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The effect of cottonseed condensed tannins on the ileal digestibility of amino acids in casein and cottonseed kernel.

The effect of adding cottonseed hulls to casein- and cottonseed-kernel-based diets on the apparent and true ileal digestibility of N and amino acids, and the proportion of this effect accounted for by condensed tannin (CT), were determined using the growing rat. Sixty rats were allocated randomly to ten semipurified diets, containing either casein (four diets) or purified unheated solvent-extracted cottonseed kernel (six diets) as the sole protein source, with Cr2O3 added as an indigestible marker. Two of the casein diets contained no hulls whilst the remaining two diets contained 70 g cottonseed hulls/kg. Two of the cottonseed-kernel-based diets contained no hulls, with two containing 23 g hulls/kg and the remaining two containing 46 g hulls/kg. For each pair of diets, PEG was either included or excluded. The effect of CT was quantified by comparing control rats (-PEG; CT acting) with PEG-supplemented rats (+PEG; CT inactivated) at each level of dietary hulls. The rats were given their respective experimental diets for 14 d. Each rat was given the food ad libitum for 10 min hourly from 08.00 to 18.00 hours. On day 14, samples of digesta were collected at death from the terminal 150 mm of ileum at 7 h from the first meal. Apparent and true ileal digestibilities were calculated for DM, N and the individual amino acids. The principal finding was that the inclusion of hulls depressed the apparent and true ileal digestibilities of N and amino acids, but with the response differing between diets. With the casein-based diet the mean apparent and true ileal amino acid digestibilities were significantly depressed from 0.89 and 0.96 to 0.85 and 0.92 respectively, by the inclusion of 70 g hulls/kg in the diet, and addition of PEG then restored these to 0.89 and 0.95. All of the depression could be explained by the CT content of the hulls. However, with the cottonseed-kernel-based diet the responses fell into three categories. The apparent and true ileal digestibilities of the essential amino acids cystine and methionine were not affected by hull addition, ileal digestibilities of leucine, isoleucine, lysine, threonine and valine were markedly depressed by hull addition with approximately 50% of the depression being explained by CT, whilst the ileal digestibilities of histidine, arginine and phenylalanine were depressed by hull addition but little or none of this effect could be explained by CT. Thus the effect of hulls on protein digestion clearly differed with source of protein. With the cottonseed-kernel-based diet it seems that components of the hulls other than CT also depressed the apparent and true ileal digestibilities of N and amino acids. The identity of these components is unknown.

Amino Acids↗

Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite.

Protein, generally agreed to be the most satiating macronutrient, may differ in its effects on appetite depending on the protein source and variation in digestion and absorption. We investigated the effects of two milk protein types, casein and whey, on food intake and subjective ratings of hunger and fullness, and on postprandial metabolite and gastrointestinal hormone responses. Two studies were undertaken. The first study showed that energy intake from a buffet meal ad libitum was significantly less 90 min after a 1700 kJ liquid preload containing 48 g whey, compared with an equivalent casein preload (P<0.05). In the second study, the same whey preload led to a 28 % increase in postprandial plasma amino acid concentrations over 3 h compared with casein (incremental area under the curve (iAUC), P<0.05). Plasma cholecystokinin (CCK) was increased by 60 % (iAUC, P<0.005), glucagon-like peptide (GLP)-1 by 65 % (iAUC, P<0.05) and glucose-dependent insulinotropic polypeptide by 36 % (iAUC, P<0.01) following the whey preload compared with the casein. Gastric emptying was influenced by protein type as evidenced by differing plasma paracetamol profiles with the two preloads. Greater subjective satiety followed the whey test meal (P<0.05). These results implicate post-absorptive increases in plasma amino acids together with both CCK and GLP-1 as potential mediators of the increased satiety response to whey and emphasise the importance of considering the impact of protein type on the appetite response to a mixed meal.

Adult↗

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins.

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.

Adenosine Triphosphate↗

TGF beta suppresses casein synthesis in mouse mammary explants and may play a role in controlling milk levels during pregnancy.

Mammary explants from 14-15-d-pregnant mice synthesize and secrete milk proteins in culture in response to insulin, hydrocortisone, and prolactin. Here we demonstrate that transforming growth factor beta (TGF beta) treatment suppresses, in a dose dependent and reversible manner, the ability of explants to synthesize and secrete milk caseins. TGF beta does not affect the level of casein mRNA within explants but inhibits casein synthesis posttranscriptionally. We also show increased expression of TGF beta 2 and TGF beta 3 in intact mammary gland as pregnancy progresses, with reduced expression of all three TGF betas at the onset of lactation. These findings suggest that endogenously produced TGF beta may limit the accumulation of milk caseins that are produced in the mammary gland during pregnancy.

