Digestibilities of casein and soya-bean protein in relation to their effects on serum cholesterol in rabbits

Diets containing isolated soya-bean protein induce lower levels of serum cholesterol in animals than diets containing casein. Experiments were conducted to investigate whether differences in digestibility of the proteins might explain this effect. At pH 8 with pancreatic enzymes or intestinal peptidase, soya-bean protein was hydrolysed in vitro much less rapidly than casein. However, with pepsin (EC 3.4.23.1) at acid pH, soya-bean protein was hydrolysed more rapidly than casein. These differences in digestibility may be due to pH-dependent changes in solubility of the proteins. Casein and soya-bean protein were most soluble at alkaline and acid pH respectively. Heat treatment of the proteins resulted in lower solubilities and digestibilities. Sonication of soya-bean protein at pH 7.8 increased solubility but only slightly raised digestibility. When fed to rabbits, enzymically hydrolysed soya-bean protein induced a 2.3-fold higher concentration of serum cholesterol than did intact soya-bean protein. The hypocholesterolaemic effect of soya-bean protein may be partly attributable to its low solubility and digestibility at alkaline pH.     

Dietary animal proteins and cholesterol metabolism in rabbits

The effect in rabbits of giving isonitrogenous purified diets containing casein, ovalbumin, fish protein, milk-whey protein and soya-bean protein were compared. The diets were balanced for cholesterol and for the amount and type of fat. When incorporated into low-cholesterol diets (0.08 g cholesterol/kg), casein, ovalbumin and soya-bean protein produced similar levels of serum cholesterol. With a high background of dietary cholesterol (1.5 g/kg), serum cholesterol concentrations increased with soya-bean protein, whey protein, casein and fish protein, in that order. Thus, the hypercholesterolaemic effect of casein in carefully balanced diets was only seen against a high-cholesterol background. The development of hypercholesterolaemia produced by giving fish protein was different from that produced by casein. First, less cholesterol accumulated in the very-low-density-lipoprotein fractions and more in the lipoproteins of higher density with fish protein than with casein. Second, fish protein, unlike casein, did not increase liver cholesterol. Third, transfer of rabbits from a diet containing soya-bean protein to one containing casein resulted in an immediate marked depression in neutral steroid and bile acid excretion in faeces. However, when rabbits were fed on the diet with fish protein after the diet with soya-bean protein, there was no significant depression in neutral steroid output and the depression in bile acid output was delayed. The present study suggests that different animal proteins cause hypercholesterolaemia by different mechanisms.     

Effects of soya-bean protein and casein on serum cholesterol levels in rats

1. The effect of the soya-bean protein isolate and casein, both given 200 g/kg diet for 3-4 weeks, on serum cholesterol was compared in male rats. 2. Soya-bean protein exerted a hypocholesteramic effect only in a cholesterol-free low-fat (10 g maize oil/kg) diet, when the lowering action appeared independent of the strain of the rat or the feeding pattern. The results obtained with diets containing cholesterol or higher levels of fats or both showed no definite pattern of response. 3. Although the decrease in serum cholesterol appeared greater in alpha-lipoproteins than in beta-lipoproteins, the proportion of the former to total cholesterol remained almost unchanged. The concentration of serum apo A-I was significantly lower in rats given the vegetable protein. 4. Rats given soya-bean protein excreted significantly more neutral sterols. 5. The serum amino acid pattern did not reflect the difference in dietary protein. Addition of cholesterol to the diets modified the serum aminogarm, the decrease in threonine being most marked in both protein groups. 6. This study shows that the hypocholesteraemic action of soya-bean protein is easily modified by the type of diet.     

