicic acid.* Hydrocarbon was elutedwith petroleum ether. Sterol esters, triglycerides,and free sterol were eluted with, respectively, 1,12, and 40 per cent diethyl ether in petroleumether, monoglycerides (?) with chloroform, andphosphatides with methanol. Free fatty acids were

separated from the triglyceride fraction either bythin-layer chromatography on silica gel G, using

*J. T. Baker silicic acid was used after removalof fine particles by suspension in methanol. Thesilicic acid was slurried into 2-3 volumes of methanol.The mixture was allowed to stand 20 minutes andthe methanol was siphoned off. After three suchtreatments, the sedimented silicic acid consistedmostly of 80-100 mesh material.

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DAYTON ET AL.

as developing solvent 83 per cent petroleumether (B.P. 60-90 C.), 16 per cent diethyl ether,and 1 per cent acetic acid,7 or by columnchromatography on Florisil.8 Sterol esters andphosphatides were saponified overnight at roomtemperature with 2 per cent sodium hydroxide in95 per cent ethanol. Benzene was added whennecessary to aid in dissolution of these lipids.After saponification, an equal volume of waterwas added, and nonsaponifiable material was ex-tracted with petroleum ether. The aqueous etha-nol solution was acidified with hydrochloric acid,and fatty acids were extracted with petroleumether. Fatty acids and triglycerides were methyl-ated with boron trifluoride methanol.9 Methylesters were analyzed on a Barber-Coleman Model-10 gas-liquid chromatograph equipped with an

ionization chamber detector, with tritium foil as

the radiation source. Stationary phase was 12per cent ethylene glycol succinate polyester andthe carrier gas was argon. A single sample sizewas used, suitable for the major components, andcurrent amplification was increased 10-fold forcomponents emerging after methyl arachidonate.No effort was made to quantitate trace peaks or

components that did not appear on the singlechromatogram. Further information concerningchromatographic conditions and reproducibilityof results has been published.10 Reproducibilityof the gas-liquid chromatographic (GLC) de-termination of linoleic acid, not reported inthe reference just cited, was evaluated by repli-cate analysis of a mixture of methyl esters on

11 separate days. Mean and standard deviationfor linoleic acid were 34.1 + 1.5 per cent of thetotal fatty acid.For calcium determination, dried, lipid-free

aortic pieces were ground with a mortar andpestle. One to five grams of this material were

digested by wet combustion with nitric and per-chloric acids.'" Calcium was precipitated as theoxalate at pH 3.5,12 and converted to the car-

bonate in a muffle furnace at 475-500 C. Calciumwas determined by titration with EDTA usingCalcon, 1- (2-hydroxy-1-naphthylazo) -2-naphthol-4-sufonic acid, as an indicator.13

Results

Except as noted, results of all analysesshowed no significant correlation with ad-herence or with period of time on the study.For this reason, data for each diet group are

pooled and presented, for the most part, as

means and standard deviations.Results of analyses of the undissected half-

aortas are shown in tablle 3. Variances of allcomponents were large, in keeping with what

might be expected in subjects exhibiting dif-ferent degrees of atherosclerosis. There was nodifference between the two diet groups withregard to total lipid and the major lipidfractions. The two-fold difference in meanaortic calcium levels was not statistically sig-nificant (O.O5<p<O.10 by both t test and ranksum test) .

Concentrations of major lipid componentsin isolated atheromata are shown in table 4.These figures are derived from gravimetricdata on the eluted fractions from silicic acidchromatography. Since isolated atheroma morenearly reflects a single tissue type than doesintact aorta (table 3), variances are smaller.Phosphatide consistently accounts for aboutone fourth of atheroma lipid. Sterol ester frac-tions are slightly larger than free sterol. Sincethese are gravimetric data and thus includethe fatty acid portion (about 40 per cent) ofthe sterol ester, there is more unesterified thanesterified sterol in all types of lesion.

