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Recently the American Heart Association has reported that favorable effects of soy protein on blood lipids were characteristic only for high amounts of soy protein and not observed for an intake less than 30 g/d. However, the period of the studies with the smaller amount was 4-6 wk and we thought a longer study was necessary for the conclusion. The death rate by heart disease is very high in Russia; therefore, we have done this study in Russian subjects with hyperlipidemia. Prior to the study we tried to find a favorable method for subjects to take 30 g protein a day from soybean protein isolate (SPI) or skimmed curd protein (SMP) and decided to use Russian style cookies. Thirty subjects with hyperlipidemia were recruited; however, due to the 5-mo long study 28 of them (19 females and 9 males aged 50+/-2 y) could complete the trial. They were randomly assigned to two groups and were given either cookie for 2 mo separated by a month-long washout interval in a cross-over design. Fasting blood samples were drawn before and after the dietary treatments. Fasting blood samples at 1 mo were also measured as a health check and to observe the trends of the blood parameters in the middle of the study period. Serum samples were used for the lipid and other biochemical measurements. Every month for 3 non-consecutive days, energy and nutrient intakes were assessed and physical activity was estimated by pedometer. With the consumption of SPI for 2 mo, concentrations of total-cholesterol changed from 280+/-7 to 263+/-8 mg/dL (-6.5%, p=0.0099), HDL-cholesterol from 57.4+/-2.5 to 62.6+/-2.9 mg/dL (+9%, p=0.0047), non-HDL-cholesterol (total-cholesterol-HDL-cholesterol) from 223+/-7 to 201+/-8 mg/dL (-11%, p=0.0023) and triglycerides from 204+/-23 to 173+/- 19 mg/dL (-18%, p=0.022). There were no significant changes with SMP (p>0.05). Thus, administration of 30 g SPI a day for 2 mo confirmed its favorable effects on serum lipids in Russians with hyperlipidemia.

Soy protein isolate (SPI) can elicit various physiological effects such as cholesterol lowering and antiobesity effects. To examine whether hepatic gene expression is altered by SPI intake, rats were fed an SPI or casein diet for 8 weeks. After 8 weeks of feeding, liver weight and plasma triglyceride and cholesterol levels were significantly lower in the SPI group than in the casein group. Hepatic gene expression was investigated using DNA microarrays. The expression profiles and statistical analysis showed clear and significant differences between the SPI and casein groups (p < 0.05); in the SPI group, 63 genes were up-regulated and 57 genes were down-regulated, most involved in various physiological functions such as lipid metabolism, antioxidant activity, transcriptional regulation, and energy metabolism. Especially in lipid metabolism, the down-regulated genes are related to fatty acid synthesis and the up-regulated genes are related to cholesterol synthesis and steroid catabolism. These results suggest that SPI intake could maintain homeostasis primarily by modulating lipid and energy metabolism.

Soy protein has various biological functions, such as cholesterol-lowering effect, anti-obese, in addition to that nutritive value, and may relieve some lifestyle-related diseases, cancer, osteoporosis, menopausal disorder and so on. Because of the cholesterol-lowering effect of soy protein had been studied extremely and internationally for mechanism and clinical proof, the Ministry of Health and Welfare in Japan had approved soy protein product as a food for special health use (FOSHU) in 1994, and also the FDA in the United States also approved the food labeling for that in 1999. Although, some reports suggested that soy protein changes in expression of the genes concerned with lipid metabolism in liver and in adipose tissue, there are few reports that it is concerned with the comprehensive influence of the intake of soy protein on gene expression. The gene expression of liver in soy protein fed animal has been analyzed with the Affymetrix DNA-microarray. About 33% of 8000 genes in soy fed animals differed compared with those of casein fed. After cluster analysis, significant differences were observed in the gene cluster concerned with lipid metabolism, also in the gene related to energy metabolism, transcription factor, and anti-oxidization enzymes.

Adipose tissue secretes various bioactive molecules called adipocytokines. Dysregulation of adipocytokines plays an important role in the development of atherosclerotic vascular diseases. Consumption of vegetable protein reduces the risks of atherosclerotic vascular diseases. Here, we investigated the effects of 10-day dietary soy protein isolate (SPI) on the regulation of adipocytokines in Wistar rats. No significant difference in body weight was observed between SPI and Casein (animal protein) group. Expression of adipose PAI-1 was lower and expression and plasma concentration of adiponectin were higher in SPI than Casein group. Triglyceride content was lower and fatty acid synthase mRNA level in adipose tissue was lower in SPI than Casein group. Although SREBP-1 mRNA expression was decreased in the liver of SPI group, adipose SREBP and PPARγ mRNA levels remained unchanged. Our data suggest that dietary SPI alters the gene expressions in adipose tissue and has beneficial effects on the expression of adipocytokines.

