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Prevention of hypercholesterolemia in cholesterol-fed chickens by high-casein and high-soybean protein diets
Arherosclerosis, 46 (1983) 95- 104 Elsevier Scientific Publishers beland,
95 Ltd.
Prevention of Hypercholesterolemia in Cholesterol-fed Chickens by High-Casein and High-Soybean Protein Diets Anthony
H.M.
Terpstra
‘, J. Ben Schutte
* and Clive
E. West
’
’Departmeni of HumanNutrition, Agricultural University, De Dreven 12, 6703 BC Wageningen and ’Institute for Animal Nurrition Research (ILOB), Haarweg 8, 6709 PJ Wageningen (The Netherlandr) (Received 5 May, 1982) (Revised, received 16 July, 1982) (Accepted 16 August. 1982)
Chickens were fed cholesterol-enriched semipurified diets containing two levels (20% and 50%, w/w) of casein or soybean protein for 29 days. The ingestion of the cholesterol-enriched diets containing 20% casein or soybean protein resulted in markedly elevated levels of serum cholesterol, compared with the feeding of a cholesterol-free control diet containing 20% soybean protein. However, this hypercholesterolemic response could be prevented by feeding high-casein and high-soybean protein diets (50%). Similarly, lower levels of serum triglycerides and phospholipids were observed in the chickens fed the 50% protein diets compared with those fed the 20% protein diets. The excess of serum cholesterol and phospholipids in the 20% protein groups was mainly carried in the VLDL and to a lesser extent in the IDL fraction; this was associated with a decrease of cholesterol and phospholipids in the LDL fraction. The amount of cholesterol in the liver paralleled the cholesterol concentrations in the serum; the chickens fed the 20% protein diets exhibited significantly higher concentrations of liver cholesterol than those on the 50% protein diets. Thus, in chickens, the proportion of protein in the diet significantly affects the
Address for correspondence: Dr. CE. West, Department of Human Nutrition. Agricultural University, De Dreijen 12, 6703 BC Wageningen, The Netherlands. Abbrevations: VLDL, very low density lipoproteins (d CZ1.006 g/ml); IDL, intermediate density lipoproteins (1.006 < d < 1.019 g/ml); LDL, low density lipoproteins (1.019 < d c 1.063 g/ml); HDL, high density lipoproteins (1.063 < d .C 1.210 g/ml); VHDL, very high density lipoproteins (d > 1.210 g/mJ). 0021-9150/83/0000-0000/$03.00
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
96
levels of serum and liver cholesterol, whereas there is practically no differential cholesterolemic effect of casein and soybean protein in the diet. Key words: Casein - Chickens - Cholesterol - Lipoproteins
- Soybean protein
Introduction Studies in man and experimental animals have shown that dietary protein can affect cholesterol metabolism [ll. When diets containing casein are fed to mice [2], rats [3,4], rabbits [5] and pigs [6], severe hypercholesterolemia results, whereas low levels of serum cholesterol are maintained on soybean protein diets. Similarly, in hypercholesterolemic patients the incorporation of soybean protein in the diet has been reported to be effective in lowering the levels of serum cholesterol [7]. In previous studies, we have examined the effect of dietary protein on the levels of serum cholesterol and the lipoprotein composition in rabbits [5,8], rats, [4,9] and guinea pigs [lol. Al1 these experiments confirmed the differential cholesterolemic effect of dietary casein and soybean protein. However, in chickens fed cholesterol-free semipurified diets, no changes in serum cholesterol were observed when casein in the diet was substituted for soybean protein [ 111. Furthermore, we found that chickens could be readily made hypercholesterolemic by feeding cholesterol [ 111. The present study was designed to examine whether there might be an effect of both the proportion and nature of dietary protein on the levels of serum and liver lipids, when chickens are fed cholesterol-enriched semipurified diets. Materials and Methods 100 female broiler chickens 1 day old (Shaver-Starbro) were wing banded and randomly divided among 10 electrically heated battery cages in an artificially lit, heated and ventilated room. An ILOB semipurified standard diet containing 20% soybean protein isolate [12] was fed to them for the first 5 weeks. After this period, the birds were selected to obtain 5 groups of 10 birds each with similar mean body weights and plasma cholesterol concentrations. Subsequently, 4 groups were transferred to cholesterol-enriched semipurified diets containing 20% and 50% of either casein or soybean protein. The remaining group continued to receive the cholesterol-free diet containing 20% soybean protein. The composition of the diets is given in Table 1. Methionine was added to the soybean protein diets and arginine to the casein diets, as supplementation of the proteins with these amino acids is necessary for optimal growth [12,13]. The animals were housed per group and the diets, offered in the form of dry mash, and water were provided ad libitum. Blood samples were collected from a wing vein into heparizined tubes after an overnight fast. After an experimental period of 29 days, the chickens were decapitated and the livers removed. The serum samples taken at the end of the experiment were pooled per group and the plasma lipoproteins were isolated by density gradient ultracentrifugation [14]. Liver lipids were extracted according to Folch et al. [15].
97 TABLE
1
COMPOSITION Results
OF THE SEMIPURIFIED
are expressed
as g/kg
DIETS
feed. Cholesterol-free Soybean
Casein a L-Arginine Soybean protein b DL-Methionine Maize starch Dextrose Coconut oil Soybean oil Cellulose (Akufloc) Vitamin and mineral KHCO, NaCl Mg0 CaCO, CaHPO,.2 Cholesterol
H,O
premix ’
Cholesterol-enriched
protein
(20%)
Casein 20%
50%
200
500
8 _ _
200 2 317 300 40 10 65 10 15 4 2 5 30
Soybean
296 300 40 10 66 10 15 6 2 5 30 12
20 _ _ 153 150 40 10 50 10 15 6 2 12 20 12
20%
protein 50%
_ 200 2 305 300 40 10 65 10 15 4 2 5 30 12
500 5 171 150 40 10 50 10 15 3 2 12 20 12
a Acid casein, 865 g protein/kg. b Soy isolate, 880 g protein/kg. ’ Supplied per kg diet: thiamine hydrochlotide, 2.5 mg; riboflavine, 5.5 mg; pyridoxine hydrochloride, 3 mg; nicotinic acid, 50 mg; Ca pantothenate, IS mg; biotin, 0.15 mg; p-aminobenzoic acid, 2.5 mg; folie acid, 0.75 mg; inositol, 100 mg; chohne chloride, 1850 mg; vitamin B,,, 0.015 mg; a-tocopheryl acetate, 30 mg; menadione, 5 mg; ascorbic acid, 50 mg; retinyl aeetate, 15000 IU; cholecalciferol, 3000 IU; MnO,, 150 mg; ZnSO,.HzO, 200 mg; CuSO.,.S H,O, ISO mg; FeSO,. H,O, 200 mg; KI. 5 mg; Na,Se03, 0.2 mg; Avoparcine (antibiotic), 15 mg; ethoxyquin (antioxidant), 100 mg.
