Quantitative Analysis of Sugars in Plant Extracts by Ion-exchange Chromatography,with Special Reference to the Examination of Conditions for Preparing the Sample Sugar Solutions

Critical examinations were made on the conditions for preparing the sugar solutions to be analyzed by ion-exchange chromatography of sugar-borate complexes by the method of Khym and Zill. A procedure was proposed which gave the best recovery of sugars with minimum hydrolysis of sucrose. By means of this procedure, sugar solutions were prepared from potato tubers which had been stored at a high (30°C) or low temperature (6°C). Results of the chromatographic separation and determination of component sugars showed that main sugars present in potato tubers were sucrose, glucose, and fructose. Maltose and pentoses could not be detected. The contents of sucrose, glucose, and especially, fructose were far greater in potatoes stored at a low temperature than in those stored at a high temperature.     

A new method for the quantitative determination of monosaccharides, amino sugars and N-acetylneuraminic acid and of 6-deoxyhexose (fucose) in the presence of other sugars

1. Monosaccharides, amino sugars and N-acetylneuraminic acid were determined by using an original colorimetric assay procedure, based on the detection of formaldehyde released after periodate oxidation. A range of these compounds was investigated by this method and they were all found to obey Beer's law within the concentration range 0-0.6mumole/ml. 2. A simple method for the determination of 6-deoxyhexose concentration in the presence of other monosaccharides is also described. 3. The optimum pH for the release of formaldehyde from sugars by periodate oxidation was 7.0-7.5. 4. The methods described have considerable advantages over existing assay systems and their particlar value in automatic colorimetry, where the use of concentrated acids is undesirable, is discussed.     

The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.     

Determination of the reducing terminal sugars of glycosaminoglycans using 2-aminopyridine

The fluorescence labeling method (Takemoto, H. et al. (1985) Anal. Biochem. 145, 245-250) has been shown to have high sensitivity for measuring the sugar composition of glycoproteins. In the present study, its applicability for analysis of the reducing terminal sugars of glycosaminoglycans was investigated. The procedure involved coupling of glycosaminoglycans with 2-aminopyridine, followed by hydrolysis and N-acetylation, and then analysis by high-performance liquid chromatography on a reverse-phase column. The method was found to be useful for simultaneous determination of acidic, neutral and amino sugars at the reducing termini of glycosaminoglycan moieties.     

Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

An automated spectrophotometric flow-injection procedure for the determination of cellulase activity in bioreactor preparations.

A spectrophotometric procedure for the determination of cellulase activity in precipitated bioreactor preparations and culture filtrates is described. It is based on the determination of reducing sugar produced by the action of the enzyme on carboxymethylcellulose. The reducing sugar is derivatized with p-aminobenzoyl-hydrazide and permits a limit of detection of 0.1 U ml-1 cellulase in the presence of background sugar, with a sampling rate of 5 h-1. The method can readily be applied to the determination of any carbohydrase acting on soluble substrates and producing reducing sugars, e.g. amylase, dextranase, xylanase, glucanase and polygalacturonase.     

Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives.

A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.     

Simultaneous determination of glucose, fructose and lactose in food samples using a continuous-flow chemiluminescence method with the aid of artificial neural networks.

In this study, a simple continuous-flow chemiluminescence (CL) system was developed for simultaneous determination of glucose, fructose and lactose in ternary mixtures of reducing sugars without previous separation. This method was based on the different kinetics of the individual sugars in the oxidation reaction with potassium ferricyanide. The known luminol-K(3)Fe(CN)(6) CL system was used to measure the kinetic data of the system. The CL intensity was measured and recorded every second from 1 to 300 s. The data obtained were processed chemometrically using an artificial neural network. The relative standard errors of prediction for three analytes were <5%. The proposed method was successfully applied to the simultaneous determination of the three sugars in some food samples.     

利用荧光手性试剂（S）—TBMB甲酸分析氨基糖的D—和L—构型

D、L型氨基糖经荧光手性试剂 (S) ( ) 2 叔丁基 2 甲基 苯并 1 ,3 二氧杂环戊烷 4 甲酸 [(S) TBMB甲酸 ]标记、完全乙酰基化得到非对映的氨基糖完全乙酰基化N (S) TBMB羧酸衍生物。其1 HNMR图谱 ,特别是强的叔丁基及甲基信号峰被用于分析氨基糖的D、L构型。此外 ,还利用反相HPLC及同样的荧光标识方法创立了简便的高灵敏度氨基糖D、L构型分析方法。其全部分析操作时间在 2h内 ,检测极限为 0 .2 pmol     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

A method for the microanalysis of hexoses in glycoproteins

A procedure has been developed by which less than micrograms quantities of hexoses in glycoproteins can be determined by g.l.c.-m.s. with selected-ion monitoring of the alditol acetates derived from the sugars released by acid hydrolysis. The effectiveness of the method was demonstrated by determination of the hexose and hexosamine composition of 5-micrograms samples of antithrombin III and von Willebrand factor, respectively.     

