Quantitative Effects of Dietary Fat on

Serum Cholesterol in Man

D. M. HEGSTED, PH.D.,* R. B. MCGANDY, M.D.,t I. L. MYERS, S.M.t AND F. J. STARE, M.D.

LTHOUGH many studies demonstrate that of dietary fat were not assessed. More re-
changes in the serum cholesterol level cently Gunning et al.2 suggested that the
may be induced by modifying the amount or square root of the iodine number rather than
composition of dietary fat, the causal factors the iodine number itself is inversely related to
in oils and fats have not been clearly identified. the level of serum cholesterol.
T\4ajor emphasis has been placed upon the (2) Keys et al.3 in the most comprehensive

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polyunsaturated fatty acids, both in the studies yet reported fed groups of men diets
scientific and the popular press. Not all the containing various kinds and amounts of fats.
evidence, however, supports this presumed From changes in serum cholesterol induced
primary role of polyunsaturated fatty acids with the various diets, the multiple regression
nor will the conclusions of this paper. equation
The principal conclusions regarding the re-
. cholesterol = 2.76 S +
lationship between various fat components and
.03 M - 1.33 L P - 1.68
the level of serum cholesterol may be sum-
marized as follows: was derived in which S, M, and P
(1) Ahrens et al) demonstrated a rough in- are the changes in the per cent of dietary
verse relationship between the serum choles- calories derived from saturated, mono- and
terol level and the iodine number (total degree polyunsaturated fatty acids. Since the co-
of unsaturation) of various oils. This con- efficient of M and the constant 1.68 were not
clusion implies that monounsaturated acids are statistically significant these were eliminated
half as effective as the diene, linoleic acid, the and the predictive equation became
primary polyunsaturated acid in vegetable oils. . cholesterol = 2.74 S - 1.31 P
The data seem to demonstrate that minimal
levels of serum cholesterol were achieved with These workers concluded that the equation

oils with an iodine number of about 100, and demonstrated that “saturated fatty acids have,

that more highly unsaturated oils were not per gramme, about twice as much effect on

more effective. Since all fats were fed at the serum cholesterol as do the poly-ethenoids

same fat level, the effects of varying the amount which, moreover, act in the opposite direction.
Whether the mono-ethenoids (essentially oleic
From the I)epartment of Nutrition, Harvard School acid here) have any effect is uncertain, but
of Public Health, Boston, Massachusetts.
if there is an effect it is certainly small.” In
* Professor of Nutrition; t Assistant Professor of
later reports, concentrates of oleic acid were
Nutrition; Instructor in Nutrition, § Professor of
Nutrition and Chairman, Department of Nutrition. fed and the apparent lack of an effect was con-
This study was supported in part by grants-in-aid firmed.4 Modifications in the predictive equa-
from the Jots,, A. Hartford Memorial Fund, the Special tion to account for differences in response in
Dairy Industry Board, the U. S. Public Health Service
various degrees of hypercholesterolemia were
(5-KG-AM 18,455 and AM-06245), the Nutrition Foun-
also introduced.5
dation, Inc., an(l the Fund for Research and Teaching,
Department of Nutrition, Harvard School of Public (3) Kinsell et al.6 emphasized the role of the
Health. polyunsaturated or essential fatty acids al-

.1 ,neruan Journal of Qinfral Nutrition 281 Vol. 17, November 1965

282 Hegsted, McCandy, Myers and Stare

TABLE I mandatory, in view of the implications for

Food Pattern of 2,600 Calorie Diet
public health and the economic impact on the
food industries, that the changes made be those
Food gm/day
which are most effective. This cannot be
accomplished until the causal factors are clearly
Fruits, vegetables, potatoes 730
Eggs 56 defined and their effects quantitatively assessed.
Lean meat, fish, cheese 204 The data to be presented consist of the re-
Skim milk powder 71
sponses in serum cholesterol of groups of men,
Bread 49
Other cereals 125 ten men per group, who were fed a low fat diet
Flour 54 to which various test fats were added. In some
Honey, syrup, jelly 27
test periods, the level of dietary cholesterol was
Sugar 62
Soups 88 also varied. The data have been analyzed to
Test oil 93 determine the serum cholesterol response to
various dietary fatty acids and to dietary
cholesterol. An unusually strong feature of
though quantitative data have not been ob- these studies is that the comparisons have been

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tamed relating response to dose. Ahrens and made with the same men throughout the study.
his associates7 have demonstrated a cholesterol
MATERIALS AND METHODS
lowering effect of the fish oils containing large
The diets used in these studies were based upon a
amounts of polyunsaturated acids which have
mixture of low fat foods which were calculated to
little essential fatty acid activity for the rat,
supply a minimal amount of fat, to provide 15 to 16
but Keys et al.8 found that highly unsaturated
per cent of the calories as protein, to provide a rela-
fish oils do not appear to be more hypo-
tively constant daily level of cholesterol intake and
cholesterolemic than oils containing primarily to allow menu planning that would represent a
linoleic acid. Moreover the work of McOsker variety of foods which could be easily standardized
et al.9 and Erickson et al.,’#{176}in which oils sub- and reproduced. To this pattern of foods, the oils
jected to varying degrees of hydrogenation were to be tested were added at appropriate levels, and
studied, indicated no significant effect on serum additional calories as needed were supplied by carbo-
cholesterol even with substantial changes in the hydrate sources-starch, sugars, potatoes, cereals
and breads.
polyunsaturated fatty acid content.
The diets were designed to supply sufficient
(4) Although the role of dietary cholesterol
calories to maintain the weight of the subjects and
was generally discounted several years
to hold the percentage of calories supplied from fat
practically all recent investigations demon-
and protein approximately constant for all the men
strate an effect.’#{176}12’6
in any particular test. It was necessary, therefore,
(5) Numerous factors other than the dietary to devise diets yielding approximately 2,200, 2,600
fat are known to influence serum cholesterol and 3,000 calories per day. These levels were deter-
levels. These include the phytosterols,’7 pec- mined by measurement of the food eaten by the sub-
tins,18 the kind of dietary carbohydrate,’9 die- jects before the studies began, and by observing the
tary protein20 and probably other factors. It weights of the men throughout the experimental
should be recognized that the role such factors period. The foods used in the 2,600 calorie, 38 per