Serum and lipoprotein cholesterol were estimated enzymatically [16], using the commercial Catalase kit of Boehringer Mannheim and liver cholesterol with the method of Abel1 et al. [ 171. Triglycerides were measured according to Soloni [ 181 and phosphorus in a lipid extract [ 151 as described by Bartlett [19] and modified by Böttcher et al. [20]. The concentration of protein in the lipoprotein fractions was determined with a modification 1211 of the method of Lowry et al. 1221. The results were analysed statistically using a modified Student’s two-tailed t-test
Resuits Serum lipids The ingestion
of cholesterol-enriched
semipurified
diets containing
20% casein or
98
20% soybean protein resulted in a rapid increase in the levels of serum cholesterol. However, when high-protein diets were fed, low levels of serum cholesterol were maintained (Table 2). The concentration of triglycerides and phospholipids in the serum showed a similar tendency, the animals fed the 50% protein diets exhibited significantly lower levels of triglycerides and phospholipids than those on the 20% protein diets. NO significant differences in serum lipids were observed between the corresponding groups fed casein and soybean protein. Composìtion of the lipoproteìns
The density profile and composition of the plasma lipoproteins in the various dietary groups are presented in Fig. 1 and Table 3, respectively. The feeding of the cholesterol-enriched diets containing 20% protein resulted in a shift of cholesterol from the LDL to the IDL and VLDL (Table 3) compared with the groups fed 50% protein diets. Most of the excess of serum cholesterol and phospholipids was transported in the VLDL fraction and to a lesser extent in the IDL. These findings were reflected in the visual impressions of the density profile of the serum lipoproteins (Fig. 1). The introduction of cholesterol in the diet resulted in a HDL band with a higher mean density (Fig. 1). This might be partly explained by a decrease in the phospholipid concentration and a lower ratio of phospholipids to protein (Table 3). Furthermore, the groups on the cholesterol-enriched diets containing 50% protein had a higher cholesterol concentration and a higher ratio of cholesterol to
VLDL IDL LDL F
c
HOL
cn
z w
m
VHDL A
B
C
D
E
F
Fig. 1. Photograph of the density profile of prestained serum lipoproteins from pooled sera of chickens fed various diets compared with that of a normocholesterolemic human. Normocholesterolemic human (A), chickerts fed a cholesterol-free diet containing 20% soybean protein (B), chickens fed a cholesterol-enriched diet containing 20% casein (C), 50% casein (D), 20% soybean protein (E) or 50% soybean protein (F).
2
are expressed
a-d Comparison
by a modified
Student’s
85.3
0.8 =
0.19 0.11 0.12 0.10 0.15 0.02 0.11
two-tailed
f
3.71 f 4.08k 3.86+ 3.75 f 3.45+ 0.18* 2.92k 72.8
k
3.83* 9.74* 10.63 f 13.01+ 11.23+ 0.33 f 3.11 +
74.49+ 8.84t 39.52+
r-test [23]: horizontal
a a = a a.b =
9.87 f 0.37 = 11.75f 2.91’ 36.38 + 0.57 ’
735 2 8 1993 _+36a 43 f l.2a 36.46f 1.41 a
b b b b a.C ’
‘ril f 62 a.b I 2.2 a.h tr 1.70 a
_+
_+
1.8 bx
0.13 0.69 h 1.03 h 0.83 ’ 0.98 b 0.06 ’ 0.17’
are significantly
77.5
3.63rt 8.29& 9.48k 9.59* 11.03+ 0.38 & 2.91 +
6.39 h 1.1 1 ‘.’ 1.06“
different
f
= ’ a ’ d b 3.0 b.d
0.20 0.21 0.26 0.19 0.25 0.01 0.11
(P < 0.05).
81.5
3.73* 3.78+ 4.55 + 4.09+ 3.81 & O.O6t_ 2.141
17.23 + 1.22’ 6.10+ 0.79a.h 39.30; 0.76 h
724 f 13 1791 +65h 31 f 2.1h 33.37+ 1.26’
50%
AND TRIGLYCERIDES OF ElTHER CASEIN
f 15 + 63 ‘.’ i2.1d f 1.63 b
protein
77.31 + 5.20+ 33.632
729 1975 43 46.21
superscript
1.o <.“
0.14 0.16 ’ 0.18’ 0.68 ’ 0.80 a 0.04 b.d 0.17 h
1.22’ 1.12 h 0.73 c
a common
80.9
3.69+ 4.07 f 4.32+ 4.67 i 5.07 + 0.10+ 2.22+
21.16+ 4.56k 31.54+
731 1821 38 36.35
values not sharing
2.8 h
0.13 0.71 0.86 1.02 1.65 0.07 0.24
5.75 b 1.75 a.h 1.03 ’
724 *9 1918 f 52 a.b 41 f 1.7 &b 44.28+ 2.07 ’
20%
50%
20%
-
Soybean
Casein
(20%)
Soybean
protein
Cholesterol-enriched
per group).
PHOSPHOLIPIDS PROPORTIONS
Cholesterol-free
as mean f SEM ( 10 animals
Initial body weight (g) Final body weight (g) Weight gain (g/day) Liver weight (g) Liver lipids (gmol/g wet liver) cholesterol triglycerides phospholipids Serum lipids (mmol/l) cholesterol initial 7 days 15 days 22 days 29 days triglycerides (29 days) phospholipids (29 days) Proportion (R;) of serum cholesterol esterified (29 days)
Results
BODY WEIGHT, WEIGHT GAIN, LIVER WEIGHT AND CONCENTRATION OF CHOLESTEROL, LIVER AND SERUM OF CHICKENS FED SEMIPURIFIED DIETS CONTAINING DIFFERENT SOYBEAN PROTEIN
TABLE
IN OR
Cholesterol VLDL IDL LDL HDL VHDL Phospholipids VLDL IDL LDL HDL VHDL Ratio cholesterol to protein VLDL IDL LDL HDL Ratio phospholipid to protein VLDL IDL LDL HDL
The lipoproteins were separated of cholesterol and phospholipids weight basis.
