PH Wendell H Griffith

PH.D Ada Marie Campbell

PH.D Marian E Swendseid

PH.D. Tuttle

Stewart G D4

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A merican Journal of Oinial Nutrition 83 Vol. 17, August 1965
Serum Lipids of Men Fed Diets Differing in
Protein Quality and Linoleic Acid Content
ADA MARIE CAMPBELL, PH.D. , MARIAN E. SWENDSEID, PH.D.,t WENDELL H. GRIFFITH, PH.D4
AND STEWART G. TUTTLE, PH.D.
T HERE is experimental evidence that dietary
alterations in each of the three major food
components, fat, carbohydrate and protein, can
affect serum lipid levels in man.’  Of the
studies carried out with protein, variable re-
sults have been reported. Olson et  found
decreased serum cholesterol in subjects when
the daily protein content of their diet was
reduced from 100 gm. of a mixture of animal
and vegetable proteins to 25 gm. of vegetable
proteins. Walker et al.4 demonstrated sig-
nificant decreases in serum cholesterol and
phospholipids during some test periods when
subjects received protein mainly from vegetable
foods as compared with test periods when the
chief source of protein was animal products.
Albanese et al.’ added a casein-containing sup-
plement to the diet of older convalescent men
and observed frequent increases in serum
cholesterol. However, Keys and Anderson6
increased the protein content of an experimen-
tal diet by an isocaloric substitution of skim
milk powder for carbohydrate and reported no
effect on the serum cholesterol level of subjects
receiving this diet. Beveridge et al. ,7a substi-
tuting calcium caseinate for carbohydrate in a
formula-type diet, obtained similar results.
From the School of Public Health, University of Cali-
fornia and the Veterans Administration Center, Los
Angeles, California.
* Present address: Associate Professor, College of
Home Economics, University of Tennessee, Knoxville,
Tennessee; t Professor of Nutrition and Biological
Chemistry;  Emeritus Professor of Chemistry and
Biological Chenmistry, University of California. Present
address: Federation of American Societies for Experi-
mental Biology, Bethesda, Maryland;  Deceased
October 1963.
This study was supported by a contract from the
Human Nutrition Research Division, Agricultural
Research Service, U. S. Department of Agriculture.
Beveridge7’ has pointed out that since animal
and vegetable foods have a variable lipid con-
tent, purified proteins must be used in experi-
mental diets if changes observed in serum lipids
are to be attributed solely to the protein pa-
rameter. To avoid the difficulties inherent in
using food as a protein source and also to avoid
changing the carbohydrate content of the diet,
we attempted to alter the protein nutritive
quality of the diet by substituting purified
animal-type proteins for a purified vegetable
protein. The possible effect of the kind of pro-
tein on serum lipids was tested by comparing a
diet containing wheat gluten as the chief source
of nitrogen with a diet containing an isonitrog-
enous amount of a mixture of casein and
lactalbumin as a replacement for wheat gluten.
The vegetable and animal proteins were each
tested in diets with an assortment of fats having
a 12 per cent linoleic acid content for one period
of study and a 40 per cent linoleic acid content
for another period.
SUBJECTS AND METHODS
Seven men ranging in age from fifty-three to
seventy years and in weight from 59 to 84 kg. were
selected to receive the diets. They resided in a meta-
bolic balance ward during the study and their level
of physical activity was relatively low. A medical
examination showed each subject to be in a satis-
factory state of health. Five men participated in all
the experimental dietary periods and two men each
in a different half of the study, giving a total of six
subjects for each dietary treatment.
The entire study consisted of four experimental
diet treatments, each of the two proteins being fed
with each of the two fat mixtures. Subjects were
maintained on a self-selected diet of ordinary foods
for five days. For a succeeding fifty day period,
subjects were fed the experimental diet, containing
one of the fat assortments, supplemented with gluten
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* Gluten : Vicrum, Hercules Powder Co. Casein-
lactalbumin : 80 per cent high-nitrogen casein, Shef-
field Chemical Company. 20 per cent lactalbumin,
Nutritional Biochemicals Co.
84 Campbell, Swendseid, Griffith and Tuttle
TABLE I
Daily Menus for Experimental Dietary Periods
Breakfast* Dinner* Supper*
Item Amount
(gm.)
Item Amount
(gm.)
Item Amount
(gm.)
Orange juice
Biscuitst
“Blend B”
Canned applesauce
100 Hash brown potatoest 117
86 Raw celery 25
15 Canned green beans 50
100 Biscuitst 86
“Blend B” 5
Canned pears 100
Mints 0-25
Bouillon 1
Noodles with tomatoest 120
Lettuce 25
Canned peaches 100
White bread 20
“Blend B” 20
Puddingf 175
* Each meal also included Sanka, sugar, jelly and 1.5 gm. of a mineral mixture prepared according to Rose.”
One Unicap (Upjohn) multivitamin capsule was given per day.
t Containing “Blend A,” the fats other than butter and margarine.