play may be partially dependent upon the com- cent fat level diet are listed in Table . Food por-
tions were proportionally increased or decreased to
position of the dietary fat or that the response
yield the other calorie and fat levels.
to dietary fat may be partially dependent upon
A seven day menu cycle was planned which was
factors such as those mentioned. In general,
repeated weekly for the entire experiment. Meats,
quantitative data indicating such relationships
the principal source of nontest fat, were carefully
are lacking. selected and trimmed and purchased in as large
Undoubtedly, the composition of the usual quantities as possible. The test oils were used pri-
diet in the United States is now being changed marily by incorporating them into recipes for many
by the emphasis upon the role of dietary fat in products such as waffles, muffins, cakes, cookies, pie
atherosclerotic heart disease. It would seem crust, biscuits, salad dressings and spreads for bread.
 Quantitative Effects of Dietary Fat 283

This necessitated considerable experimental work TABLE II

to obtain satisfactory products with certain oils. Composition of Foods in the 2,600 Calorie Diet
Without the Test Oil
Filled milk and ice creams, prepared from nonfat
milk and the appropriate oil, were used in all tests.
Data Calculation Analysis
Several weekly composites of the diets were pre-
pared during the study. These were homogenized Calories 1,907 ...

and aliquots extracted according to the A.O.A.C. Protein (gm.) 110 115
procedure.21 The total extract was taken to dry- Carbohydrates (gm.) 315 ...

nes , redissolved in ligroine and the weight of fat de- Fat 26 16.3
Saturated 9.1 5.9
termined. The 2,600 calorie diet plan as described
Monounsaturated 9.4 7.5
in Table I, but without the “test” fat, contained 5.7 2.9
Polyunsaturated
16.3 gin. of fat according to this procedure, whereas Cholesterol (mg.) 555 306
calculations from food tables indicated a content of
26 gm. (Table ii). Based upon the analytical value,
the diets finally developed contained approximately institution. The purposes of the study and the
22 and 38 per cent of the calories as fat rather than need for adherence to the diet were continually
the 25 and 40 per cent levels actually planned. The stressed to attendants and subjects, and failure to
fatty acid composition of the extract was determined comply meant removal from the study.

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by gas chromatography after methylation of the Three men were replaced after the third month
fatty acids.22 Cholesterol determinations23 on the and the group has been nearly constant since that
crude extract indicated 306 mg. per day whereas a time. Their desire to remain on the study un-
value of 555 mg. was calculated from published doubtedly reflects the quality of the food supplied
figures. 24 25 It is recognized that the colorimetric and the continuous attention they receive. Oppor-
procedure for cholesterol is not entirely specific and tunities for obtaining additional foods are very
various plant sterols and other products undoubtedly limited and infractions of the rules have been mini-
influence the results obtained to some extent.26 mal. The uniformity of the data obtained support
Values calculated from the literature are similarly this conclusion.
untrustworthy. The exact cholesterol content is Each test period was of four weeks’ duration.
therefore unknown. However, the amount of choles- Blood samples were obtained before breakfast in
terol supplied can be considered essentially constant heparinized capillary tubes by ear puncture two
in all test periods and all control periods. weeks after the start of each diet and again at three
The “control” period diet was designed to approxi- and one-half and four weeks. Samples were analyzed
mate a usual American diet. It was the same as the for total serum cholesterol,27 beta-lipoprotein choles-
experimental diet, except that milk and ice cream terol by a micro modification in which cholesterol is
contained butterfat and the oils and fats used in measured in the precipitate with a sulfated poly-
food preparation and service were margarine, mayon- galacturonic acid,H29 total fatty acids and lipid
naise, lard and high P:S ratio vegetable shorten- phDsphorus.3’ From these values, total serum tn-
ing. Analysis of this diet at the 2,600 calorie level glyceride was calculated by subtracting cholesterol
indicated a content of 331 mg. cholesterol per day ester fatty acid (assuming cholesterol to be 70 per
as compared to a calculated value of 660 mg. cent estenified) and phospholipid fatty acids from
The subjects were selected from a rather large the total fatty acids. In accordance with previous
group in the mental institution. Most of the men experience, the major cholesterol response is oh-
were chronic schizophrenic patients without evidence tamed within the first two weeks. The average of
of physical disease and were from thirty-eight to the two terminal values at three and one-half and
fifty-seven years of age. Those with serum choles- four weeks was used in all calculations.
terol values above 300 or lower than 200 mg. per During the first year of the study the men re-
cent were excluded. The final selection was based ceived the “test” and “control” diets in alternating
primarily upon the probability that they would be four week periods. During the first months, the
available and cooperative over a long period of serum cholesterol levels resulting from the control
time. diet gradually fell. Change in serum cholesterol (
While all the men were housed and ate in an iso- cholesterol) was calculated by comparing the mean
lated ward which included a recreation room in value at the termination of the test diet with the
addition to the kitchen and dining facilities, they average of the values on the control diets imme-
continued their usual supervised activities in the diately preceding and following the test period.
284 Hegsted, McGandy, Myers and Stare

After the first year, the values on the control diet Harvard Computing Center. All possible equations
stabilized and the control diet was given only at for predicting changes in serum cholesterol using
widely spaced intervals. The fatty acid and choles- each variable singly and in all combinations were de-
terol content of the experimental diets, arranged in termined. With eight dietary variables, the various
the order in which they were fed, and the choles- saturated acids, the mono- and polyunsaturated
terol values are listed in Table III. acids and dietary cholesterol, there are 256 equa-
Safflower oil, olive oil and coconut oil supply the tions. The data were also coded with the fatty acids
largest amounts of polyunsaturated fatty acid expressed as percentage of the dietary fat, rather
(linoleic acid) , monounsaturated (oleic acid) and than as percentage of total calories, and the com-
saturated acids, respectively, that can be obtained putations repeated.
with the ordinarily available edible oils. Coconut
oil, although highly saturated, is well utilized be- RESULTS
cause it contains relatively large amounts of laurie
The mean serum cholesterol of the group of
and myristie acids. These acids are not major
constituents of many edible oils and the possibility men finally selected was 225 mg. per cent while
was considered that these saturated acids may have consuming the house diet. When the diet was
properties not typical of other saturated acids. Tn- changed to the “control” diet, the level rose to
glycenides of longer chain fatty acids are not well 250 mg. per cent. In the following six months,

