Salivary gland metabolism in an animal model of chronic kidney disease.

OBJECTIVE: The aim of this study was to determine the effects of experimental CKD into the metabolism of parotid and submandibular glands of rats. CKD was induced by 5/6 nephrectomy. DESIGN: Serum analyses of BUN (Blood Urea Nitrogen) and creatinine concentrations were performed. Major salivary glands metabolism was investigated in vivo, both at rest and during salivary stimulation conditions by NMR isotopomer analysis, using [U-13C]glucose as metabolic tracer. RESULTS: CKD increases BUN and serum creatinine concentrations (p < 0.001). Multiple metabolic alterations were detected in the parotid glands of this animal model, including decreased concentrations of alanine (p < 0.05) and creatine (p < 0.05) and increased lactate/alanine ratios (p < 0.05). The salivary stimulus fostered accumulations of acetate at both analyzed glands of the CKD model (p < 0.05), indicative of disruption of the oxidative metabolic process. CONCLUSIONS: Experimental CKD induced by 5/6 nephrectomy altered the parotid salivary gland function, since glucose metabolism is clearly affected after stimulation for salivation in this gland.

Intravenous infusion of tridocosahexaenoyl-glycerol emulsion into rabbits. Effects on leukotriene B4/5 production and fatty acid composition of plasma and leukocytes.

Leukotriene (LT) B4 is a major chemical activator of PMN. Inhibitory effects of oral administration of docosahexaenoic acid (DHA) on LTB4 synthesis by PMN are known. We intravenously infused tridocosahexaenoyl-glycerol (DHA-TG) emulsion into rabbits in three different doses, namely 0.8, 0.4, or 0.2 g DHA/kg, and investigated the changes in LTB4/5 production by ionophore-activated PMN. The averaged LTB4 production by PMN was significantly reduced to 57 and 59% of baseline at 6 h after the infusion of 0.8 and 0.4 g DHA/kg, respectively (P < 0.05), but not after the infusion of 0.2 g DHA/kg or 0.8 g soybean oil/kg. The combined concentrations of both DHA and eicosapentaenoic acid in the PMN phospholipid fraction were significantly increased at 6 h after the infusion of 0.8 or 0.4 g DHA/kg but not after the infusion of 0.2 g DHA/kg or 0.8 g soybean oil/kg. Oral administration of 0.8 g DHA/kg did not increase DHA or eicosapentaenoic acid in the PMN phospholipid fraction and did not decrease LTB4 production by PMN at 6 h after administration. We suggest that the infusion of 0.4-0.8 g DHA/kg might be beneficial to patients who suffer from diseases that are related to the acute elevation of LTB4 production.

Purification and partial characterization of a protein proteinanse inhibitor isolated from eggplant exocarp.

A protein proteinase inhibitor was isolated and purified from eggplant exocarp by heat treatment, ammomium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-25 and G-50. The final purified preparation of the inhibitor was found homogeneous by electrophoretic analysis. The inhibitor showed strong and stoichiometric inhibition on trypsin whereas it showed weak inhibition on alpha-chymotrypsin. It displayed no inhibiting characteristics on pepsin. The molecular weight of the inhibitor was estimated to be approximately 6000. This finding, with the trypsin inhibition data, suggested that the inhibitor combined trypsin in the molar ratio of 1:1. The amino acid analysis indicated that the inhibitor is rich in half-cystine, glycine and aspartic acid, and contains no tryptophan, histidine, methionine or valine.

Changes in fatty acid composition in rat blood and organs after infusion of docosahexaenoic acid ethyl ester.

An infusible emulsion of docosahexaenoic acid ethyl ester (DHA-EE) was prepared. One hundred milliliters of the emulsion contained 10 g DHA-EE (90% pure). Three milliliters of the emulsion was infused into tail veins of 22 Wistar rats weighing approximately 300 g. They were killed 1, 6, and 24 h and 3 and 7 d after the infusion, and fatty acid composition of various organs and plasma was analyzed along with that of control rats. DHA concentrations reached their peaks within 24 h after DHA infusion in plasma lipid fractions and in the phospholipid fraction of liver and lung. DHA did not increase at all in cardiac phospholipid fraction. However, DHA concentrations increased markedly (from 0.7% to 11%) in the free fatty acid fraction of heart 1 h after the infusion. DHA emulsion might be useful for patients in whom a rapid increment in DHA in tissues is beneficial.

Infusion of fish oil emulsion: effects on platelet aggregation and fatty acid composition in phospholipids of plasma, platelets, and red blood cell membranes in rabbits.