Aortic atheroma, whether complicated or un-complicated, contains much less triglyceridethan does coronary atheroma. Aortic plaquesare richer than coronary lesions in cholesterolester and, less consistently, in unesterifiedcholesterol. Composition of aortic atheroma isstrikingly independent of whether the lesionsare uncomplicated or complicated.There are no significant differences between

the control and experimental groups with re-gard to concentrations of major fractions as

Table 3Lipid and Calcium Concentrations of Intact Half-aortas

Total lipidTotal cholesterolTriglyceridePhosphatideCalcium

Concentration,per cent of dry aortic weightControl Experimental

(14 cases) (10 cases)

9.4+3.4 8.8+4.14.0+1.2 3.6+2.41.4±0.7 1.6+0.81.9t0.7 1.7 +0.86.4 + 5.0* 3.1 + 2.3t

Data are given as mean : standard deviation.*Eleven cases. Includes one sample with aneurysm,

containing 7.8 per cent calcium.tNine cases. Includes one samuple witb anieurysmrt,

containing 1.4 per cent caleium.Circulation, Volume XXXII, December 1965

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determined by silicic acid chromatography. Asin the case of the half-aorta samples, concen-

trations of individual fractions of atheromalipid did not correlate with time in the studyprior to death nor with adherence.As indicated by the footnote in table 4, one

control subject was omitted from the tabula-tion of data from complicated aortic atherom-ata. The complicated aortic atheroma sampleincluded material from a fusiform abdominalaneurysm whose surface was covered by or-

ganized thrombus. Fortuitously, two atheromasamples from this aorta were submitted to thelaboratory and analyzed separately. The rep-

licate results were sterol ester 0.8 and 0 per

cent; triglyceride 1.6 and 0.4 per cent; freecholesterol 23.2 and 20.5 per cent; phosphatide69.4 and 77.0 per cent. Analysis of this subject'shalf-aorta likewise yielded the lowest totalsterol (24 per cent of total lipid) and thehighest phosphatide (31.5 per cent of totallipid) encountered in the group. It is plainthat these unique results do not reflect labora-tory error, and it is also apparent that this is,chemically at least, a different type of lesionfrom those encountered in the rest of the group.

It seems probable that these samples were

dominated by platelet lipid of the thrombusrather than by atheroma lipid. Yet the fattyacids of these fractions were not noticeablydifferent from those of other samples in thisgroup. The remainder of the samples studiedincluded three other aortic aneurysms, one

syphilitic and two arteriosclerotic, one of thesecontaining gross mural thrombus. None ofthese appeared chemically distinguishable fromthe other samples, and they are included in thedata analyses. However, it is probable thatinclusion of varying amounts of thrombus ac-

counts for some of the variation in results ofsilicic acid fractionation.

Tables 5 through 7 summarize the results ofGLC analysis of fatty acids in the major es-

terified lipid fractions of the atheromata. Cho-lesterol ester is dominated by oleate (18:1)and linoleate (18:2) at all sites and in bothdiet groups. However, coronary atheromataconsistently contained more cholesteryl oleateand less linoleate than did aortic atheromata

915



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of the same individuals. These data, withstatistical evaluation based on paired values,are summarized in figure 2.

In atheroma triglyceride (table 6), oleicacid (18:1) accounts for nearly half of thefatty acid, with palmitate (16:0) and linoleate(18:2) constituting most of the remainder. Thedifference between coronary and aortic lesionsin regard to triglyceride 18:1 and 18:2 is quitesmall, as compared with cholesterol ester, andis not statistically significant.GLC analyses of phosphatide are shown in

table 7. The dominant fatty acids are palmitate(16:0), stearate (18:0), and oleate (18:1).Here, too, there is a suggestion that coronaryatheroma contains more oleate and less lino-leate than do aortic lesions, but analysis aspaired observations fails to reveal significantdifferences.The major influence of the experimental diet

upon atheroma composition was an increase inmean linoleate content of all fractions. Thiseffect is seen best, together with statisticaldata, in figure 3. Increased linoleate contentwas most pronounced, and most consistent, intriglyceride, but similar changes of lesser de-gree are apparent in other fractions as well.The rise in linoleate content occurred largelyat the expense of oleate, and to a lesser extentat the expense of saturated fatty acids andpalmitoleate (table 5 through 7). It is ofinterest that the changes in complicated ath-eroma were at least as great as in uncom-plicated aortic atheroma and coronary ather-omata (fig. 3).