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utilized. Cocoa butter with over Go per cent of pal- the values obtained during the control periods
mitic and stearic acid contains the maximal amount gradually fell to approximately 220 mg. per
which can be incorporated into a fat without the cent. They remained essentially stable and at
formation of poorly utilized saturated triglycerides.
this level while the men were on the control
These factors determined the oils chosen for tests
diet during the following periods. The causes
during the first year of the experiment which in-
of these changes are unknown and represent
eluded the maximal spread in saturated (S), mono-
one of the limitations of studies of this kind,
unsaturated (M) and polyunsaturated (P) acids
which could be achieved readily. Each of these oils
i.e., even though the diet is as carefully con-
as well as a mixture which provided equal amounts trolled as possible, the level of serum cholesterol
of saturated, monounsaturated and polyunsaturated is not strictly reproducible. As has been ex-
acids were tested at two levels of dietary fat pro- plained, the change in serum cholesterol (A
viding 22 and 38 per cent of the calories as fat. Tests cholesterol) was calculated by subtracting the
with safflower oil and, later, with olive oil, were re- mean values of each man in the immediately
peated. preceding and succeeding control periods from
Butterfat is known to be more hypercholester- the values obtained while they were on the
olemic than most oils. Studies during the second
experimental diet.
year included a variety of mixtures of butterfat and
The mean A cholesterol values for each test
other oils to study the influence of the latter upon
diet are given in Table in. The standard er-
hypercholesterolemia induced by butterfat. All of
rors of these values are of the order of 5 or 6 mg.
these tests were made with fat providing 38 per
cent of the total calories.
per cent and it may be concluded that the
The addition of butterfat to the diet and substi- values obtained are expected to be reproducible
tution of part of the butterfat by other oils resulted within about 10 mg. per cent. Greater ac-
in substantial changes in the level of dietary choles- curacy could be achieved only with larger
terol. Studies were therefore made with safflower, groups of men.
olive and coconut oil in which the dietary choles- We were unable to demonstrate differences
terol was deliberately varied. A relatively low in the response of serum cholesterol in men with
cholesterol diet was achieved by eliminating egg different inherent levels of serum cholesterol.
yolk from the diet. The higher cholesterol diet was
That is, men who had higher levels of serum
achieved by adding additional egg yolk. The fatty
cholesterol did not show greater changes than
acid and cholesterol content of the diets after these
those with lower levels. This is probably be-
modifications are also given in Table III.
cause the men were selected to give a rela-
A total of thirty-six different test diets have been
studied. All the data in Table In were coded for tively narrow range of values at the beginning
machine computation utilizing the IBM 7094 at the of the study. All results are recorded as
 Quantitative Effects of Dietary Fat 285

TABLE III
Composition of the Diets Studied and the Changes Induced in Serum Cholesterol Levels

Fatty Acid Content (% of total calories)

H Dietary rum
% Saturated Fatty Acids
Period Diet No. Total Choles- Choles-
Fat I Total Total Total t&ol terol
(mg/day)
Calories
<12:0 12:0 14:0 16:0
18:0 and 5 M P (mg.%
over
(Sto) (Si,) (Sit) (Sio)
(St8)

Group I

Control . . . 38 0.47 0.25 0.96 5.65 6.28 13.61 15.97 8.83 331
1 Safflower i 10 22 0.00 0.01 0.10 2.52 1.09 3.72 4.85 12.98 306 -38.7
2 Coconutoil 10 38 4.46 15.49 6.15 4.45 1.46 32.01 4.91 1 .30 306 + 56.2
3 Olive I 10 22 0.00 0.01 0. 10 3.30 0.96 4.37 14.40 2.96 306 -36.5
4 Cocoa butter 10 38 0.00 0.04 0. 13 9.46 12.49 22.12 13.56 2.04 306 - 2.1
5 Mixed-SMP 10 22 0.00 0.01 0.10 3.83 3.28 7.22 7. 18 7.14 306 -12.3
6 Safflower ii 10 22 0.00 0.01 0.10 2.48 0.99 3.58 4.78 13.81 306 -28.9
7 Coconut and
olive 10 38 2.93 10.55 4.30 4.74 1 .53 24.05 1 1 .30 2.67 306 + 28.6
8 Butterfat

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100% 9 38 2.60 1.31 4.00 10.83 4.78 23.52 12.69 1.95 552 +61.8
9 BF-Cotton
50-50 9 38 1.30 0.66 2.20 9.45 3.19 16.80 10.79 10.57 437 + 9.4
10 BF-Corn
50-50 9 38 1.30 0.66 2.05 8.00 3.12 15.13 12.36 10.72 437 - 5.0
11 BF-Safflower
85-15 9 38 2.21 1 . 12 3.46 9.74 4.41 20.94 11.84 5.69 529 + 46.4
12 BF.Olive
70-30 9 38 1.82 0.92 2.83 9.10 3.77 18.44 17.01 2.67 489 +35.0
13 I BF-MCT
50-50 9 38 17.23 0.98 2.05 6.11 2.66 29.03 7.65 1.46 437 +32.1
14 Butterfat
100% 9 . :18 2.60 1.31 4.00 10.83 4.78 23.52 12.69 1 .95 552 + 60.1
15 Safflower low ‘

cholesterol 10 37 0.00 0.01 0.10 3.15 1.49 4.75 6.30 25.83 116 -51.5
16 Safflower high
cholesterol 10 40 0.00 0.01 0.10 4.57 1 .88 6.56 8.97 24.88 686 -22.3
17 Coconut high I
cholesterol 10 40 4 . 29 14.91 5.93 5.35 1 .78 32.26 6.75 1 .56 686 + 67.3
18 Olive Ii 10 22 0.00 0.01 0. 10 3.30 0.96 4.37 14.40 2.96 306 -14.1

