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Effects of a soy protein product on serum and tissue cholesterol concentratins in swine fed high-fat, high-cholesterol diets
EXPElUhIENTAL
AND
MOLECULAH
29, 385-399
PATflOLO(;Y
( 1978)
Effects of a Soy Protein Product on Serum and Tissue Concentrations in Swine Fed High-Fat, High-Cholesterol D. N. KIM, K. T. LEE, J. M. REINER, Department
of Pathology, The Neil Albany Medical College,
Received
June
26,1978,
Hellman Albany,
AND
W. A. THOMAS
Medical Research New York 12208
and in revised
form
Cholesterol Diets
July
Building,
24, 1978
Hypocholesterolemic effect of a soy protein product was studied in swine fed a high-fat, high-cholesterol diet. In the first experiment, a group of swine were fed 42% butter (by calories) and 1055 mg cholesterol daily, with casein as the source for protein, for 6 weeks and this diet resulted in moderately high serum cholesterol concentrations (219 2 33 mg/dl). Another group fed the same diet except with soy protein product as the protein source instead of casein showed virtual normocholesterolemia at the end ( 107 -C 3 mg/dl). Cholesterol balance was studied under non-steady state conditions using methods designed for this purpose. Reflecting the serum cholesterol concentration, the total body cholesterol concentration (excluding CNS ) was also significantly lower in soy protein group. However, parameters of cholesterol balance, such as fecal neutral and acidic steroid excretions, dietary cholesterol absorption, and whole body cholesterol synthesis were studied and no differences were demonstrated between the caseinand soy protein-fed swine. The experiment was repeated and in Experiment II virtually the same results were obtained. When swine were given the same high-fat, high-cholesterol diets with + casein + 3 soy protein or casein + soy protein, hypocholesterolemic effects were also observed. Therefore, such action is probably caused principally by soy protein per se rather than simply by replacement of casein by soy protein. Addition of DLmethionine to soy protein containing diet did not alter the hypocholesterolemic effect of soy protein indicating that the effect was not the result of methionine deficiency. In conclusion, we can state that the hypocholesterolemic action of soy protein was clearly demonstrated in swine fed a high-fat, high-cholesterol diet, but that the mechanism of action is yet to be established.
INTRODUCTION Hypocholesterolemic role of vegetable proteins has been reported sporadically for the last few decades in rabbits (Meeker and Kesten, 1940, 1941; Howard et aE., 1965; Carroll and Hamilton, 1975) and in humans (Olson et al., 1958; Walker et al., 1960). However, such an effect in humans has not been confirmed by all investigators (Keys and Anderson, 1957; Anderson et al., 1971). Hamilton and Carroll (1976) have observed that hypercholesterolemia develops in rabbits when a semi-purified diet containing casein, carbohydrate, lowfat, and cellulose without cholesterol is fed. The hypercholesterolemic effect disappears and a low serum cholesterol level is maintained when casein is replaced by vegetable proteins. Among the vegetable proteins, soybean protein 385 0014-4800/78/0293-0385$02.00/O All
Copyright @ 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
35%
KIM
ET AL.
exhibited the most strikiu g eifect in the rabbit. Since soy protein coutains less methionine than casein (animal proteins), the above investigators supplemelltcd the soy protein containing diet with methionine. Such supl>lementation of methionine resulted in a small increase in serum cholesterol levels. However, the overall results indicated that a relative lack of methionine in soy protein was not the principal reason for the hypocholesterolemic action of soy protein in rabbits fed a cholesterol-free semi-purified diet. No explanation for such action was given. Recently, Sirtori et al. (1977) reported hypocholesterolemic effects in type II hyperlipidemics fed a moderately low-fat, low-cholesterol diet with soy protein as the protein source. Similar results were obtained when cholesterol was added to the diet (500 mg/day ). Studies of the effect of soy protein in diets which are high in both fat and cholesterol have not been reported to our knowledge for either man or experimental animals. Therefore, the current study was designed to observe whether or not such hypocholesterolemic effects could be demonstrated in swine fed high-fat, high-cholesterol diets. In addition, we wanted to gain some insight into the mechanism of action. Swine were chosen for study because they respond to high-fat, high-cholesterol diets in much the same way as man and because we have accumulated considerable data on cholesterol turnover in this species that would serve as background for judging the effect of soy protein. In the current study we have used the term “soy protein” to describe a specific product from Miles Laboratories called Pro-Lean 45’“‘. This is a mixture of “soy protein concentrate, spun isolated soy protein, isolated soy protein, and hydrolyzed vegetable protein (1.93% ) with sugar and salt.” The dietary fibers have been largely eliminated and the product contains only 4.33% as neutral detergent fiber. Analysis of the lot used for the current experiment by Miles Laboratories shows 62.5’;r’ protein, 0%’ fat, 6.8% moisture, 8.6% ash, and 22,1c/ carbohydrate (Lot #450-2467-l). In the future, we plan to study effects of purified components, especially the protein component. However, the current study is aimed specifically at studying the Miles Pro-Lean mixture. MATERIALS Experimental
AND METHODS
Design
Two experiments were carried out in succession (see Table I). Experiment 1. Fifteen swine were divided into three dietary groups: one fed a commercial mash, and two semi-purified hyperlipidemic (HL) diets containing fat and cholesterol, one with casein and the other soy protein as the sources for proteins. These groups will be called mash, casein, and soy protein groups. Swine were fed the respective diets for 6 weeks. Initial and weekly determinations of body weight and serum cholesterol concentrations were carried out. Feces were collected during the terminal week for the analyses of neutral and acidic steroids. Terminally, swine were sacrificed and triplicate samples of liver, small intestine, muscles, adipose tissue, skin, and aorta were obtained for cholesterol determinations. The remainder of the carcasses (excluding CNS) were homogenized to obtain total body cholesterol concentra-
SOY
PROTEIN
-4ND
SERUM
TABLE Groups, Experiments I
II
Groups
Number
of Swine,
designations
Duration No. of swine 5 6 5
HL HL HL HL HL
6 5 6 5 6
casein soy protein tC + $3 C + S” soy protein
+ methe
387
I
Mash HLa + casein HL + soy protein
+ + + + +
CHOLESTEROL
and Measrrrements Duration (week)
A
Measurements
Serum and tissue cholesterol Body weights Cholesterol balance excretion, retention, synthesis Cholesterol”
4
absorption
Serum cholesterol Body weights
a Semi-purified hyperlipidemic diet containing fat and cholesterol. D Cholesterol absorption was determined only in three swine each from Experiment II, HL + Casein and HL + Soy protein. c One-half dose of casein and + dose of soy protein. d Casein and soy protein in full doses. E DL-methioninc supplemented.
the first
two
groups
of
tions (Kim et al., 1974). Whole body cholesterol synthesis was calculated from the cholesterol balance equation under non-steady state conditions: synthesis + dietary intake = retention + excretion. Experiment II. Twenty-eight swine were divided into five groups. Two groups were a repeat of the casein and soy protein groups of Experiment I. A third group received the same amount of protein as the first two groups but from two sources, one-half from casein and the other half from soy protein ($ casein + * soy protein). A fourth group was given full amounts of casein and soy protein, hence doubling the protein content of the diet as compared to the other groups. The fifth group was given the soy protein diet with DL-methionine. The swine were fed the respective diets for 4 weeks. Body weights and serum cholesterol concentrations were monitored weekly. At 5 weeks, three swine each from the first two groups, casein and soy protein, were used for the determinations of dietary choIestero1 absorption. Animals. Forty-three young male Yorkshire swine weighing approximately IO kg were housed individually in slat-bottomed cages. They were placed on a commercial mash diet for 1 to 2 weeks while they became acclimated to the laboratory. Then they were assigned to the respective dietary groups. Diets. The compositions of the HL diets with casein and soy protein and mash diet are shown in Table II. Both HL diets are nearly isocaloric. The amounts of protein in both casein and soy protein are based on the analyses Casein contained 90% protein and Prodata supplied by the manufacturers. Lean contained 62% protein, 076 fat and 2270 carbohydrates. Total cholesterol in both diets is the same (1055 mg) with I33 mg from butter and the remainder Other supplements include plant sterols from added crystalline cholesterol. (Cytelline, Lilly, IN) and chromium oxide. Plant sterols, total 570 mg including 336 mg p-sitosterol, were added to all HL diets as the marker for the loss of
3ss
.KIM
ET AL.
TABLE Composition
II of Diets -~~
Masha Corn oil Butter Corn starch Casein* Pro-Leant Salt mix Vitamin mix Non-nutritive Total Total
fiber” 7UO
(g) calories
Percent calories Fat Protein Carbohydrate
(&al)
HI,
+ rasein
HL
+ soy protein
10 R 90 a00 130
10 90 150 -
4 2 104
180 4 2 104
540
540
Sl(iY
2110
as 41.5 21.6 30.9
42.7 2 1.3 X.0
a Hog mash (Agway). * 9O7oproteins. Miles Laboratories, Inca., c Textured soy protein prodrlc%. Pro-Lean 4n-TjI, Lot No. 450-24137-1, Elkhart, IN. Protein W2.3yc, fat OS;, carbohydrate X2.17& moisture 1;.8(;<, and ash S.67; with ~.XYS~~ neutral detergent fibers. d Cellulose type. Both HL diets were supplrmentrd with sufficient cholesterol to make the daily total 1055 mg. In addition, 570 mg of plant sterol and 1 y of chromium oxide were added.
