Proposed standard for human blood vitamin B1 value using HPLC. The Committee for Vitamin Laboratory Standards, Japan.

Standard reference ranges for all laboratory test values are mandatory. This study was designed to establish a reference range for blood vitamin B1 levels, since the normal range has not been determined in the Japanese population. We founded the Japan Committee for Vitamin Laboratory Standards, which was incorporated with the Vitamin Society of Japan and the Japanese Society of Nutrition and Food Science. We standardized whole blood vitamin B1 levels using three HPLC techniques (post-column reverse-phase HPLC, pre-column reverse-phase HPLC, and precolumn GP-HPLC). The reference range was obtained in 54 volunteers administered a 1,800 kcal diet with 2 mg of vitamin B1 (1.74 mg measured) daily to avoid marginal vitamin B1 deficiency in the population. The range for each assay was 26-47, 28-51, and 28-56 ng/ml, respectively. Our data suggest that 26-28 ng/ml is the lower limit of normal for whole blood vitamin B1, but further studies in a larger population are needed in order to obtain more definitive results.

Alteration of the substrate specificity of Aspergillus oryzae beta-galactosidase by modification with polyethylene glycol.

beta-Galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae was modified with 2,4,6-trichloro-s-triazine derivatives of polyethylene glycol (activated BPEG) having molecular weights of 600, 1500, 2000, and 4000. Polyethylene glycol derivatives were attached to 6 of the 12 amino groups exposed on the surface of the enzyme. Upon modification, the enzymatic activity for a water-soluble substrate, o-nitrophenyl beta-D-galactopyranoside, was reduced with increasing molecular weight of the activated BPEG. On the contrary, the enzymatic activity for another substrate, 4-methylumbelliferyl beta-D-galactopyranoside, was increased upon modification. The Michaelis constants of native and modified enzymes for these two substrates were virtually the same. The effect of the modification was more marked in the enzymatic hydrolysis of the beta-galactosidic bond of amphipathic substrates. A fluorescent analog of naturally occurring galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, was hydrolyzed more rapidly by the modified enzyme than by the native one. The enzyme modified with activated BPEG of 1500 Da had the highest activity for this substrate. The beta-galactosidic bond of the terminal galactose of GM1-ganglioside (II3NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosyl -glucosylceramide) was cleaved by the modified but not by the native enzyme.

l-Tryptophan Production by Achromobacter liquidum.

Conditions for the production of tryptophanase from Achromobacter liquidum and for the conversion of l-serine and indole to l-tryptophan were studied. The enzyme could be produced in amounts as great as 0.750 U/ml (degradation) and 0.294 U/ml (synthesis) by shaking cultures at 30 degrees C in a medium containing dextrin, yeast extract, l-tryptophan, and l-glutamic acid. l-Tryptophan was produced most efficiently by shaking the cells at 37 degrees C in a reaction mixture containing 60 mg of l-serine per ml, 60 mg of indole per ml, and 0.5 mM pyridoxal phosphate. After 3 days, 96 mg of l-tryptophan per ml was formed, and l-tryptophan was easily isolated to 85.4% yield by concentration of the reaction mixture.

Characteristics of immobilized histamine for pyrogen adsorption.

The characteristics of immobilized histamine for pyrogen adsorption were investigated. The adsorbent showed a high affinity for pyrogen at low ionic strength, at around neutral pH, at high temperature and at low flow-rates of a solution containing pyrogen. The adsorption capacity per millilitre of the adsorbent was 0.9 mg pyrogen. Immobilized histamine could be completely regenerated by washing with 0.2 M sodium hydroxide solution containing 10--30% ethanol followed by 1.5 M sodium chloride solution, or 0.2 M sodium hydroxide solution followed by 0.5% sodium deoxycholate solution, 0.2 M sodium hydroxide solution and 1.5 M sodium chloride solution.

l-Serine Production by a Mutant of Sarcina albida Defective in l-Serine Degradation.