Group II

Control . . 38 0.47 0.25 0.96 5.65 6.28 13.61 15.97 8.83 331
19 Safflower I 9 38 0.00 0.01 0. 10 3.64 1.49 5.24 7.09 24.85 306 -47.7
20 Coconut oil 9 22 2 . 23 7.75 3.12 2.92 1.01 17.03 3.75 1.15 306 +40.9
21 Olive i 9 :38 0.00 0.01 0.10 5.13 1 .69 6.93 26.11 4.91 306 -30.9
22 Cocoa butter 9 I 22 0.00 0.03 0. 12 5.43 6.52 12.10 8.17 1 .53 306 + 7.0
23 Mixed.5MP 11 38 0.00 0.01 0.10 6.27 6.02 12.40 12.04 13.66 306 -12.3
24 Safflower Ii 11 38 0.00 0.01 0. 10 3.57 1.43 5.11 6.86 26.02 306 -40.8
25 Coconut and
safflower 10 38 2.70 10.26 4 .62 4.55 1 .50 23.60 5.84 8.55 306 + 15.0
26 Butterfat I I
100% :38 2.60 1.31 4.00 10.83 4.78 23.52 12.69 1 .95 552 + 50.0
27 BFSafflower
50-50 9 38 1.30 0.66 2.07 7.20 3.10 14.33 9.72 14.12 437 - 6.1
28 BF-Olive .

50-50 9 38 I 1.30 0.66 2.05 7.95 3.15 15.11 19.88 3.16 437 + 24.5
29 BFSafflower
70-30 9 38 1.82 0.92 2.93 8.65 4.04 18.36 10.65 9.42 489 + 29.4
30 BFO1ive
85-15 9 I 38 2.21 1.12 3.41 9.96 4.27 20.97 14.85 2.31 529 + 34 . 1
31 BF-Fish
85-iS 9 38 2.21 1.12 3.90 10.09 5.02 22.34 12.40 3.36 . 529 +37.5
32 Butterfat
100% 9 38 2.60 1.31 4.00 10.83 4.78 23.52 12.69 1 .95 552 + 64.6
33 Olive high
cholesterol 10 40 0.00 0.01 0.10 6.01 1 .94 8.06 27 . 13 5.05 686 +28.1
34 Olive low
cholesterol 10 37 0.00 0.01 0. 10 4.67 1 .55 6.33 25.56 4.81 116 + 1.0
35 Coconut low
cholesterol 10 37 4.55 15.80 6.27 3.98 1 .39 31 .99 3.94 1 . 12 116 +45.6
36 Oliveir 10 38 0.00 0.01 0.10 5.13 1 .69 6.93 26.11 4.91 306 + 6.6
 286 Hegsted, McGandy, Myers and Stare

changes in milligrams per cent rather than as easily interpreted since this is the per cent of the
per cent of the serum cholesterol level. total variance in serum cholesterol which is
The cholesterol level of the beta lipoproteins accounted for by the regression equation.
was found to parallel changes in total choles- The standard deviation from regression is the
terol levels. The values, therefore, are not re- measure of the deviation about the multiple
ported. Changes in total serum triglycerides regression line.
and serum phospholipids were found to be in
the same direction as the serum cholesterol
Cakulations Based upon Fatty Acids Expressed
levels. However, the changes were less con-
as Per Cent of Total Calories
sistent than those of the serum cholesterol and
the standard errors of the differences were In the calculations first made the role of the
large. They did not appear to contribute use- total saturated, S, monounsaturated, M, and
fully in this study and are also not reported at polyunsaturated, P, fatty acids and dietary
this time. cholesterol, C, were assessed. The multiple
As shown in Table III, the dietary fatty acid regression equation including these variables is
components were divided into five classes of
ch = 2.32 S + 0.32 M - 1.46 P +
saturated fatty acids (S10-518), the monoun-

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6.51 C + 0.83 (2)
saturated acids (M), and the polyunsaturated
acids (P). The monounsaturated acids con- As observed by Keys et the regression co-
sisted almost entirely of oleic acid with small efficient for S is large and positive indicating
amounts of palmitoleic acid. The polyun- that increases in S raise serum cholesterol. P
saturated acid in most oils was linoleic acid. has a negative coefficient and the coefficient of
Fish oil and butterfat contained a variety of M is relatively small. The coefficient for
other polyunsaturated acids. The change in changes in dietary cholesterol is 6.51, suggesting
intake of each of these classes of fatty acids as an average increase in serum cholesterol of 6.5
well as dietary cholesterol was determined by mg. per cent when the dietary cholesterol is
comparing the intake to that obtained from the raised 100 mg. per day. As shown in the first
control diet. line of Table iv the correlation coefficient is
The complete regression equation, including high, 0.937, and approximately 88 per cent
all the fatty acid classes and dietary cholesterol, (R2 = 0.879) of the total observed variation in
would have the form serum cholesterol is explained by this equation.
The standard deviation about the regression
\ch = aS10 +bSi2+cASi4+ d zS,6 +
line is 12.98 mg. per cent.
eAS,8+fM+gAP+hAC+constant (1)
The succeeding lines in Table iv contain
The problem is to determine the significance of the regression coefficients for the equations of
the regression coefficients a to h. In the dis- best fit as variables are deleted. When M is
cussion which follows, the notation in Table not considered, line 2, the fit is substantially
iii is utilized in the equations and all com- the same and R = (1.936 (Fig. 1). However,
putations have been made based upon differ- when dietary cholesterol is omitted the ability
ences in the values in the control diet and the of the equation to account for the observed
experimental diet. Thus, the equations appear changes falls appreciably (p <0.001). Dietary
as cholesterol is therefore a significant variable.
Finally, with the omission of P, the predictive
A ch = a Sio + b S14
ability falls still further. Nevertheless it
the A signs which appear in equation (1) being should be emphasized that changes in the
omitted. saturated acids alone account for 72 per cent
The multiple correlation coefficient, R, is the of the total variation in serum cholesterol ob-
statistical parameter most commonly used to served. Although the regression coefficient for
indicate the ability of the regression equation P is approximately as large as that of S in line 3,
to predict the experimental data. R2 is more the addition of P to the equation only increases
 Quantitative Effects of Dietary Fat 287