Animals↗

Involvement of vesicle coat material in casein secretion and surface regeneration.

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.

Animals↗

Localization of the casein gene family to a single mouse chromosome.

A series of mouse-hamster somatic cell hybrids containing a variable number of mouse chromosomes and a constant set of hamster chromosomes have been used to determine the chromosomal location of a family of hormone-inducible genes, the murine caseins. Recombinant mouse cDNA clones encoding the alpha-, beta-, and gamma-caseins were constructed and used in DNA restriction mapping experiments. All three casein cDNAs hybridized to the same set of somatic cell hybrid DNAs isolated from cells containing mouse chromosome 5, while negative hybridization was observed to ten other hybrid DNAs isolated from cells lacking chromosome 5. A fourth cDNA clone, designated pCM delta 40, which hybridized to an abundant 790 nucleotide poly(A)RNA isolated from 6-d lactating mouse mammary tissue, was also mapped to chromosome 5. The chromosomal assignment of the casein gene family was confirmed using a mouse albumin clone. The albumin gene had been previously localized to mouse chromosome 5 by both breeding studies and analogous molecular hybridization experiments. An additional control experiment demonstrated that another hormone-inducible gene, specifying a 620 nucleotide abundant mammary gland mRNA, hybridized to DNA isolated from a different somatic cell hybrid line. These studies represent the first localization of a peptide and steroid hormone-responsive gene family to a single mouse chromosome.

Animals↗

Comparison of actions of soy protein and casein on metabolism of plasma lipoproteins and cholesterol in humans.

Several reports claim that vegetable proteins cause a lowering of plasma cholesterol when substituted for animal proteins. The present study was carried out to determine whether soy proteins alter plasma lipoproteins or cholesterol balance when compared to casein protein in humans investigated on a metabolic ward. Fourteen men were studied in two periods, each of 1 month's duration. In period I the diet contained 30% of calories as fat (in the form of lard), 55% as carbohydrate and 15% as casein protein. Period II was identical except that soy protein was used instead of casein. Ten patients had normal levels of plasma triglycerides with varying concentrations of plasma cholesterol. The substitution of soy protein for casein protein produced no consistent change in plasma concentrations of cholesterol, triglycerides, low-density lipoprotein, or high-density lipoprotein. Estimated synthetic rates of cholesterol and bile acids also were unchanged. In contrast, soy protein caused a significant lowering of plasma triglycerides in three of four patients with hypertriglyceridemia. Thus, the only significant alteration induced by soy proteins in this study was a reduction of plasma triglycerides when levels were elevated; soy proteins had no discernable effects on the metabolism of cholesterol.

Adult↗

Hypocholesterolemic effect of dietary soy protein versus casein in rhesus monkeys (Macaca mulatta).

Ten mature female rhesus monkeys were alternately fed semipurified diets containing casein or soy protein for periods of 13 to 17 wk. Each diet was fed for two periods. When the animals were changed from the commercial diet to the semipurified diet containing soy protein, a significant elevation in serum cholesterol occurred. The serum cholesterol levels gradually increased further, when the soy protein in the diet was replaced by casein. However, when subsequently the casein in the diet was replaced by soy protein, a significant decrease in serum cholesterol levels was observed. Finally, when the animals were changed back to the casein diet, a significant increase in serum cholesterol again occurred. Changes in serum cholesterol due to modulations in the type of protein in the diet were reflected mainly by changes in low-density lipoprotein cholesterol. Thus, the results of this study clearly show that the type of protein in the diet markedly affects serum cholesterol levels not only in experimental animals such as the rabbit, as is well known, but also in the rhesus monkey, which is more akin to man.

Animals↗

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.

Animals↗

Trace analysis of ethinyl estradiol in casein diet using gas chromatography with electron capture detection.

Ethinyl estradiol (EE2) is an extremely potent synthetic estrogen and a common component in oral contraceptives. The drug has a well-characterized pharmacological profile and is used as a positive control in toxicological investigations of compounds having estrogenic activity. An analytical method developed for the determination of low microg/kg levels of EE2 in a casein-based rodent diet is presented. A methanol extract of casein diet is purified for instrumental analysis by a 3-fold solid-phase extraction process. The sample extract is derivatized with pentafluoropropionic anhydride to the pentafluoropropionyl product and analyzed by capillary gas chromatography with electron-capture detection. Recoveries of EE2 from casein diet fortified at 5, 10, and 50 microg/kg average 88.8% and have a relative standard deviation (%) of 7.2. The method limit of detection in a casein-based diet is 1 microg/kg.

Animal Feed↗

Milk production and nitrogen utilization in response to postruminal infusion of sodium caseinate in lactating cows.