neutral steroids during intestinal transit. The mash group did not receive these plant sterol supplements since the mash diet contained 569 mg of plant sterols with 426 mg of p-sitosterol. One gram daily of chromium oxide was given to all swine in Experiment I as a marker for fecal flow and recovery. Such markers were not required in Experiment II, but plant sterol was added to make the dietary conditions of Experiment I and II the same. The fifth group of Experiment II received a soy protein containing HL diet supplemented with DL-methionine to make the total methionine content equal to that contained in the casein diet (2.5 g/day or 2.2% of protein in the diet). Feeding. All swine were fed once daily at 9 AM. Crystalline cholesterol, plant sterol mixture, and chromium oxide were weighed out separately. These three ingredients were added to each diet bowl with approximately l/3 of the daily diet mixture plus water. As soon as this first bowl was consumed, the remaining diet mixture was given. Adequate amounts of water and repeated mixing is essential to prevent waste of the diet. When all of the diet was consumed, the bowl was filled with water. Total daily water intake was approximately 1.5 liters. Cholesterol Balance Stucly A method was developed previously for the study of cholesterol balance of swine under non-steady state conditions (Kim et al., 1974, 1975). This method enables us to calculate whole body cholesterol synthesis by direct measurements
SOY
PROTEIN
AND
SERUM
389
CHOLESTEROL
of steroid excretion and cholesterol retention by the following equation: synthesis = dietary intake + retention - excretion. Daily excretion was determined from the feces with minor correction for the losses through routes other than feces. Daily cholesterol retention was obtained from the terminal total body cholesterol concentration using the body cholesterol concentration of comparable swine at two different points in time. Fecal collection and analyses. Feces were collected during the terminal week in daily lots, processed, and pooled; neutral and acidic steroids were analyzed by the method described previously using thin layer chromatography and gasliquid chromatography (Grundy et al., 1965; Miettinen et al., 1965; Marsh et al., 1972; Kim et al., 1974). Neutral steroid loss during the intestinal transit was corrected for by p-sitosterol recovery (Grundy et al., 1968) and fecal flow was corrected for by the chromium oxide recovery (Davignon et al., 1968). Total body cholesterol concentrations. Whole carcass, minus CNS and intestinal contents, was homogenized by the method described previously (Kim et al., 1974) and multiple aliquots were taken for analyses. Cholesterol &terminations. Serum total cholesterol was determined by the method of Leffler ( 1959). The tissue cholesterol was measured by the same method after Folch extraction (Folch et al., 1957). Cholesterol concentration was expressed as free cholesterol. Cholesterol
Absorption
Cholesterol absorption was measured by the double isotopic single feeding method similar to that described by Sodhi et al. ( 1974), Borgstriim ( 1969), Method IV of Quintso et al. (1971) and the fecal radioactivity method of Samuel et al. (1978). A mixture of 8 &i of ( 3H)22, 23-P-sitosterol (sp. act. 58 Ci/mmole; Amersham/Searle) 4 &i of (l’C)4-cholesterol ( sp. act. 55.5 mCi/ mmole; NEN, Boston), and 500 mg of red Carmin was used to mark the diets at usual meal time, 9 AM. Red Carmin was used to mark the fecal buIk (Sodhi TABLE Body
III
Weights and the Terminal after 6 Weeks of Feeding Rlash
No.
of swine
Body weights Initial Final Liver Liver/body k/W
Liver Weights in b:xperiment HL
-5
of Swine I
+ casein
HL
+ soy protein
5
5
(kg) 9.7 f 2.ou 19.6 z+z 1.3
(g)
377
zk 24
10.0 f 16.0 + 291
f
1.7 2.3 42
10.5 zt 25.8 f 401
f
1.8 0.6D 24~
weight 19.2 f
0.2
a Mean f standard error of mean. * Significantly different from both mash and casrin mash and casein groups. c Differences among three groups arr not significant. d Significantly different from both mash and casein mash and casein groups.
15.3 f
0.7
15.5 +
0.P
grollp
(P < 0.01).
No difference
between
groups
(P
No difference
between
< 0.05).
KIM
390
ET AL,.
et al., 1974). Twenty-four hours later, swine were sacrificed by intravenous injections of Pentobarbital sodium. Stomach, small intestine, and large intestinal contents were collected in two fractions; proximal fraction stained with Carmin and the distal unstained fraction. A sufficient amount of borderline fraction was included in the proximal fraction to ensure that all radioactivity was recovered in that fraction. All intestinal contents were processed and neutral steroids extracted as described under fecal steroid analyses. Both (l,C) and ( 3H) radioactivities were measured. The distal fraction from the large intestine contained a negligible amount of radioactivity. Absorption was calculated from the equation (1 - 14Crecovery) x loo 3H recovery
Absorption =
Beta-sitosterol recovery was used to correct for the loss during the intestinal transit as described earlier. The radioactivity in the bile acid fraction of the large intestine was 7.570 of total 14C activity. RESULTS Experiment I. AII swine consumed the entire daily rations and grew well. Body and the terminal liver weights of swine after 6 weeks of feeding are shown in TabIe III. The terminal body weights were greater (P < 0.01) in HL + soy protein group (25.8 t 0.6 kg) than mash ( 19.6 -t 1.3 kg) and HL + casein (16.0 -C 2.3 kg) groups. Difference between mash and HL + casein groups was not significant. The terminal liver weights among the three groups were not significantly different, although the average value for the HL + soy protein group (401 -t 24 g) was larger than both mash (377 * 24 g) and HL + casein (29lk 42 g) groups. When the liver weights were expressed as g/kg body weight, HL + soy protein group (15.5 2 0.8 g/kg) was significantly lower (P < 0.05) than both mash (19.2 * 0.2 g/kg) and HL + casein (18.3 * 0.7 g/kg) groups.
250
-
200
-
60 f ‘0
12
I
FIG. 1. Serumcholesterolconcentrations(mg/dl, (n=5),
HL -t soy protein
(n=5)
and
3I 4 WEEKS
mean
5I
6
I+ standard
HL + casein groups (n=5)
error
of mean)
in Experiment
of mash
I.
SOY
PROTEIN
AND
SERUM
TABLE Initial
No.
of swine
HL + soy protein C
5
85 z!z 8’ 9.5* 4
intake
(mg/day) (mg/day)
0
Total (mg/day)
Comparisons 24 ~8 B
-4 “8 c
B “S c
5
error
CNS)
97% 3 219 + 33
s5* 107
5 f
3
-
P <
1055
1055
P < P <
-
624 f 372 *
14 38
617 312
832
f
58
996
f
47
929
f
57
n.s.
* 61
203
3~ 54
236
f
62
P < 0.001
f
964
f
61
P < 0.05
f
23
1233
47
28 36
-
P < 0.05
-
f35 rt 32
1078
i f
0.01
284 547
1121
Whole body cholesterol (mg/kg) (excluding = Mean ziz standard 6 Neutral steroids. c Bile acids.
+ casein B
Concentrations and Cholesterol Week (Experiment I)
(mg/dl)
Fecal excretion NS B.4c
Synthesis
HL
5
Serum cholesterol Initial Final Dietary
IV
and Terminal (6 Weeks) Serum Cholesterol Balance Parameters during the Terminal Mash A
391
CHOLESTEROL
0.001 0.01
P < P <
-
0.001 0.01
n.s.
P <
P < 0.01
0.001
n.s.
n.s. n.s. n.s. n.s.
P < 0.02
of mean
Serum cholesterol concentrations (Fig. 1 and Table IV). Serum total cholesterol concentrations during the 6 weeks feeding period are shown in Fig. I. HL + casein group showed significant increase in cholesterol level after 1 week and remained elevated, whereas HL + soy protein group showed fluctuation in serum cholesterol at lower levels. The terminal levels for HL + casein, HL + soy protein, and mash groups were 219 +- 33, 107 k 3, and 95 + 4 mg/dl, respectively. The value for the HL -+ casein group was significantly higher (P < 0.01) than that of either the HL + soy protein or the mash group. A statistically significant (P < 0.05) though small (12 mg/dl) difference was noted between HL + soy protein and mash groups. Fecal excretions of neutral and acidic steroids (Table IV). Neutral steroid excretions were greater (P < 0.001) in both HL + casein (624 * 14 mg/day) and HL + soy protein (617 2 28 mg/day ) groups than in the mash group (284 t 35 mg/day ). Bile acid excretion in the mash group (547 + 32 mg/day) was significantly greater (P < 0.01) than in both HL + casein (372 * 38 mg/day ) and HL + soy protein (312 * 36 mg/day ) groups. No difference was noted in total fecal steroid excretions among HL + casein (996 2 47 mg/day ), HL + soy protein (929 f 57 mg/day ), and mash (832 * 58 mg/day) groups. Whole body cholesterol spthesis (Table IV). Whole body cholesterol synthesis of both HL + casein (203 2 54 mg/day) and HL + soy protein (236 * 62 mg/day ) were significantly lower (P < 0.001) than in the mash group (1121 * 61 mg/day ). Whole body cholesterol concentrations (Table IV). Whole body cholesterol concentrations (excluding CNS) of I-IL + casein group ( 1233 * 47 mg/kg) was significantly (P < 0.05) higher than that of the mash group (1078 * 23 mg/kg) and HL + soy protein (964 +- 61 mg/kg). The difference between HL + soy protein and mash groups was not significant.
392
KIM
ET
AL.
TABLE Terminal
Tissue
Cholesterol
V
Concentrations
after
6 Weeks
Mash No.
of swine
HI,
(tSxperiment
+ caascin
.5
HL
LiveP Ileum Skin Muscle Fat
1.78 2.12 1.37 0.68 0.96
Aorta Arch Thora& Abdominal
0.94 * 0.08 0.9s It 0.0s 1.04 zt 0.07
a-&
I)
+ soy protein
5
mg/g * + zt dz It
of Feeding
5
weight
0.10* 0.12 0.06 0.03 0.06
zk 0.11
2.44
f
0.24
+
0.04
1.90 f
0.18
2.03 f 1.3.5 f
0.16 0.07
0.77 0.9“
0.06 o.or,
0.66 0.90
f f
0.03 0.09
0.96 & 0.05 I .I 1 + 0.01
0.90
+
0.07
I.18
1.10
2.76 2.22
n The only statistically significant differences are cholesterol (P < 0.01) and mash vs HL + soy protein (P < 0.05). * Mean & standard error of mean.
f *
1.00 ?lz O.Oti
f 0.15 - ~-~.
conumtrations
f
mash vs HL
0.06
+ casein
Cholesterol concentrations in selected tissues ( Table V). The only significant difference in the selected individual tissue concentrations among the three groups was observed in the liver. Both HL + casein (2.76 f 0.41 mg/g ) and HL + soy protein (2.44 * 0.24 mg/g) groups showed a significant increase (P < 0.05) as compared to the mash group (1.78 k 0.10 mg/g). The difference was not significant between casein and soy protein groups. The other organs, ileum, skin, muscle, and adipose tissue, did not exhibit any significant differences among the three groups. Aortic cholesterol concentrations also were not different. Experiment
II
All swine consumed all of the daily rations and grew well. The initial and terminal body weights are shown in Table VI. All groups showed fairly uniform growth during the experiment except for HL + casein group. The net inTABI,IS Initial
(;roaps
VI
and Terminal (4 Weeks) Serum Cholesterol Conccntratiorls and Body Weights (li;xpcriment II) No. of swine
Body
wclight
(kg)
8rrlun
cholesterol
Initial
Terminal
Initial
Terminal
HL + casrin HL + soy protein HL + +C + $5 HL+C+% HI, + soy protein + mcthioninr n Mean b Valllrs
f
(mg/dl)
--
standard rrror of mean. for all grol,ps were significantly:
owcr
t,h:m
HL
+ cnscxin gro,qj
(I’
< 0.05).