TABLE iv

Characteristics of Regression Equations Based Upon Total Saturated (S), Monounsaturated (M) and
Polyunsaturated (P) Fatty Acids and Dietary Cholesterol (C)

Variable

S.D. from
Line Constant R R2
S M C Regression
(% cal.) (% cal.) (% cal.) (mg. X 100)

1 2.32 0.32 -1.46 6.51 0.83 0.937 0.879 12.98

2 2.16 ... -1.65 6.77 -0.53 0.936 0.877 12.89
3 2.49 ... -1.62 ... 3.04 0.894 0.799 16.20
4 3.27 ... ... ... 3.38 0.848 0.719 18.87

the predictive ability of the equations 8 per only four of the dietary variables is 0.951 and
cent, i.e., from 72 to 80 per cent. 90 per cent of the total variation in serum
In the next step in the analyses the saturated cholesterol is explained by this equation. The
acids were divided into the classes indicated in fit is substantially better than any of the equa-

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Table iii. From the many regression equa- tions shown in Table iv. In lines 2 to 5, the
tions obtained, only a few of particular interest effects of deleting each of these variables is
can be presented. The most important, how- shown. It will be seen that the elimination of
ever, is the equation palmitic acid, S16, has the least effect, the correla-
tion coefficient fallingonly to 0.941 (line 4). Var-
. ch = 8.45 S14 + 2.12 S,6 - 1.87 P +
iance analyses,32 however, demonstrate this to
5.64 C - 6.24 (3)
be a significant change (p = 0.02). The great-

As shown in Figure 2 and in Table v, the cor- est change is observed when Si4, myristic acid,

relation coefficient with this equation involving is deleted. Deletion of polyunsaturates or

dietary cholesterol have lesser but significant
effects. Line 6 shows that changes in myristic

ch
m% /
60 845S4 ‘ 2 2S - ‘5 642-6 23 u /
.0951

40
0
LU

20
#{176}

-20

-40 11)

/@4

FIG. 1. Scatter diagram indicating the correlation be- -q -o 0 20 40 6Och,m%

FOUND
tween the cholesterol values found and those pre-
dicted by the regression equation. S refers to the total FIG. 2. Scatter diagram indicating the correlation be-
saturated fatty acids, P to total polyunsaturated fatty tween the cholesterol values found and those pre-
acids expressed as per cent of dietary calories. C refers dicted by the regression equation. S indicates mynis-
to dietary cholesterol in 100 mg. units. Individual tic acid, S,6 palmitic acid and P total polyunsaturated
points indicate the test periods described in Table in. fatty acids.
288 Hegsted, McGandy, Myers and Stare

TABLE v

Characteristics of Regression Equations When the Fatty Acids Are Expressed as Per Cent of Calories

\‘aniable
S.D. from
Line Constant R1 R2
5j4* Regression
S16t P C
(% cal.) (% cal.) (% cal.) (mg. X 100)

1 8.45 2.12 -1.87 5.64 -6.24 0.951 0.905 11.52

2 9.50 3.24 -1.63 ... -1.73 0.935 0.874 13.02
3 11.58 3.66 ... 3.02 -4.80 0.891 0.793 16.70
4 8.78 ... -2.10 7.50 -8.02 0.941 0.886 12.40
5 ... 2.80 -2.86 9.28 -2.51 0.864 0.746 18.50
6 14.22 ... ... ... 3.25 0.830 0.689 19.85

* Mynistic acid.

t Palmitic acid.
All polyunsaturated acids.
§ Dietary was expressed
cholesterol in units of 100 mg.
#{182}
Multiple regression coefficient.

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R2 is a measure of the total variance explained by the regression equation.

acid alone account for 69 per cent of the total be concluded that it is unlikely that considera-
variance in serum cholesterol. tion of Sio, S12, 518 and M assist in predicting
The equation including all variables is serum cholesterol a/’ler S14, S16, P and C are
considered.
. ch = 0.66 S + 1.03 S,2 + 4.98 Si4 + 3.76 S,6 -

It is most important to note that the regres-

0.49 S18 - 0.24 M - 1.89 P + 5.70 C - 9.44 (4) sion coefficients may change appreciably for all
The multiple correlation coefficient is then variables depending upon the particular van-

0.953. This equation (Fig. 3) is not a signifi- ables included in the equation. For example,

cantly better fit than equation 3 and it may the coefficient for cholesterol is tniplcd in line 5
as compared to line 3 and the coefficient of P is
nearly twice as high in line 5 as compared to
line 2. This is due to the fact that the van-
ables are not independent and that the coeffi-
cients are intercorrelated. Snedecon32 stresses
the fact that ‘ ‘statements made about the pre-
dictive value of a variable are not unique; they
depend upon the other variables being used in
the regression.”

