187

Atherosclerosis, 28 (1977) 187-195

0 Elsevier/North-Holland Scientific Publishers, Ltd.

PLASMA CHOLESTEROL LEVELS IN RABBITS FED LOW FAT,

CHOLESTEROL-FREE, SEMIPURIFIED DIETS: EFFECTS OF
DIETARY PROTEINS, PROTEIN HYDROLYSATES AND
AMINO ACID MIXTURES *

M.W. HUFF **, R.M.G. HAMILTON *** and K.K. CARROLL 5

Department of Biochemistry, University of Western Ontario, London, Ont. N6A 5Cl

(Canada)

(Received 16 May, 1977)

(Accepted 6 June, 1977)

Summary

Rabbits become hypercholesterolemic when fed a low fat, cholesterol-free,

semisynthetic diet containing casein as the dietary protein. This did not occur
when the casein was replaced by soy protein isolate or any one of seven other
plant protein preparations. Doubling the amounts of either the casein or soy
protein isolate from 25 to 50% by weight of the diet, made no significant dif-
ference to their effects on plasma cholesterol. Soy protein isolate was effective
in counteracting the hypercholesterolemic response to casein when mixtures of
the two proteins were fed. There appeared to be no relationship between body
weight gains and plasma cholesterol levels in rabbits fed the different diets.
Animals fed the higher level of casein failed to gain weight, whereas growth was
not significantly impaired by doubling the level of soy protein isolate in the
diet. Better growth was obtained with mixtures of casein and soy protein
isolate than with either protein alone. An enzymatic hydrolysate of casein or a
mixture of L-amino acids equivalent to casein gave elevated plasma cholesterol
levels similar to those obtained with the intact protein. Plasma cholesterol
levels remained low in rabbits fed an enzymatic digest of soy protein. A
moderate, but not significant, increase in plasma cholesterol was observed when
a mixture of L-amino acids equivalent to soy protein isolate was fed. The
results of these experiments indicate that the level of plasma cholesterol can be
influenced by the amino acids supplied in the diet.

* Supported by the Ontario Heart Foundation and the Medical Research Council of Canada.
** Rwipient of an Ontario Graduate Scholarship.
*** Prrsmt address: Animal Research Institute. Agriculture Canada. Ottawa. Ont. KlA OC6, Canada.
# Medical Rvsrarch Associate of the Medical Research Council of Canada.
188

Key words: Amino acids - Casein - Cholesterol-free, semipurified diets -Dietary protein
-Plasma cholesterol -Protein hydrolysates - Rabbits -Soy protein isolate

Introduction

Although experimental atherosclerosis is commonly produced by feeding

cholesterol, it has been known for some time that hypercholesterolemia and
atherosclerosis can also be produced in rabbits by feeding cholesterol-free,
semipurified diets [l-3]. These do not normally occur in rabbits on diets
commercially formulated from natural ingredients, and the effects were at first
attributed to a deficiency of essential fatty acids, because they were largely
prevented by including substantial amounts of polyunsaturated fat in the semi-
purified diets [3]. As pointed out by Kritchevsky [4], however, it was dif-
ficult to explain the differing effects of semipurified and commercial diets on
the basis of their fat content alone. This conclusion was substantiated by later
experiments of Kritchevsky and Tepper [5], who showed that extracting the
fat from commercial feed and adding it to a semipurified diet made little dif-
ference to the effects of these diets on either serum cholesterol or atherogenesis.
More recently, experiments in our laboratory have provided evidence that
the protein component of cholesterol-free, semipurified diets is primarily
responsible for their ability to produce hypercholesterolemia in rabbits [6,7].
Casein or other proteins from animal sources produced an elevation of plasma
cholesterol when fed in a cholesterol-free, semipurified diet, whereas the plas-
ma cholesterol remained low when the dietary protein requirement was
supplied by soy protein isolate or other plant proteins.
The present studies were undertaken to investigate in more detail the effects
of dietary protein on plasma cholesterol levels in rabbits. A number of addi-
tional plant protein preparations were tested and feeding trials were carried out
with diets containing mixtures of casein and soy protein isolate in different
proportions. The results of increasing the level of these proteins in the diet
were investigated and the effects of enzymatic hydrolysates of casein and soy
protein were compared with those of the intact proteins. Mixtures of L-amino
acids equivalent to casein or soy protein isolate were also fed to compare their
effects on plasma cholesterol levels.

