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The concentration of glycosaminoglycans, in an aqueous solution was determined by observing the fluorescent intensity of the ion of 2-hexadecyl-9H-pyrido[4,3b]indole at 450 nm by irradiating 253-nm light after dissolution of the insoluble salt in ethanol, which was formed by the reaction between a solution of a sample of glycosaminoglycan and an aqueous solution of the binary mixture of fluorescent 2-hexadecyl-9H-pyrido[4,3b]indolium bromide and hexadecyl pyridinium chloride. The fluorescent quaternary ammonium salt, which was slightly soluble in water, was solubilized in water by forming mixed micelles with a nonfluorescent soluble quaternary ammonium salt. The present method showed good linearity for the calibration curves between 5 and 1000 micrograms/ml in all samples of glycosaminoglycan except keratan sulfate. The relative standard deviation in determination was less than 3% for the whole calibration range.

To improve the efficiency of the preliminary experiments for the JEM, the telescience capabilities have been implemented into the parabolic flight experiments on the small jet plane. In this paper, we will describe the results of the 1st telescience support experiments using the two commercial airline telephone system. In the experiments, these lines have been used not only for the voice communication, but also for the data transfer, such as FAX, still TV image transmission, and data transfer. Utilizing these capability, human physiological experiment was tested as the sample experiment. The parabolic flights were done in the two major training/testing area, that are C & K area.

Sensitivities of the following derivatives of oligosaccharides in MS and LC/MS with frit FAB and ESI interfaces were compared under the conditions where samples were supplied to the probes in solutions at constant rates: 2-aminopyridine, 4-aminobenzoic acid ethyl ester (ABEE), 1-phenyl-3-methyl-5-pyrazolone (PMP) as well as 4-methoxyphenyl analogue, 2-aminoethanethiol, and 2-aminobenzenethiol. In FAB-MS, maltopentaose labeled with ABEE gave the most abundant [M + H]+ ions in positive ion mode when ionized in thioglycerol or glycerol matrix supplied in 50% (v/v) aqueous methanol. The lowest limit of detection was obtained for this compound in these matrices in water. The [M - H]- ion in negative ion mode was also abundant in all matrices examined in both 50% (v/v) aqueous methanol and water. On the other hand PMP derivatives gave the highest sensitivities in ESI-MS under the optimized conditions. On the basis of these findings, authentic oligosaccharides and oligosaccharides derived from a few glycoprotein samples were analyzed by LC/MS with frit FAB and ESI interfaces. The results indicated the usefulness of these derivatives for simultaneous microanalysis of oligosaccharides.

Recent developments in the analysis of glycoproteins by high-performance capillary electrophoresis are reviewed, with emphasis on their carbohydrate chains. Glycoforms of glycoproteins were directly separated from each other by careful optimization of the analytical conditions. Glycopeptides in tyrptic digests were separated and the peptides carrying glycosylation sites were differentiated from others. Released oligosaccharide chains were separated from each other by direct or modified zone electrophoresis and directly detected by measuring the UV absorption at a low wavelength. Precolumn derivatization by various methods extended the utility of both the separation mode and detection technique. Dual mode analysis after derivatization permitted reliable identification and quantification without references.

Familiarity ratings for a large number of aurally and visually presented Japanese words wer measured for 11 subjects, in order to investigate the modality dependency of familiarity. The correlation coefficient between auditory and visual ratings was .808, which is lower than that observed for English words, suggesting that a substantial portion of the mental lexicon is modality dependent. It was shown that the modality dependency is greater for low-familiarity words than it is for medium- or high-familiarity words. This difference between the low- and the medium- or high-familiarity words has a relationship to orthography. That is, the dependency is larger in words consisting only of kanji, which may have multiple pronunciations and usually represent meaning, than it is in words consisting only of hiragana or katakana, which have a single pronunciation and usually do not represent meaning. These results indicate that the idiosyncratic characteristics of Japanese orthography contribute to the modality dependency.

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE). O- and N-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection. N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.