Calculations Based upon Fatty Acids Expressed

as Per Cent of Dietary Fat

Calculations similar to those shown in Tables

iv and v were also made with the dietary fatty
acids expressed as per cent of total dietary fat.
Generally speaking, the results are quite similar.
The coefficients for the important regression
equations are shown in Table vi. Analysis of
FIG. 3. Scatter diagram showing the correlation be-
variance demonstrates that each of these vari-
tween the values found and those predicted by the re-
ables contributes significantly to the ability of
gression equation. S,0, S,2, etc., refer to the components
indicated in Table IH. All except C (dietary choles- the equation to account for changes in the
terol) are expressed as per cent of dietary calories. serum cholesterol. It is most important, how-
 Quantitative Effects of Dietary Fat 289

TABLE vi
Characteristics of Regression Equations \Vhen the Fatty Acids Are Expressed as Per Cent of Dietary Fat*

Variable

Line Constant R R2 S.D. from

S,4 S16 P C Regression
( of fat) (% of fat) (%of fat) (mg. X 100)

1 3.27 0.89 -0.61 5.88 - 6.70 0.955 0.911 11.11

3.68 1 .23 -0.56 - 2.80 0.934 0.872 13.14
3 4.42 1.73 4194 -10.64 0.914 0.835 14.90
4 3.14 -0.75 6.94 - 5.39 0.945 0.892 12.05
0.53 -1.08 9.14 + 0.25 0.859 0.738 18.59
6 5.28 - 4.03 0.817 0.668 20.11
4.69 1 96 - 7.04 0.898 0.807 15.88
3.60 -0.76 + 0.21 0.913 0.833 14.76
9 + 4.61 0.538 0.289 29.79
10 -1 .25 + 10.58 0.780 0.608 22.14
11 13.27 - 1.39 0.450 0.202 31.25

See footnotes of Table v for explanation of column headings.

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*

ever, to note that the equation given in line 1 of effects are not additive. For example, in
Table vi and Figure 4 Table vi, line 6, the per cent myristic acid is
shown to correlate highly, R = 0.817. This
ch = 3.27 5, + 0.89 516 0.61 P +
variable has the highest correlation singly of
5.88 C - 6.7 (5)
any variable we have considered. However,
is actually slightly superior to equation 3 P alone is also highly correlated (line 10,
given in line 1 of Table v. Thus with these R = 0.780), whereas palmitic acid is correlated
data, consideration of only the proportions to a much lesser degree (line 9, R = 0.538).
of fatty acids in the dietary fat without con- Nevertheless, the combination of mynistic and
sideration of the amount does not impair
the ability to predict serum cholesterol change.
Lines 6 to 11 in Table vi indicate the ability
of individual and some pairs of variables to
predict changes in serum cholesterol. The rela-
tively high correlations with myristic acid and
polyunsaturated acids are again apparent as
is the limited effect of palmitic acid. The large
changes in the size of the regression coefficients
depending upon the variables included in the
equation should also be noted.

COMMENTS

The major difficulty in studies of this kind in

arriving at firm conclusions as to the physio-
logic role of dietary components should be
clearly understood. When naturally available
FOUNI
oils are used, the dietary variables are corre-
lated to a greater or lesser Much extent. of FIG. 4. Scatter diagram showing the correlation be-
tween the values found and those predicted by the re-
the same information is imparted regardless of
gression equation. 814, Si6, and P are expressed as per
which variable is considered and practically
cent of dietary fat. Compare to Figure 2 in which these
any variable may show considerable correlation components were expressed as per cent of dietary
with change in serum cholesterol. But these calories.
290 Hegsted, McGandy, Myers and Stare

palmitic acid (line 7) provides nearly as good the C18 saturated acid, has little if any effect
a fit for the data as does the combination of upon serum cholesterol. Our data confirm this
myristic and P together (line 8). finding. Changes in dietary stearic acid have
Further, as has already been indicated, the a very low correlation with changes in serum
size of the regression coefficients depends upon cholesterol (R 0.245) = and inclusion of stearic
the other variables included in the equation. acid in the regression equation with the four
Snedecor32 has explained why this is true. important variables (myristic, palmitic, poly-
These equations are primarily descriptive of the unsaturates and dietary cholesterol) does not
information from which they are derived. improve the fit. Furthermore, the equation
Should new variables be included, such as a containing only myristic and palmitic acid,
dietary carbohydrate component, the regression rather than total saturated acids (Fig. 2 and
coefficients for the fatty acids might be ex- 4) , predicts the response to cocoa butter at the
pected to change. It is somewhat hazardous high level (period 4, Table ‘II) much better
to attach as much functional significance to than does the equation including the total
the regression equations as Keys et have saturated acids (Fig. 1). The reports of
done. Horlick,36 Malmrosn and Erickson et
It is surprising indeed to find that myristic well as the data of Ahrens et a!.’ are all

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as
acid appears to be a most important variable consistent with the conclusion that stearic acid
in influencing serum cholesterol. The satu- is essentially without effect on serum choles-
rated acids containing 10 carbons and less are terol. However, Keys has concluded that
known to have essentially no hypercholesterol- since stearic acid is not involved, palmitic acid
emic action33’34 and this was confirmed in must be the saturated acid of primary interest.
period 13 (Table III) when a 50 to 50 mixture This does not automatically follow as we have
of butterfat and medium-chain triglycerides shown.
was fed. The shorter chain fatty acids are Keys et al.4 have emphasized that mono-
known to be absorbed primarily via the portal unsaturated acids have no effect upon serum
system whereas the longer chain acids en- cholesterol whereas Ahrens et ‘ and Gunning
ter the lymphatic system. If dietary fats et al.2 believe that total unsaturation may be
influence cholesterol absorption, it might have the more important variable. In general, our
been suspected that the distinction between data support the conclusion of Keys and his
effective and noneffective fatty acids would group ; yet, we do not consider any of the data
occur between the C10 and C12 acids since available entirely adequate to settle this point.
the latter, lauric acid, appears to be pri- The first trials with olive oil (periods 3 and 21.
marily absorbed via the lymph. We found no Table in) indicated that this oil was nearly as
evidence, however, that lauric acid is a signifi- effective as safflower oil. Repetition of these
cant variable in these studies. groups (periods 18 and 36) gave lower results
Another factor which must be considered is more in line with expectation. Nevertheless,
the degree of variation obtained in the dietary it must be emphasized that as shown in the
component under study. Although a sub- triangular plot (Fig. 5), the oils we studied,
stance may influence serum cholesterol, this except for olive oil, most often fall in the range
will not be evident unless it is varied in the of 15 to 40 per cent of monounsaturates. The
experiment. Conversely, a component which saturated and polyunsaturated levels were more
is varied greatly in the experimental design has uniformly distributed. A similar plot of the
a better opportunity to manifest its influence. oils studied by Keys et al.3 will show the same
Inspection of Table in, however, does not re- tendency. It can be shown in fact that with
veal any specific reason why myristic acid either Keys’ data or our own, when selected
(S14) should be particularly important. The diets are studied, regression equations with
C16 acid was present in larger amounts and significant regression coefficients for M are
subjected to substantial variation as were the found. Furthermore, since the proportion of
C12 and C18 saturated acids. myristic, palmitic and polyunsaturated acids in
Keys et al.35 have concluded that stearic acid, an oil seems as important and perhaps more
 Quantitative Effects of Dietary Fat 291