Linoleate content of these various fractionsin subjects on experimental diet displayed littlecorrelation with either length of time on dietor with adherence. The only significant excep-tion was the relationship between adherenceand cholesteryl linoleate of uncomplicatedplaques (r = 0.78; p < 0.05). However, therewas a distinct relaitionship to the linoleate con-centration in corresponding serum lipid frac-tions. In most but not all of the subjects therewas available a serum sample obtained withinthe 4-month period immediately precedingdeath. Figure 4 contains plots of serum choles-terol ester linoleate against atheroma choles-


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terol ester linoleate, and of serum triglyceridelinoleate against atheroma triglyceride linole-ate, for the three different types of atheroma.Correlation coefficients, shown in figure 4, arepositive in all instances, and in all but one ofthe comparisons (cholesterol ester in uncom-plicated aortic atheroma) the correlations arestatistically significant. In the case of phospha-tide (not shown in figure 4), corresponding

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Comparison of coronary and aortic atheromata tvithregard to content of cholesteryl oleate (18:1) andlinoleate (18:2). P values were calculated by t testapplied to paired observations; the asterisks refer tocomparison of the two adjacent values. In no instancewas there a significant difference between uncom-

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correlation coefficients ranged from +0.46 to+0.59, but with p > 0.05 in each case.

Discussion

The analytical data of greatest interest in astudy of this type are those which might yieldevidence that atherosclerosis had progressedat different rates in the two groups of partici-pants. From this point of view, total lipid con-centration of intact arterial tissue (table 3) istheoretically of considerable importance. Un-fortunately, the likelihood of getting usefulaffirmative information from this source isextremely small. Because the total lipid con-tent of an individual's aorta on entry into thestudy is unknown, one is limited to a simplecomparison of the terminal values for the con-trol and experimental subjects. Because vari-ances in the terminal total lipid concentrationsare very large (table 3), statistical sig-nificance can be established only if the meansof the two groups differ greatly or if data areavailable on very large numbers of subjects.The comparison is further hampered by the factthat data are derived not from a random sam-ple of each dietary group but rather from menwho died during the period of follow-up, afact which would almost certainly bias thevalues in both groups of subjects. For all thesereasons, it seems unlikely that prolonged ac-cumulation of data such as those in table 3will provide substantial evidence concerningpossible effectiveness of the experimental diet.The values for total aortic lipid and for the

major subfractions reported in table 3 arecomparable to Bottcher's data on "Stage II"atherosclerotic aortaiS and to the figures re-ported by Mead and Gouze for aortas ofgrades 1 through 4.1i The analyses of dis-sected atheromata by silicic acid chromatog-raphy (table 4) show strikingly little differ-ence between complicated and uncomplicatedaortic lesions. Comparison with published dataon comparable material reveals a larger per-centage of phosphatide than reported bySmith for aortic atheromata at various stages'7or by Luddy et al.'8 The observation thatcoronary atheroma contains more triglycerideand less sterol than does aortic atheroma con-

Circulalion, Volume XXXII, December 1965

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C.E. CON. ]

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LINOLEIC ACID %

Figure 3Comparison of atheromata of control and experimental subjects in regard to linoleic acid con-tent of various lipid fractions (left, uncomplicated aortic atheroma; center, complicatedaortic atheroma; right, coronary atheroma). C.E., cholesterol ester; T.G., triglyceride; P.L.,phosphatide. *P < 0.05, **p < 0.01, ***p < 0.001-for comparison of control and experimentalgroups (by t test).

AORTICUNCOMPL ICATED

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Figure 4

Relationship of atheroma cholesterol ester linoleic acid and triglyceride linoleic acid to thelinoleic acid concentration in corresponding fractions of the last antemortem serum samples insubjects on experimental diet.