SOY
PROTEIN
AND
SERUM
lEOl 1 250
of
I 0
393
CHOLESTEROL
HL+cosein t
1
2
3
4
Weeks FIG. 2. Serum cholesterol casein, HL + soy protein, periment II.
concentrations (mg/dl, mean 2 standard error of mean) of HL + HL + C + S, HL + ?C + $S, HL + S + methionine groups in Ex-
crease in body weight during 4 weeks of study of HL + casein group was 6.7 kg and the remaining groups had values ranging from 9.9 to II.7 kg. Serum cholesterol concentrations are shown in Fig. 2 and Table VI. Serum cholesterol concentrations of HL + casein group were elevated significantly after 1 week and continued to rise, reaching 238 I+ 33 mg/dl at the end of 4 weeks. The HL + soy protein group showed a slight elevation of serum cholesterol level after 1 week but gradually declined to 106 k 9 mg/dl at the end. These responses are essentially the same as those observed in Experiment I. The HL + ‘,C + $S and HL + C + S groups revealed a fluctuation and more variation in their serum cholesterol levels, but the terminal values (142 t 11 and 146 f 24 mg/dl) were significantly lower than HL + casein group (P < 0.05). The group supplemented with methionine (HL + soy protein + nL-methionine) also showed a significantly lower serum cholesterol concentration than HL + casein group (P < 0.01). Dietary cholesterol absorption was measured in three swine from each of HL + casein and HL + soy protein groups terminally (Table VII). Although the
TABLE Dietary
Cholesterol Casein
Absorption in Swine Fed HL Diet or Soy Protein (Experiment II) HL
No. cd swine ‘x, absorption I* Mean
& standard
error
VII
of mean ; difference
+ casein 3
50 *
HL
with
+ soy protein 3
3
not statistically
Either
42 It 5 significant.
394
KIM
ET AL.
average value of the IIL + soy protein group (4‘2 * 5(;/,!) was lower than for the HL + casein group ( 56 5 3% ), the difference was not significant. DISCUSSION The main question we proposed to answer in this study was whether the substitution of a soy protein product for milk protein would prevent hypercholesterolemia in swine fed a high-fat, high-cholesterol diet. A profound hypocholesterolemic effect was demonstrated in two separate experiments when the soy protein product was substituted for milk protein (casein) in the diet, A second question was whether the soy protein product would prevent the increase in concentration of cholesterol in the whole carcass that we have previously noted when casein was the source of protein in high-fat, high-cholesterol diets. The results in Experiment I, where carcass cholesterol contents were determined, indicate that there was no increase over mash concentration in the soy protein product group in contrast to the significant increase observed with the casein. A third question that we hoped to answer was which of the parameters of cholesterol balance was (were) altered by the soy protein product in such a way as to produce the observed effect on concentration of cholesterol in the serum and whole carcass. We were not entirely successful in answering this question, but we did gain some insights that should form a basis for planning future experiments. The balance parameters to be considered are (1) whole body cholesterol synthesis, (2) absorption, (3) excretion of bile acids, (4) escretion of neutral steroids, and (5) retention in the carcass.
1. Whole bony cholesterol synthesis. Synthesis is profoundly and equally depressed by both the casein and the soy protein high-fat, high-cholesterol diets. Thus, alteration in synthesis cannot account for the observed effects of the soy protein product. 2. Cholesterol ahsorptiorl from the gut. Cholesterol absorption was not significantly altered by the soy protein diet as compared to the casein diet even though there was a numerical difference. However, the number of swine was small and this aspect needs to be further studied in future esperiments. With low-fat mash diets practically no cholesterol is absorbed even when a large amount is added to the diet (Marsh, et al., 1972). 3. Excretion of bile acids in the feces. Increase in excretion of bile acids is one of the ways in which drugs such as cholestyramine produce their hypocholesterolemic effect. No such effect on bile acid excretion was observed in the current study with soy protein. This aspect should perhaps be pursued in other types of esperiments, but it seemsunlikely that increases in bile acid escretion will be found. 4. Excretion of neutrul steroids in the feces, Neutral steroids in the feces can come from unabsorbed dietary or excreted endogenous (absorbed and synthesized) cholesterol. Increase in excretion of unabsorbed cholesterol can result only if absorption is decreased. Thus, the comments regarding absorption apply equally to escretion of unabsorbed cholesterol. Endogenous and unabsorbed dietary cholesterol could not l)e separated with the design used in the current studies. The fact that the total cholesterol escre-
SOY PROTEIN
AND
SERUM
CHOLESTEROL
395
tioli iii the feces was not increased makes it seem unlikely that cscretioii of either endogenous or unabsorbed dietary cholesterol was increased. IIowever, this aspect needs to be investigated further for reasons that will be given in the discussion of whole body cholesterol retention. 5. Whole body cholesterol retention. We have already stated that the soy protein product prevented the increase in concentration of cholesterol in the carcass that was observed with casein high-fat, high-cholesterol diet. However, for unknown reasons in the experiment in which the balance study was done, the soy protein product-fed swine grew considerably more than the casein-fed. As a result, the total amount of cholesterol retained as part of normal tissue was considerably greater in the soy protein product-fed than in the casein-fed swine. This was the only factor in the balance study that was significantly different between the two groups. We might conclude from this that the hypocholesterolemic effect resulted entirely from normal utihzation of cholesterol to form the greater tissue mass. However, in Experiment II (where a balance study was not done) there was somewhat less disparity between growth rates and yet the same profound hypocholesterolemic effect of the soy protein product was observed as in the first. This aspect needs to be investigated in the future in pair-fed experiments designed to maintain similarity in growth rates. We may find in such experiments that the rate of excretion of endogenous cholesterol into the feces is increased as it is in clofibrate-treated swine on high-fat, high-cholesterol diets (Kim et al., 1975). If such were found we might conclude that the “efficiency” in clearing cholesterol from the body is increased by the soy protein diet. Viewing the results from another aspect, the differences in effects on cholesterol concentrations of the soy protein product as compared to casein might be a result of: (1) positive depressant effect of soy protein product with casein being inactive, (2) positive elevating effect of casein with soy protein product being inactive, or (3) active effect of each in opposite directions. The groups fed mixtures of the two proteins were chosen in an attempt to differentiate among the above possibilities. The results are not entirely conclusive. Mixing casein and soy protein product with amounts of each half of that used when each was given alone resulted iu significantly lower serum cholesterol levels than when casein was given alone in full amount. A similar result was obtained when the two were combined with each given in the quantity as when given alone. If the results are taken literally, this would seem to mean that the soy protein product was exerting a positive cholesterol lowering effect and that casein was inactive in this regard. However, the serum cholesterol values with the combination were numerically (though not significantly) higher than with mash or soy protein product alone. More experiments with larger numbers of swine need to be done before we can conclude that casein is inactive in regard to serum cholesterol levels and that the hypocholesterolemic effect is produced entirely by direct action of the SOY protein product. A number of possibilities regarding mechanism of action of soy protein on cholesterol balance have been entertained by others in studies of the effect of soy protein on serum cholesterol levels in rabbits and man. Factors that have been considered include: (1) fiber content, (2) relative lack of methionine and/
396
KIM
ET AL.
or imbalance in other amiiro acids, ( 3) plaint sterols, (4) sapoiiin, and (5) otlrer factors native to soy beans. Helm (1977) upon reading the report of Sirtori et (11. ( 1976) in man on the effect of a low lipid modified diet containing soy protein product similar to Pro-Lean used here, suggested that the hypocholesterolemic effect may have resulted from the presence of “indigestible non-cellulose polysaccharides,” which could cause an increase in bile acid excretion. Gatti and Sirtori (1977) dismissed such a possibility on the ground that the quantity of such fiber was too According to the analysis supplied by the manusmall to be of significance. facturer, the soy protein product used in the current study, contained 4.55% of neutral detergent fiber and this quantity (8.2 g daily in the current study) in our previous experience with swine, is too small to produce any effect on bile acid excretion. Also, in the current experiments, there was no difference in fecal bile acid excretion between casein and soy protein groups. Another point frequently considered in various studies with soy protein is a relative lack of methionine in soy protein. However, the current study showed that a methionine supplementation in swine did not increase serum cholesterol o b served some increase in serum levels. In rabbits, Hamilton and Carroll (1976) cholesterol levels when either soy protein concentrate or isolate were supplemented with methionine, but such supplementation resulted in an increase of serum cholesterol levels only up to the level of those rabbits fed commercial chows only. Therefore, they concluded that a relative lack of methionine in soy protein may be part of but not the principal reason for the hypocholesterolemic effect. Similarly, Gatti and Sirtori (1977) observed a lack of effect on serum cholesterol of methionine supplementation to the soy protein diet. It becomes obvious when one inspects the amino acid compositions of casein and soy protein, that a relative lack of methionine is not the only difference between these two proteins. Kritchevsky et al. (1978) h ave noted the fact that soy protein contained more arginine than casein and that the arginine/lysine ratio g-fold in soy protein as compared to casein. They observed was approximately that atherogenicity of semi-purified diets can be affected by addition of specific amino acids in rabbits. Another possible factor is the plant sterol content. Although we have not demonstrated in swine that plant sterols affect serum cholesterol levels, such effects in humans have been reported repeatedly (Best et al., 1955; Farquhar et al., 1956; Farquhar and Sokolow, 1958; Grundy and Mok, 1977). In the current study all of the diets, except for the mash diet, were supplemented with 570 mg of plant sterols of which 336 mg was ,&sitosterol. This quantity is similar to that contained in the mash diet. On analysis, the soy protein product contained 4 mg/lOO g of plant sterols and 3 mg/lOO g of p-sitosterol. Therefore, plant sterol content in the soy protein containing diet in the current study was 7.2 mg. It seems very unlikely that such a quantity can be of any significance. Soybeans in their native state contain “antinutritional factors” for rats. These can be divided into heat labile and heat stable factors. Among heat labile factors are trypsin inhibitor, hemagglutinins, phytate, goitrogens, and anti-vitamin factors. The heat stable factors included saponins, estrogens, flatulence factors, and 1977). Since the isolation process for the soy protein lysinoalanine (Leiner, various heat labile factors are destroyed. product involves heat treatment,
SOY
PROTEIN
AND
SERUM
CHOLESTEROL
387
Therefore, such factors can be omitted from the list of potential factors. Among the heat stable factors that arc considered to be “antinutritional,” estrogens and flatulence factors can be omitted because the estrogenic activity is present in too low a concentration to be of any physiological significance, and processed soy protein products are essentially devoid of flatus activity. Remaining factors, thus, are saponin and lysinoalanine. The term “antinutritional” was used by Leiner (1977) to include a poor protein efficiency ratio, other specific deficiency states and pathologic changes. In certain respects, such “antinutritional” effects might be exploited to advantage, if reduction in utilization of certain dietary ingredients is desired. The role of saponin as an “antinutritional” factor has been disclaimed in other studies with chicks, rats and mice. However, recent studies with alfalfa saponins in rats and monkeys by Malinow et al. (1977a, 1977b) compel us to look further into the role of saponin; they have demonstrated reduction in dietary cholesterol absorption in rats fed alfalfa top or root saponins, in both native and hydrolyzed states. The current study does not permit us to speculate regarding such effect on absorption, because there was no significant difference in cholesterol absorption between casein fed and soy protein fed groups. According to the manufacturer, the saponin content of the soy protein concentrate is approximately 0.4%. Therefore, the approximate content of saponin in the soy protein containing diet in the current study is 720 mg. Since the saponin content in the studies of Malinow et al. (1977a, 1977b) was ten times that of dietary cholesterol, 720 mg of saponin in the current study with 1055 mg of cholesterol probably was not sufficient to affect the cholesterol absorption. In mice, Ishaaya et al. (1969) have shown that an addition of soybean saponin extract did not affect the plasma cholesterol levels, whereas alfalfa saponin seemed to lower them. Other reports from the same group (Birk, 1969; Gestetner et al., 1972) indicated that such a difference is due to the lack of “affinity” of soybean saponin to cholesterol, although such views are not uniformly shared by others (Morgan et al., 1972). A heat stable factor, lysinoalanine, is produced when soy protein products are alkali-treated. Such lysinoalanine attached to protein in peptide linkage is not absorbed but is excreted in feces. Such loss through the feces may accentuate the “imbalance” already existing in the amino acid composition of soy protein as compared to casein. ACKNOWLEDGMENTS We are grateful to Dr. C. J. O’Donovan of Miles Laboratories, Inc., Elkhart, Indiana for the supply of Pro-Lean and his interest. We are also grateful to Mr. James Baker, Mrs. Maureen Gennett, Miss Susan Szawiela, Mr. Paul Sargent, and Mrs. Diana Surgick for their technical assistance. This study was supported by U.S. Public Health Service Grant HL-20993.