20 0

40 I Q

M P

6 40

8 0

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31

20 40 60 80

FIG. 5. A triangular plot showing the proportions of saturated, monoun-

saturated, and polyunsaturated fatty acids in the total dietary fat supplied
in the test periods. Points are numbered according to Table III. Filled circles
refer to diets containing only 22 per cent of calories as fat. Other diets sup-
plied 38 to 40 per cent of calories as fat.

important than the per cent of calories of these much easier to obtain diets of this kind when
acids consumed, we would definitely not agree the total fat intake is high. Furthermore,
with the conclusion that “Oleic acid, and per- since a low proportion of myristic and palmitic
haps other natural mono-enes, is neutral in this acid is of primary importance, the inclusion of
respect and may be isocalorically exchanged monosaturated acids, stearic and short chain
for starch in the diet without affecting the acids as well as polyunsaturated acids is help-
cholesterol in the blood. “38 ful in achieving these kinds of diets. Dietary
It is of considerable interest that the changes advice to lower the total fat intake is likely to
in serum cholesterol are explained as well or be self-defeating.
slightly better by the percentage composition of Unfortunately, analytical data on the myris-
the dietary fat than by the fatty acids ex- tic acid content of food fats is limited. It ap-
pressed as per cent of calories. This conclusion pears to be a constituent of all animal fats
is limited, of course, to the range of fat intake although the amount may vary from 1 to 12 per
studied which was 22 and 38 to 40 per cent of cent. Most of the common vegetable oils
the calories. It seems likely to us that this other than coconut oil contain very little.
would not be true when very low levels of fat Dietary cholesterol is obviously an important
are used. Nevertheless, the range of fat in- variable in determining the serum cholesterol
takes we studied included the limits of prac- level. All recent work appears to support this
tically acceptable diets in the United States. conclusion. There does not appear to be ade-
The most effective diets appear to be those in quate quantitative information on the serum
which the percentage of myristic and palmitic cholesterol response to different dietary levels.
acid in the dietary fat is low and the percentage As shown in Figure 6, our data indicate an es-
of polyunsaturated acids is high. Clearly it is sential linear response to dietary cholesterol
292 Hegsted, McGandy, Myers and Stare

zch
mg.#{176}

60
ONUT OIL
SLOPE 4.5mg/I 00mg.

40

20
OIL
SLOPE = 5.Omg./IQOmg.

-20

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-40

SLOPE SOmg./IOOmg.

2#{212}0 400 600 rng./day

Dietary Cholesterol
FIG. 6. Serum cholesterol response at various levels of dietary cholesterol
with three different dietary fats. Lines were drawn by inspection.

and an average increase of about 5 mg. per cent It must be stressed that although the correla-
serum cholesterol for each additional 100 mg. tion coefficients for various equations presented
of dietary cholesterol although there is one here and by Keys et al. are extremely high for
aberrant value for olive oil. This value is biological material, the error of the estimated
consistent with the regression coefficients in cholesterol change of the best equations is
the equations in Figures 2, 3 and 4 and is about 10 mg. per cent. This is a mean value
similar in magnitude to the responses obtained obtained with ten men per group. In our data
by Connor et al.’2 Since the three lines in the mean values from which the data are
Figure 6 are essentially parallel, there appears derived also have standard deviations of about
to be little or no interaction between the die- 5 mg. per cent. It is clear, therefore, that
tary oils and dietary cholesterol. Although equations of this kind are of little value in
Anderson et al. 14. 39 concluded that the serum predicting what may happen to the serum
cholesterol response is proportional to the cholesterol of an individual. From this it also
square root of the dietary cholesterol, it appears follows that changes in the serum cholesterol
more satisfactory to consider the response as of two or three patients are of little value in
proportional to the absolute intake at the levels attempting to elucidate the action of various
we have investigated. We do not agree that oils or their components. The cause of this
serum cholesterol levels are “essentially inde- rather large biologic variation is unknown but
pendent” of cholesterol intakes at the levels must be recognized.
which occur in usual diets. It should be emphasized that the differences
Following Jolliffe’s suggestion, P: 540 ratio in the levels of serum cholesterol of various
has been widely accepted as a useful measure population groups cannot be adequately ex-
of the efficacy of dietary fat in lowering serum plained by differences in fat intake.43 This is
cholesterol.41 42 The P: S ratio is not closely particularly evident in recent reports on some
related to serum cholesterol response. It of the nomadic tribes44 in which low levels of
should be discarded even though it has the serum cholesterol are found even though
merit of simplicity. large amounts of butterfat are consumed.
 Quantitive Effects of Dietary Fat 293