Cerculation, Volume XXXII, December 1965

CORONARY

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DAYTON ET AL.

firms a similar finding by Bottcher et al.19 ob-tained on less extensively dissected tissue. Themetabolic basis for this difference is not at allapparent from available information. A num-ber of possibilities can be proposed, includingdifferential uptake of different lipid classesfrom plasma, differences in amounts of sterolor triglyceride contributed by local synthesiswithin the arterial wall, or quantitative differ-ences in mechanisms for disposing of steroland triglyceride.

Fatty acid compositions of cholesterol esterand triglyceride found in atheromata of con-trol subjects of the present study were in gen-eral similar to data reported by previous in-

CONTROL

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vestigators.16' 19-21 However, we found onlytraces of compounds with 22 and 24 carbonatoms in phosphatide, in contrast to the ma-terial of Bottcher et al.,19 which containedabout 15 per cent of these higher acids in"Stage 11/111" lesions from aortas and coronaryarteries. Like the latter authors, we found asmall free fatty acid fraction in atheromata;but as indicated under Methods, a significantpart of this was due to postmortem hydrolysisof esterified lipid.

Since animal tissues are not known to syn-thesize linoleic acid (18:2), the presence ofincreased amounts of this compound in ath-eromata of men on the experimental diet very

EXPERIMENTAL

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Figure 5Ratio of linoleic acid in atheroma lipid fractions to linoleic acid in correspond-ing fractions of the last antemortem serum samples, plotted against time on

experimental diet. C.E., cholesterol ester; T.G., triglyceride; P.L., phosphatide.


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likely represents the result of exchange of lipidsbetween atheroma and plasma. As in the caseof experiments involving use of isotopicallylabeled lipids,22 it is difficult to be entirelycertain that rising linoleic acid concentrationis a result of a physiologic process as opposedto a passive physicochemical exchange be-tween tissue lipid and the lipoprotein lipid ofplasma. An alternative possibility-simple ac-cretion of new, unsaturated atheroma on topof old-might have contributed to the incre-ment in linoleic acid. However, it seems un-likely that this is a major reason for the rise in18:2. To account for the increase in triglyc-eride linoleate by this mechanism, one wouldhave to postulate that atheroma lipid masshad increased by 55 to 80 per cent in less than3 years. A rise of this magnitude would (iflimited to the group on experimental diet) beapparent in the total lipid data of table 3. Anestimate of the mean rate of increase in aorticlipid in "normal" men at this age, based onmeasurements of cholesterol concentration,23 is6 per cent per year. Additional evidence thatlarge-scale accretion of lipid did not occur isprovided by the fact (see below and fig. 5)that there was no progressive rise in ratio ofatheroma linoleic acid to plasma linoleic acidover the period from 495 days to 1,351 days. Itis striking that the changes in complicatedaorta atheroma were at least as great as in thesmaller and presumably younger lesions. Thisdoes not imply, however, that the changes oc-curred at the same rate in both types of ath-eroma.

Linoleic acid concentrations of atheromatriglyceride and cholesterol esters in the ex-perimental subjects were clearly related tothe corresponding linoleic acid concentrationsof the last antemortem sera (fig. 4). Consider-ing that the single serum sample may well havebeen unrepresentative of the situation over theentire period of the study, the correlations ap-pear satisfactorily clear, but they do not neces-sarily indicate a linear relationship. Phospha-tide also shows a positive correlation, but notat a statistically significant level. Atheroma lin-oleic acid showed surprisingly little correla-tion with the individual's record of adherenceCirculation, Volume XXXII, December 1965

or with time on the experimental diet prior todeath. (This observation is in contrast to thesituation in adipose tissue, in which linoleicacid concentrations were still rising at 5years.*) Lack of correlation with time on thediet appears (see below and fig. 5) to be duesimply to the fact that the entire rise in linoleicacid concentration occurred prior to the deathof the first man in this group at 495 days (ex-cept for cholesterol ester in uncomplicatedaortic atheroma). A number of factors verylikely contribute to the lack of correlation withadherence: the over-all adherence figure maynot be representative of the few months im-mediately before death, which would havethe greatest influence on atheroma composi-tion; nonadherence might in part reflect mealsmissed completely rather than deviation fromthe diet; the relationship, if any, would be ob-scured by differences in linoleic acid concen-trations of both serum and atheroma lipids atthe start of the study (cf. variances in controldata of tables 5 to 7); and there is appreciableexperimental error in the linoleic acid deter-minations.