High-performance liquid chromatography of N,O-acylated sialic acids

A rapid, isocratic high-performance liquid chromatographic method for the analysis of N-acetylneuraminic acid, N-glycolylneuraminic acid, and their O-acetylated derivatives is described. Separation of sialic acids and of other monosaccharides as sugar-borate complexes is achieved on an anion-exchange resin. The sialic acids elute as individual peaks after the other sugars tested. The method allows quantitative determination, for example, of amounts of N-acetylneuraminic acid as small as 10 nmol. On cation-exchange resin sialic acids cannot be differentiated, but can be separated from neutral and amino sugars, allowing the determination of as little as 3 nmol of total sialic acids.     

Fructose determination with a specific fructokinase

A method for the determination of fructose is described. It is based on the phosphorylation of this sugar by a highly specific fructokinase and the coupling of this reaction to NADP reduction by means of glucosephosphate isomerase and glucose-6-phosphate dehydrogenase. Because of the lack of interference by other sugars, the procedure is appropriate for fructose determination in biological samples where a high content of glucose interferes in other methods currently in use. Even a 100-fold excess glucose causes no detectable interference (less than 1%). The new method has been applied for the estimation of fructose in a series of normal human sera. A mean value of ca. 1 mg per 100 ml was obtained, most serums being within the range 0.5–2.     

Simplified fluorometric assay for sphingosine bases

A simplified procedure for fluorometric determination of sphingosine bases is presented. Nitrogenous bases are reacted with fluorescamine and then extracted into a lower phase solvent, obviating the need to transfer the reaction products prior to quantitation. Consequently, the entire procedure may be carried out in a single set of screw-capped reaction tubes. Chloroform was found to be the best solvent of those tested for the maximal extraction of derivatized sphingosine bases with negligible extraction of nitrogenous contaminants derived from phospholipids and amino sugars. The simplified method retains excellent sensitivity and selectivity and allows for simultaneous processing of large numbers of samples. Furthermore, quantitation of glycolipid-derived hexosamine can be obtained by measuring the fluorescence of the aqueous phase after partitioning.     

Automation of a fluorometric method for the determination of 2-deoxy-D-glucose

A continuous-flow fluorometric procedure for the determination of 2-deoxy-D-glucose (2DG) is described. The method utilizes Technicon Autoanalyzer equipment and modules, and is based on the acid-catalyzed condensation of 3,5-diaminobenzoic acid with 2DG. The procedure permits analysis of 20 samples/h, is sensitive to concentrations of 2DG as low as 0.2 mg/100 ml, and requires sample volumes of only 0.25 ml. 2DG can be quantitatively measured in serum samples or tissue extracts without requiring deproteinization. Glucose does not interfere with the assay while 2-deoxy-D-ribose develops a fluorescence which is about 15% of that produced by the same amount of 2DG and is additive when both deoxy sugars are present together. The procedure is accurate, reproducible, and fast, and can be run continuously.     

Determination of aldoses and uronic acid content of vegetable fiber.

The factors affecting the stability, hydrolysis, reduction, acetylation, quantitation, and identification of the neutral sugars from vegetable fiber preparations have been studied critically and optimized. The recommended method offers a consolidation of the recent modifications of the alditol acetate procedure for the estimation of neutral sugars. The recovery of the sugars was tested by glc and ion-exchange chromatography. Also, the modified carbazole method of Bitter and Muir was adapted to make it applicable for the estimation of uronic acid content of fiber because uronic acid cannot be estimated quantitatively by the acetylation procedure. It is emphasized that the proposed method is applicable only to highly purified fiber preparations which are free of coprecipitated intracellular compounds. Also, the levels of pentoses and hexoses in the fiber must be well defined and a suitable correction made for their interference in the assay.     

Determination of Monosaccharides,Disaccharides, and Oligosaccharides in the Same Plant Sample by High-Performance Liquid Chromatography

A method for determining low-molecular carbohydrates based on the use of HPLC with refractometric detection was modified. In contrast to previous methods, the determination of the qualitative and quantitative composition of sugars in plant extracts was performed without their preliminary separation into monosaccharides, disaccharides, and oligosaccharides. The exclusion of an additional separation stage made it possible to reduce twofold the time of analysis of a single sample, to save expensive materials necessary for carbohydrate HPLC, and to increase the useful life of the analytical column.     

Fluorometric analysis of amino sugars and derivatized neutral sugars

A rapid and sensitive procedure for the analysis of neutral and amino sugars is presented. Neutral sugars are separated after conversion to the corresponding glycamines, while the amino sugars are analyzed without modification, using an automatic amino acid analyzer and fluorometric detection. The method has been applied for the analysis of glycoproteins and oligosaccharides of the complex and high-mannose types.     

An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.     

A lipid linked oligosaccharide which contains hexosamine,mannose and glucose

Summary Previous work showed that liver microsomes catalyze the transfer of glucose from dolichol monophosphate glucose to an endogenous acceptor believed to be a dolichol pyrophosphate derivative of an oligosaccharide. This oligosaccharide has now been prepared on a larger scale so as to permit the determination of its sugars. The purification procedure includes, as a last step, a thin layer chromatography on kieselguhr-silica gel to obviate glucose-containing contaminants. After complete hydrolysis mannose, glucose and a small amount of hexosamine were detected.Abbreviations DolMp dolichyl monophosphate - DolDP dolichyl diphosphate - GEA glucosylated endogenous acceptor solvent 1103, chloroform-methanol-water (1:1:0.3) This paper is dedicated to Dr.Luis Federico Leloir on the occasion of his 70th birthday.