Less striking differences are seen in the corn- tion of the amounts of fatty acids consumed.
parison of brothers in Boston and Ireland45 Thus, the proportions of fatty acids in the
where the consumption of larger amounts of dietary fat rather than the percentage of cab-
animal fats in Ireland is associated with ries they supply is thought to be of primary im-
somewhat lower levels of serum cholesterol. portance.
Evidently other environmental factors are in- 5. Dietary cholesterol appeared to be
volved. It appears likely that total calorie linearly related to the serum cholesterol. An
consumption and physical activity may mod- increase of 100 mg. in dietary cholesterol pro-
ify the effects of the dietary fatty acids. yokes a rise in serum cholesterol of approxi-
mately 5 mg. per cent. This response was
SUMMARY AND CONCLUSIONS
independent of the effects induced by dietary
Data are presented concerning the re- fat.
sponse of serum cholesterol in two groups of 6. In view of these results, the most effec-
men fed a low fat diet to which a wide variety tive practical diets for lowering the serum
of oils were added. The total dietary fat sup- cholesterol should be those relatively high in
plied was either 22 or 38 to 40 per cent of the total fat with (a) a small proportion of myristic
calories. Dietary cholesterol supplied as egg and palmitic acids, particularly myristic acid;

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yolk was also varied. Test periods were of (b) a high proportion of polyunsaturated acids;
four weeks’ duration. The same men were and (c) a small amount of dietary cholesterol.
repeatedly tested throughout the study. The
ADDENDUM
following conclusions are drawn:
1 . A multiple regression equation involving In the discussion of this paper, we indicated

only the changes in intake of myristic acid, the rather severe limitations of multiple regres-
palmitic acid, polyunsaturated fatty acids and sion analysis as a means of determining the

dietary cholesterol was adequate to explain 91 specific effects of the individual variables.

per cent of the total variance in the levels of Presumably the effects of each fatty acid can

serum cholesterol. Inclusion of other variables only be determined with security by changing

did not significantly improve the fit. each fatty acid independently and keeping the
2. Approximately 67 per cent of the total rest of the diet constant. This cannot be done

variance in the level of serum cholesterol was with natural oils.

explained by changes in dietary myristic acid As this paper goes to press, we have prelim-

alone. This appears to be the most important inary results obtained with partially synthetic

of the fatty acid components affecting serum triglycerides. Trimyristin and tripabmitin

cholesterol levels. were added to safflower oil and incorporated

3. Palmitic acid has significant but much into the fat by transesterification. This is
lesser effects upon the level of serum cholesterol necessary since tripalmitin is not well digested.

than does niyristic acid. Increases in the poly- One oil contains 26 per cent myristic acid and
unsaturated acids lower serum cholesterol. 6 per cent palmitic acid; the other 7 per cent
No specific effects on serum cholesterol could myristic acid and 27 per cent palmitic acid.
be detected for stearic, lauric or shorter chain The remainder of the fatty acids are
essentially
saturated acids or for monounsaturated acids the same in both oils and consist of 53 per cent
except that their presence in fats lowers the linoleic, 1(1 per cent oleic and 2 per cent stearic
proportions of myristic, palmitic and poly- acid.* These were incorporated in the basal
unsaturated fatty acids. diet described to supply a total fat intake of
4. The amount of dietary fat, tested be- 38 per cent of the calories. The experiment was
tween 22 and 40 per cent of the total calories, designed, of course, to study the specific effect
appeared to be without influence upon the level of substituting myristic acid for palmitic acid.
of serum cholesterol. Consideration of only To our surprise the preliminary results sug-
the percentage composition of the dietary fat gest that there is no substantial difference in the
was somewhat more effective in predicting * These oils were generously supplied by Dr. F. H.
serum cholesterol response than was considera- Mattson of the Proctor and Gamble Company.
294 Hegsted, McGandy, Myers and Stare

effect of these oils on the serum cholesterol of the Hood Milk Company, Boston, Massachusetts,

level. Further studies are underway. with the cooperation of Dr. H. L. Wildasin and Mr.
Wallace Fogg. Dr. Jane Worcester and Miss Margaret
It will be recognized that oils of this kind,
E. Drolette of the Department of Biostatistics, Har-
i.e. relatively
, high in linoleic acid and myristic yard School of Public Health, provided valuable con-
acid, do not apparently occur in nature, and sultative services in developing and imiterpretating the
these preparations may represent an unfortu- data for machine computation. Laboratory assistance
was supplied by Miss Rosemary Bonanno and Mrs.
nate choice. There may be complex interac-
Anna Gallagher. Supervision of the meals and their
tions between several fatty acids on serum
preparation was performed by Mrs. Elaine S. Kenneally
cholesterol which are not revealed by the mul- and Miss Margaret Brown.
tiple regression analysis. However, it may be
REFERENCES
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T. T. and PETERSON, TSALTAS,
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somehow yield the regression equations ob- 3. KEYS, A. , ANDERSON, J. T. and GRANDE, F. Pre-
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tamed.
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are relatively inaccurate and a single test can- feet on serum cholesterol in many of mnono-ene
not be decisive. Nevertheless, should the pre- fatty acids (oleic acid) in the diet. Proc. Soc.
Exper. Biol. & Med., 98: 387, 1958.
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5. KEYS, A., ANDERSON, J. T. and GRANDE, F. Serum
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of myristic, palmitic and linoleic acid that we and SPLITTER, S. D. Essential fatty acids, lipid
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7. AHRENS, E. H., JR., INSULL, W., JR., HIRSCH, J.,
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simply descriptive and the equations we have effect of human
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dietary oils act as though the specific fatty acids rated fatty acids and of linoleic acid on man’s
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their regression coefficients. The equations do
9. MCOSKER, D. E., MATTSON, F. H., SWERINGEN,
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ACKNOWLEDGMENT
hydrogenation of dietary fats, of the ratio of
We are indebted to a number of people and organiza- polyunsaturated to saturated fatty acids, and of
tions for assistance in these studies. The facilities and dietary cholesterol upon plasma lipids in man.
patients at the Danvers State Hospital were made J. Clin. Invest., 43: 2017, 1964.
available by Dr. Peter P. Hagopian, Superintendent, 11. KEYS, A., ANDERSON, J. T., MICKELSEN, 0.,
and his support made these studies possible. The fats ADELSON, S. F. and FIDANZA, F. Diet and
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Miami Valley Laboratories, Procter and Gamble Com- tary cholesterol. J. Nutrition, 59: 39, 1956.
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