OF CHOLESTEROL TO PROTEIN AND PHOSPHOLIPIDS DIETS CONTAINING DIFFERENT PROPORTIONS OF
4.36 3.65 2.80 0.42 1.58 1.83 1.68 0.48
2.32 3.3 1 2.45 0.43 0.92 1.68
3.28 3.61 2.94 0.32 1.20 1.84 1.77 0.41
_
_ 1.35 0.39
_ 1.42 0.71
0.55
1.37
1.43 0.58
1.27
0.33
0.75 2.33 2.24 0.45
0.17
1.39
0.08 0.12 0.29
1.09 0.25 0.18 1.30 0.15
1.30 0.39 0.19 1.10 0.15
0.04 0.02 0.31 2.39 0.20
0.16 0.18 0.39 1.32 0.16
50%
0.36 0.44 0.91 2.15 0.12
protein
6.01 1.00 0.60 2.32 0.15
20%
7.12 1.53 0.63 1.69 0.11
0.8 1 0.7 1 1.40 2.05 0.09
50%
Soybean
0.02 0.02 0.59 2.65 0.14
20%
Casein
(20%)
Soybean
protein
Cholesterol-enriched
Cholesterol-free
by density gradient ultracentrifugation from pools of 10 animals in each group, after 29 days on the diets. The concentrations are expressed in mmol/l of whole serum. The ratio of cholesterol to protein and phospholipids to protein are calculated on a
CONCENTRATION OF CHOLESTEROL AND PHOSPHOLIPIDS AND THE RATIO TO PROTEIN IN SERUM LIPOPROTEINS OF CHICKENS FED SEMIPURIFIED EITHER CASEIN OR SOYBEAN PROTEIN
TABLE 3 B
101
protein in the LDL, together group fed the cholesterol-free
with a lower mean density diet.
of the LDL band
than the
Liver lip&The concentration of cholesterol in the liver paralleled the levels of cholesterol in the serum. The 50% protein groups exhibited markedly lower levels of liver cholesterol than the 20% protein groups (Table 2). Nevertheless, the groups receiving the cholesterol-enriched diets containing 50% protein had stil1 higher levels than the chickens fed the cholesterol-free diet. Similarly, significantly lower concentrations of triglycerides and phospholipids were measured in the liver of the 50% casein group compared with the 20% casein group. However, in the chickens fed cholesterol-enriched soybean protein diets, the lowest phospholipid concentration was found in the animals fed the 20% soybean protein diet, whereas no significant differences in liver triglycerides were observed. Discussion This study shows that in cholesterol-fed chickens the replacement of casein in the diet by soybean protein does not significantly affect the levels of serum cholesterol. These results are in contrast with findings in other animal species, showing a hypercholesterolemic effect of dietary casein compared with soybean protein [ 1-61. The present study also revealed that high protein diets are effective in lowering serum cholesterol levels in cholesterol-fed chickens, irrespective of whether casein or soybean protein is fed. These findings are in agreement with data from other studies with chickens, in which a hypocholesterolemic effect was observed with diets containing increasing proportions of casein or soybean protein [ll. In rats, highsoybean protein diets also have a hypocholesterolemic effect [9]. However, when casein diets are fed, it has been reported that in rats [4,9], rabbits [24] and pigeons [25], increasing the proportion of protein in the diet resulted in elevation of serum cholesterol levels. An explanation for these contrasting results obtained in chickens and other animal species is not readily available. It is possible that, as wil1 be discussed later, differences between animal species in the digestion of the protein play a role. In the present study, the casein and soybean protein in the diet was enriched with arginine and methionine, respectively. Soybean protein is low in methionine [ 121 and chickens fed casein diets not supplemented with arginine exhibit poor growth [ 131. Although there is evidente that addition of amino acids to the diet can affect the levels of serum cholesterol [ 11, it seems unlikely that this has occurred in the present study. Johnson et al. [13] reported that in chickens fed 10% protein diets, supplementation of casein with arginine and soybean protein with methionine resulted in a decrease of serum cholesterol levels. However, such effects were not found when 25% or 40% protein diets were used. Similarly, Leveille et al. [26] and Nishida et al. [27] observed that in chickens the addition of methionine to a soybean protein diet had a cholesterol-lowering effect only with diets containing marginal levels of this amino acid.
102
The excess of cholesterol in the serum of the chickens fed the cholesterol-enriched diets containing 20% protein was found to be carried mainly in the VLDL and the IDL lipoproteins. This was associated with a lower concentration of LDL cholesterol, compared with the animals fed the cholesterol-enriched diets containing 50% protein. A similar pattern has been found in rats made hypercholesterolemic by feeding cholesterol-enriched casein-diets [4]. However, in rats, the LDL fraction cannot be clearly discerned [4,28] and an increase in serum cholesterol was found to be associated with elevated cholesterol levels in the VLDL and IDL together with lower levels of cholesterol in the HDL. In rabbits [5] and guinea pigs [lol, on the other hand, hypercholesterolemia is primarily associated with elevated LDL levels. It is not yet clear how dietary protein can affect cholesterol metabolism and how, in chickens, high-casein and high-soybean protein diets are able to lower serum cholesterol levels. However, it has been shown that in chickens the feeding of high-protein diets resulted in an increase in cholesterol synthesis [29,30], fecal steroid excretion [31-331 and cholesterol turnover rate [32]. The major pathway for the disposal of steroids from the body is the excretion of neutral steroids and bile acids in the feces [34]. The secretion and reabsorption of bile acids and cholesterol by means of the enterohepatic circulation plays an important role in the regulation of cholesterol metabolism. Cholesterol can be converted to bile acids and subsequently excreted in the bile. However, most of the bile acids are reabsorbed. Furthermore, there is evidente that a less efficient reabsorption of bile acids can lower serum cholesterol levels. We have found that the feeding of cholestyramine, a bile acid sequestrant, to hypercholesterolemic rabbits effectively reduced the levels of serum cholesterol [35]. On the other hand, rabbits starved for several weeks and in which fecal production and hence the fecal excretion of bile acids and neutral steroids are markedly reduced, are known to develop severe hypercholesterolemia [36]. In chickens, Sklan and coworkers [37,38] reported that high-casein diets depressed the absorption of bile acids and found a highly significant correlation between the bile acid, absorption, which mainly occurs in the ileum, and the concentration in the ileum of both insoluble nitrogen and the high molecular weight peptide fractions. They also observed that intact casein, albumin or soybean protein caused an inhibition of the mucosal uptake of taurocholic acid from chick intestinal duodenum loops; this effect could be enhanced by increasing the amount of protein. However, predigestion of casein released this inhibitory effect. These authors also reported that intact casein was able to bind bile acids. Therefore, they suggested that the presence of undigested protein in the intestine of the chicken might interfere with the bile acid reabsorption and subsequently affect the serum cholesterol levels. Chickens react differently from other species of animals in their cholesterolemic response to diets containing various proportions of casein and soybean protein. This could possibly be explained by a different mode of digestion of dietary protein between various animal species. For example, in chickens, a reflux of digesta from the duodenum to the gizzard occurs [39], which might also affect the manner and rate of digestion of the protein in the diet. Further studies have to be done to elucidate the mechanism of the cholesterolemic properties of dietary protein.