Materials and methods

The experimental diets were fed for 28 days to male, New Zealand White
rabbits weighing initially 1.0 to 1.5 kg. The rabbits were housed individually in
metal cages with wire mesh bottoms, in an air-conditioned room with con-
trolled temperature and lighting. All rabbits were fed ground Master Feed
Rabbit Pellets for 4 to 5 days after arrival, and were transferred gradually to
the experimental diets over a 3-4-day period, as in earlier experiments [7].
Diets and water were provided ad libitum, animals were weighed weekly and
feed intake was measured over the 2%day period of the experiment.
The basal, low fat, cholesterol-free diet was similar to that used in earlier
 189

studies [7]. It consisted of casein 27, dextrose 60, salt mixture IV and cellu-
flour 5 parts by weight, to which were added 3 parts by weight of molasses
diluted 1 : 1 (v/v) with water. The diet also contained a supplement of water-
soluble vitamins, prepared as described previously [8], and a mixture of fat-
soluble vitamins, added in corn oil to provide a source of essential fatty acids
[71.
The vitamins, salt mixture (Phillips and Hart salt mixture IV [9], plus
cobalt) and “vitamin-free” casein were obtained from ICN Life Sciences Group,
Nutritional Biochemicals Division, Cleveland, Ohio. Dextrose monohydrate was
obtained from Teklad Test Diets, ARS/Sprague-Dawley Division of the Mogul
Corporation, Madison, Wis., and Celluflour from the Chicago Dietetic Supply
House, Chicago, Ill. Molasses was obtained from a local feed mill. The isolated
soy protein (Promine-R) was generously provided by Dr. E.W. Meyer of Central
Soya, Chemurgy Division, Chicago, Ill. Other plant protein preparations were
obtained as follows: detoxified rapeseed flour was kindly donated by Dr. J.D.
Jones, Food Research Institute, Agriculture Canada, Ottawa, Ontario; the oat
protein by Dr. R.E. Smith, Quaker Oats Co., Barrington, Ill.; the cottonseed
protein was obtained from the late Dr. C.M. Cater, Texas A and M University,
Oil Products Division, College Station, Texas; the sesame protein was kindly
provided by Mr. R.H. Anderson, Sesame Products, Inc., Paris, Texas; the sun-
flower protein by Dr. F.W. Sosulski, Crop Science Department, University of
Saskatchewan, Saskatoon, Sask.; and the pea protein and faba bean protein

TABLE 1

COMPOSITION OF THE CASEIN- AND SOY PROTEIN-EQUIVALENT AMINO ACID MIXTURES

The figures for casein were a composite derived from a number of different sources, and those for soy
protein were based on data provided for soy protein isolate by Dr. E.W. Meyer of Central Soya. In each
case, the values are reasonably close to those quoted by FAO [ 101.

Amino acid Composition (wt %)

Soy protein

L-alanine 2.1 3.9

Garginine 3.5 7.7
L-aspartic acid 6.5 11.7
L-cystine 0.4 1.2
L-glu tamic acid 20.2 20.2
Glycine 2.5 4.0
L-histidine 2.8 2.5
L-isoleucine 5.6 4.8
L-leucine 8.2 7.6
L-lysine HCl 7.3 6.0
L-methionine 2.6 1.1
L-phenylakmine 4.5 5.3
L-proline 10.0 5.2
L-serble 5.8 5.4
L-threonine 4.2 3.7
L-tryptophan 1.2 1.4
Gtyrosine 5.6 3.7
L+aliIle 6.5 4.7

100.1 100.1
190

preparations by Dr. C.G. Youngs, Prairie Regional Laboratory, National

Research Council, Saskatoon, Sask.
Enzymatic hydrolysates of casein and soy protein were obtained from the
Nutritional Biochemicals Division of ICN. According to the supplier, pancreatin
was used to prepare these hydrolysates. Total nitrogen and amino nitrogen
were reported as 12.7 and 4.9% respectively for the casein hydrolysate and
12.7 and 4.7% for the soy protein hydrolysate. Amino acid mixtures equivalent
to casein or soy protein isolate were formulated with L-amino acids obtained
either from General Biochemicals (now Teklad Mills) or from Ajinomoto,
U.S.A., Inc., N.Y. The composition of the mixtures is shown in Table 1.
Non-fasting blood samples were obtained by cardiac puncture from unanes-
thetized rabbits. Total plasma cholesterol was measured by the method of
Sperry and Webb [ 111. Total nitrogen of the protein preparations was deter-
mined by the micro Kjehldahl method [ 121, and their protein content was cal-
culated as Nz X 6.25. Statistical analyses were carried out as described
previously [ 71.