Response of hormones modulating plasma cholesterol to dietary casein or soy protein in minipigs

To elucidate the mechanism mediating the effect of dietary casein or soy protein on serum cholesterol concentrations we followed the endocrine response to the intake of these dietary proteins. The hormones analyzed were those known to modulate serum cholesterol concentration. A 7-wk crossover nutrition study was performed with six adult G?ttingen minipigs consuming semisynthetic diets based on either 20 wt% casein or soy isolate. At d 42 and 49, concentrations of six hormones were determined in 22 blood samples taken over the whole day. There were no significant differences in insulin, glucagon, the insulin/glucagon ratio, hydrocortisone or triiodothyronine among dietary groups. In the late postprandial phase (5 h after the meal and later) there were significantly higher growth hormone concentrations in soy-fed animals. At all times of the day, total and free thyroxine concentrations were higher after soy feeding than after casein feeding. On average, total and free thyroxine concentrations were 34 and 26% higher with soy protein feeding than with casein feeding. Our data agree with other reports of protein-dependent changes of thyroid hormones and may explain why different dietary proteins have different effects on serum cholesterol levels in sensitive species.     

Digestibility of casein, formaldehyde-treated casein and soya-bean protein in relation to their effects on serum cholesterol in rabbits

Adult male rabbits were fed on semi-purified diets containing soya-bean protein isolate, casein or formaldehyde-treated casein as the protein source and 1 g cholesterol and 5 g of the non-absorbable marker chronic oxide/kg diet. The concentration of cholesterol in serum and in liver was increased on both the casein and formaldehyde-treated-casein diets. Excretion of bile acids and their concentration in faeces were lower in rabbits fed on casein or formaldehyde-treated casein when compared with rabbits fed on soya-bean protein. Apparent digestibility of nitrogen was lowest when formaldehyde-treated casein was fed, and highest on the casein diet. In rabbits fed on casein treated with formaldehyde, higher proportions of N were found in the water-soluble and trichloroacetic acid-insoluble protein fractions of the gastrointestinal tract contents compared with rabbits on the other two diets. Absorption of phosphate from the gastrointestinal tract was higher in rabbits fed on casein than in rabbits fed on soya-bean protein or formaldehyde-treated casein. The results indicate that, in rabbits, protein digestibility may not be an important determinant of serum cholesterol.     

The phosphorylation state of casein and the species-dependency of its hypercholesterolaemic effect

1. The present study concerns the question whether the hypercholesterolaemic effect of casein, a phosphorylated protein, is modified in species differing both in glycine-taurine conjugation of bile acids and in intestinal alkaline phosphatase (EC 3.1.3.1). 2. Since these two variables are entirely different in rabbits and rats, identical (cholesterol-free) semi-purified diets containing either casein or soya-bean protein were given to both species. 3. In rabbits casein, as compared with soya-bean protein, did not affect calcium absorption but immediately increased phosphate absorption and decreased faecal excretion of bile acids. These effects preceded the accumulation of apo B-cholesterol in serum, which indicates a cause-and-effect relation. 4. In contrast, none of these casein-specific effects were observed in rats. 5. These results suggest that the hypercholesterolaemic potential of casein is expressed mainly in species (like the rabbit) with a low activity of intestinal phosphatase and with a high glycine conjugation of bile acids. This might explain why species (like rat and man) are rather insensitive to dietary casein.     

Effects of addition of sulfur-containing amino acids and glycine to soybean protein and casein on serum cholesterol levels of rats

The effects of the supplementation of methionine (Met), cystine (Cys), and glycine (Gly) to soybean protein or casein on serum and liver lipid levels were studied in rats. Rats were fed cholesterol-free diets containing 25% soybean protein or casein supplemented with 0.75% Met, 2.5% Gly, or a combination of these two for 4 weeks. The addition of Met to soybean protein caused a significant increase in serum cholesterol and this was slightly ameliorated when Gly was given simultaneously. In rats fed casein diets, serum cholesterol tended to decrease when Gly, or Met and Gly were added. A simultaneous supplementation of Met and Gly to casein resulted in a reduction of hepatic cholesterol. Cystine added at the 0.6% level did not cause demonstrable changes in lipid concentrations except for a drop in serum triglyceride of the casein group. When 2.0% Gly was added to cholesterol-enriched diets containing 20% protein, serum cholesterol decreased significantly only when the protein source was casein and the level attained was comparable to that observed in rats fed soybean protein. Liver cholesterol was also markedly decreased by the addition of Gly to casein. The results suggest a possible role of Gly in the regulation of serum cholesterol levels by dietary protein.     