REFERENCES ANDERSON, J. T., GRANDE, F., and KEYS, A. (1971). Effect on man’s serum lipids of two proteins with different amino acid composition. Am. J. Clin. Nutr. 24, 524-530. BEST, M. M., DUNCAN, C. H., VAN LOON, E. J., and WALTHEN, J. D. (1955). The effects of sitosterol on serum lipids. Am. J. Med. 19, 61-70. BIRK, Y. (1969). Saponin. In “Toxic Constituents of Plant Foodstuffs” (I. E. Leiner, ed.), Academic Press, New York.
398
KIM
ET AL.
Uonc:sw~iinr, the kc&g
1%. ( 1960). (~it;ultificaticl~~ of c~llol~~stc~rcJ alxsorpticuk ill m;uk 11,. fcyd ;ctdysi5 ;lltc~i of a sin& isotope-labclcd mcsal. J. LilGtl Rrs. 10, 331-337. CAHHOLL, K. IL, and I~UILTON, R. M. G. ( 1975). Eft‘ccts of dietary protein and c;~rbohydrate on plasma cholesterol lcvcls in relation to atherosclerosis. J. Footl Sci. 40, 18-23. DATWZNON, J., SIhih%oNI%, \\‘. J., and AIIHENS, E. H., JH. ( 1968). Usefulness of chromic oxide as an internal standard for balance studies in formula-fed patients and for assessment of colonic function. J. Clin. Inuest. 47, 127-138. FAHQUHAR, J. W., SMITH, R. E., and DEZIPSEI., hl. (1936). The effect of p-sitosterol on serum lipids on young men with arteriosclerotic heart discax. Circulation 14, 77-82, 1956. FAHQUHAH, J. W., and SOSOLOW, M. ( 1958). Response of serum lipids and lipoproteins of man to /.%sitosterol and safflower oil: A long term study. Circthfiorl 17, 890-899. FOLCH, J., LEES, h4., and SLOANE-STANLEY, G. H. ( 1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Bio. Chem. 226, 497-509. GATTI, E., and SIRTORI, C. R. ( 1977). Soybean-protein diet and plasma cholesterol. (Reply) Lancet I (8015), 805-806. GESTETNEH, B., ASSA, Y., HEMIS, Y., TEXER, I-., RO,IXAX, M., BIHK, I’., and BONIX, A. (1972). Interaction of lucernc saponins with sterols. Bi~chitt~. Bio~hys. Actcl 270, 181-187. GRUNDY, S. M., AHHENS, E. H., Jn., and MIETTIXES, T. A. ( 1965). Quantitat;ve isolation and gas-liquid chromatographic analysis of total fecal bile acids. J. Lipid Res. 6, 397-410. GHUP\TDY, S. M., AHHENS, E. H., JH., and SALEN, G. (1968). Dietary /+sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J. Lipid Res. 9, 374-388. GRUXDY, S. M., and Mos, H. II. I. ( 1977). Determination of cholesterol ahsorption in man by intestinal perfusion. .l. Lipid Res. 18, 263-271. HAMILTON, R. M. G., and CAHHOLL, K. K. ( 1976). Pl a\ma ,’ cholesterol levels in rabbits fed low fat, low cholesterol diets. Effects of dietary proteins, carbohydrates, and fiber from different sources. Atherosclerosis 24, 47-62. HELMS, P. ( 1977). Soybean-protein diet axed plasma cholesterol. Lancct I ( 8015 ), 805-8’36. HODGES, R. E., KHEHL, W. A., STONE, D. B., and LOPEZ, A. (1967). Dietary carbohydrates and low choleatcrol diets: effects on serum lipids of man. Am. J. C/in. Ntcfr. 20, 198-208. HOWAHD, A. N., GHESHAM, G. A., JONES, D., and JESR.IR’GS, I. \\I. ( 1953). The pwvention of rabbit atherosclerosis by soybean. J. Atherosclerosis Res. 5, 330-337. IWAAYA, I., BIHK;, Y., BONDI, A., and TENCER, Y. (1969). %)-bean saponins. IS. Studies of their effect on birds, mammals, and cold-blooded organisms. J. Sci. Fd. Agric. 20, 43%136. KEYS, A., and ANDEHSON, J. T. (1957). Dietary protein and the strum cholesterol level. Am.
J. Clin. Nutr. 5, 29-34. KIM, D. N., LEE, K. T., REISM~, J. hl., and THOMAS, W. A. (1974). Rcatraint of cho!estcrol accumulation in tissue pools in hypercholesterolemic swine associated \vith drastic shortterm lowering of serum cholesterol levels by clofibrate or cholcstyramine. J. Lipid Rcs. 15, 326-331. \\‘. A. ( 1975). Effect of combined KIM, D. N., LEE, K. T., REIXER, J. hf., and TH~XM, clofibrate-cholestyramine treatment on serum and tissue cholesterol pools and on cholesterol synthesis in hypcrcholcsterolemic swine. EXAM/. MO~CC. Pathd. 23, 83-93. KHITCHEVSKY, D., TEPPEH, S. A., and STWW, J. A. (1978). of soy protein and I n fl ucncc casein on atherosclerosis in rabbits. Fed. Proc. 37, 747. LEFFLEH, H. H. ( 1959). Estimation of cholesterol in serum. AI~I. .I. Clin. Path. 31, 310-31:3. LEINEH, I. E. (1977). Nutritional aspects of soy protein products. J. Am. Oil Chem. Sot. 54, 454A-472A. MALINOW, M. R., MCLAUGHLIX, I?., KOHLEH, G. O., and LIVINGSU)N, A. L. (1977a). Prevention of elevated cholesterolemia in monkeys by alfalfa saponins. Steroids 29, 105-110. MALINOW, h4. R., MCLAUGHLIN, P., PAPU’ORTH, L., STAFFORD, C., KOHLER, G. O., LWISGSTOS, A. L., and CHEEKE, P. R. (1977b). Effect of alfalfa saponins on intestinal cholesterol absorption in rats Am. J. Clin. Nutr. 30, 2061-2067. MARSH, A., KIM, D. N., LEE, 6. T., HEWER, J. M., and THONAS, W. A. ( 1972). Cholesterol turnover, synthesis and retention in hypercholesterolemic growing swine. J. Lipid Res. 13, 600-615. MEEKER, D. R., and KESTEN, H. D. ( 1940). Experimental atherosclerosis and high protein diets. Proc. Sot. Exp. Biol. S Mccl. 45, 543-545 ( Abstract ).
SOY
PROTEIN
AND
SERUM
CHOLESTEROL
399
MEEKER, D. R., and KESTEN, H. D. ( 1941). Effects of high protein diets on experimental atherosclerosis of rabbits. Arch. Pathol. 31, 147-162. MIETTINEN, R. A., AHRENS, E. H., JR., and CRUNDY, S. M. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. .I. Lipid Res. $411424. MORGAN, B., HEALD, M., BROOKS, S. G., TEE, J. L., and GREEN, J. (1972). The interaction between dietary saponin, cholesterol, and related sterols in the chick. Pot& Science 51, 677-682,. OLSON, R. E., VESTEH, J. W., GURSEY, D., DAVIS, N., and LONC,AIAN, D. (1958). The effect of low protein diets upon serum cholesterol in man. Am. .I. Clin. Notr. 6, 310-324. QUINT~O, E., GRUNDY, S. M., and AHREA-S, E. H., JR. ( 1971). An evaluation of four methods for measuring cholesterol absorption by the intestine in man. J. Lipid Res. 12, 221-232. SAMUEL, P., CROUSE, J. R., and AHRENS, E. H., Jn. (1978). Evaluation of an isotope ratio method for measurement of cholesterol absorption in man. .I. Lipid Res. 19, 82-93. SIRTORI, C. R., AGRADI, E., CONTI, F., and MOXTERO, 0. (1977). Soybean-protein diet in the treatment of Type II hyperlipoproteinemia. Luncet I, 275-277. SODHI, H. S., KUDCHODKAR, B. J., VARUGHESE, P., and DUNCAN, D. (1974). Validation of the ratio method for calculating absorption of dietary cholesterol in man. Proc. Sot. Exp. Biol. and Med. 145,107-111. WALKER, G. R., MORSE, E. H., and OVEIILEY, V. A. ( 1960). The effect of animal protein and vegetable protein diets having the same fat content on the serum lipid levels of young women. J. Nutr. 72, 317-321.