Results

In agreement with earlier findings [6,7], rabbits fed the low fat, casein-con-
taining, semipurified diet developed a significant hypercholesterolemia (P <
0.05) over a 28day feeding period, whereas in rabbits fed a similar diet con-
taining isolated soy protein, the plasma cholesterol levels remained low, as in
animals on a natural ingredient diet (Table 2). Both weight gain and feed intake
were significantly lower on the semipurified diets than on the commercial feed.
The results of experiments in which the casein in the semipurified diet was
replaced by proteins from various plant sources are also shown in Table 2. The
average plasma cholesterol levels of rabbits maintained on these diets for 28
days were all low, ranging from 30 to 100 mg/dl. Weight gains were variable
and there appeared to be no relationship between growth rate and level of
plasma cholesterol.
The effects of feeding different proportions of casein and isolated soy
protein in a semipurified diet are shown in Table 3. No elevation in plasma
cholesterol was observed with a 50 : 50 mixture of these proteins. The level in
rabbits fed a 75 : 25 mixture was intermediate between those obtained with
casein or soy protein isolate alone. The animals fed mixtures of these proteins
grew better than those fed either protein alone.
The results of doubling the amount of casein or soy protein isolate in the
semipurified diet at the expense of carbohydrate are shown in Table 4. Increas-
ing the casein gave a higher average level of plasma cholesterol, but the differ-
ence was not significant. Increasing the soy protein isolate had no effect on
plasma cholesterol level. Rabbits on the high casein diet failed to gain weight,
although there was no reduction in feed intake. Doubling the amount of soy
protein isolate in the diet did not cause any significant reduction in growth
rate.
Other experiments were carried out to investigate whether the differing
effects of casein and soy protein isolate on plasma cholesterol levels were
related to the structure or amino acid composition of the proteins themselves.
TABLE 2

EFFECT OF PLANT PROTEIN FROM DIFFERENT SOURCES ON PLASMA CHOLESTEROL LEVELS AND GROWTH PERFORMANCE OF RABBITS

Results given as mean ? SEM. Each dietary group contained 6 rabbits.

Diet % of protein a Initial Weight Feed Plasma cholesterol (mg/dl)

prep’n in diet weight gain intake
(g) (g/day ) (g/day> Initial 1’4 days 28 days

COlltWlS
ChS52iIl 27 1180f 40 14 * 1 58f I 91 f 21 137 2 16 204 ? 44
soy protein
isolate 27.5 1235? 45 15+ 1 642 3 42+ 7 63? 9 67? 7
Ground rabbit b
pellets 1212+ 88 23 ? 1 104? 5 642 7 65? 5 74? 9

Plant protein c
Rapeseed 42 1130? 60 6?2 64 ? 3 74? 9 96 + 13 99 * 11
oat 30 1654 ? 100 10 + 2 67 f 3 57f 8 74 ? 14 77 * 14
Cottonseed 41 1043 * 57 17 + 4 53 ? 6 126 + 28 88? 15 76 ? 14
Sesame 42 1508f 88 18? 2 83 f 2 43? 6 59+ 8 70f 5
SUnflOWe1 38 1308? 75 15+ 2 64 f 2 48? 7 57 +_ 14 53 ? 12
Pea 46 1245? 43 7+2 53 + 3 ,57 * 7 52? 8 41 + 11
Faba bean 42 1279 * 44 8?2 54 ? 6 53? 11 47 t 12 30?: 4

a Indicates the amount of nutrient present in the diet to give a protein content of 25% by weight. Associated non-protein material was added at the expense of car-
bohydrate.
b Master Feeds Division of Maple Leaf Mills, Ltd.. Toronto, Ont. According to the manufacturer. the pellets contained 15% protein (min). For further details of
composition see [ 71.
C Pea and faba bean protein preparations contained approximately 5% of naturaIly-occurring lipid. AU other protein preparations contained < 3% lipid.
192

TABLE 3

PLASMA CHOLESTEROL LEVELS AND GROWTH PERFORMANCE OF RABBITS FED DIETS CON-
TAINING MIXTURES OF CASEIN AND SOY PROTEIN ISOLATE

Results given as mean + SEM.