Effect of soy protein and casein intake on intestinal absorption and lymphatic transport of cholesterol and oleic acid

Rats were fed for 4 wk on defined diets containing either casein or soy as the protein source, or diets in which the lysine/arginine ratios were modified by addition of arginine to casein, and lysine to the soy diet. During this period, weight gains and food intakes were comparable in the four dietary groups. Animals were subjected to cannulation of the left thoracic lymphatic duct, and after an overnight fast, were given a single intragastric dose of a lipid emulsion containing oleic acid and cholesterol. The overall 24-h recoveries of cholesterol and fatty acid in lymph were similar in the four groups, as were the distribution of lipids among the major lipid fractions and lipoprotein classes of thoracic duct lymph. However, analysis of timed lymph collections indicated that absorption of lipids was more rapid in casein-fed rats than in those fed soy protein. Furthermore, addition of arginine to the casein diet resulted in a slowed rate of lipid absorption, and addition of lysine to the soy diet markedly increased the rate of lipid absorption.     

The importance of the non-protein components of the diet in the plasma cholesterol response of rabbits to casein. Zinc and copper

To characterize further the effect of protein-mineral interaction on plasma cholesterol, four groups of rabbits were placed on cholesterol-free, semi-purified diets for 12 weeks. The diets were similar in proximate composition with either casein or soya-bean protein isolate as the protein source (250 g/kg). The dietary zinc content of the casein diets was increased to 22 mg/kg to equal that of the soya-bean diets. The copper content was set at 2 and 6 mg/kg. There were no differences in weight gain; all animals grew at a similar rate. When the dietary Zn content was equalized, the plasma and liver cholesterol responses were similar regardless of the source of protein. Differences in liver Cu and Zn were observed. As dietary Cu increased, Cu decreased and Zn increased in the liver. Hepatic Zn was also higher in soya-bean-fed than in casein-fed animals. No effects on plasma Cu and Zn were seen. The addition of Zn to the casein diet resulted in a similar plasma cholesterol to that of animals fed on the soya-bean diet, suggesting that casein-induced hypercholesterolaemia is due to a marginal Zn intake.     

Apolipoprotein E polymorphism and plasma lipid, lipoprotein, and apolipoprotein levels in Italian children.

We have investigated the effect of apolipoprotein (apo) E polymorphism on serum lipid, lipoprotein, and apolipoprotein levels in a sample of 195 children, aged 8-11 years, from Sezze, Central Italy. The relative frequencies of e2, e3, and e4 alleles were 0.062, 0.867, and 0.072, respectively. Variation at the apo E gene locus explained 5.1% of the sample variance in serum total cholesterol levels, 7.6% in low-density lipoprotein (LDL) cholesterol levels, 7.3% in apo B levels, and 14.1% in high-density lipoprotein-apo E (HDL-E) levels. The effect of the e2 allele was to lower levels of total cholesterol, LDL-cholesterol, and apo B and to raise levels of HDL-E, while the effect of the e4 allele was the opposite. Variation at the apo E gene locus was not associated with differences in serum triglyceride, HDL-cholesterol, or apo AI levels. The effects of common apo E polymorphisms and genetic variation associated with the PvuII RFLP of the apo B gene on serum apo B levels were additive, explaining 11.3% of the phenotypic variance in this sample. When the effect of apo E polymorphism on serum lipid traits was estimated in boys and girls separately, variation at the apo E gene locus explained 10.4, 13.3, 13.3, and 13.5% of the phenotypic variance in serum total cholesterol, LDL-cholesterol, apo B, and HDL-E levels, respectively, in boys, while in girls only the effect on HDL-E levels (19.3%) reached statistical significance. This study has demonstrated that genetic variations at the apo E locus contribute to the determination of serum lipid, lipoprotein, and apolipoprotein levels in youths and that the effects are gender specific.     