An emulsion of fish oil was manufactured to contain 10 g of fish oil/100 mL. Of this, 3 g were eicosapentaenoic acid (EPA) and 1 g was docosahexaenoic acid (DHA). We administered 100 mL of the emulsion into six rabbits intravenously on days 1, 4, 7, 10, and 13. Blood samples were taken on days 0 and 16. The EPA content in phospholipids of plasma, platelets, and red blood cell (RBC) membranes increased 16, 4, and 5 times, respectively. The DHA content in phospholipids of plasma and RBC membranes increased two times whereas that in platelet phospholipids did not increase significantly. Platelet aggregation induced by collagen (10 micrograms/mL) and ADP (5 microM) was depressed significantly after infusion of the fish oil emulsion. In control experiments with soybean oil emulsion, there were almost no significant changes. Therefore, fish oil emulsion may be beneficial to patients who cannot take n-3 fatty acids orally but need them.

The complete amino acid sequence of rice bran trypsin inhibitor.

The complete amino acid sequence of a double-headed trypsin inhibitor (RBTI) from rice bran was determined by a combination of limited proteolysis of the native inhibitor with Streptomyces griseus trypsin at pH 3 and conventional methods. RBTI consists of 133 amino acid residues including 18 half-cystine residues which are involved in 9 disulfide bridges in the molecule. The limited proteolysis at pH 3 produced a major split of Lys(83)-Met(84) and a minor split of Arg(107)-Val(108) together with a non-enzymatic hydrolysis of Asp(19)-Pro(20) in the molecule. The established sequence showed that RBTI is composed of 4 domains, domains I and III, and domains II and IV being homologous to the first and the second domains of soybean Bowman-Birk inhibitor, respectively, indicating that RBTI has a duplicated structure of the Bowman-Birk type inhibitor.

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.

The amino acid sequence of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI0 but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence comparison with other Bowman-Birk type inhibitors.

Infusion of emulsified trieicosapentaenoyl-glycerol into rabbits--the effects on platelet aggregation, polymorphonuclear leukocyte adhesion, and fatty acid composition in plasma and platelet phospholipids.

Using 90%-pure free eicosapentaenoic acid, we synthesized 1,2,3-trieicosapentaenoyl-glycerol (EPA-TG) and manufactured an emulsion of EPA-TG with purified phosphatidylcholine from krill as an emulsifier. After two intravenous injections of the EPA-TG emulsion into rabbits, the EPA content in plasma and platelet phospholipids increased markedly. ADP- and collagen-induced platelet aggregation and polymorphonuclear leukocyte adhesion to glass beads were depressed significantly. No significant changes were observed in serum lipids and liver function. In control experiments which were performed in exactly the same manner except that soybean oil emulsion was used instead of the EPA-TG emulsion, there were almost no significant changes. Our results suggest that an EPA-TG emulsion is applicable to those patients who need both intravenous alimentation and preventive care of thrombosis, such as postoperative patients.

Injection of tridocosahexaenoyl-glycerol emulsion and fatty acid composition of blood cells.

An injectable emulsion of docosahexaenoic acid (DHA) was prepared. One hundred ml of the emulsion contained 3 g of 93%-pure 1,2,3-tridocosahexaenoylglycerol (DHA-TG), 1.2 g of 93%-pure 2-docosahexaenoyl-phosphatidylcholine as an emulsifier and 2.5 g of glycerol. Thirty ml of the emulsion of DHA-TG was injected into three rabbits on days 1 and 4 of the study. Blood was taken on day 0, on day 4 just before the second injection and on day 7. The percentage of DHA in the total phospholipid fraction of platelets increased from 0.46% (day 0) to 1.88% (day 4, p less than 0.05) and 3.66% (day 7; p less than 0.02 vs day 0). The percentage of eicosapentaenoic acid (EPA) increased from 0.46% (day 0) to 1.03% (day 4, p less than 0.02) and 1.63% (day 7: p less than 0.05 vs day 0). The percentage of arachidonic acid (AA) decreased from 9.45% (day 0) to 4.31% (day 4, p less than 0.05) and 6.68% (day 7; p less than 0.02 vs day 0). The percentage of DHA in the total phospholipid fraction of erythrocyte membranes increased from 0.23% (day 0) to 0.91% (day 4, p less than 0.05) and 1.52% (day 7; p less than 0.005 vs day 0); that of EPA increased from 0.21% (day 0) to 0.34% (day 4, p less than 0.005%) and 0.52% (day 7, p less than 0.01 vs day 0); that of AA was unchanged. Blood lipids were the same before and after the two injections of the emulsion, except that free fatty acids decreased markedly from 0.32 to 0.06 mEq/1 (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary proteins and/or their digestive products on intestinal taurocholate absorption.