Dietary protein a Plasma cholesterol (mg/dl)

~_._~

Casein Isolated No. of Initial Weight Feed Initial 14 days 28 days

(W) SOY animals weight gain intake
protein (g) (g/day) (g/day)
(%)

100 0 6 1220? 46 14? 1 60? 2 52f 8 167 + 20 239 ?: 28

75 25 5 1423 + 109 20 f 1 65 f 3 76 f 27 168 f 21 138 + 20
50 50 5 1175? 71 21 * 2 73 f 4 52? 10 98 + 23 70 * 15
0 100 6 1200 + 53 16i 2 61 f 2 66 + 14 78 + 19 67 + 12

a Dietary protein represented 25% (w/w) of the total diets.

TABLE 4

PLASMA CHOLESTEROL CONCENTRATIONS AND GROWTH PERFORMANCE IN RABBITS FED

DIETS CONTAINING TWO DIFFERENT LEVELS OF CASEIN OR ISOLATED SOY PROTEIN

Results are given as mean f SEM for groups of 6 rabbits.

Dietary % of the Initial Weight Feed Plasma cholesterol (mg/dl)

protein diet weight gain intake
tg) (g/day) (g/day) Initial 14 days 28 days

Casein 27 1331? 47 18f 4 58 t 2 60f 15 185? 48 313 t 74

54 a 1325 + 57 -3 f 1 59 + 9 42?r 9 198 + 51 369 ? 88

Isolated
soy protein 27.5 1147 2 36 14 + 1 63 ? 3 60? 7 71+ 10 62 + 10
55 a 1179 f 77 11 ? 3 67 + 4 39 2 10 62? 15 58 ? 13

a Protein was increased at the expense of carbohydrate.

TABLE 5

EFFECT OF ENZYMATIC HYDROLYSATES AND AMINO ACID MIXTURES ON PLASMA CHOLES-

TEROL LEVELS AND GROWTH PERFORMANCE IN RABBITS

Results are given as mean f. SEM.

Diets a No. of Initial Weight Feed Plasma cholesterol (mg/dl)

animals weight gain intake
(g) (g/day) (g/day) Initial 14 days 28 days

Casein

Intact 6 1113? 69 14? 2 58? 3 402 5 165 f 29 213 + 53

Enzymatic
hydrolysate 6 808 + 36 11 ? 2 47+ 6 66 f 15 130? 18 178 + 30
Amino acid
mixture 9 1206f 69 13 f 2 60? 2 54f 8 159 f 39 213 f 42

SOY protein
Intact 6 1287 t 33 14 ? 1 60+ 2 61? 7 51r 8 69 ? 12
Enzymatic
hydrolysate 6 1251 f 110 12? 4 63 f. 18 6Oi 9 81 ? 11 41? 8
Amino acid
mixture 10 1254f 82 13 f 2 61f 2 56 ? 27 127t20 124f30

a The proteins. enzymatic hydrolysates and amino acid mixtures were added in amounts calculated to
provide dietary nitrogen equivalent to 25% protein.
 193

The results of feeding enzymatic digests of casein or soy protein were not sig-
nificantly different from those obtained with the intact proteins (Table 5). A
mixture of amino acids equivalent to casein (Table 1) also gave a result similar
to that obtained with casein itself. A mixture of amino acids corresponding to
soy protein isolate gave a higher average value than either the intact protein or
the enzymatic hydrolysate, but the differences were not statistically significant
(Table 5). The growth rates of rabbits fed the enzymatic hydrolysates or amino
acid mixtures were similar to those obtained with the intact proteins.