Addition of arginine but not glycine to lysine plus methionine-enriched diets modulates serum cholesterol and liver phospholipids in rabbits.

Previous experiments from our laboratory showed that in rabbits fed an amino acid diet corresponding to 30% casein, enrichment of the diet with L-lysine and L-methionine caused a marked increase in serum total and LDL cholesterol levels as well as a substantial body weight loss. Both effects were partially prevented by supplementation with L-arginine. The present studies were designed to extend this earlier observation by assessing the role of different dietary amino acids in modulation of cholesterolemic responses and body weights. In the first experiment, the original lysine and methionine-enriched diet was supplemented with glycine in an attempt to modify methionine metabolism, and thus to reduce body weight loss. In addition, the mechanism of action of lysine and methionine was investigated by quantitation of major liver phospholipids. The results showed that glycine addition had no effect on weight loss or hypercholesterolemia, nor did it alter plasma levels of homocyst(e)ine, an intermediate in methionine metabolism. However, enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus may explain the hypercholesterolemia. A second experiment showed that similar effects on body weights and serum cholesterol could be obtained by adding lysine and methionine to a diet containing amino acids equivalent to only 15% casein, or 15% intact casein. This approach is more physiologic and also reduces the expense of experiments designed to study the effects of lysine and methionine in more detail.     

Comparison of the hypocholesterolemic effects of dietary soybean protein with those of formaldehyde-treated casein in rabbits

Treatment of casein with formaldehyde changes its tertiary structure and decreases its hypercholesterolemic properties in rabbits. To investigate whether formaldehyde-treated casein exerts this hypocholesterolemic effect in the same manner as soybean protein, rabbits were fed high or low cholesterol diets containing soybean protein, casein, formaldehyde-treated casein or a mixture of casein and formaldehyde-treated casein. Formaldehyde-treated casein was hypocholesterolemic when fed in a low, but not in a high, cholesterol diet. The hypocholesterolemic effect of soybean protein was independent of the amount of cholesterol included in the diet. In contrast to rabbits fed soybean protein, steroid absorption in those fed formaldehyde-treated casein did not differ from that in rabbits fed native casein. Furthermore, the absorption of phosphorus and nitrogen was lower in rabbits fed formaldehyde-treated casein than in those fed native casein, whereas the absorption found in rabbits fed soybean protein resembled that of their casein-fed counterparts. The diets containing soybean protein and formaldehyde-treated casein produced a comparable ratio of lysine to arginine in serum. The results presented in this paper indicate that the hypocholesterolemic action of dietary formaldehyde-treated casein does not resemble that of soybean protein.     

The effects of guar gum, sugar beet fibre and wheat bran supplementation on serum lipoprotein levels in normocholesterolaemic volunteers

The effect of dietary supplementation with guar gum, sugar beet fibre (SBF) and wheat bran on serum lipoprotein leveh was assessed in normocholesterolemic men. Each fibre preparation was incorporated into bread and subjects ate half a loaf a day which provided 20 g of the fibre preparation. Each bread was eaten for 14 days with a 21-dey rest period between each stage of the study. Twelve subjects consumed each type of fibre-supplemented bread.
During fibre supplementation dietary non-starch polysaccharide (NSP) intake increased significantly (P < 0.025) and the mean daily fat and energy intakes increased but not significantly. Serum total cholesterol levels were reduced by an average of 11.7% (s.e. 1.8) (P<0.01) during guar supplementation and 4.6% (s.e. 1.9) (P < 0.025) during SBF supplementation. Bran supplementation was without effect. Mean HDL cholesterol, serum triglycerides and plasma glucose and insulin levels were unaffected by supplementation with any of the fibres. Mean serum total bile acids were significantly reduced by guar (P < 0.01) and SBF (P < 0.025) but not bran supplementation. Mean apo B levels were only significantly reduced by guar (P < 0.05). Apo A1 levels were not affected by any of the fibres. There was a significant correlation between apo B and total cholesterol levels (P<0.01) and between apo A1 and HDL cholesterol levels (P < 0.01) throughout each stage of the trial.
This study demonstrates that dietary supplementation with 20 g guar or sugar beet fibre has a favourable effect on both cholesterol and apoprotein levels.     