103
Ref erences 1 Terpstra, A.H.M., Hermus, R.J.J. and West, C.E., The role of dietary protein in cholesterol metabolism, World Rev. Nutr. Diet., 39 (1982) In press. 2 Roy, D.M. and Schneeman, B.O., Effect of soy protein, casein and trypsin inhíbitor on cholesterol, bile acids and pancreatic enzymes in mice, J. Nutr., 111(1981) 878. 3 Yadav, N.R. and Liener, I.E., Reduction of serum cholesterol in rats fed vegetable protein or an equivalent amino acid mixture, Nutr. Rep. Int., 16 (1977) 385. 4 Terpstra, A.H.M., van Tintelen, G. and West, C.E., The effect of semipurified diets containing different proportions of either casein or soybean protein on the concentration of cholesterol in whole serum, serum Iipoproteins and Iiver in male and female rats, Atherosclerosis, 42 (1982) 85. 5 Terpstra, A.H.M., Woodward, C.J.H., West, C.E. and van Boven, H.G., A Iongitudinal cross-over study of serum cholesterol and lipoproteins in rabbits fed semi-purified diets containing either casein or soy-bean protein, Brit. J. Nutr., 47 (1982) 213. 6 Kim, D.N., Lee, K.T., Reiner, J.M. and Thomas, W.A., Effects of a soy protein product on serum and tissue cholesterol concentrations in swine fed high-fat, high-cholesterol diets, Exp. Mol. Path.. 29 (1978) 385. 7 Sirtori, C.R., Gatti, E., Montero, 0.. Conti, F., Agradi, E., Tremoli, E., Sirtori, M., Fraterrigo. L., Tavazzi, L. and Kritchevsky, D., Clinical experience with the soybean protein diet in the treatment of hypercholesterolemia, Amer. J. Clin. Nutr., 32 (1979) 1645. 8 Scholz, K.E.. Beynen, A.C. and West, CE., Comparison between the hypercholesterolemia in rabbi& induced by semipurified diets containing either cholesterol or casein, Atherosclerosis. 44 (1982) 85. 9 Terpstra, A.H.M., Van Tintelen, G. and West, C.E., The hypocholesterolemic effect of dietary soy protein in rats, J. Nutr., 112 (1982) 810. 10 Terpstra, A.H.M., Van Tintelen, G. and West, C.E., Dietary protein and serum cholesterol in guinea pigs, Nutr. Rep. Int., 25 (1982) 725. 11 Mol, M.A.E., De Smet, R.C., Terpstra, A.H.M. and West, CE., The effect of dietary protein and cholesterol on cholesterol concentration and lipoprotein pattern in the serum of chickens, J. Nutr., 112 (1982) 1029. 12 Schutte, J.B. and Van Weerden, E.J., Effectiveness of the Iiquid methionine product DL-methionine-Na in comparison with DL-methionine in broilers, Feed Management, 33 (1982) 43. 13 Johnson, D., Leveille, G.A. and Fisher, H., Influence of amino acid deficiencies and protein leve1 on the plasma cholesterol of the chick, J. Nutr.. 66 (1958) 367. 14 Terpstra, A.H.M., Woodward, C.J.H. and Sanchez-Muñiz, F.J., Improved techniques for the separation of serum Iipoproteins by density gradient ultracentrifugation - Visualization by prestaining and rapid separation of serum Iipoproteins from smal1 volumes of serum, Anal. Biochem., 11 l (1981) 149. 15 Folch, J., Lees, M. and Stanley, G.H.S., A simple method for the isolation and purification of total lipids from animal tissues, J. Biel. Chem., 226 (1957) 497. 16 Röschlau, P.. Bernt, E. and Gruber, W.. Enzymatische Bestimmung des Gesamt-Cholesterlns im Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403. 17 Abel], L.L.. Levy, B.B., Brodie, B.B. and Kendall, F.E., A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biel. Chem., 195 (1952) 357. 18 Soloni, F.G.. Simplified manual micromethod for determination of serum triglycerides, Clin. Chem., 17 (1971) 529. 19 Bartlett. G.R., Phosphorus assay in column chromatography. J. Biel. Chem., 234 (1959) 466. 20 Böttcher, C.J.F., Van Gent, C.M. and Pries, C., A rapid and sensitive sub-micro phosphorous determination, Anal. Chim. Acts, 24 (1961) 203. 21 Markwell, M.A.K., Haas, S.M., Bieber, L.L. and Tolbert. N.E., A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples, Anal. Biochem.. 87 ( 1978) 206. 22 Lowry, O.H.. Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (195 1) 265. 23 Snedecor, G.W. and Cochran, W.G., Statistical Methods. Iowa State University Press, Ames, IA. 1967. p. 115.