Five lactating, rumen-fistulated Holstein cows were used to obtain additional information concerning the effects of postruminal infusion of sodium caseinate on milk production and amino acid utilization. A 7-day continuous abomasal infusion of approximately 450 g/day of sodium caseinate was preceded and followed by 7-day infusions of an isonitrogenous-isocaloric solution of glucose, monosodium glutamate, and urea. Total collections of milk, urine, and feces were obtained during the last 5 days of each infusion period. On the last day of each period, arterial and mammary venous blood samples were obtained for analysis of plasma free amino acids. During infusion of sodium caseinate, milk production, milk protein (N times 6.38) production, and efficiency of nitrogen utilization for milk crude protein production were increased. Arterial plasma concentrations of free histidine, isoleucine, phenylalanine, valine, and total essential amino acids were elevated above control levels during infusion of sodium caseinate, while ornithine and tryrosine were decreased. Calculation of the relative concentration of essential amino acids in arterial plasma and in milk protein indicated that methionine and lysine were least abundant in plasma relative to their requirement for milk protein synthesis. A high precentage extraction from arterial plasma by the mammary gland also suggested that methionine and lysine may have been the essential amino acids in most critical supply for milk protein synthesis. Calculation of uptake to output ratios of individual plasma amino acids by the mammary gland suggested that significant quantities of extracted arginine, isoleucine, leucine, threonine, and valine were utilized in pathways other than direct incorporation into milk protein.

Amino Acids↗

Effect of dietary mineral composition on nutritional equivalency of amino acid mixtures and casein in rats.

The effect of dietary mineral composition on the quality of amino acid mixtures and intact casein was examined in growing rats fed for 28 days under "conventional" conditions. When 25% demineralized casein (DC) was the sole dietary protein source, the widely-used mineral mixture by Harper (MMH) supported growth poorly (3.2 g/day), but a new formula mineral mixture (MM2) containing sufficient amounts of zinc gave a growth rate of over 8 g/day for 21 days. With a crude casein (CC) diet, rats grew at the same rate regardless of mineral mixtures. The growth-supporting power of amino acid mixtures, one of which simulated casein (AA-A) and another patterned after Rogers and Harper to give maximal growth of rats (AA-B), was compared with those of DC and CC with addition of MMH or MM2. When the protein source was DC, AA-A or AA-B, MM2 significantly improved the growth over that of rats fed MMH. The growth-stimulating effect of MM2 was greater when added to DC diet than when added to AA-A diet. When MM2 was added to AA-B diet, the growth rate greatly increased and reached that of rats fed DC diet containing MM2 (over 8 g/day), showing a nutritional equivalency between the amino acid mixture and intact protein. Drawbacks inherent in MMH did not appear with CC diet, because CC contained considerable amounts of zinc. When a highly purified amino acid-sucrose diet is used, dietary minerals become most limiting for growth even under conventional conditions. Thus, the previous conflicting results concerning nutritional equivalency of amino acid mixtures and protein can be explained by inadequate mineral sources.

Amino Acids↗

Dietary minerals modify the food intake suppressing effects of high casein diets fed to rats.

Food intake, growth, and urinary urea and ammonia excretion were studied in young rats undergoing adaptation to high protein diets (70% casein) containing varying amounts of potassium, sodium and chloride. Two commercial mineral mixtures were used. The Bernhart-Tomarelli (BT) mineral mixture is low in K, Na and Cl compared to the TD (modified Williams-Briggs) mineral mixture. Rats consumed significantly less food and had poor growth when fed 70% casein diets containing the BT mineral mixture. Food intake and feed efficiency improved significantly when the BT diets were supplemented with KCl and NaCl, Na acetate or K acetate but not 3-chloropropionate. Urinary urea and ammonia excretion were directly proportional to food (protein) intake. However, body weight gain during the last 3 days of the 9-day study (experiment 3) was negatively correlated with urinary ammonia nitrogen (milligrams per gram food eaten) but not with urinary urea nitrogen. It is concluded that dietary K and/or Na content affects food consumption in rats fed high casein diets. Alterations in renal capacity for handling ammonia may be responsible for the food intake enhancing effect of K or Na in rats fed a high casein diet.

Ammonia↗

Effects on plasma amino acid concentrations and hepatic branched-chain alpha-keto acid dehydrogenase activity of feeding rats diets containing 9 or 50% casein.

