A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

A simple and rapid preparation of alditol acetates for monosaccharide analysis

A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.     

Molecular recognition of carbohydrates by a resorcinarene. Selective transport of alditols through a supported liquid membrane

A supported liquid membrane (SLM) containing a resorcinarene carrier has been used for the selective transport of erythritol, threitol, ribitol and xylitol from concentrated (1.0-0.01 M) aqueous solutions. The membrane is made of a microporous polytetrafluoroethylene film impregnated with a 0.01 M solution of the carrier in CCl4. The permeabilities of the SLM for all alditols were calculated. On the basis of the flux dependence on the initial concentrations of carrier and alditol, the rate-determining step in the transport mechanism is shown to be the migration of the 1:1 carrier-carbohydrate complex in the immobilized organic phase. The flux of sugar is related to the initial concentration of alditol in the feed phase by a saturation law, which allowed the determination of the apparent diffusion coefficients and the stability constants of the resorcinarene complexes of alditols formed in the liquid membrane.     

Automated analysis of alditols by anion-exchange chromatography with photometric and fluorimetric postcolumn derivatization

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.     

Regioselective synthesis of alditol vicinal bis-cyclic thionocarbonates via alditol stannylene acetal complexes as a short and efficient route to alpha,omega-diiodoalditol derivatives.

The bis-cyclic thionocarbonates of alditols (pentitols and hexitols) were quickly and easily obtained from alditol stannylene complexes and phenyl chlorothionoformate (PhOC(S)Cl) in good yields. Acetylation of isolated free alditol bis-thionocarbonates and subsequent iodination using methyl iodide under pressure led to alpha,omega-diiodo derivatives of alditols in good to excellent isolated yields (67-93%).     

A quantitative method to determine the points of O-(2-hydroxypropyl) substitution in O-(2-hydroxypropyl)-guar

A method is described for locating the O-(2-hydroxypropyl) groups in O-(2-hydroxypropyl)-substituted guar. Per-O-methylation of the O-(2-hydroxypropyl)guar yielded guar that was partially O-methylated and partially O-(2-methoxypropyl)ated. This polymer was hydrolyzed, to afford a mixture of partially O-methylated monosaccharides and partially O-(2-methoxypropylated), partially O-methylated monosaccharides. These monosaccharide derivatives were reduced, and the alditols acetylated, to give a mixture of partially O-acetylated, partially O-methylated alditols with partially O-acetylated, partially O-(2-methoxypropyl)ated, partially O-methylated alditols. These alditol derivatives were identified by gas-liquid chromatography-mass spectrometry, and quantitated by gas-liquid chromatography.     

The specific nature of plant cell wall polysaccharides

Polysaccharide compositions of cell walls were assessed by quantitative analyses of the component sugars. Cell walls were hydrolyzed in 2 n trifluoroacetic acid and the liberated sugars reduced to their respective alditols. The alditols were acetylated and the resulting alditol acetates separated by gas chromatography. Quantitative assay of the alditol acetates was accomplished by electronically integrating the detector output of the gas chromatograph. Myo-inositol, introduced into the sample prior to hydrolysis, served as an internal standard.     

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.     

Accumulation of sugar alcohols by filamentous fungi

Five hundred isolates of different xerophilic and non-xerophilic fungi belonging to 10 genera and 74 species were screened for alditol (sugar alcohol) accumulation. Ninety-two of the isolates failed to grow on a salt medium, most of the isolates (408) produced alditols; 348,44 and 16 of them produced low, moderate and high levels of alditols, respectively. The high alditol producers belonged to five species ofAspergillus, six species ofEurotium andFennellia flavipes. Glycerol andd-mannitol were the main constituents of alditol pools of the 16 high alditol producers.d-Arabinitol andmeso-erythritol were also formed but at low concentrations by several of the tested isolates.     

Separation of neutral oligosaccharide alditols from human meconium using high-pH anion-exchange chromatography

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0–500 mM sodium acetate—80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.     

Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol derivatives by electrospray tandem mass spectrometry

Maltopentaose and olive pulp xylo-oligosaccharides and the correspondent alditol derivatives were analysed by ESI-MS and ESI-MS/MS. The ESI-MS spectrum of maltopentaose and maltopentaose alditols showed [M+Na]⁺and [M+H]⁺ ions. ESI-MS spectrum of xylo-oligosaccharides and their alditols showed [M+Na]⁺of neutral (Xyl_3–6) and acidic (Xyl_2–3MeGlcA and Xyl_2–3GlcA) xylo-oligosaccharides. The ESI-MS/MS spectra of maltopentaose and underivatised xylo-oligosaccharides presented fragments of glycosidic cleavages attributed to B/Z and C/Y ions. On the other hand, MS/MS spectra of the correspondent alditols showed glycosidic cleavages unambiguously identified as B-type and Y-type ions. Y-type fragment ions showed higher abundance in the MS/MS spectra of the alditol derivatives when compared to the non-reduced samples. The study of the oligoxylosyl alditols fragmentation permits to distinguish fragmentation pathways that occur both from the reducing end and from the non-reducing end of the xylan chain, allowing to obtain more information about the localization of the acidic substituent along the glucuronoxylan backbone.     

Removal of boric acid and related compounds from solutions of carbohydrates with a boron-selective resin (IRA-743)

A boron-selective resin (IRA-743) was used to remove boric acid from synthetic solutions of carbohydrates. A commercially available polystyrene-divinylbenzene resin, which contains (covalently attached) 1-deoxy-1-methylamino-d-glucitol functional groups, absorbed > 11 mg of boric acid per mL of resin. Optimum conditions are described for use of this resin in a model system and in two practical applications: the quantitative removal of boric acid from a preparative-scale ( > 100 g) ketose-synthesis mixture and in the removal of boric acid from a solution of borohydride-reduced sugars (alditols) prior to acetylation and g.l.c. analysis.     