For improved l-serine production, an l-serine dehydratase-defective mutant of Sarcina albida IAM 1012 was obtained. In the mutant, the activities of the enzymes responsible for l-serine production were as high as those in the parent strain, and, at a low glycine concentration, the mutant accumulated l-serine more efficiently than the parent. Under optimum conditions, 21 mg of l-serine per ml accumulated from 100 mg of glycine per ml. l-Serine was isolated from a reaction mixture as l-serine m-xylene-4-sulfonate, and free amino acid was obtained in high yields by use of an ion-exchange resin. Residual glycine was recovered at a yield of 61%.

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases.

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.

Purification of high molecular weight urokinase from human urine and comparative study of two active forms of urokinase.

An improved method for the purification of high molecular weight urokinase to homogeneity from human urine was established. A yield of 32% with a 3,100-fold purification was obtained by Hyflo Super-Cel treatment, heat treatment at 60 degrees C for 10 hr, serial column chromatography on DEAE-Sepharose CL-6B and O-[3-(p-sulfophenylamino)-2-hydroxypropyl]-cellulose (SFOP-cellulose), and gel filtration on Ultrogel AcA 54. The low molecular weight form of urokinase was also purified to homogeneity by chromatography on hydroxyl apatite and gel filtration on Sephadex G-75 after the SFOP-cellulose column step. The high molecular weight urokinase had only one isoelectric form with a pI of 9.7, whereas the low molecular weight form had six isoelectric subforms with pI values between 9.4 and 6.4. The absorption coefficients at 280 nm of both urokinase forms were 13.61 and 13.50, respectively. Fibrinolytic and esterolytic activities of the two urokinase forms were compared in various assay methods.

Nutritional responses of tumor-bearing rats to oral or intravenous feeding.

Two experiments were conducted with male rats weighing 170 to 190 grams. In experiment 1, some nutritional parameters were determined in tumor-bearing (TB) (Walker 256 carcinosarcoma) rats fed a 23.6% casein diet for 4 weeks after the tumor inoculation. Cumulative weight gain and food intake were less in TB rats than in nontumor-bearing (NTB) rats. At 3 and 4 weeks after the tumor inoculation, plasma histidine, alanine, and glycine levels were higher in TB rats than in NTB animals. The arginine level was lower in the plasma of TB rats at 4 weeks after the inoculation. The significance of decrease in plasma arginine with regard to tumor growth is discussed. In experiment 2, the effects of total parenteral nutrition (TPN) on TB rats were evaluated as compared with those of 5% glucose (Glc) solution. Body weights of TPN rats were maintained and their nitrogen (N) balances were positive during a 7-day experimental period, while 5% Glc animals showed severe body weight loss and apparent negative N balance. After the end of infusion, the plasma urea level of the TPN group was within normal range, whereas that of 5% Glc group showed a markedly high value. The plasma albumin level was higher in the TPN group. Liver and spleen weights were increased in TPN rats. Absolute tumor weight was somewhat greater in TPN rats than in 5% Glc rats, but the difference in tumor weight:body weight ratios became more slight. These results indicate that TPN was effective for maintaining the nutritional status of TB host without significant acceleration in tumor growth.

Production of l-Phenylalanine from trans-Cinnamic Acid with Rhodotorula glutinis Containing l-Phenylalanine Ammonia-Lyase Activity.

An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid.

Mechanism of l-Glutamine Production by an l-Glutamine-Producing Mutant of Flavobacterium rigense.

Properties of some enzymes involved in l-glutamine biosynthesis in an l-glutamine-producing mutant of Flavobacterium rigense were examined. Glutamate-oxaloacetate transaminase in the mutant was nearly at the same level as that in the parent strain and was the most active among the enzymes participating in glutamate biosynthesis from alpha-ketoglutarate. Glutamine synthetase formation in the mutant was enhanced by increasing the concentration of (NH(4))(2)-fumarate in the medium, but the activity of this enzyme in the parent strain was very low, and its formation was not influenced by the concentration of (NH(4))(2)-fumarate. Glutaminase formation by both strains was similar and was not influenced by the levels of (NH(4))(2)-fumarate. Glutaminase activity of the mutant was inhibited by ammonia and fumarate. Intracellular amino acids and extracellular free amino acids in the mutant were compared with those of the parent strain. It seems reasonable to conclude that l-glutamine leaks out specifically through the cell membrane of strain 703 and that this specific excretion of l-glutamine probably allows a continuous conversion of l-glutamate to l-glutamine inside the cell.

beta-methylnorleucine, an antimetabolite produced by Serratia marcescens.