AND
MOLECULAH
29, 385-399
PATflOLO(;Y
( 1978)
Effects of a Soy Protein Product on Serum and Tissue Concentrations in Swine Fed High-Fat, High-Cholesterol D. N. KIM, K. T. LEE, J. M. REINER, Department
of Pathology, The Neil Albany Medical College,
Received
June
26,1978,
Hellman Albany,
AND
W. A. THOMAS
Medical Research New York 12208
and in revised
form
Cholesterol Diets
July
Building,
24, 1978
Hypocholesterolemic effect of a soy protein product was studied in swine fed a high-fat, high-cholesterol diet. In the first experiment, a group of swine were fed 42% butter (by calories) and 1055 mg cholesterol daily, with casein as the source for protein, for 6 weeks and this diet resulted in moderately high serum cholesterol concentrations (219 2 33 mg/dl). Another group fed the same diet except with soy protein product as the protein source instead of casein showed virtual normocholesterolemia at the end ( 107 -C 3 mg/dl). Cholesterol balance was studied under non-steady state conditions using methods designed for this purpose. Reflecting the serum cholesterol concentration, the total body cholesterol concentration (excluding CNS ) was also significantly lower in soy protein group. However, parameters of cholesterol balance, such as fecal neutral and acidic steroid excretions, dietary cholesterol absorption, and whole body cholesterol synthesis were studied and no differences were demonstrated between the caseinand soy protein-fed swine. The experiment was repeated and in Experiment II virtually the same results were obtained. When swine were given the same high-fat, high-cholesterol diets with + casein + 3 soy protein or casein + soy protein, hypocholesterolemic effects were also observed. Therefore, such action is probably caused principally by soy protein per se rather than simply by replacement of casein by soy protein. Addition of DLmethionine to soy protein containing diet did not alter the hypocholesterolemic effect of soy protein indicating that the effect was not the result of methionine deficiency. In conclusion, we can state that the hypocholesterolemic action of soy protein was clearly demonstrated in swine fed a high-fat, high-cholesterol diet, but that the mechanism of action is yet to be established.
INTRODUCTION Hypocholesterolemic role of vegetable proteins has been reported sporadically for the last few decades in rabbits (Meeker and Kesten, 1940, 1941; Howard et aE., 1965; Carroll and Hamilton, 1975) and in humans (Olson et al., 1958; Walker et al., 1960). However, such an effect in humans has not been confirmed by all investigators (Keys and Anderson, 1957; Anderson et al., 1971). Hamilton and Carroll (1976) have observed that hypercholesterolemia develops in rabbits when a semi-purified diet containing casein, carbohydrate, lowfat, and cellulose without cholesterol is fed. The hypercholesterolemic effect disappears and a low serum cholesterol level is maintained when casein is replaced by vegetable proteins. Among the vegetable proteins, soybean protein 385 0014-4800/78/0293-0385$02.00/O All
Copyright @ 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
35%
KIM
ET AL.
exhibited the most strikiu g eifect in the rabbit. Since soy protein coutains less methionine than casein (animal proteins), the above investigators supplemelltcd the soy protein containing diet with methionine. Such supl>lementation of methionine resulted in a small increase in serum cholesterol levels. However, the overall results indicated that a relative lack of methionine in soy protein was not the principal reason for the hypocholesterolemic action of soy protein in rabbits fed a cholesterol-free semi-purified diet. No explanation for such action was given. Recently, Sirtori et al. (1977) reported hypocholesterolemic effects in type II hyperlipidemics fed a moderately low-fat, low-cholesterol diet with soy protein as the protein source. Similar results were obtained when cholesterol was added to the diet (500 mg/day ). Studies of the effect of soy protein in diets which are high in both fat and cholesterol have not been reported to our knowledge for either man or experimental animals. Therefore, the current study was designed to observe whether or not such hypocholesterolemic effects could be demonstrated in swine fed high-fat, high-cholesterol diets. In addition, we wanted to gain some insight into the mechanism of action. Swine were chosen for study because they respond to high-fat, high-cholesterol diets in much the same way as man and because we have accumulated considerable data on cholesterol turnover in this species that would serve as background for judging the effect of soy protein. In the current study we have used the term “soy protein” to describe a specific product from Miles Laboratories called Pro-Lean 45’“‘. This is a mixture of “soy protein concentrate, spun isolated soy protein, isolated soy protein, and hydrolyzed vegetable protein (1.93% ) with sugar and salt.” The dietary fibers have been largely eliminated and the product contains only 4.33% as neutral detergent fiber. Analysis of the lot used for the current experiment by Miles Laboratories shows 62.5’;r’ protein, 0%’ fat, 6.8% moisture, 8.6% ash, and 22,1c/ carbohydrate (Lot #450-2467-l). In the future, we plan to study effects of purified components, especially the protein component. However, the current study is aimed specifically at studying the Miles Pro-Lean mixture. MATERIALS Experimental
AND METHODS
Design
Two experiments were carried out in succession (see Table I). Experiment 1. Fifteen swine were divided into three dietary groups: one fed a commercial mash, and two semi-purified hyperlipidemic (HL) diets containing fat and cholesterol, one with casein and the other soy protein as the sources for proteins. These groups will be called mash, casein, and soy protein groups. Swine were fed the respective diets for 6 weeks. Initial and weekly determinations of body weight and serum cholesterol concentrations were carried out. Feces were collected during the terminal week for the analyses of neutral and acidic steroids. Terminally, swine were sacrificed and triplicate samples of liver, small intestine, muscles, adipose tissue, skin, and aorta were obtained for cholesterol determinations. The remainder of the carcasses (excluding CNS) were homogenized to obtain total body cholesterol concentra-
SOY
PROTEIN
-4ND
SERUM
TABLE Groups, Experiments I
II
Groups
Number
of Swine,
designations
Duration No. of swine 5 6 5
HL HL HL HL HL
6 5 6 5 6
casein soy protein tC + $3 C + S” soy protein
+ methe
387
I
Mash HLa + casein HL + soy protein
+ + + + +
CHOLESTEROL
and Measrrrements Duration (week)
A
Measurements
Serum and tissue cholesterol Body weights Cholesterol balance excretion, retention, synthesis Cholesterol”
4
absorption
Serum cholesterol Body weights
a Semi-purified hyperlipidemic diet containing fat and cholesterol. D Cholesterol absorption was determined only in three swine each from Experiment II, HL + Casein and HL + Soy protein. c One-half dose of casein and + dose of soy protein. d Casein and soy protein in full doses. E DL-methioninc supplemented.
the first
two
groups
of
tions (Kim et al., 1974). Whole body cholesterol synthesis was calculated from the cholesterol balance equation under non-steady state conditions: synthesis + dietary intake = retention + excretion. Experiment II. Twenty-eight swine were divided into five groups. Two groups were a repeat of the casein and soy protein groups of Experiment I. A third group received the same amount of protein as the first two groups but from two sources, one-half from casein and the other half from soy protein ($ casein + * soy protein). A fourth group was given full amounts of casein and soy protein, hence doubling the protein content of the diet as compared to the other groups. The fifth group was given the soy protein diet with DL-methionine. The swine were fed the respective diets for 4 weeks. Body weights and serum cholesterol concentrations were monitored weekly. At 5 weeks, three swine each from the first two groups, casein and soy protein, were used for the determinations of dietary choIestero1 absorption. Animals. Forty-three young male Yorkshire swine weighing approximately IO kg were housed individually in slat-bottomed cages. They were placed on a commercial mash diet for 1 to 2 weeks while they became acclimated to the laboratory. Then they were assigned to the respective dietary groups. Diets. The compositions of the HL diets with casein and soy protein and mash diet are shown in Table II. Both HL diets are nearly isocaloric. The amounts of protein in both casein and soy protein are based on the analyses Casein contained 90% protein and Prodata supplied by the manufacturers. Lean contained 62% protein, 076 fat and 2270 carbohydrates. Total cholesterol in both diets is the same (1055 mg) with I33 mg from butter and the remainder Other supplements include plant sterols from added crystalline cholesterol. (Cytelline, Lilly, IN) and chromium oxide. Plant sterols, total 570 mg including 336 mg p-sitosterol, were added to all HL diets as the marker for the loss of
3ss
.KIM
ET AL.
TABLE Composition
II of Diets -~~
Masha Corn oil Butter Corn starch Casein* Pro-Leant Salt mix Vitamin mix Non-nutritive Total Total
fiber” 7UO
(g) calories
Percent calories Fat Protein Carbohydrate
(&al)
HI,
+ rasein
HL
+ soy protein
10 R 90 a00 130
10 90 150 -
4 2 104
180 4 2 104
540
540
Sl(iY
2110
as 41.5 21.6 30.9
42.7 2 1.3 X.0
a Hog mash (Agway). * 9O7oproteins. Miles Laboratories, Inca., c Textured soy protein prodrlc%. Pro-Lean 4n-TjI, Lot No. 450-24137-1, Elkhart, IN. Protein W2.3yc, fat OS;, carbohydrate X2.17& moisture 1;.8(;<, and ash S.67; with ~.XYS~~ neutral detergent fibers. d Cellulose type. Both HL diets were supplrmentrd with sufficient cholesterol to make the daily total 1055 mg. In addition, 570 mg of plant sterol and 1 y of chromium oxide were added.