Analysis of derivatives of carbohydrates by high-pressure liquid chromatography

The behaviour of benzoylated derivatives of alditols, monosaccharides, disaccharides, amino sugars, and methyl glycosides in high-pressure liquid chromatography (h.p.l.c.) has been investigated. A system was devised, using the most basic equipment of a single pump and fixed-wavelength u.v. detector, which gave good separations of the components of mixtures of derivatised methyl glycosides. Fractionation of complex mixtures of many of the other benzoylated carbohydrates was achieved in less than 30 min. The 4-nitrobenzoates were less useful for routine analyses.     

Structure of tumor-associated carbohydrate antigen Ca 19-9 on human seminal-plasma glycoproteins from healthy donors

The monoclonal antibody-defined, tumor-associated antigen Ca 19-9, chemically identical with the sialylated Lewisa-carbohydrate determinant of a monoganglioside and a mucin, was demonstrated by radioimmunoassay to be present in large amounts as component of fucose-rich sialoglycoproteins, which had been extracted from human seminal plasma of healthy donors. The carbohydrate antigen of these glycoproteins (m greater than 205 kDa and m 115 kDa), which are presumably secreted by the prostatic gland, was absent in seminal plasma from blood-group-Lewis-negative men. The Ca 19-9 active sialyl-oligosaccharide was cleaved from the proteins by mild alkaline borohydride treatment and was shown to chromatograph on gradient elution from DEAE-Sephadex with the fraction of monosialylated saccharide alditols (MS-SP). The asialo derivative of the major saccharide alditol in this fraction was composed of L-fucose, D-galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosaminitol in the molar proportions 1:2:1:1 and chromatographed on Bio-Gel P2 according to approximately seven hexose units. A methylation analysis of the sialylated saccharide alditol in fraction MS-SP, which had been purified by high-pressure liquid chromatography, revealed the presence of terminal, non-reducing L-fucose, 3-O-substituted D-galactose, 3,4 di-O-substituted N-acetyl-D-glucosamine and 3-O-substituted N-acetyl-D-galactosaminitol. The presented data and the fragmentation pattern obtained on direct probe EI and FAB+ mass spectrometry of the permethylated asialo derivative are in accordance with the structure of a sialylated pentasaccharide alditol (formula; see text).     

Structure of some oligosacharides derived from rat-intestinal glycoproteins

Glycoproteins derived from intestinal mucus were isolated by phenol-water extraction of feces from germ-free rats. The water-soluble glycoproteins were subjected to alkaline-borohydride degradation, and three different oligosaccharide alditols were isolated. The structures of these were determined by using methylation analysis and n.m.r. spectroscopy as the principal methods. One disaccharide alditol and one trisaccharide alditol were characterised as β-D-GlcNAcp-(1→3)-D-GalNAcol and α-L-Fucp-(1→2)-β-D-Galp-(1→3)-D-GalNAcol. The third oligosaccharide alditol was a blood-group A-active tetrasaccharide alditol for which the structure α-D-GalNAcp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-(1→3)-D-GalNAcol is proposed.     

Structural studies on fetal mucins from human amniotic fluid. Core typing of short-chain O-linked glycans.

Mucins in human amniotic fluid are represented by two distinct molecular species, FM-1 and FM-2, with apparent molecular masses of 700 and 570 kDa, respectively, in SDS/polyacrylamide gradient gels. FM-1 and FM-2 were isolated by preparative SDS/PAGE to apparent homogeneity and subjected to structural studies on their carbohydrate portions. The carbohydrate compositions of the mucin species differed only marginally and exhibited significant amounts of mannose. O-linked core-region glycans on human amniotic mucin-derived pronase-stable glycopeptides were analyzed after reductive beta-elimination and purification on HPLC by a combination of methylation analysis, electron-impact mass spectrometry of permethylated oligosaccharide alditols and fast-atom-bombardment mass spectrometry of acetylated or methylated alditols (positive-ion mode) or alditol-derived neoglycolipids (negative-ion TLC-MS). The primary structures of major monosaccharides to tetrasaccharides have been established which exhibit at their reducing termini core 1, core 2 and core 3 sequences, as follows. [Table; see text]     

Chromatographic separation of glucose and mannose on cation-exchange resin

An analytical technique has been developed for separating mixtures of monosaccharides by partition chromatography in aqueous ethanol on columns of anion- (1) and cation- (2) exchange resins in the inorganic counterion forms. One disadvantage of the cation resin was that glucose could not be separated from mannose. This present report describes the optimum conditions for separating these sugars from each other and from other commonly occurring monosaccharides on cation-exchange resin in the potassium and sodium forms.     

Conformations of acyclic sugar derivatives : Part II. Determination of the conformations of alditol acetates in solution by the use of 250-MHz n.m.r. spectra

The p.m.r. spectra of all the fully acetylated pentitols and several fully acetylated hexitols have been analysed. Computation by iterative analysis and recourse to 250-MHz spectra were required in several cases. The vicinal coupling constants were used to determine the conformations of these compounds in solution. The planar zigzag conformation was found to be predominant only in those configurations (arabino, manno) which do not possess parallel 1,3-interactions between acetoxyl groups on alternate carbon atoms. The other compounds were found to be mixtures of two or more conformers, none of which has the planar zigzag conformation, except in the case of hexa-O-acetylallitol. The conformations of alditols in the crystalline state and of the alditol acetates in solution are compared.     

Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. (c) 1993 John Wiley & Sons, Inc.