Malonyl isoflavone glucosides are water-soluble derivatives of soybean hypocotyls. This study compared the hydrolysis and absorption of malonyl isoflavone glucosides and nonmalonyl isoflavone glucosides in rats. Plasma concentrations of isoflavones were measured after oral administration of malonyl isoflavone glucosides or isoflavone glucosides. After fasting, the duodenum, jejunum, ileum, and colon were excised, and homogenates were prepared. The extent of hydrolysis of each glucoside by each intestinal homogenate was measured. Plasma levels of isoflavone aglycones, such as daidzein and glycitein, were higher in rats administered malonyl isoflavone glucosides than in those administered isoflavone glucosides. The area under the curve of daidzein in plasma of rats administered malonyl isoflavone glucosides was also significantly greater than that in those administered isoflavone glucosides. A transport experiment using Caco-2 cells suggested that degradation of malonyl glucosides to aglycones is necessary for intestinal absorption. Malonyl isoflavone glucosides were hydrolyzed only in the colon, whereas hydrolysis of isoflavone glucosides occurred in the jejunum, ileum, and colon. These results indicated more effective absorption of malonyl isoflavone glucosides than of nonmalonyl isoflavone glucosides. Moreover, effective absorption of malonyl isoflavone aglycones in the colon contributed to the significant increase in plasma isoflavone levels.

The effect on genetically obese mice of a milk whey protein isolate (WPI) and soy protein isolate (SPI) and their hydrolysates (WPI-H, SPI-H) on the rate of body fat disappearance was investigated. Male yellow KK mice were made obese by feeding with a high-fat diet containing 30% fat from 6 to 10 weeks of age. They were then fed with an energy-restricted low fat (5.0%) and high protein (35% WPI, WPI-H, SPI or SPI-H) diet for 2 weeks at the 60% level of energy intake by mice on laboratory feed. During the weight reduction period, the body weight of the WPI, WPI-H, SPI and SPI-H groups changed by -9.1, -9.1, -10.0 and -11.1 g/14 days, respectively, the reduction being significantly lower in the SPI-H group than in the WPI and WPI-H groups. The plasma total cholesterol level was significantly lower with the SPI diet, and the plasma glucose level was lower with the SPI and SPI-H diets than with the WPI and WPI-H diets. Although the body protein content was comparable in all the groups, the body fat content was significantly lower with the SPI diet than with the WPI diet, and was also significantly lower with the SPI-H diet than with the WPI and WPI-H diets. The weight of the perirenal fat pads was significantly lower with the SPI-H diet than with the WPI and WPI-H diets. These results indicate that SPI and SPI-H are suitable protein sources in an energy-restricted diet for treating obesity.

The effect of dietary phytate-free soybean protein (PFS) on intestinal mineral absorption and retention was examined in growing male rats using a three-day mineral balance technique. The rats were fed diets containing PFS, soybean protein isolate (SPI) or casein at a 20% level for 5 wk. Total calcium (Ca), magnesium (Mg), phosphorus (P), iron (Fe) and zinc (Zn) contents in diets were adjusted to 0.35, 0.05, 0.7, 0.0035 and 0.003%, respectively, by supplementation of the diet with their salts. Mineral absorption and retention ratios in rats fed the PFS diet were significantly higher than those in rats fed either the SPI or casein diet. These results suggest that PFS may be a promising dietary protein source for improving the mineral bioavailability in humans.

The effect of trypsin inhibitor (TI) in soybean on the prevention of cancer has been reported in animal studies. However, the actual intake of active TI in daily life is not known. In this experiment we measured TI activity in various soybean products and the effects of some factors of processing on the TI activity. Average TI activity in whole soybean was 4,819 U/100 g, and the average percentages of remaining activity in products against that of whole soybean were momen-tofu 2.5%, yose-tofu 3.4%, kinugoshi-tofu 4.3%, jyuten-tofu 7.9%, soymilk 13.0%, natto 0.7%, soy sauce 0.8% and miso 0.3%. By heating soymilk at 100 degrees C, TI activity decreased to 11% at 10 min and 5% at 20 min. After heating soymilk in a water-bath for 15 min at 75 and 100 degrees C, TI activity decreased to 35 and 12%, respectively. The TI activity of tofu was proportional to the remaining whey. The effects of chemicals used for the coagulation of soy protein and foam-removal on TI activity were little. The results suggest that soybean products retain 2.5-12.5% TI activity of the whole soybean and that humans are consuming some active TI in their daily lives.

