Yukifumi Kokuba

Degradation kinetics of l-glutamine in aqueous solution

The degradation kinetics of L-glutamine (Gln) in aqueous solution was studied as a function of buffer concentration, pH and temperature. Stability tests were performed using a stability-indicating high-performance liquid chromatographic assay. The degradation product of Gln was 5-pyrrolidone-2-carboxylic acid. The reaction order for Gln in aqueous solution followed pseudo-first-order kinetics under all experimental conditions. The maximum stability of Gln was observed in the pH range from 5.0 to 7. 5. The pH-rate profile described by specific acid-base catalysis and hydrolysis by water molecules agreed with the experimental results. Arrhenius plots showed the temperature dependence of Gln degradation, and the apparent activation energy at pH 6.41 was determined to be 9.87 x 10(4) J mol(-1).

The effect of α-linolenic acid-rich emulsion on fatty acid metabolism and leukotriene generation of the colon in a rat model with inflammatory bowel disease

The purpose of this study was to investigate the efficacy of α-linolenic acid-rich perilla oil emulsion (POE) in a rat model with trinitrobenzenesulfonic acid (TNB)-induced inflammatory bowel disease. Three different isocaloric solutions, which are glucose solution (FF), soybean oil emulsion (SOE) and POE, were infused for 14 days after instillation of TNB. After infusion, total cholesterol and phospholipid concentration in the plasma in the POE group were significantly decreased compared with the FF and SOE groups. Arachidonic acid level in the colonic phospholipids was significantly decreased and eicosapentaenoic acid level was significantly increased in the POE group compared with the FF and SOE groups. Thickness, damage score and leukotriene B4 content in the colon in the POE group were the lowest among the infusion groups. These results suggest that α-linolenic acid suppresses the synthesis of leukotriene B4 in the colon by changing the fatty acid composition in the colonic phospholipids and that POE may be effective in the improvement of inflammation in the colon.

Degradation kinetics of L-alanyl-L-glutamine and its derivatives in aqueous solution

The degradation kinetics of five glutamine dipeptides in aqueous solution, i.e. glycyl-L-glutamine (Gly-Gln), L-alanyl-L-glutamine (Ala-Gln), L-valyl-L-glutamine (Val-Gln), L-leucyl-L-glutamine (Leu-Gln) and L-isoleucyl-L-glutamine (Ile-Gln), were studied. Stability tests were performed using a stability-indicating high-performance liquid chromatographic assay. Two different Ala-Gln degradation routes, i.e. the cleavage of a peptide bond and the deamination of an amide group, were observed. The degradation was adequately described by pseudo-first-order kinetics. The maximum stability of Ala-Gln was obtained at an approximate pH of 6.0. The pH-rate profile described by specific acid-base catalysis and hydrolysis by water molecules agreed with the experimental results. The activation energy of Ala-Gln at pH 6.0 was determined to be 27. 1kcal mol-1, and the shelf-life (90% remaining) at 25 and 40 degrees C was predicted to be 5.3 years and 7.1 months, respectively. The rate constants of the glutamine dipeptides were influenced by the N-terminal amino acid residue and decreased in the order: Gly-Gln, Ala-Gln, Leu-Gln, Val-Gln and Ile-Gln.

Magnetic resonance image and blood manganese concentration as indices for manganese content in the brain of rats

Neurological disorders similar to parkinsonian syndrome and signal hyperintensity in brain on T1-weighted magnetic resonance (MR) images have been reported in patients receiving long-term total parenteral nutrition (TPN). These symptoms have been associated with manganese (Mn) depositions in brain. Although alterations of signal intensity on T1-weighted MR images in brain and of Mn concentration in blood are theoretically considered good indices for estimating Mn deposition in brain, precise correlations between these parameters have not been demonstrated as yet.Male Sprague-Dawley rats received TPN with 10-fold the clinical dose of the trace element preparation (TE-5) for 7 d. At 0, 2, 4, 6, and 8 wk post-TPN, the cortex, striatum, midbrain, and cerebellum were evaluated by MR images, and Mn concentration in blood and Mn content in these brain sites were measured by atomic absorption spectrometry. Immediately after TPN termination, signal hyperintensity in brain sites and elevated Mn content in blood and brain sites were observed. These values recovered at 4 wk post-TPN. A positive correlation was observed between either the signal intensity in certain brain sites or Mn content in blood and the relevant brain sites.Our observations suggest that the Mn concentration in blood and signal intensity in the brain sites on T1-weighted MR images are reliable indices for monitoring Mn contents in brain.

Determination of D-amino acid oxidase activity in tumour cells.

Background We evaluated the assay for determining D-amino acid oxidase (DAAO) activity in tumour cells, rat liver and rat kidney for studying the effects of D-amino acid-containing solution on cancer patients. Methods and Results In this method the amount of ammonia produced by the DAAO activity after removal of endogenous ammonia using a Sephadex G25 column was determined. The highest activity was observed in rat kidney, which was almost eight times that found in rat liver. As compared with host tissues, the DAAO activity in tumour cells was considerably less. Conclusions This DAAO assay may be useful for analysis of various tissue samples as well as tumour cells.

Preparation of fine emulsified fat particles without glycerol for intravenous nutrition.