 Containing 1.6 gm. nitrogen. The weight for biscuits is weight unbaked.
§ The mixture of butter and margarine.
for the first twenty-five days and with a casein-
lactalbumin mixture for the second twenty-five days.
After another five-day period on ordinary foods, sub-
jects were given the other fat assortment containing
a different amount of linoleic acid for a fifty day
l)eriOd, twenty-five days with the gluten supplement
and twenty-five days with the casein-lactalbumin
mixtures. Because of limited space in the metabolic
ward, only one or two subjects were studied at a
time. Some subjects were given the fat assortment
with the lower linoleic acid level first and some
began with the higher level. The order in which the
two proteins were given at each level of linoleic acid
intake also was varied.
The experimental proteins* were obtained at the
beginning of the study and the casein and lactal-
humin were mixed in a large mixer. Throughout the
study, the level of dietary nitrogen was maintained at
6.5 gm rer day, of which the experimental protein
l)rovided 4.8 gm.
Fat provided 40 per cent of the total calories
throughout the experiment. Two fat assortments
differing in linoleic acid content were prepared at
the beginning of the study. Assortment I was
planned to approximate the fat composition of the
average diet in the United States and contained in
per cent : butter, 25 ; margarine, 7 ; rendered beef
fat, 17 ; h’drogenated vegetable oil, 12; lard, 31;
cottonseed oil, 9. For preparing assortment II, 32
gm. of safflower oil was added to 68 gm. of assort-
ment I. In the interests of palatability, butter and
margarine were mixed separately and used as a
spread. At the beginning of the study the assort-
ments were prepared in an institutional type mixer,
packaged in one and a half gallon tubs and stored at
approximately - 20#{176}c. The linoleic acid concentra-
tion as determined by gas liquid chromatography
was 12 per cent in assortment i and 40 per cent in
assortment u.
Menus for the study were unchanged from day to
day and are shown in Table i. Biscuits served as
carrier for the experimental protein at the morning
and noon meals, and a starch-thickened pudding
contained the protein at the evening meal. Daily
caloric intakes varied slightly, ranging from 2,250 to
2,360 kcal. When caloric adjustment was necessary
to maintain weight, it was achieved by changing the
amount of experimental fat to the level providing 40
per cent of the calories and by varying the amount
of wheat starch in the biscuits. All subjects re-
ceived the same number of calories from sugars.
Nitrogen balance studies were carried out by the
Kjeldahl method.7’ Dietary nitrogen was determined
on one-day food composites prepared every five days.
Urinary nitrogen was determined daily and fecal
nitrogen for each five-day period.
Fat in food and feces was analyzed by the Van de
Kamer method.8 Dietary fat was determined for
one-day food composites prepared in alternating
five-day periods and fecal fat for each five-day col.
lection.
Fasting blood samples were drawn at least once
during the five-day foreperiod and at the conclusion
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Effects of Dietary Alterations on Serum Lipids 85
* 20 per cent ethylene glycol succinate polyester on
,I iconized Chromosorb, 80-100 mesh.
TABLE II
Serum Lipid Values* for the Various Experimental Diets
12% Linoleic Acid 40% Linoleic Acid
Self-SelectedSerum Comnponent (mg./100 ml.)
(mg./100 ml.) (mg./100 ml.)
..tuten Casein- .
Lactalbummn
uten
Casein-
.
Lactalbumin
Total lipid 815 ± 135 756 ± 210 685 ± 173 785 ± 293 667 ± 211
Cholesterol 258 ± 28 232 ± 33 250 ± 32 218 ± 26 209 ± 37
Sterol ester 217 ± 23 195 ± 36 200 ± 33 192 ± 45 169 ± 26
Glyceride 207 ± 51 204 ± 71 169 ± 62 268 ± 131 210 ± 87
Phospholipid 299 ± 62 272 ± 95 238 ± 72 237 ± 94 208 ± 78
Unesterified fatty acidst (in  Eq/-
liter serum)’ 762 ± 192t 795 ± 202t 788 ± 166t 804 ± 114t 818 ± 143f
* Each value is the mean value for six men ± standard deviation.
t Based on palmitic acid. Values given in SEq. per L. serum.
of each twenty-five day period. The serum was
frozen at - 20#{176}c.in tubes with screw caps. When a
serum sample was thawed, portions were used im-
mediately for preparation of filtrate for cholesterol
determination and for extraction of total lipids.
Cholesterol was measured by the procedure of
Sperry and \Vehbm and lipids were extracted and
fractionated by the procedure described by Fillerup
and Mead.’#{176} The remainder of the serum (under
nitrogen) was refrozen immediately and held in the
freezer until a convenient number of samples was on
hand for determination of unesterified fatty acids by
Dole’s method.”
Fatty acid methyl esters of the glyceride, sterol
ester and phospholipid fractions were prepared by
the method of Stoffel et al.” and subjected to gas
liquid chromatographic analysis. A Barber-Colman
chromatograph, Model 10, with a packed column*
and an argon ionization detector with a radium
source was used.
RESULTS
The substitution of wheat gluten for the