This paper proposes a new method for simultaneous analysis of unsaturated disaccharides derived from glycosaminoglycans by enzymatic digestion with chondroitinase ABC, based on high-performance capillary electrophoresis (HPCE) of their 1-phenyl-3-methyl-5-pyrazolone derivatives. The O-sulphate group is stable in this derivatization, and this method allows reproducible microdetermination of glycosaminoglycans. This paper also demonstrates the applicability of this method to estimation of urinary chondroitin sulphates. Urinary creatinine as an inherent internal standard could also be estimated by HPCE, though in another mode of separation, i.e. ion-exchange electrokinetic chromatography.

N-Glycosidically linked oligosaccharides were released from glycoproteins by digestion with trypsin followed by hydrazinolysis and subsequently re-N-acetylated and reductively pyridylaminated. Derivatives of sialic acid-containing oligosaccharides were further desialylated with neuraminidase. The final derivatives of asialo-oligosaccharides were analyzed by capillary zone electrophoresis in two carriers, an acidic phosphate buffer and an alkaline borate buffer. The former carrier allowed direct zone electrophoresis as cationic immonium ions, accordingly size-dependent separation, whereas the latter realized indirect electrophoresis as anionic borate complexes, i.e., separation based on the structural variation in outermost monosaccharide residues. Two-dimensional plots of relative mobilities of the derivatives in these dual separation modes to reductively pyridylaminated glucose provided a good tool for identification of oligosaccharides.

Protein-sugar interaction was observed by capillary zone electrophoresis, using a few beta-galactose-specific lectins and lactobionic acid as protein and sugar models, respectively. The lectin peaks were retarded in a concentration-dependent manner by addition of lactobionic acid in a carrier, and association constants of monovalent mode interactions could be obtained from t1 (migration time of protein), t2 (migration time of complex, obtainable as the migration time at the plateau) and the slope of the (t-t1)-1 vs. [S]-1 plots, where t and [S] are the migration of protein in the presence of lactobionic acid and the concentration of lactobionic acid, respectively. The values for Ricinus communis agglutinin, peanut agglutinin and soy bean agglutinin at pH 6.8 were 3.3 . 10(3), 9.1 . 10(2) and 1.1 . 10(2)1 mol-1, respectively. This method required only small amounts of protein samples and was reproducible. The amount of the sugar could be minimized under the conditions that the carrier was a buffer containing the sugar whereas the electrode solutions consisted only of the buffer.

Cefixime (CX), an oral cephalosporin antibiotic, and its metabolites in human digestive organs were separated by various modes of high-performance capillary electrophoresis. The zone electrophoresis mode in phosphate buffer (pH 6.8) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate gave the best separation, permitting the complete resolution of CX and all of five metabolites. On the other hand, the plain zone electrophoresis mode in phosphate buffer (pH 6.8) offered a simple procedure for the direct determination of urinary CX concentration using intact urine samples.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.

N-Glycosidically bound sialooligosaccharides in a model glycoprotein of serum type (porcine thyroglobulin) were released by pronase digestion, followed by hydrazinolysis. The resulting oligosaccharides were re-N-acetylated and reduced with sodium borohydride. On the other hand, O-glycosidically bound sialooligosaccharides were released from a mucin-type glycoprotein (bovine submaxillary mucin) with alkali in the presence of sodium borohydride. The reduced oligosaccharides thus obtained from both types of glycoproteins were analysed by high-performance liquid chromatography on a column of a latex-type pellicular anion-exchange resin with strong alkali as eluent. These sequential procedures were useful for mapping of oligosaccharides in glycoproteins.

The oligosaccharides in ovalbumin as a glycoprotein model were released with anhydrous hydrazine, and reductively pyridylaminated after re-N-acetylation. The derivatives were analyzed by capillary zone electrophoresis (CZE) with on-column fluorometric detection. Direct CZE could separate the derivatives on the basis of the degree of polymerization, giving five peaks of hepta-, octa-, nona-, deca-, and undecasaccharides. Coelectrophoresis with the standard mixture of isomaltooligosaccharide derivatives was effective for peak assignment. CZE as borate complexes allowed separation on the basis of structural difference, especially in the peripheral monosaccharide residues. Peaks were tentatively assigned to the derivatives of reported oligosaccharides by comparing their relative mobilities with those of the chromatographic fractions obtained by using the ODS and Dowex 50W x 2 columns. These two modes gave excellent separation and were complementary to each other. Although the actual amount analyzed in the capillary tube was quite small (ca. 5 ng as carbohydrates), a larger amount (ca. 25 micrograms as carbohydrates) was required to make sample concentration sufficiently high to be detected by a modification of a commercial fluoromonitor for HPLC.