neutral steroids during intestinal transit. The mash group did not receive these plant sterol supplements since the mash diet contained 569 mg of plant sterols with 426 mg of p-sitosterol. One gram daily of chromium oxide was given to all swine in Experiment I as a marker for fecal flow and recovery. Such markers were not required in Experiment II, but plant sterol was added to make the dietary conditions of Experiment I and II the same. The fifth group of Experiment II received a soy protein containing HL diet supplemented with DL-methionine to make the total methionine content equal to that contained in the casein diet (2.5 g/day or 2.2% of protein in the diet). Feeding. All swine were fed once daily at 9 AM. Crystalline cholesterol, plant sterol mixture, and chromium oxide were weighed out separately. These three ingredients were added to each diet bowl with approximately l/3 of the daily diet mixture plus water. As soon as this first bowl was consumed, the remaining diet mixture was given. Adequate amounts of water and repeated mixing is essential to prevent waste of the diet. When all of the diet was consumed, the bowl was filled with water. Total daily water intake was approximately 1.5 liters. Cholesterol Balance Stucly A method was developed previously for the study of cholesterol balance of swine under non-steady state conditions (Kim et al., 1974, 1975). This method enables us to calculate whole body cholesterol synthesis by direct measurements
SOY
PROTEIN
AND
SERUM
389
CHOLESTEROL
of steroid excretion and cholesterol retention by the following equation: synthesis = dietary intake + retention - excretion. Daily excretion was determined from the feces with minor correction for the losses through routes other than feces. Daily cholesterol retention was obtained from the terminal total body cholesterol concentration using the body cholesterol concentration of comparable swine at two different points in time. Fecal collection and analyses. Feces were collected during the terminal week in daily lots, processed, and pooled; neutral and acidic steroids were analyzed by the method described previously using thin layer chromatography and gasliquid chromatography (Grundy et al., 1965; Miettinen et al., 1965; Marsh et al., 1972; Kim et al., 1974). Neutral steroid loss during the intestinal transit was corrected for by p-sitosterol recovery (Grundy et al., 1968) and fecal flow was corrected for by the chromium oxide recovery (Davignon et al., 1968). Total body cholesterol concentrations. Whole carcass, minus CNS and intestinal contents, was homogenized by the method described previously (Kim et al., 1974) and multiple aliquots were taken for analyses. Cholesterol &terminations. Serum total cholesterol was determined by the method of Leffler ( 1959). The tissue cholesterol was measured by the same method after Folch extraction (Folch et al., 1957). Cholesterol concentration was expressed as free cholesterol. Cholesterol
Absorption
Cholesterol absorption was measured by the double isotopic single feeding method similar to that described by Sodhi et al. ( 1974), Borgstriim ( 1969), Method IV of Quintso et al. (1971) and the fecal radioactivity method of Samuel et al. (1978). A mixture of 8 &i of ( 3H)22, 23-P-sitosterol (sp. act. 58 Ci/mmole; Amersham/Searle) 4 &i of (l’C)4-cholesterol ( sp. act. 55.5 mCi/ mmole; NEN, Boston), and 500 mg of red Carmin was used to mark the diets at usual meal time, 9 AM. Red Carmin was used to mark the fecal buIk (Sodhi TABLE Body
III
Weights and the Terminal after 6 Weeks of Feeding Rlash
No.
of swine
Body weights Initial Final Liver Liver/body k/W
Liver Weights in b:xperiment HL
-5
of Swine I
+ casein
HL
+ soy protein
5
5
(kg) 9.7 f 2.ou 19.6 z+z 1.3
(g)
377
zk 24
10.0 f 16.0 + 291
f
1.7 2.3 42
10.5 zt 25.8 f 401
f
1.8 0.6D 24~
weight 19.2 f
0.2
a Mean f standard error of mean. * Significantly different from both mash and casrin mash and casein groups. c Differences among three groups arr not significant. d Significantly different from both mash and casein mash and casein groups.
15.3 f
0.7
15.5 +
0.P
grollp
(P < 0.01).
No difference
between
groups
(P
No difference
between
< 0.05).
KIM
390
ET AL,.
et al., 1974). Twenty-four hours later, swine were sacrificed by intravenous injections of Pentobarbital sodium. Stomach, small intestine, and large intestinal contents were collected in two fractions; proximal fraction stained with Carmin and the distal unstained fraction. A sufficient amount of borderline fraction was included in the proximal fraction to ensure that all radioactivity was recovered in that fraction. All intestinal contents were processed and neutral steroids extracted as described under fecal steroid analyses. Both (l,C) and ( 3H) radioactivities were measured. The distal fraction from the large intestine contained a negligible amount of radioactivity. Absorption was calculated from the equation (1 - 14Crecovery) x loo 3H recovery
Absorption =
Beta-sitosterol recovery was used to correct for the loss during the intestinal transit as described earlier. The radioactivity in the bile acid fraction of the large intestine was 7.570 of total 14C activity. RESULTS Experiment I. AII swine consumed the entire daily rations and grew well. Body and the terminal liver weights of swine after 6 weeks of feeding are shown in TabIe III. The terminal body weights were greater (P < 0.01) in HL + soy protein group (25.8 t 0.6 kg) than mash ( 19.6 -t 1.3 kg) and HL + casein (16.0 -C 2.3 kg) groups. Difference between mash and HL + casein groups was not significant. The terminal liver weights among the three groups were not significantly different, although the average value for the HL + soy protein group (401 -t 24 g) was larger than both mash (377 * 24 g) and HL + casein (29lk 42 g) groups. When the liver weights were expressed as g/kg body weight, HL + soy protein group (15.5 2 0.8 g/kg) was significantly lower (P < 0.05) than both mash (19.2 * 0.2 g/kg) and HL + casein (18.3 * 0.7 g/kg) groups.
250
-
200
-
60 f ‘0
12
I
FIG. 1. Serumcholesterolconcentrations(mg/dl, (n=5),
HL -t soy protein
(n=5)
and
3I 4 WEEKS
mean
5I
6
I+ standard
HL + casein groups (n=5)
error
of mean)
in Experiment
of mash
I.
SOY
PROTEIN
AND
SERUM
TABLE Initial
No.
of swine
HL + soy protein C
5
85 z!z 8’ 9.5* 4
intake
(mg/day) (mg/day)
0
Total (mg/day)
Comparisons 24 ~8 B
-4 “8 c
B “S c
5
error
CNS)
97% 3 219 + 33
s5* 107
5 f
3
-
P <
1055
1055
P < P <
-
624 f 372 *
14 38
617 312
832
f
58
996
f
47
929
f
57
n.s.
* 61
203
3~ 54
236
f
62
P < 0.001
f
964
f
61
P < 0.05
f
23
1233
47
28 36
-
P < 0.05
-
f35 rt 32
1078
i f
0.01
284 547
1121
Whole body cholesterol (mg/kg) (excluding = Mean ziz standard 6 Neutral steroids. c Bile acids.
+ casein B
Concentrations and Cholesterol Week (Experiment I)
(mg/dl)
Fecal excretion NS B.4c
Synthesis
HL
5
Serum cholesterol Initial Final Dietary
IV
and Terminal (6 Weeks) Serum Cholesterol Balance Parameters during the Terminal Mash A
391
CHOLESTEROL
0.001 0.01
P < P <
-
0.001 0.01
n.s.
P <
P < 0.01
0.001
n.s.
n.s. n.s. n.s. n.s.
P < 0.02
of mean
Serum cholesterol concentrations (Fig. 1 and Table IV). Serum total cholesterol concentrations during the 6 weeks feeding period are shown in Fig. I. HL + casein group showed significant increase in cholesterol level after 1 week and remained elevated, whereas HL + soy protein group showed fluctuation in serum cholesterol at lower levels. The terminal levels for HL + casein, HL + soy protein, and mash groups were 219 +- 33, 107 k 3, and 95 + 4 mg/dl, respectively. The value for the HL -+ casein group was significantly higher (P < 0.01) than that of either the HL + soy protein or the mash group. A statistically significant (P < 0.05) though small (12 mg/dl) difference was noted between HL + soy protein and mash groups. Fecal excretions of neutral and acidic steroids (Table IV). Neutral steroid excretions were greater (P < 0.001) in both HL + casein (624 * 14 mg/day) and HL + soy protein (617 2 28 mg/day ) groups than in the mash group (284 t 35 mg/day ). Bile acid excretion in the mash group (547 + 32 mg/day) was significantly greater (P < 0.01) than in both HL + casein (372 * 38 mg/day ) and HL + soy protein (312 * 36 mg/day ) groups. No difference was noted in total fecal steroid excretions among HL + casein (996 2 47 mg/day ), HL + soy protein (929 f 57 mg/day ), and mash (832 * 58 mg/day) groups. Whole body cholesterol spthesis (Table IV). Whole body cholesterol synthesis of both HL + casein (203 2 54 mg/day) and HL + soy protein (236 * 62 mg/day ) were significantly lower (P < 0.001) than in the mash group (1121 * 61 mg/day ). Whole body cholesterol concentrations (Table IV). Whole body cholesterol concentrations (excluding CNS) of I-IL + casein group ( 1233 * 47 mg/kg) was significantly (P < 0.05) higher than that of the mash group (1078 * 23 mg/kg) and HL + soy protein (964 +- 61 mg/kg). The difference between HL + soy protein and mash groups was not significant.
392
KIM
ET
AL.
TABLE Terminal
Tissue
Cholesterol
V
Concentrations
after
6 Weeks
Mash No.
of swine
HI,
(tSxperiment
+ caascin
.5
HL
LiveP Ileum Skin Muscle Fat
1.78 2.12 1.37 0.68 0.96
Aorta Arch Thora& Abdominal
0.94 * 0.08 0.9s It 0.0s 1.04 zt 0.07
a-&
I)
+ soy protein
5
mg/g * + zt dz It
of Feeding
5
weight
0.10* 0.12 0.06 0.03 0.06
zk 0.11
2.44
f
0.24
+
0.04
1.90 f
0.18
2.03 f 1.3.5 f
0.16 0.07
0.77 0.9“
0.06 o.or,
0.66 0.90
f f
0.03 0.09
0.96 & 0.05 I .I 1 + 0.01
0.90
+
0.07
I.18
1.10
2.76 2.22
n The only statistically significant differences are cholesterol (P < 0.01) and mash vs HL + soy protein (P < 0.05). * Mean & standard error of mean.
f *
1.00 ?lz O.Oti
f 0.15 - ~-~.
conumtrations
f
mash vs HL
0.06
+ casein
Cholesterol concentrations in selected tissues ( Table V). The only significant difference in the selected individual tissue concentrations among the three groups was observed in the liver. Both HL + casein (2.76 f 0.41 mg/g ) and HL + soy protein (2.44 * 0.24 mg/g) groups showed a significant increase (P < 0.05) as compared to the mash group (1.78 k 0.10 mg/g). The difference was not significant between casein and soy protein groups. The other organs, ileum, skin, muscle, and adipose tissue, did not exhibit any significant differences among the three groups. Aortic cholesterol concentrations also were not different. Experiment
II
All swine consumed all of the daily rations and grew well. The initial and terminal body weights are shown in Table VI. All groups showed fairly uniform growth during the experiment except for HL + casein group. The net inTABI,IS Initial
(;roaps
VI
and Terminal (4 Weeks) Serum Cholesterol Conccntratiorls and Body Weights (li;xpcriment II) No. of swine
Body
wclight
(kg)
8rrlun
cholesterol
Initial
Terminal
Initial
Terminal
HL + casrin HL + soy protein HL + +C + $5 HL+C+% HI, + soy protein + mcthioninr n Mean b Valllrs
f
(mg/dl)
--
standard rrror of mean. for all grol,ps were significantly:
owcr
t,h:m
HL
+ cnscxin gro,qj
(I’
< 0.05).