Discussion

The present experiments provide further evidence that the hypercholesterol-

emia normally obtained by feeding a cholesterol-free, semipurified diet to
rabbits can be prevented by using plant protein rather than casein as the dietary
protein (Table 2). They also show that soy protein isolate can partially counter-
act the hypercholesterolemic response to the casein-containing diet (Table 3).
An enzymatic hydrolysate of casein or a mixture of L-amino acids equivalent
to casein produced elevations of plasma cholesterol similar to those obtained
with the intact protein (Table 5). The result with the amino acid mixture
indicates that the hypercholesterolemia obtained with the casein-containing
diet is not due to any non-protein component of the casein preparation. It is
less clear, however, whether the ability of plant proteins to maintain low
plasma cholesterol levels can be attributed entirely to their amino acid com-
position. Rabbits fed a mixture of L-amino acids equivalent to soy protein
isolate showed a tendency to develop a moderate hypercholesterolemia relative
to those fed the intact protein or the protein hydrolysate, although the differ-
ences were not statistically significant with the numbers of animals used (Table
5).
A number of different factors must be considered in evaluating these results.
For example, it is unlikely that digestion of an intact protein will provide
amino acids in exactly the same proportions and time sequence as feeding an
equivalent amino acid mixture. Thus, the consistently low plasma cholesterol
levels obtained with soy protein isolate may be related to the way in which the
amino acids are released during digestion. If dietary amino acids are responsible
for the observed effects on plasma cholesterol, it seems more probable that
these effects are due to an imbalance of amino acids, rather than a deficiency
of any particular amino acid, since doubling the amount of soy protein isolate
in the diet made no difference to the results obtained (Table 4). Methionine is
the first limiting amino acid in soy protein, but supplementation of soy protein
diets with methionine seemed to have relatively little effect on the plasma
cholesterol level [ 6,7].
It is perhaps unwise to make a clear distinction between dietary proteins of
plant and animal origin with respect to their effect on plasma cholesterol levels
in rabbits. In our earlier studies, some animal proteins failed to give a signifi-
cant hypercholesterolemia [7] and Hermus [ 131 observed that rabbits fed a
mixture of casein, milk and fish protein had lower serum cholesterols than
rabbits fed casein as the sole source of protein.
It should also be kept in mind that the protein content of many of the plant
194

preparations used in our experiments was only 55 to 65% of their total weight.
Although the preparations contained little or no fat, it cannot be concluded
that the protein itself was necessarily responsible for the low levels of choles-
terol observed. Most of the associated non-protein material is probably carbo-
hydrate and studies in our own and other laboratories have shown that dietary
carbohydrate can influence the level of plasma cholesterol [6,7,14,15].
In the present studies, as in earlier experiments [7], there appeared to be
little or no relationship between weight gain and plasma cholesterol levels in
rabbits on the different diets. The growth rates with some of the plant protein
preparations were equal to or better than those obtained with casein (Table 2).
In other cases, the growth rate was only about half that of rabbits fed the
casein diet. Mixtures of casein and soy protein isolate gave better growth than
either preparation alone (Table 3) in agreement with other reports [ 13,161 that
rabbits grow better on mixtures of proteins from different sources.
Doubling the amount of casein in the diet to provide 50% by weight of
protein led to weight loss associated with a high plasma cholesterol level (Table
4). It is possible that the weight loss may have had some influence on plasma
cholesterol since Swaner and Connor [ 171 observed a hypercholesterolemia in
rabbits losing weight during a period of starvation. The reason for the weight
loss in our experiments is not clear, since doubling the amount of soy protein
isolate had little effect on either growth performance or plasma cholesterol
level. Excessive amounts of certain amino acids can depress weight gain in
rabbits [ 181 and casein is richer in some of these amino acids than soy protein
isolate. It is possible than an imbalance of amino acids in casein is accentuated
at this higher level.
The unusual mineral requirements of rabbits [13,19,20] may also have had
an influence on the growth rate of animals fed the different diets. However, in
our earlier studies a change to a salt mix recommended for rabbits [21] had no
apparent effect on either weight gain or plasma cholesterol level [7].
It is evident that more work is required to determine which specific compo-
nents of the different dietary protein preparations are influencing the level of
plasma cholesterol in rabbits. The present experiments suggest that the amino
acid composition of the proteins is one of the factors involved, but do not rule
out the possibility that non-protein constituents of these preparations may also
be at least partly responsible for some of the observed effects.

Acknowledgements

Technical assistance by L.F. McPhee, K. Pamplin and R.M. Rasmussen is

gratefully acknowledged.

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