A method of preparing fine emulsified fat particles without glycerol for intravenous nutrition was investigated. The factors assessed were the oil phase ratio, the glucose level of the aqueous phase and the temperature of high-pressure homogenization. The particle size decreased with an increase in the oil phase ratio and it went below 250 nm only in the emulsion with a 50% oil phase ratio. The weight-weighted particle size (dw)/number-weighted particle size (dn) value reflected the particle size distribution. The emulsion with a 50% oil phase ratio had a very narrow distribution of particle sizes and the dw/dn value was below 1.1. With the use of glucose solutions for the aqueous phase, smaller particle sizes and narrower distributions were obtained with increasing glucose concentrations. The controlled temperature of 50 degrees C was appropriate for high-pressure homogenization, producing particles below 160 nm. The rate of the layer separation was a function of particle size. The particle sizes below 180 nm can be expected to suppress the separation of the formulation which consisted of 10.0% soybean oil, 1.2% phospholipids and 5.0% glucose. The stability studies were conducted at 40 degrees C for 3 months and the fat emulsion was stable during storage. These investigations contribute to the preparation of a new caloric source for peripheral parenteral nutrition.

Significance of magnetic resonance image and blood manganese measurement for the assessment of brain manganese during total parenteral nutrition in rats

In this study, we report on the influence of trace elements (TE) on signal intensities of nuclear magnetic resonance images (MRI), both in vivo and in vitro. Optimal parameters for the assessment of Mn concentration in the brain of rats on total parenteral nutrition were established.For the in vitro study, Mn and trace element solutions, one containing Zn, Cu, Fe, and I (TE-4) and another containing the above elements plus Mn (TE-5), were diluted with physiological saline or with rat brain homogenate and used to measure signal intensities in MRI. Concentration-dependent signal hyperintensity was observed in both cases in the Mn and the TE-5 solutions, but no effect was observed with the TE-4 solution. The signal increase was greater for brain tissue homogenates.In the in vivo study, the experimental animals were maintained under total parenteral nutrition (TPN) with a standard clinical dose of TE-5 and/or with 10-fold the clinical dose of TE-4 and TE-5 for 1 wk. Only rats that were receiving the increased TE-5 dose showed signal hyperintensity on MRI. Positive correlations were observed among the signal hyperintensity, the blood Mn concentrations, and that of the rat brain.Our results suggest that Mn in TE preparations may be the cause of signal hyperintensity on MRI in a concentration-dependent fashion, and that MRI and measurement of blood Mn may be used to estimate Mn accumulation in brain tissue.

Amino Acid Supplementation to Hyperalimentation in Uremic Rats: Effects of Amount and Composition of Amino Acids on Nutrition and Uremia

We evaluated amount and composition of amino acids in supplementation of hyperalimentation from the standpoint of whether it may improve nutrition and/or reduce the indexes of uremia such as BUN. Rats with established uremia, by 5/6 nephrectomy, were treated with various isocaloric solutions containing different amount of essential amino acids and histidine (EAA) or standard amino acids (SAA) which were formulated to provide Cal/N ratios of 300, 600, and 900. The BUN was lower and the nutritional index was better in rats infused with EAA compared with those administrated SAA, while severe distortion of plasma amino acid concentration, hyperammonemia, and fatty liver were observed at the Cal/N 300 condition. Rats infused with SAA gained positive nitrogen balance at the condition of Cal/N 300; however, plasma amino acid distortion was still observed. These results indicate that administration of EAA alone for treatment of renal failure needs high-calorie and low-nitrogen conditions such as Cal/N 600 for avoiding complications. Administration of standard amino acid solution is safe and nutritionally effective in the Cal/N 300 condition, but there are a few problems concerning nitrogen availability and plasma amino acid pattern.

New Peritoneal Dialysis Model in Rats with Bilateral Nephrectomy

Many peritoneal dialysis (PD) patients with chronic renal failure (CRF) suffer from metabolic and nutritional abnormalities. However, these abnormalities have been not sufficiently investigated. At present, the resolution of these issues in this field has been hindered by the lack of suitable PD models. We attempt to develop a rat model of PD under no constraints and under non-anesthetization to evaluate amino acid solution as suitable nutritional therapy for renal failure. In our model, bilateral nephrectomy rats were dialyzed 6 h per day for three days. The dialysate was infused and removed continually via a metering pump. Under fasting, rats were infused with 5% glucose or amino acid solution for renal failure, and they remained alive. This model can be used to examine bilateral nephrectomy in rats for three or more days. We were also able to determine protein and calorie malnutrition, negative nitrogen balance, abnormalities in the plasma amino acid pattern, and calcium and phosphorus metabolism. Thus, this model has the characteristics of renal failure in humans and may be used to easily examine the metabolic changes due to loss of kidney function.

Morphological studies on the central nervous system of rats with portacaval anastomosis which were continuously infused with ammonium acetate

Abstract Morphological changes in the striatum of portacaval anastomosis (PCA) rats which were continuously infused with ammonium acetate for 6 days have been studied. PCA rats with the infusion of ammonium acetate showed no neurological symptoms. However, ammonia concentration in the plasma was significantly higher than that in PCA rats without the infusion of ammonium acetate. Histologically, numerous atrophic neurons have been observed in the striatum. Moreover, glial fibrillary acidic protein (GFAP)-positive astrocytes in the same area became hypertrophied and had enlarged processes in hyperammonemic PCA rats. These results suggest that long-term hyperammonemia, which did not produce any visible neurological symptoms, might induce the morphological changes in the brain of PCA rats. These changes might be related to excessive ammonia metabolism in astrocytes.