104
24 Terpstra, A.H.M., Harkes, L. and Van der Veen, F.H., The effect of different proportions of casein in semipurified diets on the concentration of serum cholesterol and the lipoprotein composition in rabbits, Lipids, 16 (1981) 114. 25 Lofland, H.B., Clarkson, T.B. and Goodman, H.O., Interactions among dietary fat, protein and cholesterol in atherosclerosis-susceptible pigeons - Effect on serum cholesterol and aortic atherosclerosis, Circ. Res., 9 (1961) 919. 26 Leveille, G.A., Shockley, J.W. and Sauberlich, H.E., Influence of dietary protein leve1 and amino acids on plasma cholesterol of the growing chick, J. Nutr., 76 (1962) 321. 27 Nishida, T., Takenaka, F. and Kummerow, F.A., Effect of dietary protein and heated fat on serum cholesterol and beta-lipoprotein levels, and on the incidence of experimental atherosclerosis in chicks, Circ. Res., 6 (1958) 194. 28 Terpstra, A.H.M., Sanchez-Muñiz, F.J., West, CE. and Woodward, C.J.H., The density profile and the cholesterol concentration of serum lipoproteins in domestic and laboratory animals, Comp. Biochem. Physiol., 71B (1982) 669. 29 Yeh, Y.-Y. and Leveille, G.A., Effects of dietary protein on hepatic lipogenesis in the growing chick, J. Nutr., 98 (1969) 356. 30 Yeh, S.-j.C. and Leveille, G.A., Cholesterol and fatty acid synthesis in chicks fed different levels of protein, J. Nutr., 102 (1972) 349. 31 Kenney, J.J. and Fisher, H., Effect of medium chain triglycerides and dietary protein on cholesterol absorption and deposition in the chicken, J. Nutr., 103 (1973) 923. 32 Yeh, S.-j.C. and Leveille, G.A., Influence of dietary protein leve1 on plasma cholesterol turnover and fecal steroid excretion in the chick, J. Nutr., 103 (1973) 407. 33 Leveille, G.A., Shockley, J.W. and Sauberlich, H.E., Influence of dietary factors on plasma lipid relationships in the growing chick, Proc. Sec. Exp. Biol. Med., 108 (1961) 313. 34 Dietschy, J.M., Mechanism for the intestinal absorption of bile acids, J. Lipid Res., 9 (1968) 297. 35 Terpstra, A.H.M., Hermus, R.J.J. and West, C.E., Dietary protein and cholesterol metabolism in rabbits and rats. In: D. Kritchevsky and M.J. Gibney (Eds.), Animal and Vegetable Protein in Lipid Metabolism, Alan R. Liss Inc., New York, NY, In press. 36 Swanor, J.C. and Connor, W.E., Hypercholesterolemia of total starvation - Its mechanism via tissue mobilization of cholesterol, Amer. J. Physiol., 229 (1975) 365. 37 Sklan, D., Budowski, P. and Hurwitz, S., Absorption of oleic and taurocholic acids from the intestine of the chick, Biochim. Biophys. Acts, 573 (1979) 31. 38 Sklan, D., Digestion and absorption of casein at different levels in the chick - Effect on fatty acid and bile acid absorption, J. Nutr., 110 (1980) 989. 39 Sklan, D., Shachof, B., Baron, J. and Hurwitz, S., Retrograde movement of digesta in the duodenum of the chick - Extent, frequency and nutritional implications, J. Nutr., 108 (1978) 1485.
95 Ltd.
Prevention of Hypercholesterolemia in Cholesterol-fed Chickens by High-Casein and High-Soybean Protein Diets Anthony
H.M.
Terpstra
‘, J. Ben Schutte
* and Clive
E. West
’
’Departmeni of HumanNutrition, Agricultural University, De Dreven 12, 6703 BC Wageningen and ’Institute for Animal Nurrition Research (ILOB), Haarweg 8, 6709 PJ Wageningen (The Netherlandr) (Received 5 May, 1982) (Revised, received 16 July, 1982) (Accepted 16 August. 1982)
Chickens were fed cholesterol-enriched semipurified diets containing two levels (20% and 50%, w/w) of casein or soybean protein for 29 days. The ingestion of the cholesterol-enriched diets containing 20% casein or soybean protein resulted in markedly elevated levels of serum cholesterol, compared with the feeding of a cholesterol-free control diet containing 20% soybean protein. However, this hypercholesterolemic response could be prevented by feeding high-casein and high-soybean protein diets (50%). Similarly, lower levels of serum triglycerides and phospholipids were observed in the chickens fed the 50% protein diets compared with those fed the 20% protein diets. The excess of serum cholesterol and phospholipids in the 20% protein groups was mainly carried in the VLDL and to a lesser extent in the IDL fraction; this was associated with a decrease of cholesterol and phospholipids in the LDL fraction. The amount of cholesterol in the liver paralleled the cholesterol concentrations in the serum; the chickens fed the 20% protein diets exhibited significantly higher concentrations of liver cholesterol than those on the 50% protein diets. Thus, in chickens, the proportion of protein in the diet significantly affects the
Address for correspondence: Dr. CE. West, Department of Human Nutrition. Agricultural University, De Dreijen 12, 6703 BC Wageningen, The Netherlands. Abbrevations: VLDL, very low density lipoproteins (d CZ1.006 g/ml); IDL, intermediate density lipoproteins (1.006 < d < 1.019 g/ml); LDL, low density lipoproteins (1.019 < d c 1.063 g/ml); HDL, high density lipoproteins (1.063 < d .C 1.210 g/ml); VHDL, very high density lipoproteins (d > 1.210 g/mJ). 0021-9150/83/0000-0000/$03.00
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
96
levels of serum and liver cholesterol, whereas there is practically no differential cholesterolemic effect of casein and soybean protein in the diet. Key words: Casein - Chickens - Cholesterol - Lipoproteins
- Soybean protein
Introduction Studies in man and experimental animals have shown that dietary protein can affect cholesterol metabolism [ll. When diets containing casein are fed to mice [2], rats [3,4], rabbits [5] and pigs [6], severe hypercholesterolemia results, whereas low levels of serum cholesterol are maintained on soybean protein diets. Similarly, in hypercholesterolemic patients the incorporation of soybean protein in the diet has been reported to be effective in lowering the levels of serum cholesterol [7]. In previous studies, we have examined the effect of dietary protein on the levels of serum cholesterol and the lipoprotein composition in rabbits [5,8], rats, [4,9] and guinea pigs [lol. Al1 these experiments confirmed the differential cholesterolemic effect of dietary casein and soybean protein. However, in chickens fed cholesterol-free semipurified diets, no changes in serum cholesterol were observed when casein in the diet was substituted for soybean protein [ 111. Furthermore, we found that chickens could be readily made hypercholesterolemic by feeding cholesterol [ 111. The present study was designed to examine whether there might be an effect of both the proportion and nature of dietary protein on the levels of serum and liver lipids, when chickens are fed cholesterol-enriched semipurified diets. Materials and Methods 100 female broiler chickens 1 day old (Shaver-Starbro) were wing banded and randomly divided among 10 electrically heated battery cages in an artificially lit, heated and ventilated room. An ILOB semipurified standard diet containing 20% soybean protein isolate [12] was fed to them for the first 5 weeks. After this period, the birds were selected to obtain 5 groups of 10 birds each with similar mean body weights and plasma cholesterol concentrations. Subsequently, 4 groups were transferred to cholesterol-enriched semipurified diets containing 20% and 50% of either casein or soybean protein. The remaining group continued to receive the cholesterol-free diet containing 20% soybean protein. The composition of the diets is given in Table 1. Methionine was added to the soybean protein diets and arginine to the casein diets, as supplementation of the proteins with these amino acids is necessary for optimal growth [12,13]. The animals were housed per group and the diets, offered in the form of dry mash, and water were provided ad libitum. Blood samples were collected from a wing vein into heparizined tubes after an overnight fast. After an experimental period of 29 days, the chickens were decapitated and the livers removed. The serum samples taken at the end of the experiment were pooled per group and the plasma lipoproteins were isolated by density gradient ultracentrifugation [14]. Liver lipids were extracted according to Folch et al. [15].