Plasma concentrations of amino acids and alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate decarboxylation activities in livers of rats trained to eat 9 or 50% casein diets for 5 hours/day, were measured one-half hour before and one-half and 3 hours after the start of the feeding period. Decarboxylation of both alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate by liver increased significantly within one-half hour after rats had ingested either the 9 or the 50% casein diet. Liver decarboxylation activity of rats fed the 50% casein diet was from two- to fivefold higher than that of rats fed the 9% casein diet. The greatest difference was observed when calcium, NAD and coenzyme A were included in the decarboxylation assay medium. Although the activity of the branched-chain alpha-keto acid dehydrogenase increased in response to food ingestion, plasma concentrations of branched-chain amino acids also increased greatly after the ingestion of food. The similarity in the responses of alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate decarboxylation in rats fed diets differing in protein content and subjected to different feeding regimens allows us to suggest that the branched-chain alpha-keto acid dehydrogenase is responsible, in part, for the oxidative decarboxylation of the alpha-keto acid analog of methionine. J. Nutr . 114: 1025-1034, 1984.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

Comparison of dietary casein or soy protein effects on plasma lipids and hormone concentrations in the gerbil (Meriones unguiculatus).

The effects of dietary animal protein (casein) or soy protein (soy isolate) on plasma lipids and hormones were investigated in the gerbil. Diets, fed to male gerbils (initial weight, 60 g) for 4 wk, contained either 18% casein or soy isolate as the protein source. The dietary fat sources were lard (16%) and safflower oil (1%). The cholesterol content of the diet was 0.1%. Plasma total cholesterol concentrations were lower in gerbils fed the soy protein diet (159 mg/dl) than in the gerbils fed the casein diet (190 mg/dl). Absolute HDL-cholesterol concentrations were unaffected by the protein source, but LDL-cholesterol concentrations were lower in the soy-fed gerbils. Thus, the ratio of LDL to HDL cholesterol was lower in the soy-fed gerbils (0.42) compared with the casein-fed gerbils (0.70). Plasma insulin levels were higher in the soy-fed gerbils as were plasma thyroxine and thyroid-stimulating hormone levels. The results indicate that the gerbils can be used to study dietary effects on cholesterol parameters. These data also suggest that changes in plasma thyroxine levels may in part account for the hypocholesterolemic effect of soy protein.

Animals↗

Effects of casein or soybean protein on plasma cholesterol level in jejunectomized or ileectomized rats.

The contribution of enterohepatic circulation of cholesterol and bile acids in the hypocholesterolemic activity of soybean protein isolate (SPI) was compared with the activity of casein. Intact, sham-operated, jejunectomized or ileectomized adult rats were fed a cholesterol-free, purified diet containing either 20% casein or 20% SPI for 7 or 10 d. For the subsequent 7 d the diets were reversed. In intact rats the plasma cholesterol concentration (p-chol) was significantly higher when the casein diet was fed than when the SPI diet was fed. Within 3 d after the diet crossover, p-chol in each group of intact rats reached rapidly the same level as that in its previous counterpart. These rapid inherent responses of p-chol to the casein and SPI diets remained unchanged even when the jejunum or the ileum was resected, and p-chol in jejunectomized or ileectomized rats was similar to that found in sham-operated rats. The extent of the change in p-chol induced by exchanging dietary proteins was almost the same among intact, sham-operated, jejunectomized and ileectomized rats. These findings indicate that the interruption of enterohepatic circulation of cholesterol and bile acids is not the major factor involved in the differential effects of dietary proteins on p-chol.

Animals↗

Aqueous ethanol extraction of dietary soy protein isolate improves 59Fe absorption by the rat from a casein-based test meal.

A commercial soy protein isolate (SPI) was further processed in an attempt to understand how a diet based on SPI can cause decreased iron retention by the rat from a separately administered casein-based test meal. Groups of eight rats were fed either a casein-based diet or a diet based on SPI. The acid-precipitated SPI was incorporated into diets as such, after neutralization, after 60% (v/v) ethanol extraction and neutralization, or after 60% ethanol exposure and neutralization. All dietary SPI was heat-treated by exposure to steam at 108 degrees C for 30 min. Rats were fed their respective diets, each containing 25 mg Fe/kg, for 13 d, and then all rats were fed a 59Fe-radiolabeled 2.5-g casein test meal containing 64 micrograms of iron. Ingested radioactivity was determined following the meal, and retained radioactivity over the subsequent 10-d period. Absorption was not distinguishable for groups fed the casein-based (78.3 +/- 3.6%) and the ethanol-extracted, SPI-based diet (80.2 +/- 5.4%). Absorption was lower (P less than 0.01) for groups fed each of the other SPI-based diets: SPI as such (68.3 +/- 8.9%), neutralized SPI (69.8 +/- 5.0%) and ethanol-exposed SPI (67.6 +/- 4.8%). An ethanol-extractable component of SPI may be responsible for decreased iron absorption by animals fed SPI prior to a radiolabeled test meal.

Absorption↗

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 Göttingen 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.

Amino Acids↗