The effect of amino-acid-supplemented wheat gluten on cholesterol metabolism in the rat

The effect of lysine- and threonine-supplemented wheat gluten on cholesterol metabolism was studied using male weanling rats. Animals were fed on cholesterol-free diets containing 100 or 200 g gluten/kg with or without amino acid supplementation, and compared with animals given 50, 100 and 200 g casein/kg diets, for 3 weeks. A hypocholesterolaemic effect observed with the wheat gluten-fed rats, compared with the animals given 100 and 200 g casein/kg diets, was accompanied by increased turnover of cholesterol as expressed by enhanced cholesterol biosynthesis and increased faecal excretion of cholesterol and bile acids. This effect was not abolished by lysine and threonine supplementation. Low levels of blood cholesterol were also observed in the rats fed on the 50 g casein/kg diet. However, a different mechanism, related to impairment of cholesterol transport from the liver, was most likely responsible for the hypocholesterolaemia found in these protein-malnourished animals. The effect on cholesterol metabolism produced by dietary wheat gluten was independent of the low quality of the protein and of its specific deficiency in lysine and threonine.     

Dietary protein effects on cholesterol and lipoprotein concentrations: a review

Different dietary proteins exert different effects on plasma cholesterol concentrations. Animal studies have shown that animal proteins, most notably casein, increase plasma total cholesterol concentrations compared with vegetable proteins, such as soy. Soy protein has been shown to be hypocholesterolemic in rats, swine, primates, and rabbits. Epidemiologic studies have disclosed that vegetarians have lower mean plasma cholesterol concentrations than populations consuming diets of mixed proteins, but it is unclear whether this effect results specifically from the animal or vegetable nature of the protein. In human clinical experiments, substituting soy protein for mixed protein reduces plasma total cholesterol concentration in hypercholesterolemic subjects, but it causes only a small, nonsignificant change in persons with normal plasma cholesterol concentrations. The mechanism responsible for the effects of different proteins on plasma cholesterol concentrations has not been established. One hypothesis suggests that animal proteins, which have a greater content of phosphorylated amino acids than vegetable proteins, interfere with bile acid reabsorption. Another hypothesis suggests that the amino acid content of the protein affects cholesterol absorption, tissue storage, synthesis, and excretion. The dietary protein may also alter cholesterol metabolism by affecting plasma hormone concentrations, either postprandially or over weeks to months. Among the hormones thought to be affected by dietary protein source are insulin, glucagon, and thyroid hormones. Gastrointestinal hormones, such as gastrointestinal inhibitory polypeptide, may also be affected by dietary protein.     

Dietary protein effects on cholesterol and lipoprotein concentrations: a review.

Different dietary proteins exert different effects on plasma cholesterol concentrations. Animal studies have shown that animal proteins, most notably casein, increase plasma total cholesterol concentrations compared with vegetable proteins, such as soy. Soy protein has been shown to be hypocholesterolemic in rats, swine, primates, and rabbits. Epidemiologic studies have disclosed that vegetarians have lower mean plasma cholesterol concentrations than populations consuming diets of mixed proteins, but it is unclear whether this effect results specifically from the animal or vegetable nature of the protein. In human clinical experiments, substituting soy protein for mixed protein reduces plasma total cholesterol concentration in hypercholesterolemic subjects, but it causes only a small, nonsignificant change in persons with normal plasma cholesterol concentrations. The mechanism responsible for the effects of different proteins on plasma cholesterol concentrations has not been established. One hypothesis suggests that animal proteins, which have a greater content of phosphorylated amino acids than vegetable proteins, interfere with bile acid reabsorption. Another hypothesis suggests that the amino acid content of the protein affects cholesterol absorption, tissue storage, synthesis, and excretion. The dietary protein may also alter cholesterol metabolism by affecting plasma hormone concentrations, either postprandially or over weeks to months. Among the hormones thought to be affected by dietary protein source are insulin, glucagon, and thyroid hormones. Gastrointestinal hormones, such as gastrointestinal inhibitory polypeptide, may also be affected by dietary protein.     

Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA:cholesterol acyltransferase activity in liver of Zucker rats

There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of Delta 5 and Delta 6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein-fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient.     

Effect of soya-bean protein on meat iron solubility and absorption in rats

1. Soya-bean proteins were used to replace 30 and 50% of the protein from 59Fe-labelled pigeon (Columba L.) and chicken meat, and the solubility of the meat 59Fe in vitro and its absorption in vivo in rats in the presence and absence of soya-bean proteins were measured. 2. Replacement of part of the chicken meat by soya-bean proteins reduced 59Fe solubility from chicken meat at all stages during simulated in vitro digestion. 3. 59Fe absorption from 59Fe-labelled chicken meat when given to both Fe-replete and Fe-deficient rats was reduced in the presence of soya-bean proteins but was unaffected by the presence of casein or bovine serum albumin. 59Fe-absorption from pigeon meat in the presence of soya-bean proteins was not reduced to the same extent as that from chicken meat. 4. There was no significant effect of soya-bean proteins on 59Fe-labelled haemoglobin Fe absorption in vivo in Fe-replete rats. 5. Absorption of 59Fe from the isolated haemoproteins from chicken meat was unaffected by soya-bean proteins but 59Fe absorption from the main non-haem-Fe fractions was strongly inhibited, particularly from haemosiderin.     

Comparative contributions of dietary protein quality and quantity to growth during gestation, lactation and postweaning in the rat

The growth promotion by feeding purified diets containing 25%, 12% or 7% of either casein or wheat gluten (W.G.) and the supplementation value of adding cystine to the low casein diets, and lysine or lysine plus threonine to the wheat gluten diets ascertained in rats by comparing the weight achieved at birth, after 3 weeks suckling or 4 weeks postweaning. Birth weight was depressed by 30% with 7% casein; by 20% with 7% W.G.; by 15% with 25% W.G. compared to controls and there was no change when lysine alone was added to 7% W.G. Addition of lysine plus threonine to 7% W.G., lysine to 25% W.G. and cystine to 7% casein increased birth weight. The weight gain of dams reflected birth weight of pups in most cases. Weaning weight was depressed by 60% with either 12% casein or 12% W.G., and by 40% with 25% W.G. compared to controls. Addition of cystine to 12% casein and lysine to 25% W.G. increased the weaning weight slightly. Addition of lysine, lysine plus threonine or lysine plus glutamic acid (G.A.) to 12% W.G. had no effect on weaning weight. Maternal weight loss was decreased by amino acid supplementation of the diet. Four-week postweaning body weight was equal to the control when cystine was added to 12% casein and lysine was added to 25% W.G. Addition of lysine to 12% W.G. improved the growth rate, and addition of lysine plus threonine further improved the growth rate. Food intake was not decreased by protein deficiency during gestation. Food intake was significantly lowered by protein deficiency during both lactation and postweaning, however, supplementation of amino acid partially restored food intake to normal levels.     

Dietary animal proteins and cholesterol metabolism in rats

The effects on cholesterol metabolism in rats of diets containing various animal proteins or soy protein were studied. The animal proteins tested were casein, whey protein, fish protein, hemoglobin, plasma proteins, ovalbumin, egg-yolk protein, beef protein and chicken-meat protein. The semi-purified diets were isonitrogenous and balanced for residual fat and cholesterol in the protein preparations. The nature of the dietary protein had no effect on serum cholesterol concentration. Group mean liver cholesterol concentration was increased and fecal excretion of bile acids was decreased by all animal proteins when compared with soy protein. This study suggests that carefully balancing diets for components other than protein in the protein preparations prevents protein effects on serum cholesterol in rats but not on liver cholesterol and bile acid excretion.