97 TABLE
1
COMPOSITION Results
OF THE SEMIPURIFIED
are expressed
as g/kg
DIETS
feed. Cholesterol-free Soybean
Casein a L-Arginine Soybean protein b DL-Methionine Maize starch Dextrose Coconut oil Soybean oil Cellulose (Akufloc) Vitamin and mineral KHCO, NaCl Mg0 CaCO, CaHPO,.2 Cholesterol
H,O
premix ’
Cholesterol-enriched
protein
(20%)
Casein 20%
50%
200
500
8 _ _
200 2 317 300 40 10 65 10 15 4 2 5 30
Soybean
296 300 40 10 66 10 15 6 2 5 30 12
20 _ _ 153 150 40 10 50 10 15 6 2 12 20 12
20%
protein 50%
_ 200 2 305 300 40 10 65 10 15 4 2 5 30 12
500 5 171 150 40 10 50 10 15 3 2 12 20 12
a Acid casein, 865 g protein/kg. b Soy isolate, 880 g protein/kg. ’ Supplied per kg diet: thiamine hydrochlotide, 2.5 mg; riboflavine, 5.5 mg; pyridoxine hydrochloride, 3 mg; nicotinic acid, 50 mg; Ca pantothenate, IS mg; biotin, 0.15 mg; p-aminobenzoic acid, 2.5 mg; folie acid, 0.75 mg; inositol, 100 mg; chohne chloride, 1850 mg; vitamin B,,, 0.015 mg; a-tocopheryl acetate, 30 mg; menadione, 5 mg; ascorbic acid, 50 mg; retinyl aeetate, 15000 IU; cholecalciferol, 3000 IU; MnO,, 150 mg; ZnSO,.HzO, 200 mg; CuSO.,.S H,O, ISO mg; FeSO,. H,O, 200 mg; KI. 5 mg; Na,Se03, 0.2 mg; Avoparcine (antibiotic), 15 mg; ethoxyquin (antioxidant), 100 mg.
Serum and lipoprotein cholesterol were estimated enzymatically [16], using the commercial Catalase kit of Boehringer Mannheim and liver cholesterol with the method of Abel1 et al. [ 171. Triglycerides were measured according to Soloni [ 181 and phosphorus in a lipid extract [ 151 as described by Bartlett [19] and modified by Böttcher et al. [20]. The concentration of protein in the lipoprotein fractions was determined with a modification 1211 of the method of Lowry et al. 1221. The results were analysed statistically using a modified Student’s two-tailed t-test
Resuits Serum lipids The ingestion
of cholesterol-enriched
semipurified
diets containing
20% casein or
98
20% soybean protein resulted in a rapid increase in the levels of serum cholesterol. However, when high-protein diets were fed, low levels of serum cholesterol were maintained (Table 2). The concentration of triglycerides and phospholipids in the serum showed a similar tendency, the animals fed the 50% protein diets exhibited significantly lower levels of triglycerides and phospholipids than those on the 20% protein diets. NO significant differences in serum lipids were observed between the corresponding groups fed casein and soybean protein. Composìtion of the lipoproteìns
The density profile and composition of the plasma lipoproteins in the various dietary groups are presented in Fig. 1 and Table 3, respectively. The feeding of the cholesterol-enriched diets containing 20% protein resulted in a shift of cholesterol from the LDL to the IDL and VLDL (Table 3) compared with the groups fed 50% protein diets. Most of the excess of serum cholesterol and phospholipids was transported in the VLDL fraction and to a lesser extent in the IDL. These findings were reflected in the visual impressions of the density profile of the serum lipoproteins (Fig. 1). The introduction of cholesterol in the diet resulted in a HDL band with a higher mean density (Fig. 1). This might be partly explained by a decrease in the phospholipid concentration and a lower ratio of phospholipids to protein (Table 3). Furthermore, the groups on the cholesterol-enriched diets containing 50% protein had a higher cholesterol concentration and a higher ratio of cholesterol to
VLDL IDL LDL F
c
HOL
cn
z w
m
VHDL A
B
C
D
E
F
Fig. 1. Photograph of the density profile of prestained serum lipoproteins from pooled sera of chickens fed various diets compared with that of a normocholesterolemic human. Normocholesterolemic human (A), chickerts fed a cholesterol-free diet containing 20% soybean protein (B), chickens fed a cholesterol-enriched diet containing 20% casein (C), 50% casein (D), 20% soybean protein (E) or 50% soybean protein (F).
2
are expressed
a-d Comparison
by a modified
Student’s
85.3
0.8 =
0.19 0.11 0.12 0.10 0.15 0.02 0.11
two-tailed
f
3.71 f 4.08k 3.86+ 3.75 f 3.45+ 0.18* 2.92k 72.8
k
3.83* 9.74* 10.63 f 13.01+ 11.23+ 0.33 f 3.11 +
74.49+ 8.84t 39.52+
r-test [23]: horizontal
a a = a a.b =
9.87 f 0.37 = 11.75f 2.91’ 36.38 + 0.57 ’
735 2 8 1993 _+36a 43 f l.2a 36.46f 1.41 a
b b b b a.C ’
‘ril f 62 a.b I 2.2 a.h tr 1.70 a
_+
_+
1.8 bx
0.13 0.69 h 1.03 h 0.83 ’ 0.98 b 0.06 ’ 0.17’
are significantly
77.5
3.63rt 8.29& 9.48k 9.59* 11.03+ 0.38 & 2.91 +
6.39 h 1.1 1 ‘.’ 1.06“
different
f
= ’ a ’ d b 3.0 b.d
0.20 0.21 0.26 0.19 0.25 0.01 0.11
(P < 0.05).