[14C]Taurocholate was orally administered to rats together with a definite amount of either casein- or soy protein-based diet and its postprandial movement along the digestive tract was investigated. A difference was observed between both dietary groups in intraluminal transit as well as mucosal accumulation of [14C]taurocholate in the ileum; namely the soy protein intake led to a decrease in the bile acid incorporation into the ileal mucosa relative to the casein intake, although raising its intraluminal stay. In addition, the digestive products from these and other food proteins by pepsin-pancreatin digestion (peptides with molecular weights of more than 1,000) were examined for their inhibitory effects on in vitro absorption of taurocholate with ileal everted sacs. As the digestive product affinity for taurocholate increased, the rate of taurocholate absorption decreased. It thus seems likely that a food protein more abundant in hydrophobic peptides following intraluminal digestion adsorbs much more bile acids in the gut, thereby disturbing their intestinal absorption.

Effect of feeding peptic digest of soy protein isolate on rat serum cholesterol.

Growing rats were fed ad libitum soy protein isolate (SPI) or its peptic (SPI-P) or tryptic digest (SPI-T) for a month and their sera were examined for cholesterol and triglyceride levels and enzyme activities such as cholinesterase, glutamate-pyruvate transaminase (GPT) and alkaline phosphatase. The rats fed SPI-P or SPI-T were inferior in growth to those fed SPI. Similarly, the serum glyceride level was lower in the SPI-P and SPI-T groups than in the SPI group. On the other hand, a significant difference was found in the serum cholesterol level between the SPI-P and SPI or SPI-T groups but not between the SPI and SPI-T groups. A similar tendency was observed for serum GPT and alkaline phosphatase activities, although there were no significant differences among dietary groups in small intestinal enzyme activities. As for the atherogenic index being a risk factor inducing atherosclerosis, the order of its value was SPI-P less than SPI less than SPI-T.

Gelation of the heat-induced complex between kappa-casein and alpha-lactalbumin.

Whey is a by-product of the manufacture of cheese from milk. The usual practice is to dispose of it, the usage of whey being not sufficiently developed, though it contains proteins of excellent quality such as beta-lactoglobulin and alpha-lactalbumin. Interaction between kappa-casein and whey protein was examined in order to form new food materials. Gelation of the heat-induced complex between kappa-casein and alpha-lactalbumin is described in this paper. alpha-Lactalbumin was easily separated from whey protein by the gel filtration technique on a Toyopearl HW-50 column at pH 6.0. Heat treatment facilitated the hydrolysis of a mixture of kappa-casein and alpha-lactalbumin by trypsin, alpha-chymotrypsin and pronase. Heat treatment at above 75 degrees C and a protein level of over 5% were needed to form gel in 35 mM phosphate buffer, pH 7.6, containing 0.4 M NaCl. The temperature was in agreement with that at which alpha-lactalbumin denatured and formed the complex with kappa-casein. Decrease of soluble protein concentration and increase of turbidity were induced with gelation. Gel was not formed when only kappa-casein or alpha-lactalbumin was heated under the appropriate conditions. It was considered that a kappa-casein-alpha-lactalbumin gel was formed from a complex of the two proteins by heat treatment. The breaking stress of kappa-casein-alpha-lactalbumin gel was less than that of kappa-casein-beta-lactoglobulin gel. If the pH was reduced to 5.8 after complex formation by the two proteins, gel was formed at a low protein concentration compared with that with no alteration of pH.

Susceptibility of kappa-casein components to various proteases.

In order to clarify the function of the carbohydrate moiety of bovine kappa-casein, kappa-casein components having different carbohydrate contents were prepared by DEAE-cellulose chromatography. Five adsorbed fractions so obtained had an identical peptide chain and contained carbohydrate moieties of increasing size in the order of components P-2, P-3, P-4, P-5 and P-6. The subsceptibility of kappa-casein components, having different carbohydrate contents, to various proteases was examined. kappa-Casein components were subjected to calf rennin [chymosin; EC 3.4.23.4], bovine trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], pronase [EC 3.4.24.4] and human plasmin [EC 3.4.21.7]. The component containing a larger carbohydrate moiety was less susceptible to hydrolysis than the component containing a smaller carbohydrate moiety. Rennin, trypsin, alpha-chymotrypsin and pronase hydrolyzed each component with a different reaction rate. On the contrary, human plasmin hydrolyzed component P-2, but did not hydrolyze component P-5. These results indicate that the carbohydrate moiety of kappa-casein components to various proteases.

An improved method for the purification of eggplant trypsin inhibitor.

The trypsin inhibitor in eggplant, Solanum melongena L., was isolated and purified by the improved method with the techniques of dialysis using acetylated cellulose tube and ion-exchange chromatography on DEAE-Sephadex. The final preparation was found to be homogeneous by disc and SDS-polyacrylamide gel electrophoresis. This inhibitor had the molecular weight of about 6,200, the pI value of 4.7, and furthermore characteristic amino acid composition lacking in tryptophan, histidine, valine and methionine. The trypsin inhibition data indicated that the purified inhibitor combined with bovine trypsin [EC 3.4.21.4] in the molar ratio of 1:1. These properties of this inhibitor were in agreement with those of the dialyzable eggplant trypsin inhibitor previously purified, indicating that the dialyzable and non-dialyzable inhibitors in eggplant are identical.