Plots of linoleic acid concentrations of ath-eroma lipid fractions against time in the studyfor the experimental group show no consistentpatterns, presumably because of considerablevariations in initial values. Plots of the ratios oflinoleic acid content in atheroma lipid frac-tions to the concentrations in correspondingserum fractions are a good deal more informa-tive and are shown in figure 5.The ratios for the experimental subjects show

no rise over the period from 495 to 1,351days, with the possible exception of cholesterolester during the very first interval. The data ofGunning et al.24 indicate that serum cholesterolester would have reached a new, constantcontent of linoleic acid during the first monthof the study. The same is true of erythrocytephosphatide25 and therefore, presumably, ofplasma phosphatide as well. Thus the rela-tively constant ratio implies a constantconcentration in the cholesterol ester and phos-phatide of atheromata. If one views the

*Unpublished data.

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enhanced linoleic acid concentrations in ath-eromata of the experimental group as duelargely to exchange with serum, this constantlevel suggests that the various fatty acid poolswould have turned over several times duringthe first 2 years. While we cannot estimate half-times from these data, it appears that they areprobably less than 1 year. A longer figure wasestimated by Farquhar et al.26 in an abstractdescribing atheroma analyses in five men oncorn oil formulas. Their data apparently indi-cated atheroma lipid turnover rates compara-ble to those of stored adipose tissue fat which,as reported by Hirsch et al.,2 has a half-timeof I to 2 years.*

Since there exists no direct information con-cerning fatty acid kinetics of individual lipidfractions in human atheromata, it seems worth-while to seek some kinetic inferences in thedata of the present study. Figure 5 offers someclues. In all fractions, linoleic acid concentra-tion stabilizes at a level below the corre-sponding serum level. One might postulatepreferential uptake of other fatty acids andtheir esters, or particularly rapid degradationor "excretion" of linoleic acid by the atheroma.However, a number of facts appear to suggestthat we are observing mainly the results of di-lution by locally synthesized acids. Some dilu-tion of this type must occur, since fatty acidsynthesis occurs in atheromata28 and since fattyacid synthesized in atheromata would be lino-leic-free. Furthermore, the ratios seen in figure5 for cholesterol ester, triglyceride, and phos-phatide are rather similar, suggesting thatthese ratios probably originate from a processcommon to all fractions. If we assume thatdilution by fatty acids synthesized within theartery wall represents the major reason forthe difference between linoleic acid concentra-tions of atheroma and plasma, then the ra-tio of the concentration in plasma to thatin atheroma, when both are at plateau levels,constitutes an estimate of the fraction ofatheroma fatty acyl moieties derived from

*A similar figure is estimated from analyses of adi-pose tissue obtained in the present study (unpub-lished data).

plasma.* It thus appears (fig. 5) that plas-ma is the source of 70 to 90 per cent of thecholesterol ester fatty acid in the control groupand about 65 per cent in the experimentalgroup. It also appears that the percentage ishigher in uncomplicated aortic plaques of thecontrol subjects than in any other group oflesions, and that the experimental diet induceda small but significant decrease, particularly inuncomplicated plaques,t in the percentage ofcholesterol ester fatty acid derived from plas-ma. It is possible that these observations arerelated to the fact, reported from this labora-tory, that normal rat aorta takes up labeledcholesterol from plasma more rapidly in ani-mals on diets containing saturated fat (coconutoil) than in animals on unsaturated fat (saf-flower oil).30 However, these animal experi-ments involved tracing cholesterol, and theresults do not necessarily apply to the fattyacyl groups of the cholesterol esters.