81.5
3.73* 3.78+ 4.55 + 4.09+ 3.81 & O.O6t_ 2.141
17.23 + 1.22’ 6.10+ 0.79a.h 39.30; 0.76 h
724 f 13 1791 +65h 31 f 2.1h 33.37+ 1.26’
50%
AND TRIGLYCERIDES OF ElTHER CASEIN
f 15 + 63 ‘.’ i2.1d f 1.63 b
protein
77.31 + 5.20+ 33.632
729 1975 43 46.21
superscript
1.o <.“
0.14 0.16 ’ 0.18’ 0.68 ’ 0.80 a 0.04 b.d 0.17 h
1.22’ 1.12 h 0.73 c
a common
80.9
3.69+ 4.07 f 4.32+ 4.67 i 5.07 + 0.10+ 2.22+
21.16+ 4.56k 31.54+
731 1821 38 36.35
values not sharing
2.8 h
0.13 0.71 0.86 1.02 1.65 0.07 0.24
5.75 b 1.75 a.h 1.03 ’
724 *9 1918 f 52 a.b 41 f 1.7 &b 44.28+ 2.07 ’
20%
50%
20%
-
Soybean
Casein
(20%)
Soybean
protein
Cholesterol-enriched
per group).
PHOSPHOLIPIDS PROPORTIONS
Cholesterol-free
as mean f SEM ( 10 animals
Initial body weight (g) Final body weight (g) Weight gain (g/day) Liver weight (g) Liver lipids (gmol/g wet liver) cholesterol triglycerides phospholipids Serum lipids (mmol/l) cholesterol initial 7 days 15 days 22 days 29 days triglycerides (29 days) phospholipids (29 days) Proportion (R;) of serum cholesterol esterified (29 days)
Results
BODY WEIGHT, WEIGHT GAIN, LIVER WEIGHT AND CONCENTRATION OF CHOLESTEROL, LIVER AND SERUM OF CHICKENS FED SEMIPURIFIED DIETS CONTAINING DIFFERENT SOYBEAN PROTEIN
TABLE
IN OR
Cholesterol VLDL IDL LDL HDL VHDL Phospholipids VLDL IDL LDL HDL VHDL Ratio cholesterol to protein VLDL IDL LDL HDL Ratio phospholipid to protein VLDL IDL LDL HDL
The lipoproteins were separated of cholesterol and phospholipids weight basis.
OF CHOLESTEROL TO PROTEIN AND PHOSPHOLIPIDS DIETS CONTAINING DIFFERENT PROPORTIONS OF
4.36 3.65 2.80 0.42 1.58 1.83 1.68 0.48
2.32 3.3 1 2.45 0.43 0.92 1.68
3.28 3.61 2.94 0.32 1.20 1.84 1.77 0.41
_
_ 1.35 0.39
_ 1.42 0.71
0.55
1.37
1.43 0.58
1.27
0.33
0.75 2.33 2.24 0.45
0.17
1.39
0.08 0.12 0.29
1.09 0.25 0.18 1.30 0.15
1.30 0.39 0.19 1.10 0.15
0.04 0.02 0.31 2.39 0.20
0.16 0.18 0.39 1.32 0.16
50%
0.36 0.44 0.91 2.15 0.12
protein
6.01 1.00 0.60 2.32 0.15
20%
7.12 1.53 0.63 1.69 0.11
0.8 1 0.7 1 1.40 2.05 0.09
50%
Soybean
0.02 0.02 0.59 2.65 0.14
20%
Casein
(20%)
Soybean
protein
Cholesterol-enriched
Cholesterol-free
by density gradient ultracentrifugation from pools of 10 animals in each group, after 29 days on the diets. The concentrations are expressed in mmol/l of whole serum. The ratio of cholesterol to protein and phospholipids to protein are calculated on a
CONCENTRATION OF CHOLESTEROL AND PHOSPHOLIPIDS AND THE RATIO TO PROTEIN IN SERUM LIPOPROTEINS OF CHICKENS FED SEMIPURIFIED EITHER CASEIN OR SOYBEAN PROTEIN
TABLE 3 B
101
protein in the LDL, together group fed the cholesterol-free
with a lower mean density diet.
of the LDL band
than the
Liver lip&The concentration of cholesterol in the liver paralleled the levels of cholesterol in the serum. The 50% protein groups exhibited markedly lower levels of liver cholesterol than the 20% protein groups (Table 2). Nevertheless, the groups receiving the cholesterol-enriched diets containing 50% protein had stil1 higher levels than the chickens fed the cholesterol-free diet. Similarly, significantly lower concentrations of triglycerides and phospholipids were measured in the liver of the 50% casein group compared with the 20% casein group. However, in the chickens fed cholesterol-enriched soybean protein diets, the lowest phospholipid concentration was found in the animals fed the 20% soybean protein diet, whereas no significant differences in liver triglycerides were observed. Discussion This study shows that in cholesterol-fed chickens the replacement of casein in the diet by soybean protein does not significantly affect the levels of serum cholesterol. These results are in contrast with findings in other animal species, showing a hypercholesterolemic effect of dietary casein compared with soybean protein [ 1-61. The present study also revealed that high protein diets are effective in lowering serum cholesterol levels in cholesterol-fed chickens, irrespective of whether casein or soybean protein is fed. These findings are in agreement with data from other studies with chickens, in which a hypocholesterolemic effect was observed with diets containing increasing proportions of casein or soybean protein [ll. In rats, highsoybean protein diets also have a hypocholesterolemic effect [9]. However, when casein diets are fed, it has been reported that in rats [4,9], rabbits [24] and pigeons [25], increasing the proportion of protein in the diet resulted in elevation of serum cholesterol levels. An explanation for these contrasting results obtained in chickens and other animal species is not readily available. It is possible that, as wil1 be discussed later, differences between animal species in the digestion of the protein play a role. In the present study, the casein and soybean protein in the diet was enriched with arginine and methionine, respectively. Soybean protein is low in methionine [ 121 and chickens fed casein diets not supplemented with arginine exhibit poor growth [ 131. Although there is evidente that addition of amino acids to the diet can affect the levels of serum cholesterol [ 11, it seems unlikely that this has occurred in the present study. Johnson et al. [13] reported that in chickens fed 10% protein diets, supplementation of casein with arginine and soybean protein with methionine resulted in a decrease of serum cholesterol levels. However, such effects were not found when 25% or 40% protein diets were used. Similarly, Leveille et al. [26] and Nishida et al. [27] observed that in chickens the addition of methionine to a soybean protein diet had a cholesterol-lowering effect only with diets containing marginal levels of this amino acid.