Soy bean trypsin inhibitor (SBTI) belongs to the family of serpins – serine protease inhibitors widely distributed in the nature [Silverman G.A. et al., 2004; 21]. Serpins participate in the regulation of proteopytic reactions underling very important physiological and pathological processes such as digestion [16], blood clotting [3, 14, 17], immunity [44] apoptosis [36, 42], inflammation [10], dystrophy [31], carcinogenesis [22, 11] and so on. Despite the apparent importance of the correction of imbalances of the proteolysis in pathology, in fact only the pancreatic trypsin inhibitor (aprotinin) is used more or less widely as a protease inhibitor drug (Trasisol, Contrical, Gordox etc) in the treatment of some diseases [24, 13, 40]. Being the animal protein aprotinin possesses substantial disadvantages [23, 12] and attempts to de‐ velop new drug on the base of plant or recombinant proteins or peptidomimetics are under‐ taken [35, 27]. SBTI is one of the candidates for such a role [34].

beta-Conglycinin decreased blood triacylglycerol (TAG) levels in male Wistar adult rats. Liver mitochondrial carnitine palmitoyltransferase activity in the beta-conglycinin-fed group significantly increased as against the casein-fed group. Hepatic fatty acid synthase activity in the beta-conglycinin group significantly decreased as against that of the casein-fed group. Fecal fatty acid excretion in the beta-conglycinin group was significantly higher than in the casein group.

Background Soy protein and soy peptides have attracted considerable attention because of their potentially beneficial biological properties, including antihypertensive, anticarcinogenic, and hypolipidemic effects. Although soy protein isolate contains several bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptide sequences are responsible for the triglyceride (TG)-lowering effects. In the present study, we investigated the effects of soy protein-derived peptides on lipid metabolism, especially TG metabolism, in HepG2 cells and obese Otsuka Long-Evans Tokushima fatty (OLETF) rats. Results In the first experiment, we found that soy crude peptide (SCP)-LD3, which was prepared by hydrolyze of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and OLETF rats. In the second experiment, we found that hydrophilic fraction, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid-lowering effects in HepG2 cells and OLETF rats. In the third experiment, we found that Fraction-C (Frc-C) peptides, fractionated from hydrophilic peptides by gel permeation chromatography-high performance liquid chromatography, significantly reduced TG synthesis and apolipoprotein B (apoB) secretion in HepG2 cells. In the fourth experiment, we found that the fraction with 0.1% trifluoroacetic acid, isolated from Frc-C peptides by octadecylsilyl column chromatography, showed hypolipidemic effects in HepG2 cells. In the final experiment, we found that 3 di-peptides, Lys-Ala, Val-Lys, and Ser-Tyr, reduced TG synthesis, and Ser-Tyr additionally reduced apoB secretion in HepG2 cells. Conclusion Novel active peptides with TG-lowering effects from soy protein have been isolated.

Peptides are unique components of living organisms and appear in many biological capacities, nutritional activities, and fermentation processes. Many types of biologically active peptides in the body, such as peptide hormones, participate in inter- and intracellular communication and activities. Partially fermented foods contain various peptides and amino acids due to microbial activity and result in specific tastes in traditional foods. Much attention has been paid to food-derived peptides because of their good absorbability compared to amino acids. Many processing methods using enzymes, acids, and heating are used for protein hydrolysis to produce peptides. Industrialized soy peptide production by enzyme hydrolysis started in the early 1980s in Japan with the growth of soy protein manufacturing. Food manufacturers are interested in the solubility, low viscosity, and good absorbability of peptides. Practical commercial soy peptide production consists of the following processes. The soybean protein curd from whole soybean first undergoes delipidation, water extraction, and isoelectric point precipitation.