An amino acid was formed by alpha-aminobutyrate-resistant mutants of Serratia marcescens grown in a medium containing norvaline. This amino acid was identified as erythro-beta-methyl-L-norleucine [(2S,3S)-2-amino-3-methylhexanoic acid] by instrumental analyses. beta-Methylnorleucine inhibited the growth of several bacteria in synthetic medium.

Biosynthetic pathway of beta-methylnorleucine, an antimetabolite produced by Serratia marcescens.

beta-Methylnorleucine biosynthesis was examined in Serratia marcescens using regulatory mutants of branched-chain amino acid biosynthesis. The accumulation of beta-methylnorleucine from norvaline in the wild-type strain was inhibited by the simultaneous additions of isoleucine, valine and leucine, although its accumulation in the derepressed mutant of isoleucine, valine and leucine biosynthesis was markedly increased and was not inhibited by additions of these amino acid. Accumulation of this compound was not observed in an isoleucine-valine auxotroph, although its accumulation was not affected in an isoleucine or leucine auxotroph. Transaminase B catalyzed the conversion of alpha-keto-beta-methylcaproate to beta-methylnorleucine. These results suggest that beta-methylnorleucine is formed from alpha-ketovalerate, alpha-ketoacid corresponding to norvaline, by enzymes of the isoleucine-valine biosynthetic pathway.

Influence of Nutritional Conditions on Production of l-Glutamine by Flavobacterium rigense.

The nutritional conditions for the production of l-glutamine by Flavobacterium rigense strain 703 were investigated. The optimum concentration of ammonia for achieving the highest yield of l-glutamine (25 mg/ml of broth) was relatively broad, from 0.9 to 1.6%, whereas fumaric acid had a narrow optimum range, near 5.5%. High concentration of inorganic ions such as chloride or sulfate ion clearly inhibited cell growth. Therefore, ammonium salts other than (NH(4))(2)-fumarate were unsuitable for the highest production. The optimum concentration of (NH(4))(2)-fumarate was 7%. To reduce the concentration of fumaric acid in the medium, many substances were evaluated as substitutes. The fumaric acid concentration required for highest l-glutamine yield could not be replaced by any one of the compounds tested. However, part of fumaric acid could be replaced with succinic acid and cupric ion; 4% (NH(4))(2)-fumarate plus 2.5% succinic acid or 5% (NH(4))(2)-fumarate plus 1 mM cupric ion produced results similar to 7% (NH(4))(2)-fumarate in the fermentation medium.

Nutritional effects of intravenous infusion solutions on normal rats: effects of increased energy level and deletion of acidic amino acids.

Nutritional effects of intravenous infusion of an amino acid (AA) mixture enriched with the branched chain AA were previously evaluated at a daily level of 45 kcal and 200 mg N using male rats weighing approximately 200 g. The present study was conducted with male rats weighing approximately 200 g to evaluate the nutritional effects of 1) an AA infusion solution at further increased energy level, and 2) an AA solution devoid of aspartic and glutamic acids. By increasing daily energy input from 45 to 55 kcal/rat, the body weight gain of rat was markedly increased and more positive nitrogen balance was observed. Glucose, albumin, and free AA levels were unchanged in plasma of rats after the infusion period, while plasma urea level was somewhat lowered. Organ weights and liver lipid content were also unchanged. The administration of an infusion solution devoid of aspartic and glutamic acids resulted in little alteration in the amounts of urinary excretion and plasma levels of these acidic AA. Furthermore, other parameters measured showed no significant effect of the deletion of the AA. These results indicate that no advantage is expected in the use of acidic AA for parenteral nutrition.