Percentage of atheroma triglyceride fattyacid derived from plasma appears similar tothat for cholesterol ester in the experimental

*In view of reports of lipid peroxides in athero-mata,29 it is necessary to consider the possibility that";autoxidation" of linoleic acid accounts for its lowerconcentration in atheroma lipid. To evaluate thispossibility, ratios of atheroma arachidonic acid toserum arachidonic acid were calculated from the dataof the sterol ester and phosphatide fractions. Al-though the variances are relatively large, owing inpart to poor precision in estimating this component,the mean ratios were similar to those for linoleicacid. The arachidonic acid ratios, given as mean +standard deviation, are

Aorta, uncomplicatedAorta, complicatedCoronary

Sterol ester.68 ± .25.63 ± .35.56 + .24

Phosphatide.57 ± .29.47 ± .36.65 ± .42

If oxidative destruction were responsible for signifi-cant lowering of linoleic acid concentrations inatheromata, one would expect evidence of drasticlosses of arachidonic acid, which is a great dealmore susceptible to nonenzymatic oxidation. Theseconsiderations do not, of course, controvert the re-ported presence of lipid peroxides in atheromata northe possibility that they may have some pathogeneticimportance.tEven omitting the two values in the experimental

group at 495 and 666 days, p < 0.01 by t test.


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group (fig. 5). Despite poor agreement amongthe triglyceride values for the control subjects,the ratio appears closer to unity than in theexperimental group. The ratio for phosphatidefatty acid is somewhat lower, about 0.5 forboith groups. Interpretation of this value ismore difficult than in the case of cholesterolester and triglyceride, since phosphatide is amore distinctly heterogeneous group of com-pounds for which assumptions of indiscrimi-nate uptake from plasma and indiscriminatedisposal are of dubious validity. B6ttcher andvan Gent3' have reported that atheroma phos-phatide contains considerable sphingomyelin,which is almost free of linoleic acid. This frac-tion, vwhether derived from plasma or fromlocal synthesis, could account for occurrenceof a lower linoleic acid content in atheromaphosphatide as compared with plasma phos-phatide.

Summary

Detailed chemical analyses have been car-ried out on aorta, and on coronary and aorticatheromata, of men who died during a studyof prolonged use of diets rich in unsaturatedfat and of control subjects. Concentrations oftotal aortic lipid and of total aortic calciumwere not significantly different for the twogroups of subjects. Concentrations of choles-terol and cholesterol esters, triglyceride, andphosphatide showed no difference betweenthe two groups in analyses of aorta, uncom-plicated aortic atheromata, complicated aorticatheromata, and coronary atheromata. Coro-nary atheroma lipid contained substantiallymore triglyceride than did aortic atheromalipid, whether derived from complicated oruncomplicated plaques.Atheroma triglyceride in experimental sub-

jects contained more linoleic acid than in con-trol subjects, in all types of plaques. Lesserbut significant increases in linoleic acid wereseen in cholesterol ester and phosphatide ofcoronary atheromata and complicated aorticatheromata. In the case of uncomplicated aor-tic atheroma, changes in linoleic acid of cho-lesterol ester and phosphatide were smallerand not significant. In both groups of sub-Circulation, Volume XXXII, December 1965

jects, coronary atheroma contained more cho-lesteryl oleate and less linoleate than did aorticatheroma.

Linoleic acid contents of atheroma lipidfractions were positively correlated with thelinoleic acid figures for corresponding fractionsof the most recent antemortem serum samples.Ratio of linoleic acid in an atheroma lipidfraction to the linoleic acid content of the cor-responding serum fraction was, in most cases,constant after the time of the first sample, ob-tained after 495 days on experimental diet. Itis postulated that this ratio constitutes an esti-mate of the fraction of atheroma fatty acidderived from plasma. In cholesterol ester andtriglyceride, the ratio was about 0.65 in experi-mental subjects, higher in subjects on the con-trol diet. The ratio for phosphatide was about0.5 in both groups of subjects.