Presence of O-glycosidic linkage through serine residue in kappa-casein component from bovine mature milk.

This paper describes the glycosylation sites of kappa-casein component P-5 from bovine mature milk. A short glycopeptide was prepared from kappa-casein component P-5 containing two carbohydrate chains by pronase P digestion, followed by gel filtration and ion exchange chromatographies. The glycopeptide obtained corresponded to residues 128-141 (Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val-Glu-Ser) of kappa-casein A from the results of analyses with chemical and enzymatic procedures. The effect of alkaline borohydride treatment indicated the presence of serine as well as threonine as the binding site of carbohydrate moieties. From the facts of Edman degradation and carboxypeptidase P hydrolysis of glycopeptide treated with alkali, the carbohydrate moieties were considered to be attached to threonine residue No. 133 and serine residue No. 141.

Minor components of reduced bovine kappa-casein.

Bovine kappa-casein reduced with 2-mercaptoethanol was fractionated on a DEAE-cellulose column to one nonadsorbed, five major adsorbed and two minor adsorbed fractions. The properties of the nonadsorbed and five major adsorbed fractions were reported in our previous paper (4). In this paper, the characteristics of two minor adsorbed fractions (P-X and P-Y) were reported. Gel electrophoretic patterns of these fractions were similar to kappa-casein. These fractions had the same amino acid composition and the same phosphorus content as the major components. P-X contained one residue each of N-acetylneuraminic acid, galactose and galactosamine, and P-Y two residues each. Furthermore, these fractions showed the stabilizing ability for alpha s1-casein in the presence of calcium ion. These results indicate that P-X and P-Y are minor components of reduced bovine kappa-casein.

In vivo action of alpha-amylase inhibitor from cranberry bean (Phaseolus vulgaris) in rat small intestine.

An alpha-amylase inhibitor prepared from cranberry bean (Phaseolus vulgaris) was examined for its in vivo action on pancreatic alpha-amylase in rat small intestine. For this purpose, postprandial changes not only in intraluminal alpha-amylase activity but also in plasma glucose and insulin concentrations were measured at various times after administration of 2 g of 10% polyethylene glycol-containing experimental diets with and without the inhibitor. No considerable increase was observed in the levels of intraluminal alpha-amylase activity, blood sugar, and plasma insulin in the animals given the inhibitor at a dose of 10 mg each. These results suggest that the purified preparation from cranberry bean serves in fact as a potent inhibitor of rat pancreatic alpha-amylase.

Chemical composition of winged bean (Psophocarpus tetragonolobus) varieties.

The chemical composition of dry seeds of four varieties, pods, stalks and leaves of winged beans (Psophocarpus tetragonolobus) was determined. The seeds had a high range of protein (27.8-36.6%) and fat (14.8-17.9%), which were similar to soybeans. the seeds contained high phosphorus, calcium and magnesium. The leaf was highest in protein content (33.7%) of all the parts studied except for the seeds. The protein and fat content of pods decreased as pods ripened. the calcium content in the leaf was much higher than in the other parts. Protein was extracted sequentially with 2% NaCl, 30% isopropyl alcohol, 4% lactic acid and 0.5% KOH from dry seeds of four varieties of winged beans. The NaCl extract showed the highest range of protein concentration (60.2-77.6%). The NaCl extract was separated into two fractions based on solubility in water. the amino acid composition of the flour from the seeds and of the two fractions from the NaCl extract were determined. Contents of lysine, aspartic acid, glutamic acid and leucine were large, while the sulfur-amino acid content was small. Trypsin and chymotrypsin inhibitory activities of 2% NaCl extract from the seeds were determined, and chymotrypsin inhibitory activity was higher than the trypsin.

Several properties of the partially purified proteinase inhibitor in eggplant exocarp.

A proteinase inhibitor was isolated and partially purified from the exocarp of eggplant, Solanum melongena L., by means of acetate buffer extraction, heat treatment, salting-out and column chromatography on DEAE-cellulose. This preparation showed inhibitory activities on various proteinases; trypsin [EC 3.4.4.4] and Pronase were strongly inhibited while alpha-chymotrypsin [EC 3.4.4.5] and Nagarse were weakly inhibited. The inhibitor was a protein substance, and, therefore, it was gradually inactivated by the long-time incubation with Pronase. The inhibition mode was non-competitive on trypsin and competitive on Pronase on the basis of Lineweaver-Burk plots. The investigations on the inhibition behavior in the co-existence of two kinds of proteinases suggested that the inhibitor was not of multi-headed type.