The quality of commercial heparin injections was examined by 400-MHz proton nuclear magnetic resonance (1H-NMR) spectroscopy using several measuring modes. The signals of the N-acetyl protons, as well as the sugar-ring protons, attached to the sulfamino and sulfato group-bearing carbons could be easily distinguished from other proton signals and quantified. Measuring at a high temperature (60 degrees C) enabled clear isolation of the H-5 proton signal in the sulphated iduronic acid residue (Is-5) from other proton signals including that of water. The heparin contents of various heparin injections were estimated by using this signal as an index. However, the signal intensity was not parallel with anticoagulant activity. On the other hand, the N-acetyl proton signal was highly correlated to anticoagulant activity. The present method was also useful for concurrent identification of additives in heparin injections.

Sialolithiasis occurs due to the calculous concretion in salivary ducts or glands, but it is rare in childhood. In this paper, a case of sialolithiasis with a submandibular duct calculus observed in a 2-year-old girl is reported. This case is one of the youngest, based on a review of the literature regarding sialolithiasis found in Japan. The retrospective survey was made in 30 cases of sialolithiasis in children under 10 years of age which were reported in the Japanese literature with clear descriptions of age, sex and location. The summaries are as follows: 1) Sex difference: The difference between males and females was in the ratio 16/14. There was no significant difference according to sex. 2) Location of the salivary calcul: In 27 cases they were found in the duct of the submandibular glands, and 3 cases in the parotid glands, and no cases in the sublingual glands. 3) Term before treatment: Most cases were treated within a month after the patients has noticed the symptom. 4) Removal method of salivary calculi: Salivary calculi were removed by means of intraoral incision in most cases. 5) Number of the removed calculi: In each of all cases, one calculus was removed. 6) Size of the removal calculi: The diameter of the calculi was less than 5.0 millimeters long in most cases.

A convenient gas chromatographic method has been devised for the analysis of hexosamines in the presence of neutral and acidic sugars, which involves sequential derivatization reactions of nitrous acid deamination, mercaptalation, and trimethylsilylation. This method allows rapid, simultaneous determination of 0.1-1 micromole samples of hexosamines with coefficients of variation of less than 3%.

A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis.

A sensitive method for determination of sialic acids by monitoring the fluorescence produced with malononitrile in borate buffer has been established. Measurement of the fluorescence intensity of the reaction mixture at 430 nm with irradiation at 360 nm allowed determination of 3-60 nmol of sialic acids with high reproducibility. A few amino sugars and deoxy sugars, as well as catecholamines reacted with this reagent; however other carbohydrates, amino acids, amines, aldehydes, and carboxylic acids including alpha-keto acids, etc., showed little reactivity. This method was successfully applied to postcolumn fluorescence labeling of sialic acids in high-performance liquid chromatography.

Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 microns i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates.

Positive ion fast atom bombardment mass spectra of maltooligosaccharides reductively aminated with 2-aminopyridine (Gn-AP) contain abundant [M + H]+ ions. Determination of G2-AP and G3-AP produced from G5-AP by the action of alpha-amylases, based on the abundance of their [M + H]+ ions relative to that of cellobiose reductively aminated with 2-amino-6-methylpyridine as the internal standard, allowed rapid and reproducible assay of these enzymes. It was advantageous for clinical investigation that the proportion of pancreatic and salivary alpha-amylase activities could be determined.

Concanavalin A (Con A) or wheat germ agglutinin (WGA) was immobilized on a silica-based support, and the chromatographic behaviours of a series of dansylated ovalbumin-derived glycopeptides on small columns of the resultant gels were compared. These columns had high contents of lectins, and allowed differentiation of these glycopeptides. This method was rapid and reproducible, and enabled sensitive detection of these fluorescent glycopeptides. The structural requirement of these glycopeptides to manifest affinity to the immobilized lectins is also discussed, based on binding constants obtained from their retention times.