AcknowledgmentWe are grateful to Kathryn Barton, David Fisher,

Natalie Fisher, Paul Hanlon, Alvin Harry, RebeccaHeld, Richard Johnston, Loretta Karolewics, WandaMcElroy, Diane Minasian, Thomas Payne, BarbaraSingleton, Robert Stewart, Evelynn Warech, andPatricia Yelenosky for carrying out the analytical pro-cedures; to Dr. James Mead for helpful suggestionsconcerning the manuscript; to Mary Brock for sta-tistical calculations; and to Carolyn Struble for typingthe manuscript. Dietetic aspects of the program havebeen supervised by Elva Hiscock. Contributions offood were received from Corn Products Company,Edgemar Farms, Frozen Desserts Company, NationalSoybean Processors' Association, and Pitman-MooreCompany.

References1. DAYTON, S., PEARCE, M. L., HASHIMOTO, S.,

FAKLER, L. J., HISCOCK, E., AND DIXON, W.J.: A controlled clinical trial of a diet high inunsaturated fat. New England J. Med. 266:1017, 1962.

2. HisCOCK, E., DAYTON, S., PEARCE, M. L., ANDHASHIMOTO, S.: A palatable diet high in un-saturated fat. J. Am. Dietetic A. 40: 427, 1962.

3. FOLCH, J., LEES, M., AND STANLEY, G. H. S.:Simple method for isolation and purificationof total lipids from animal tissues. J. Biol.Chem. 226: 497, 1957.

4. SPERRY, W. M., AND WEBB, M.: A revision ofthe Schoenheimer-Sperry method for choles-terol determination. J. Biol. Chem. 187: 97,1950.

5. NEWMAN, H. A. I., LiU, C., AND ZILVERSMIT,

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D. B.: Evidence for the physiological occur-rence of lysolecithin in rat plasma. J. LipidRes. 2: 403, 1961.

6. CHEN, P. S., JR., TORIBARA, T. Y., AND WARNER,H.: Microdetermination of phosphorus. Anal.Chem. 28: 1756, 1956.

7. RoHEIM, P. S., HAFT, D. E., GIDEZ, L. I., WHITE,A., AND EDER, H. A.: Plasma lipoprotein me-tabolism in perfused rat livers. II. Transferof free and esterified cholesterol into the plas-ma. J. Clin. Invest. 42: 1277, 1963.

8. CARROLL, K. K.: Separation of lipid classes bychromatography on florisil. J. Lipid Res. 2:135, 1961.

9. METCALFE, L. D., AND SCHMITZ, A. A.: Therapid preparation of fatty acid esters for gaschromatographic analysis. Anal. Chem. 33:363, 1961.

10. TOMPKINS, M. J., DAYTON, S., AND PEARCE, M.L.: Effect of long-term feeding of various fatson whole blood clotting times in men. J.Lab. & Clin. Med. 64: 763, 1964.

11. BUELL, M.: A method for ashing soft tissuespreliminary to the determination of cations. J.Biol. Chem. 130: 357, 1939.

12. DELUCA, H. A.: Photometric microdeterminationof calcium. Canad. J. Res. 25: 449, 1947.

13. GOLBY, R. L., HILDEBRAND, G. P., AND REILLY,C. N.: Direct titration of calcium in bloodserum. J. Lab. & Clin. Med. 50: 498, 1957.

14. DIXON, W. J., AND MASSEY, F. J., JR.: Introduc-tion to Statistical Analysis, Ed. 2. New York,McGraw-Hill Book Co., Inc., 1957. p. 124.

15. B6TTCHER, C. J. F., WOODFORD, F. P., TER HAARROMENY, C., BOELSMA, E., AND VAN GENT,C.: Composition of lipids isolated from theaorta, coronary arteries and circulus Willisiiof atherosclerotic individuals. Nature 183: 47,1959.