Objective A feeding study in rats investigated the principal active component for the hypocholesterolemic effect of soy protein isolate (SPI) by comparing the effect before and after ethanol washing. Methods Five-week-old male Sprague-Dawley rats were fed cholesterol-enriched AIN-93G diets containing 20% casein (CAS), 20% SPI, 20% ethanol-washed SPI (EWS), 18.4% EWS plus 1.6% ethanol extract (EE), or 20% CAS plus 1.6% EE for 2 wk. Results Plasma cholesterol concentrations in rats fed EWS and SPI were comparable and were significantly lower than those in rats fed CAS. The addition of EE to EWS and CAS did not influence plasma cholesterol level. Fecal steroid excretion of the three SPI groups was higher than that of the two CAS groups. The addition of EE to EWS and CAS showed a tendency to increase acidic steroid and decrease neutral steroid. Conclusions In this experiment which used identifiable protein preparations, a significant fraction of the cholesterol-lowering effect of SPI in rats was attributed to its protein component but not to the ethanol-extractable minor constituents including isoflavones.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.

To know whether isoflavones are responsible for the hypocholesterolemic effect of soy protein, the effect on plasma cholesterol of isoflavone-free soy protein prepared by column chromatography was examined in rats. Five-week-old male Sprague-Dawley rats were fed cholesterol-enriched AIN-93G diets containing either 20% casein (CAS), 20% soy protein isolate (SPI), 20% isoflavone-free SPI (IF-SPI), 19.7% IF-SPI + 0.3% isoflavone-rich fraction (isoflavone concentrate, IC), or 20% CAS + 0.3% IC for 2 weeks. Plasma total cholesterol concentrations of rats fed SPI and IF-SPI were comparable and were significantly lower than that of rats fed CAS. The addition of IC to the CAS and IF-SPI did not influence plasma cholesterol level. Fecal steroid excretion of the three SPI groups was higher than that of the two CAS groups, whereas the addition of IC showed no effect. Thus, a significant fraction of the cholesterol-lowering effect of SPI in rats can be attributed to the protein content, but the isoflavones and other minor constituents may also play a role.

This study examined the effects of soybean beta-conglycinin, from which phytate was mostly removed, on the plasma lipids in young and adult rats. Male Wistar young (6 week-old) and adult (21 week-old) rats were fed high cholesterol diets containing 20% casein, soy protein isolate (SPI), or soybean beta-conglycinin for 10 days. In young rats, although the food intake of the beta-conglycinin group was higher than those of the casein and SPI groups, the weight gain was significantly lower than those of the other groups. However, in adult rats, the weight gain was not different among the groups. In young and adult rats, relative liver weights of SPI and beta-conglycinin groups were significantly lower than that of the casein group, and the degree of the reduction was more marked in the beta-conglycinin group than in the SPI group. In young rats, the plasma triglyceride level was significantly lower in the SPI and beta-conglycinin groups than that in the casein group. In addition, the plasma triglyceride level of the beta-conglycinin group was significantly lower than that of the SPI group. Plasma total cholesterol levels of the SPI and beta-conglycinin groups were significantly lower than that of the casein group. However, there was little difference in the lowering effect between SPI and beta-conglycinin. These results indicate that soybean beta-conglycinin may have lowering functions not only on plasma total cholesterol level, but also on plasma triglyceride level.

Antioxidative activity of isoflavones has not been shown in humans. Newly-developed isoflavone-rich soya hypocotyl tea contains about 12 mg isoflavones per liter. 15 tea drinkers and 23 control young female students were randomly selected from volunteers, and underwent physical examination, blood chemistry and urinary analysis before and after one month of tea drinking. A three-day dietary record was taken before each physical examination. The tea drinkers showed a lower level of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidyl-ethanolamine hydroperoxide (PEOOH) in the red blood cells and a significant reduction of 8-hydroxydeoxyguanine (8ohdG) in the urine compared to the controls.

This study examined in dietary obese and genetically obese rodents the effects of soy protein isolate (SPI) and its hydrolysate (SPI-H) on the rate of body-fat disappearance. Male Sprague-Dawley rats (4-16 wk old) and yellow KK mice (6-10 wk old) were made obese by feeding high-fat diets containing 30% fat. They were then fed energy-restricted, low-fat (5.0%), and high-protein (35% casein, SPI, or SPI-H) diets for 4 wk at 60% of the level of the energy intake of rodents on laboratory chow. The body-fat contents of rats and mice fed a high-fat diet were 27.3 and 33.6 g/100 g body weight, respectively, at the end of the obese period. For rats, the apparent absorbability of dietary energy and fat was significantly lower in the SPI and SPI-H groups than in the casein group, but vice-versa for nitrogen balance. Body-fat content in mice fed SPI and SPI-H diets was significantly lower than in those fed the casein diet. In rats, plasma total cholesterol level was lower with the SPI-H diet, and plasma glucose level was lower with the SPI and SPI-H diets than with the casein diet. These results indicate that SPI and SPI-H are suitable protein sources in energy-restricted diets for the treatment of obesity.