Current chromatographic methods for the analysis of a variety of carbohydrate materials in body fluids and tissues have been reviewed, from the viewpoints of clean-up of samples, separation modes, methods for detection and quantification, and degree of convenience. This review also contains several tables, listing names of samples, methods of analysis, analytical conditions, and normal as well as pathological levels reported, from representative publications.

We found that 1-phenyl-3-methyl-5-pyrazolone reacts with reducing carbohydrates almost quantitatively to yield 2:1 compounds having no stereoisomers, which strongly absorb the uv light at 245 nm and are easily oxidizable on a glassy carbon electrode. Reverse-phase partition chromatography on a column of Capcell Pak C18 with uv or electrochemical detection allowed rapid analysis of aldoses and N-acetylhexosamines with the detection limit of 1 pmol or 100 fmol, respectively. This method proved especially useful for analysis of component monosaccharides of glycorproteins. It was also shown to be valid for separation of reducing oligosaccharides; maltodextrins with a degree of polymerization up to 19 were similarly derivatized and separated on this stationary phase.

Catecholamines in the eluates of high-performance liquid chromatography were reacted with 2-cyanoacetamide in borate buffer in flow analysis mode, and the intensity of fluorescence developed was recorded. Under the optimum conditions (column: Hitachi 3011 C, 25 cm X 2.6 mm I.D., 45 degrees C; eluent: 0.05 M KH2PO4 containing 0.05% H3PO4, 0.60 ml/min; reagent solution: a mixture of 1% 2-cyanoacetamide, 0.50 ml/min, and a 0.60 M H3BO3--KOH buffer, 1.0 ml/min; size of reaction coil: 5 m X 0.5 mm I.D.; reaction temperature: 100 degrees C; wavelengths for detection: 383 nm for excitation and 486 nm for emission), this method allowed simultaneous determination of 5-500 pmol catecholamines with high reproducibility. The lower limits of detection (signal-to-noise ratio = 2) for epinephrine, norepinephrine and dopamine were 0.28, 0.11 and 0.098 pmol, respectively. Some applications of the analysis of urinary and serum catecholamines are also presented.

Oligosaccharides of ovalbumin were released by hydrazinolysis and converted to the glycamine derivatives by reductive amination. The resultant derivatives were immobilized on an epoxy-activated methacrylate polymer. Application of lectins on the column containing the resultant resin, followed by injection of the competing sugars and detection of the eluate using natural fluorescence, allowed differentiation of micro amounts of the lectins, owing to their high specificity. Stepwise elution with various competing sugars also permitted separation of lectins. Application of this method to serum samples enabled detection of various carbohydrate-binding proteins with specific affinity to the injected sugars. This method, based on two-stage separation at the adsorption and elution stages, was highly specific. It was also rapid, reproducible, and sensitive.

This paper investigates a sensitivity measurement method for carbon transmitters and a calculation method for the loudness rating of a telephone system. The sensitivity varies depending on the artificial voice used for the measurement and the conditioning procedures. The most preferable methods for obtaining values which coincide with those in the subjective loudness rating test are discussed. With respect to the loudness calculation method, the algorithm, parameter values, ear leakage loss, and voice spectrum are discussed. As a result of these investigations, the deviation between the objective and subjective rating is found to be 0.7 dB for the various types of handset telephones when the measurement conditions are optimized. This deviation is considered to be the limit of measurement accuracy. The performance of currently developed objective measurement instruments is close to this limit. Considering the fluctuations accompanying repetitive subjective measurements, the objective rating corresponds satisfactorily with the subjective rating.

The aldehydes in the periodate oxidation products of non-dialyzable urinary carbohydrate materials were determined simultaneously by gas chromatography of their diethyl dithioacetal derivatives. The yields of aldehydes for normal male urine varied considerably among subjects, but their molar ratios were almost constant. The average values for glyceraldehyde/glyoxal and lactaldehyde/glyoxal molar ratios were 0.41 and 0.27, respectively. The average amount of L-fucose at the non-reducing terminals of carbohydrate chains, as estimated from the yield of lactaldehyde, was about 5 mg/day.