16. MEAD, J. F., AND GOUZE, M. L.: Alterations inaorta lipids with advancing atherosclerosis.Proc. Soc. Exper. Biol. & Med. 106: 4, 1961.

17. SMITH, E. B.: Intimal and medial lipids inhuman aortas. Lancet 1: 799, 1960.

18. LUDDY, F. E., BARFORD, R. A., AND RIEMEN-SCHNEIDER, R. W.: Fatty acid composition ofcomponent lipides from human plasma andatheromas. J. Biol. Chem. 232: 843, 1958.

19. B6TTCHER, C. J. F., BOELSMA-VAN HOUTE, E.,TER HAAR ROMENY-WACHTER, C., WOOD-FORD, F. P., AND VAN GENT, C. M.: Lipid andfatty-acid composition of coronary and cere-bral arteries at different stages of atherosclero-sis. Lancet 2: 1162, 1960.

20. TUNA, N., RECKERS, L., AND FRANTZ, I. D., JR.:The fatty acids of total lipids and cholesterolesters from normal plasma and atheromatousplaques. J. Clin. Invest. 37: 1153, 1958.

21. SWELL, L., FIELD, H., JR., SCHOOLS, P. E., JR.,AND TREADWELL, C. R.: Fatty acid composi-tion of tissue cholesterol esters in elderlyhumans with atherosclerosis. Proc. Soc. Exper.Biol. & Med. 103: 651, 1960.

22. NEWMAN, H. A. I., AND ZILVERSMIT, D. B.:Quantitative aspects of cholesterol flux in rab-bit atheromatous lesions. J. Biol. Chem. 237:2078, 1962.

23. FABER, M., AND LUND, F.: The human aorta;influence of obesity on the development ofarteriosclerosis in the human aorta. Arch. Path.48: 351, 1949.

24. GUNNING, B., MICHAELS, G., NEUMANN, L.,SPLITTER, S., AND KINSELL, L.: Effects of a

diet high in polyunsaturated fat on the plasmalipids of normal young females. J. Nutrition79: 85, 1963.

25. FARQUHAR, J. W., AND ABHENS, E. H., JR.:Effects of dietary fats on human erythrocytefatty acid patterns. J. Clin. Invest. 42: 675,1963.

26. FARQUHAR, J. W., HIRSCH, R. L., AND AHRENS,E. H., JR.: Evidence that atheroma fatty acidsare in flux. J. Clin. Invest. 39: 984, 1960.

27. HIRSCH, J., FARQUHAR, J. W., AHRENS, E. H.,JR., PETERSON, M. L., AND STOFFEL, W.:Studies of adipose tissue in man: Microtech-nic for sampling and analysis. Am. J. Clin.Nutrition 8: 499, 1960.

28. NEWMAN, H. A. I., MCCANDLESS, E. L., AND

ZILVERSMIT, D. B.: The synthesis of C14-lipids in rabbit atheromatous lesions. J. Biol.Chem. 236: 1265, 1961.

29. GLAVIND, J., HARTMANN, S., CLEMMESEN, J.,JESSEN, K., AND DAM, H.: Studies on the roleof lipoperoxides in human pathology. II. Thepresence of peroxidized lipids in the athero-sclerotic aorta. Acta pathol. microbiol. scand.30: 1, 1952.

30. DAYTON, S., HASHIMOTO, S., AND JESSAMY, J.:Cholesterol kinetics in the normal rat aorta,and the influence of different types of dietaryfat. J. Atheroscler. Res. 1: 444, 1961.

31. BOTTCHER, C. J. F., AND VAN GENT, C. M.:Changes in the composition of phospholipidsand of phospholipid fatty acids associated withatherosclerosis in the human aortic wall. J.Atheroseler. Res. 1: 36, 1961.

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SEYMOUR DAYTON, SAM HASHIMOTO and MORTON LEE PEARCEand Atheromata in Man

Influence of a Diet High in Unsaturated Fat upon Composition of Arterial Tissue

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1965 American Heart Association, Inc. All rights reserved.

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