SOY
PROTEIN
AND
SERUM
lEOl 1 250
of
I 0
393
CHOLESTEROL
HL+cosein t
1
2
3
4
Weeks FIG. 2. Serum cholesterol casein, HL + soy protein, periment II.
concentrations (mg/dl, mean 2 standard error of mean) of HL + HL + C + S, HL + ?C + $S, HL + S + methionine groups in Ex-
crease in body weight during 4 weeks of study of HL + casein group was 6.7 kg and the remaining groups had values ranging from 9.9 to II.7 kg. Serum cholesterol concentrations are shown in Fig. 2 and Table VI. Serum cholesterol concentrations of HL + casein group were elevated significantly after 1 week and continued to rise, reaching 238 I+ 33 mg/dl at the end of 4 weeks. The HL + soy protein group showed a slight elevation of serum cholesterol level after 1 week but gradually declined to 106 k 9 mg/dl at the end. These responses are essentially the same as those observed in Experiment I. The HL + ‘,C + $S and HL + C + S groups revealed a fluctuation and more variation in their serum cholesterol levels, but the terminal values (142 t 11 and 146 f 24 mg/dl) were significantly lower than HL + casein group (P < 0.05). The group supplemented with methionine (HL + soy protein + nL-methionine) also showed a significantly lower serum cholesterol concentration than HL + casein group (P < 0.01). Dietary cholesterol absorption was measured in three swine from each of HL + casein and HL + soy protein groups terminally (Table VII). Although the
TABLE Dietary
Cholesterol Casein
Absorption in Swine Fed HL Diet or Soy Protein (Experiment II) HL
No. cd swine ‘x, absorption I* Mean
& standard
error
VII
of mean ; difference
+ casein 3
50 *
HL
with
+ soy protein 3
3
not statistically
Either
42 It 5 significant.
394
KIM
ET AL.
average value of the IIL + soy protein group (4‘2 * 5(;/,!) was lower than for the HL + casein group ( 56 5 3% ), the difference was not significant. DISCUSSION The main question we proposed to answer in this study was whether the substitution of a soy protein product for milk protein would prevent hypercholesterolemia in swine fed a high-fat, high-cholesterol diet. A profound hypocholesterolemic effect was demonstrated in two separate experiments when the soy protein product was substituted for milk protein (casein) in the diet, A second question was whether the soy protein product would prevent the increase in concentration of cholesterol in the whole carcass that we have previously noted when casein was the source of protein in high-fat, high-cholesterol diets. The results in Experiment I, where carcass cholesterol contents were determined, indicate that there was no increase over mash concentration in the soy protein product group in contrast to the significant increase observed with the casein. A third question that we hoped to answer was which of the parameters of cholesterol balance was (were) altered by the soy protein product in such a way as to produce the observed effect on concentration of cholesterol in the serum and whole carcass. We were not entirely successful in answering this question, but we did gain some insights that should form a basis for planning future experiments. The balance parameters to be considered are (1) whole body cholesterol synthesis, (2) absorption, (3) excretion of bile acids, (4) escretion of neutral steroids, and (5) retention in the carcass.
1. Whole bony cholesterol synthesis. Synthesis is profoundly and equally depressed by both the casein and the soy protein high-fat, high-cholesterol diets. Thus, alteration in synthesis cannot account for the observed effects of the soy protein product. 2. Cholesterol ahsorptiorl from the gut. Cholesterol absorption was not significantly altered by the soy protein diet as compared to the casein diet even though there was a numerical difference. However, the number of swine was small and this aspect needs to be further studied in future esperiments. With low-fat mash diets practically no cholesterol is absorbed even when a large amount is added to the diet (Marsh, et al., 1972). 3. Excretion of bile acids in the feces. Increase in excretion of bile acids is one of the ways in which drugs such as cholestyramine produce their hypocholesterolemic effect. No such effect on bile acid excretion was observed in the current study with soy protein. This aspect should perhaps be pursued in other types of esperiments, but it seemsunlikely that increases in bile acid escretion will be found. 4. Excretion of neutrul steroids in the feces, Neutral steroids in the feces can come from unabsorbed dietary or excreted endogenous (absorbed and synthesized) cholesterol. Increase in excretion of unabsorbed cholesterol can result only if absorption is decreased. Thus, the comments regarding absorption apply equally to escretion of unabsorbed cholesterol. Endogenous and unabsorbed dietary cholesterol could not l)e separated with the design used in the current studies. The fact that the total cholesterol escre-
SOY PROTEIN
AND
SERUM
CHOLESTEROL
395
tioli iii the feces was not increased makes it seem unlikely that cscretioii of either endogenous or unabsorbed dietary cholesterol was increased. IIowever, this aspect needs to be investigated further for reasons that will be given in the discussion of whole body cholesterol retention. 5. Whole body cholesterol retention. We have already stated that the soy protein product prevented the increase in concentration of cholesterol in the carcass that was observed with casein high-fat, high-cholesterol diet. However, for unknown reasons in the experiment in which the balance study was done, the soy protein product-fed swine grew considerably more than the casein-fed. As a result, the total amount of cholesterol retained as part of normal tissue was considerably greater in the soy protein product-fed than in the casein-fed swine. This was the only factor in the balance study that was significantly different between the two groups. We might conclude from this that the hypocholesterolemic effect resulted entirely from normal utihzation of cholesterol to form the greater tissue mass. However, in Experiment II (where a balance study was not done) there was somewhat less disparity between growth rates and yet the same profound hypocholesterolemic effect of the soy protein product was observed as in the first. This aspect needs to be investigated in the future in pair-fed experiments designed to maintain similarity in growth rates. We may find in such experiments that the rate of excretion of endogenous cholesterol into the feces is increased as it is in clofibrate-treated swine on high-fat, high-cholesterol diets (Kim et al., 1975). If such were found we might conclude that the “efficiency” in clearing cholesterol from the body is increased by the soy protein diet. Viewing the results from another aspect, the differences in effects on cholesterol concentrations of the soy protein product as compared to casein might be a result of: (1) positive depressant effect of soy protein product with casein being inactive, (2) positive elevating effect of casein with soy protein product being inactive, or (3) active effect of each in opposite directions. The groups fed mixtures of the two proteins were chosen in an attempt to differentiate among the above possibilities. The results are not entirely conclusive. Mixing casein and soy protein product with amounts of each half of that used when each was given alone resulted iu significantly lower serum cholesterol levels than when casein was given alone in full amount. A similar result was obtained when the two were combined with each given in the quantity as when given alone. If the results are taken literally, this would seem to mean that the soy protein product was exerting a positive cholesterol lowering effect and that casein was inactive in this regard. However, the serum cholesterol values with the combination were numerically (though not significantly) higher than with mash or soy protein product alone. More experiments with larger numbers of swine need to be done before we can conclude that casein is inactive in regard to serum cholesterol levels and that the hypocholesterolemic effect is produced entirely by direct action of the SOY protein product. A number of possibilities regarding mechanism of action of soy protein on cholesterol balance have been entertained by others in studies of the effect of soy protein on serum cholesterol levels in rabbits and man. Factors that have been considered include: (1) fiber content, (2) relative lack of methionine and/
396
KIM
ET AL.
or imbalance in other amiiro acids, ( 3) plaint sterols, (4) sapoiiin, and (5) otlrer factors native to soy beans. Helm (1977) upon reading the report of Sirtori et (11. ( 1976) in man on the effect of a low lipid modified diet containing soy protein product similar to Pro-Lean used here, suggested that the hypocholesterolemic effect may have resulted from the presence of “indigestible non-cellulose polysaccharides,” which could cause an increase in bile acid excretion. Gatti and Sirtori (1977) dismissed such a possibility on the ground that the quantity of such fiber was too According to the analysis supplied by the manusmall to be of significance. facturer, the soy protein product used in the current study, contained 4.55% of neutral detergent fiber and this quantity (8.2 g daily in the current study) in our previous experience with swine, is too small to produce any effect on bile acid excretion. Also, in the current experiments, there was no difference in fecal bile acid excretion between casein and soy protein groups. Another point frequently considered in various studies with soy protein is a relative lack of methionine in soy protein. However, the current study showed that a methionine supplementation in swine did not increase serum cholesterol o b served some increase in serum levels. In rabbits, Hamilton and Carroll (1976) cholesterol levels when either soy protein concentrate or isolate were supplemented with methionine, but such supplementation resulted in an increase of serum cholesterol levels only up to the level of those rabbits fed commercial chows only. Therefore, they concluded that a relative lack of methionine in soy protein may be part of but not the principal reason for the hypocholesterolemic effect. Similarly, Gatti and Sirtori (1977) observed a lack of effect on serum cholesterol of methionine supplementation to the soy protein diet. It becomes obvious when one inspects the amino acid compositions of casein and soy protein, that a relative lack of methionine is not the only difference between these two proteins. Kritchevsky et al. (1978) h ave noted the fact that soy protein contained more arginine than casein and that the arginine/lysine ratio g-fold in soy protein as compared to casein. They observed was approximately that atherogenicity of semi-purified diets can be affected by addition of specific amino acids in rabbits. Another possible factor is the plant sterol content. Although we have not demonstrated in swine that plant sterols affect serum cholesterol levels, such effects in humans have been reported repeatedly (Best et al., 1955; Farquhar et al., 1956; Farquhar and Sokolow, 1958; Grundy and Mok, 1977). In the current study all of the diets, except for the mash diet, were supplemented with 570 mg of plant sterols of which 336 mg was ,&sitosterol. This quantity is similar to that contained in the mash diet. On analysis, the soy protein product contained 4 mg/lOO g of plant sterols and 3 mg/lOO g of p-sitosterol. Therefore, plant sterol content in the soy protein containing diet in the current study was 7.2 mg. It seems very unlikely that such a quantity can be of any significance. Soybeans in their native state contain “antinutritional factors” for rats. These can be divided into heat labile and heat stable factors. Among heat labile factors are trypsin inhibitor, hemagglutinins, phytate, goitrogens, and anti-vitamin factors. The heat stable factors included saponins, estrogens, flatulence factors, and 1977). Since the isolation process for the soy protein lysinoalanine (Leiner, various heat labile factors are destroyed. product involves heat treatment,
SOY
PROTEIN
AND
SERUM
CHOLESTEROL
387
Therefore, such factors can be omitted from the list of potential factors. Among the heat stable factors that arc considered to be “antinutritional,” estrogens and flatulence factors can be omitted because the estrogenic activity is present in too low a concentration to be of any physiological significance, and processed soy protein products are essentially devoid of flatus activity. Remaining factors, thus, are saponin and lysinoalanine. The term “antinutritional” was used by Leiner (1977) to include a poor protein efficiency ratio, other specific deficiency states and pathologic changes. In certain respects, such “antinutritional” effects might be exploited to advantage, if reduction in utilization of certain dietary ingredients is desired. The role of saponin as an “antinutritional” factor has been disclaimed in other studies with chicks, rats and mice. However, recent studies with alfalfa saponins in rats and monkeys by Malinow et al. (1977a, 1977b) compel us to look further into the role of saponin; they have demonstrated reduction in dietary cholesterol absorption in rats fed alfalfa top or root saponins, in both native and hydrolyzed states. The current study does not permit us to speculate regarding such effect on absorption, because there was no significant difference in cholesterol absorption between casein fed and soy protein fed groups. According to the manufacturer, the saponin content of the soy protein concentrate is approximately 0.4%. Therefore, the approximate content of saponin in the soy protein containing diet in the current study is 720 mg. Since the saponin content in the studies of Malinow et al. (1977a, 1977b) was ten times that of dietary cholesterol, 720 mg of saponin in the current study with 1055 mg of cholesterol probably was not sufficient to affect the cholesterol absorption. In mice, Ishaaya et al. (1969) have shown that an addition of soybean saponin extract did not affect the plasma cholesterol levels, whereas alfalfa saponin seemed to lower them. Other reports from the same group (Birk, 1969; Gestetner et al., 1972) indicated that such a difference is due to the lack of “affinity” of soybean saponin to cholesterol, although such views are not uniformly shared by others (Morgan et al., 1972). A heat stable factor, lysinoalanine, is produced when soy protein products are alkali-treated. Such lysinoalanine attached to protein in peptide linkage is not absorbed but is excreted in feces. Such loss through the feces may accentuate the “imbalance” already existing in the amino acid composition of soy protein as compared to casein. ACKNOWLEDGMENTS We are grateful to Dr. C. J. O’Donovan of Miles Laboratories, Inc., Elkhart, Indiana for the supply of Pro-Lean and his interest. We are also grateful to Mr. James Baker, Mrs. Maureen Gennett, Miss Susan Szawiela, Mr. Paul Sargent, and Mrs. Diana Surgick for their technical assistance. This study was supported by U.S. Public Health Service Grant HL-20993.