Eight alditols were separated in ca. 80 min as their borate complexes by stepwise elution with three borate buffers on a column packed with Hitachi 2633 resin. The alditols in the eluate were derivatized automatically to colored, fluorescent products by applying sequential reactions of periodate oxidation and Hantzsch condensation, and the products were detected either photometrically or fluorimetrically. This automated method allowed simultaneous determination of 20-500 and 20-200 nmol amounts of alditols by photometric and fluorimetric monitorings, respectively. The lower limits of detection were ca. 2 and 0.5 nmol, respectively. The interference by aldoses was slight. Aldoses may be also determined as alditols by direct injection of aqueous solutions to which excess amounts of sodium borohydride have been added. This method was applied with success to urinary alditol assay and to molecular weight determination by end group analysis.

A convenient method for the analysis of sialic acids is proposed, which is based on their dissociation into pyruvate and N-acylmannosamines by the action of N-acetylneuraminate lyase, followed by gas-chromatographic analysis of the latter products as trimethylsilylated diethyl dithioacetals. Conjugated sialic acids should be freed with neuraminidase before being subjected to the action of the lyase, but these sequential enzymic reactions may be performed in one pot. N-Acetyl-, N-glycolyl- and N,O-diacetylneuraminic acids gave the corresponding mannosamines, and the dithioacetal derivatives of these mannosamines were well separated on a column of silicone OV-1. Quantitation of this enzymic and gas chromatographic method indicated that the error and coefficient of variation for free N-acetylneuraminic acid were 1.1% and 2.5%, respectively, for ten determinations at the 100 nmol level. The values for conjugated N-acetylneuraminic acid in N-acetylneuraminlactose were 2.9% and 5.9%, respectively. This method was applied to the analysis of sialic acids in some biological samples, and the results were compared with those obtained by the conventional colorimetric method. Preliminary data on urinary sialic acids indicated that cancerous patients gave significantly higher levels of urinary N-acetylneuraminic acid than normal subjects.

A few carbohydrate-immobilized resins were prepared by coupling glycamines of mono- and disaccharides to an epoxy-activated methacrylate base, and the elution profiles of various carbohydrates and lectins from columns packed with the resultant resins were compared. The introduction of glucamine, maltamine or lactamine to the methacrylate base had little effect on the separation of stereoisomers of aldoses, though it enabled group separation of pentoses as well as 6-deoxyhexoses from hexoses. On the other hand, the maltamine-immobilized resin showed strong specific affinity for concanavalin A, as well as lentil and pea lectins. The lactamine-immobilized resin was specific to ricinus agglutinin, peanut agglutinin and soy bean agglutinin. These carbohydrate-immobilized resins were also useful for the isolation of a lectin-like substance from rat liver.

A method for the high-performance liquid chromatographic assay of the relative activities of serum pancreatic and salivary alpha-amylase has been developed, using maltopentaose reductively aminated with 2-aminopyridine as a fluorescent substrate. Both enzymes showed similar modes of action, cleaving the second and the third (from the non-reducing terminal) interglycosidic linkages of this substrate. However, the relative ease of cleavage of these two sites by the pancreatic enzyme was significantly different from that by the salivary enzyme. Therefore, determination of the molar ratio of the cleavage products by HPLC could lead to estimation of the activity ratio of these enzymes. The optimum chromatographic conditions for HPLC were as follows: column, LiChrosorb RP-18 (Merck, 7 microns, 250 X 4 mm I.D.); column temperature, ambient; eluent, 0.01% orthophosphoric acid-acetonitrile (4:1, v/v) containing 2.4 mM sodium laurylsulphate; flow-rate, 1.0 ml/min; wavelengths for fluorimetric detection, 320 nm (excitation)/400 nm (emission). The problem of interference by serum alpha-glucosidase was solved by specific inhibition with tris (hydroxymethyl) aminomethane and erythritol. The data obtained by the proposed method correlated well with those produced by the conventional method based on electrophoresis.

Glucosamine and galactosamine were well separated in ca. 60 min on a Hitachi 2617 column (polystyrene sulfonate type, 4 mm X 25 cm) with a borate buffer (pH 7.5) containing sodium chloride. The hexosamines in the eluate were monitored fluorimetrically and photometrically at 331 (excitation)/383 (emission) and 276 nm, respectively, by postcolumn labeling with 2-cyanoacetamide. This simple method allowed the simultaneous, automated determination of 10-500 nmol of glucosamine and galactosamine with high reproducibility. This method was applied successfully to the analysis of hexosamines in some glycoconjugates.