casein-lactalbumin mixture appeared to have
an effect on nitrogen balance that was not in-
fluenced by the type of fat in the diet. For the
five subjects who completed the entire study,
the mean nitrogen balance values during the
last fifteen days of both wheat gluten-contain-
ing dietary periods were -0.22, -0.93, +0.49,
+0.42 and -0. 12 gm. nitrogen per day. In
the same order, the mean nitrogen balances for
similar time intervals with the casein-lactal-
bumin supplemented diet were +0.28, +0.07,
+0.85, + 1 .26 and - 0. 15 gm. nitrogen per day.
The first four of the five subjects as listed
showed a significant decrease in nitrogen reten-
tion (P < 0.001 by t test) during the adminis-
tration of the wheat gluten, an indication that
(based on the criterion of nitrogen balance)
this diet was less adequate than the diet con-
taming the casein-lactalbumin mixture in meet-
ing protein needs.
The mean fecal fat excretion was 3.8 gm. per
day with the lower level of linoleic acid and 3.2
gm. per day with the higher linoleate concen-
tration. The values were not significantly
different and are well within the normal limits
of fat excretion.
The values obtained for the serum lipid frac-
tions from blood seruni drawn at the conclusion
of the various experimental diet periods are
given in Table II. There were no significant
differences in total lipids, sterol esters, glyc-
erides, phospholipids or unesterified fatty acids
associated with any of the dietary treatments.
In the case of serum cholesterol, the values
observed with the casein-lactalbumin diet did
not differ significantly from those observed
with the wheat gluten diet, regardless of the
linoleic acid content. However, since the
change in dietary fat had a greater effect when
the diet contains casein-lactalbumin (from a
mean of 250 to 209 mg. per 100 ml.) than wheat
gluten (from a mean of 232 to 2 18 mg. per 100
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86 Campbell, Swendseid, Griffith and Tuttle
ml.), the data were also tested for an interaction
between the kind of dietary protein and the
composition of dietary fat by the method of
linear contrasts. No significant interaction was
found. When the cholesterol values obtained
during all periods in which the diets contained
the higher level of linoleic acid were compared
with those obtained during periods of self-
selected diet, there was a significant difference
(P < 0.05 by t test). This confirms the results
of other workers that use of a highly unsatu-
rated dietary fat is associated with a decrease in
serum ‘ . ‘  16
In conclusion, the ingestion of two diets
differing in kind of protein and in nutritive
quality of protein as judged by nitrogen balance
criteria could not be shown to have any effect
on the levels of various serum lipid fractions
with the number of subjects and the experi-
mental conditions used in this study.
Serum fatty acid patterns were also deter-
mined in sterol ester, glyceride, and phospho-
lipid fractions at the two concentrations of
dietary linoleic acid and with both dietary pro-
teins. The type of dietary protein had no
effect on the distribution of fatty acids in any
of the fractions and hence the results are not
tabulated. However, again in confirmation of
other workers, “  in all three fractions the
percentage of linoleic acid increased with the
increase in dietary linoleic acid from 62 to 73
per cent in the sterol esters, from 13 to 26 per
cent in the glycerides, and from 23 to 28 per
cent in the phospholipids. The increase in
linoleate was accompanied by a decrease in
the per cent of oleate in all three fractions and
by a decrease in palmitate in the phospholipid
and sterol ester fractions.
SUMMARY
When wheat gluten was substituted for a
casein-lactalbumin mixture as the chief source
of nitrogen in an experimental diet, less nitrogen
was retained in four of five subjects tested.
With six subjects studied and under the experi-
mental conditions employed, this treatment
had no significant effect on the serum content
of total lipids, sterol esters, glycerides, phos-
pholipids or unesterified fatty acids. This
situation pertained whether the diet contained
12 per cent of linoleic acid or 40 per cent of
linoleate as fat. However, serum cholesterol
levels were lower and the per cent of linoleate
in the sterol ester, glyceride and phospholipid
serum fractions was increased when the sub-
jects were fed diets containing the larger
amount of the polyunsaturated fatty acid with
either type of protein.
ACKNOWLEDGMENT
We wish to acknowledge the technical assistance of
D. Mulcare and C. Pierce. We are grateful also to J.
Mend, R. Stein and V. Slossen for helpful advice and
for the use of gas chromatographic equipment.
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Serum Lipids of Men Fed Diets Differing in Protein Quality and Linoleic Acid Content

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Serum Lipids of Men Fed Diets Differing in Protein Quality and Linoleic Acid Content

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