Yukiko Shimokawa

New Biological Function of Bovine α-Lactalbumin : Protective Effect against Ethanol- and Stress-induced Gastric Mucosal Injury in Rats

Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow’s milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23°C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, α-lactalbumin (α-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% α-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, β-lactoglobulin, α-LA, bovine serum albumin and γ-globulins (immunoglobulins), on the basis of their contents in WPI revealed that α-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. α-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of α-LA. These results indicate that α-LA has marked antiulcer activity as an active component of cow’s milk protein, and suggest that α-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.

Effects of Bovine α-Lactalbumin on Gastric Defense Mechanisms in Naive Rats

We recently investigated the effects of the major proteins in cows milk on gastric mucosal injuries in rat ulcer models. We found that α-lactalbumin (α-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of α-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of α-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, α-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of α-LA). Thus, we demonstrated that α-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.

New fluorogenic substrate for esterase activity of α-chymotrypsin and related enzymes

Abstract A new fluorogenic substrate, benzyloxycarbonyl- l -phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of α-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl- l -phenylalanine and 7-hydroxy-4-methylcoumarin in a 86% yield. The esterase activity was measured by increase of the fluorescence intensity at excitation and emission wave-lengths of 325 and 465 nm, respectively. An initial rate of hydrolysis was linear over a 100-fold range of the enzyme concentration. As little as 2 ng of α-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported.

Highly sensitive method for determination of esterase activity of α-chymotrypsin and α-chymotrypsin-like enzymes using micro high-performance liquid chromatography

Abstract A new substrate, Dns- L -phenylalanine ethyl ester, with high UV absorption has been developed for the determination of the esterase activity of α-chymotrypsin and α-chymotrypsin-like enzymes. The product, generated by the enzyme action, Dns- L -phenylalanine, was clearly separated from the ester substrate by micro reversed-phase high-performance liquid chromatography. The substrate was highly stable under the enzyme assay conditions used. As little as 0.15 ng of α-chymotrypsin and 1.49 ng of subtilisin BPN′ could be detected when a long reaction time was employed. Hydrolyses of the substrate by α-chymotrypsin and α-chymotrypsin-like enzymes were blocked by specific inhibitors of the enzymes.

Simultaneous Quantification of Iodine and Other Elements in Infant Formula by ICP-MS Following an Acid Digestion with Nitric Acid and Hydrogen Peroxide

A method for quantifying iodine in infant formula is described. Nitric acid and hydrogen peroxide converted iodine into iodate in microwave-assisted digestion and prevented iodine volatilization and memory effects. Acetic acid as a carbon source was added to both the sample and standard solutions as a countermeasure against carbon charge transfer to iodine and the addition of acetic acid helped to enhance the sensitivity. The instrument limit of quantification was 0.1 ng mL(-1) and the relative standard deviation was less than 3%. The spike recoveries were between 94.8 and 106%. Good agreement with the values obtained using the tetramethylammonium hydroxide method was obtained for infant formula sold in several countries. This method permitted the simultaneous determination of iodine and 12 other important elements (Na, Mg, P, K, Ca, Cr, Mn, Fe, Cu, Zn, Se and Mo) in infant formula.

Isolation and activities of the trypsin-modified vicia angustifolia proteinase inhibitor lacking carboxyl-terminal hexapeptide

The Vicia angustifolia proteinase inhibitor was incubated with p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-trypsin (EC 3.4.21.4) and a main product was isolated. The purified product was different to the first trypsin-modified V. angustifolia inhibitor. The C-terminal residues of the new derivative were arginine, which was also the C-terminal of the cleaved antitryptic site; lysine was a newly exposed C-terminal. These results suggest that the new derivative lacks the C-terminal portion of the native inhibitor, which has asparagine at its C-terminus. The liberated C-terminal peptide had the following amino acid sequence: H-Glu-Glu-Val-Ile-Lys-Asn-OH. The derivative lacking the C-terminal hexapeptide still possesses inhibitory activities against trypsin and alpha-chymotrypsin (EC 3.4.21.1), however, its antichymotryptic activity was inactivated by incubation with chymotrypsin at pH 8.0.