Four-week-old rats were fed on diets containing either no dietary fiber (DF) or a DF source (WSSF, ISF or cellulose) for 4 weeks. The DF level was adjusted to 5%. The WSSF diet contained 3% and 2%, respectively, of WSSF and cellulose. No rat in any group experienced diarrhea, and none of the experimental diets suppressed the growth of rats, the apparent absorption of major nutrients being almost 100%. However, the rate of degradation of DF during the digestive process was significantly different (p0.05, cellulose, 23.6%; WSSF with cellulose, 64.5% (WSSF degradation only was 91.8%); and ISF, 77.6%). The plasma and liver lipid levels were within normal ranges, although the liver cholesterol level in those rats fed on WSSF and ISF was significantly lower (p0.05) than in those fed on cellulose. The cecal organic acid contents were in the order of WSSFISFcelluloseDF-free. Furthermore, WSSF was effective in shortening the gastrointestinal transit time. The results indicate that WSSF seems to have favorable effects on the intestinal functions.

The erythrocyte ghost function method was used to introduce 125I-labeled diphtheria toxin, its fragments A and B, and two labeled crossreacting material (CRM), mutant proteins CRM176 and CRM197, into cultured mouse cells. Fragment A was relatively stable in mouse cytoplasm at 37 degrees C and at least 80% was recovered from cells after 24 hr of incubation. In contrast, wild-type fragment B and A fragments from CRM176 and CRM197 were unstable and were degraded with half-lives of about 2.5 hr under similar conditions. When a rabbit anti-fragment A IgG fraction was introduced with wild-type A, the rate of degradation of A was accelerated, whereas the rates of degradation of A176 and A197 were retarded by the same antibody. In every instance the degradation rate appeared to be that of the IgG fraction itself with a half-life of about 7.5 hr.

Elongation factor 2 (EF-2) was purified from rat liver extracts by affinity chromatography using fragment A of diphtheria toxin as the ligand. Purified EF-2 has a molecular weight of 96,000 and isoelectric point of 6.6-6.8. The sequence of the nineteen N-terminal amino acid is Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala Ile-Met-Asp-Lys-Lys-Ala-Asn and the C-terminal amino acid is leucine. Purified rat EF-2 modified with ADP-ribose was injected into rabbits to prepare antibodies against EF-2. The anti-EF-2 antibodies can immunoprecipitate with EF-2 from various eukaryotic cells.

We report here that pricking cells with a microneedle causes introduction into cells of a fixed quantity of material that is contained in the culture medium. Using fragment A of diphtheria toxin (21 500 D), the volume of injected external medium by a single prick was determined (1.4× 10 −15 l (μm 3 )). We also show that immunoglobulin G molecules of 1.6 × 10 5 D can be introduced into cells by this technique. The speed of the microinjection by this pricking method is fast (by using a modified microscope 2 000–7 000 cells/h).

Background Dietary 1(3)-behenoyl-2,3(1)-dioleoyl-rac-glycerol (BOO) has been reported to inhibit pancreatic lipase activity in vitro and suppress postprandial hypertriacylglycerolemia in humans. In the present study, the anti-obesity activities of BOO and its inhibitory effects on lymphatic triacylglycerol (TAG) absorption were investigated in rats. Methods In Experiment 1, rats were fed either BOO or soybean oil (SO) diet for 6 weeks. In the BOO diet, 20% of SO was replaced with an experimental oil rich in BOO. In Experiments 2 and 3, rats cannulated in the thoracic duct were administered an emulsions containing trioleoylglycerol (OOO) or an oil mixture (OOO:BOO, 9:1). Tri[1-14C]oleoylglycerol (14C-OOO) was added to the emulsions administered in Experiment 3. Results No observable differences were detected in food intake or body weight gain between the BOO and SO groups in Experiment 1. Plasma and liver TAG concentrations and visceral fat weights were significantly lower in the BOO group than in the SO group. The apparent absorption rate of fat was significantly lower in the BOO group than in the SO group. In Experiment 2, the lymphatic recovery of oleic and behenic acids was significantly lower at 5 and 6 h after BOO administration than after OOO administration. In Experiment 3, the lymphatic recovery of 14C-OOO was significantly lower at 5 and 6 h after BOO administration than after OOO administration. Conclusions These results suggest that BOO prevents deposition of visceral fat and hepatic TAG by lowering and delaying intestinal absorption of TAG.