102
The excess of cholesterol in the serum of the chickens fed the cholesterol-enriched diets containing 20% protein was found to be carried mainly in the VLDL and the IDL lipoproteins. This was associated with a lower concentration of LDL cholesterol, compared with the animals fed the cholesterol-enriched diets containing 50% protein. A similar pattern has been found in rats made hypercholesterolemic by feeding cholesterol-enriched casein-diets [4]. However, in rats, the LDL fraction cannot be clearly discerned [4,28] and an increase in serum cholesterol was found to be associated with elevated cholesterol levels in the VLDL and IDL together with lower levels of cholesterol in the HDL. In rabbits [5] and guinea pigs [lol, on the other hand, hypercholesterolemia is primarily associated with elevated LDL levels. It is not yet clear how dietary protein can affect cholesterol metabolism and how, in chickens, high-casein and high-soybean protein diets are able to lower serum cholesterol levels. However, it has been shown that in chickens the feeding of high-protein diets resulted in an increase in cholesterol synthesis [29,30], fecal steroid excretion [31-331 and cholesterol turnover rate [32]. The major pathway for the disposal of steroids from the body is the excretion of neutral steroids and bile acids in the feces [34]. The secretion and reabsorption of bile acids and cholesterol by means of the enterohepatic circulation plays an important role in the regulation of cholesterol metabolism. Cholesterol can be converted to bile acids and subsequently excreted in the bile. However, most of the bile acids are reabsorbed. Furthermore, there is evidente that a less efficient reabsorption of bile acids can lower serum cholesterol levels. We have found that the feeding of cholestyramine, a bile acid sequestrant, to hypercholesterolemic rabbits effectively reduced the levels of serum cholesterol [35]. On the other hand, rabbits starved for several weeks and in which fecal production and hence the fecal excretion of bile acids and neutral steroids are markedly reduced, are known to develop severe hypercholesterolemia [36]. In chickens, Sklan and coworkers [37,38] reported that high-casein diets depressed the absorption of bile acids and found a highly significant correlation between the bile acid, absorption, which mainly occurs in the ileum, and the concentration in the ileum of both insoluble nitrogen and the high molecular weight peptide fractions. They also observed that intact casein, albumin or soybean protein caused an inhibition of the mucosal uptake of taurocholic acid from chick intestinal duodenum loops; this effect could be enhanced by increasing the amount of protein. However, predigestion of casein released this inhibitory effect. These authors also reported that intact casein was able to bind bile acids. Therefore, they suggested that the presence of undigested protein in the intestine of the chicken might interfere with the bile acid reabsorption and subsequently affect the serum cholesterol levels. Chickens react differently from other species of animals in their cholesterolemic response to diets containing various proportions of casein and soybean protein. This could possibly be explained by a different mode of digestion of dietary protein between various animal species. For example, in chickens, a reflux of digesta from the duodenum to the gizzard occurs [39], which might also affect the manner and rate of digestion of the protein in the diet. Further studies have to be done to elucidate the mechanism of the cholesterolemic properties of dietary protein.
103
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24 Terpstra, A.H.M., Harkes, L. and Van der Veen, F.H., The effect of different proportions of casein in semipurified diets on the concentration of serum cholesterol and the lipoprotein composition in rabbits, Lipids, 16 (1981) 114. 25 Lofland, H.B., Clarkson, T.B. and Goodman, H.O., Interactions among dietary fat, protein and cholesterol in atherosclerosis-susceptible pigeons - Effect on serum cholesterol and aortic atherosclerosis, Circ. Res., 9 (1961) 919. 26 Leveille, G.A., Shockley, J.W. and Sauberlich, H.E., Influence of dietary protein leve1 and amino acids on plasma cholesterol of the growing chick, J. Nutr., 76 (1962) 321. 27 Nishida, T., Takenaka, F. and Kummerow, F.A., Effect of dietary protein and heated fat on serum cholesterol and beta-lipoprotein levels, and on the incidence of experimental atherosclerosis in chicks, Circ. Res., 6 (1958) 194. 28 Terpstra, A.H.M., Sanchez-Muñiz, F.J., West, CE. and Woodward, C.J.H., The density profile and the cholesterol concentration of serum lipoproteins in domestic and laboratory animals, Comp. Biochem. Physiol., 71B (1982) 669. 29 Yeh, Y.-Y. and Leveille, G.A., Effects of dietary protein on hepatic lipogenesis in the growing chick, J. Nutr., 98 (1969) 356. 30 Yeh, S.-j.C. and Leveille, G.A., Cholesterol and fatty acid synthesis in chicks fed different levels of protein, J. Nutr., 102 (1972) 349. 31 Kenney, J.J. and Fisher, H., Effect of medium chain triglycerides and dietary protein on cholesterol absorption and deposition in the chicken, J. Nutr., 103 (1973) 923. 32 Yeh, S.-j.C. and Leveille, G.A., Influence of dietary protein leve1 on plasma cholesterol turnover and fecal steroid excretion in the chick, J. Nutr., 103 (1973) 407. 33 Leveille, G.A., Shockley, J.W. and Sauberlich, H.E., Influence of dietary factors on plasma lipid relationships in the growing chick, Proc. Sec. Exp. Biol. Med., 108 (1961) 313. 34 Dietschy, J.M., Mechanism for the intestinal absorption of bile acids, J. Lipid Res., 9 (1968) 297. 35 Terpstra, A.H.M., Hermus, R.J.J. and West, C.E., Dietary protein and cholesterol metabolism in rabbits and rats. In: D. Kritchevsky and M.J. Gibney (Eds.), Animal and Vegetable Protein in Lipid Metabolism, Alan R. Liss Inc., New York, NY, In press. 36 Swanor, J.C. and Connor, W.E., Hypercholesterolemia of total starvation - Its mechanism via tissue mobilization of cholesterol, Amer. J. Physiol., 229 (1975) 365. 37 Sklan, D., Budowski, P. and Hurwitz, S., Absorption of oleic and taurocholic acids from the intestine of the chick, Biochim. Biophys. Acts, 573 (1979) 31. 38 Sklan, D., Digestion and absorption of casein at different levels in the chick - Effect on fatty acid and bile acid absorption, J. Nutr., 110 (1980) 989. 39 Sklan, D., Shachof, B., Baron, J. and Hurwitz, S., Retrograde movement of digesta in the duodenum of the chick - Extent, frequency and nutritional implications, J. Nutr., 108 (1978) 1485.