REFERENCES ANDERSON, J. T., GRANDE, F., and KEYS, A. (1971). Effect on man’s serum lipids of two proteins with different amino acid composition. Am. J. Clin. Nutr. 24, 524-530. BEST, M. M., DUNCAN, C. H., VAN LOON, E. J., and WALTHEN, J. D. (1955). The effects of sitosterol on serum lipids. Am. J. Med. 19, 61-70. BIRK, Y. (1969). Saponin. In “Toxic Constituents of Plant Foodstuffs” (I. E. Leiner, ed.), Academic Press, New York.
398
KIM
ET AL.
Uonc:sw~iinr, the kc&g
1%. ( 1960). (~it;ultificaticl~~ of c~llol~~stc~rcJ alxsorpticuk ill m;uk 11,. fcyd ;ctdysi5 ;lltc~i of a sin& isotope-labclcd mcsal. J. LilGtl Rrs. 10, 331-337. CAHHOLL, K. IL, and I~UILTON, R. M. G. ( 1975). Eft‘ccts of dietary protein and c;~rbohydrate on plasma cholesterol lcvcls in relation to atherosclerosis. J. Footl Sci. 40, 18-23. DATWZNON, J., SIhih%oNI%, \\‘. J., and AIIHENS, E. H., JH. ( 1968). Usefulness of chromic oxide as an internal standard for balance studies in formula-fed patients and for assessment of colonic function. J. Clin. Inuest. 47, 127-138. FAHQUHAR, J. W., SMITH, R. E., and DEZIPSEI., hl. (1936). The effect of p-sitosterol on serum lipids on young men with arteriosclerotic heart discax. Circulation 14, 77-82, 1956. FAHQUHAH, J. W., and SOSOLOW, M. ( 1958). Response of serum lipids and lipoproteins of man to /.%sitosterol and safflower oil: A long term study. Circthfiorl 17, 890-899. FOLCH, J., LEES, h4., and SLOANE-STANLEY, G. H. ( 1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Bio. Chem. 226, 497-509. GATTI, E., and SIRTORI, C. R. ( 1977). Soybean-protein diet and plasma cholesterol. (Reply) Lancet I (8015), 805-806. GESTETNEH, B., ASSA, Y., HEMIS, Y., TEXER, I-., RO,IXAX, M., BIHK, I’., and BONIX, A. (1972). Interaction of lucernc saponins with sterols. Bi~chitt~. Bio~hys. Actcl 270, 181-187. GRUNDY, S. M., AHHENS, E. H., Jn., and MIETTIXES, T. A. ( 1965). Quantitat;ve isolation and gas-liquid chromatographic analysis of total fecal bile acids. J. Lipid Res. 6, 397-410. GHUP\TDY, S. M., AHHENS, E. H., JH., and SALEN, G. (1968). Dietary /+sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J. Lipid Res. 9, 374-388. GRUXDY, S. M., and Mos, H. II. I. ( 1977). Determination of cholesterol ahsorption in man by intestinal perfusion. .l. Lipid Res. 18, 263-271. HAMILTON, R. M. G., and CAHHOLL, K. K. ( 1976). Pl a\ma ,’ cholesterol levels in rabbits fed low fat, low cholesterol diets. Effects of dietary proteins, carbohydrates, and fiber from different sources. Atherosclerosis 24, 47-62. HELMS, P. ( 1977). Soybean-protein diet axed plasma cholesterol. Lancct I ( 8015 ), 805-8’36. HODGES, R. E., KHEHL, W. A., STONE, D. B., and LOPEZ, A. (1967). Dietary carbohydrates and low choleatcrol diets: effects on serum lipids of man. Am. J. C/in. Ntcfr. 20, 198-208. HOWAHD, A. N., GHESHAM, G. A., JONES, D., and JESR.IR’GS, I. \\I. ( 1953). The pwvention of rabbit atherosclerosis by soybean. J. Atherosclerosis Res. 5, 330-337. IWAAYA, I., BIHK;, Y., BONDI, A., and TENCER, Y. (1969). %)-bean saponins. IS. Studies of their effect on birds, mammals, and cold-blooded organisms. J. Sci. Fd. Agric. 20, 43%136. KEYS, A., and ANDEHSON, J. T. (1957). Dietary protein and the strum cholesterol level. Am.
J. Clin. Nutr. 5, 29-34. KIM, D. N., LEE, K. T., REISM~, J. hl., and THOMAS, W. A. (1974). Rcatraint of cho!estcrol accumulation in tissue pools in hypercholesterolemic swine associated \vith drastic shortterm lowering of serum cholesterol levels by clofibrate or cholcstyramine. J. Lipid Rcs. 15, 326-331. \\‘. A. ( 1975). Effect of combined KIM, D. N., LEE, K. T., REIXER, J. hf., and TH~XM, clofibrate-cholestyramine treatment on serum and tissue cholesterol pools and on cholesterol synthesis in hypcrcholcsterolemic swine. EXAM/. MO~CC. Pathd. 23, 83-93. KHITCHEVSKY, D., TEPPEH, S. A., and STWW, J. A. (1978). of soy protein and I n fl ucncc casein on atherosclerosis in rabbits. Fed. Proc. 37, 747. LEFFLEH, H. H. ( 1959). Estimation of cholesterol in serum. AI~I. .I. Clin. Path. 31, 310-31:3. LEINEH, I. E. (1977). Nutritional aspects of soy protein products. J. Am. Oil Chem. Sot. 54, 454A-472A. MALINOW, M. R., MCLAUGHLIX, I?., KOHLEH, G. O., and LIVINGSU)N, A. L. (1977a). Prevention of elevated cholesterolemia in monkeys by alfalfa saponins. Steroids 29, 105-110. MALINOW, h4. R., MCLAUGHLIN, P., PAPU’ORTH, L., STAFFORD, C., KOHLER, G. O., LWISGSTOS, A. L., and CHEEKE, P. R. (1977b). Effect of alfalfa saponins on intestinal cholesterol absorption in rats Am. J. Clin. Nutr. 30, 2061-2067. MARSH, A., KIM, D. N., LEE, 6. T., HEWER, J. M., and THONAS, W. A. ( 1972). Cholesterol turnover, synthesis and retention in hypercholesterolemic growing swine. J. Lipid Res. 13, 600-615. MEEKER, D. R., and KESTEN, H. D. ( 1940). Experimental atherosclerosis and high protein diets. Proc. Sot. Exp. Biol. S Mccl. 45, 543-545 ( Abstract ).
SOY
PROTEIN
AND
SERUM
CHOLESTEROL
399
MEEKER, D. R., and KESTEN, H. D. ( 1941). Effects of high protein diets on experimental atherosclerosis of rabbits. Arch. Pathol. 31, 147-162. MIETTINEN, R. A., AHRENS, E. H., JR., and CRUNDY, S. M. (1965). Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. .I. Lipid Res. $411424. MORGAN, B., HEALD, M., BROOKS, S. G., TEE, J. L., and GREEN, J. (1972). The interaction between dietary saponin, cholesterol, and related sterols in the chick. Pot& Science 51, 677-682,. OLSON, R. E., VESTEH, J. W., GURSEY, D., DAVIS, N., and LONC,AIAN, D. (1958). The effect of low protein diets upon serum cholesterol in man. Am. .I. Clin. Notr. 6, 310-324. QUINT~O, E., GRUNDY, S. M., and AHREA-S, E. H., JR. ( 1971). An evaluation of four methods for measuring cholesterol absorption by the intestine in man. J. Lipid Res. 12, 221-232. SAMUEL, P., CROUSE, J. R., and AHRENS, E. H., Jn. (1978). Evaluation of an isotope ratio method for measurement of cholesterol absorption in man. .I. Lipid Res. 19, 82-93. SIRTORI, C. R., AGRADI, E., CONTI, F., and MOXTERO, 0. (1977). Soybean-protein diet in the treatment of Type II hyperlipoproteinemia. Luncet I, 275-277. SODHI, H. S., KUDCHODKAR, B. J., VARUGHESE, P., and DUNCAN, D. (1974). Validation of the ratio method for calculating absorption of dietary cholesterol in man. Proc. Sot. Exp. Biol. and Med. 145,107-111. WALKER, G. R., MORSE, E. H., and OVEIILEY, V. A. ( 1960). The effect of animal protein and vegetable protein diets having the same fat content on the serum lipid levels of young women. J. Nutr. 72, 317-321.