High Molecular Weight Carbohydrates Research Articles

Application of Aqueous Gel Permeation Chromatography with In-Line Multi-Angle Laser Light Scattering and Differential Viscometry Detectors for the Characterization of Natural Product Carbohydrate Pharmaceuticals

Abstract Natural product complex carbohydrate polymers are currently being developed as prophylactic and/or therapeutic drugs. These water-soluble carbohydrate pharmaceuticals, which are primarily β-1, 3-D-glucan polymers, belong to the class of drugs known as biological response modifiers (BRMs). A major obstacle to the development, understanding and clinical utilization of carbohydrate BRMs has been the difficulty involved in accurately characterizing high molecular weight (MW) carbohydrate polymers. Recent advances in aqueous gel permeation chromatography (GPC), differential viscometry (DV) and multi- significance of these observations are readily apparent. As stated above, the characterization and quality control of natural product carbohydrate BRMs has, in the past, relied heavily on bioassays and conventional GPC. The data presented demonstrate the applicability of high performance GPC with multiple detectors in the analysis of complex carbohydrate BRMs. This technology when combined with other analytical techniques such as high-field 13C and 1H-NMR, elemental analysis, helix coil transition analysis and exhaustive methylation/gas chromatography/mass spectrometry (12) provide a much more complete description of this novel class of pharmaceuticals.

Analysis of sucrose fatty acid esters by high temperature gas chromatography

A commercially available high temperature GC system has been investigated to determine its suitability for the automated analysis of sucrose fatty acid esters. In comparison with conventional GC, the high temperature variety broadens the scope of the technique to encompass high molecular weight carbohydrate derivatives while maintaining high separation efficiency. Despite the broad range of polarities, simultaneous quantification of educts and sucrose fatty acid ester fractions is possible in one run, after derivatization. Chromatographic requirements, sample preparation, structure verification by direct chemical ionization mass spectrometry, and advantages and limitations of the high temperature GC-analysis are discussed.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.

Acute iron poisoning. Rescue with macromolecular chelators.

Acute iron intoxication is a frequent, sometimes life-threatening, form of poisoning. Present therapy, in severe cases, includes oral and intravenous administration of the potent iron chelator, deferoxamine. Unfortunately, high dose intravenous deferoxamine causes acute hypotension additive with that engendered by the iron poisoning itself. To obviate this problem, we have covalently attached deferoxamine to high molecular weight carbohydrates such as dextran and hydroxyethyl starch. These macromolecular forms of deferoxamine do not cause detectable decreases in blood pressure of experimental animals, even when administered intravenously in very large doses, and persist in circulation much longer than the free drug. These novel iron-chelating substances, but not deferoxamine itself, will prevent mortality from otherwise lethal doses of iron administered to mice either orally or intraperitoneally. Further reflecting this enhanced therapeutic efficacy, the high molecular weight iron chelators also abrogate iron-mediated hepatotoxicity, suppressing the release of alanine aminotransferase. We conclude that high molecular weight derivatives of deferoxamine hold promise for the effective therapy of acute iron intoxication and may also be useful in other clinical circumstances in which control of free, reactive iron is therapeutically desirable.

Carbohydrates and the pharmaceutical industry

Abstract

Isolation and partial characterization of a novel keratan sulfate proteoglycan from metamorphosing bonefish (Albula) larvae

Proteoglycans (PGs) were isolated from leptocephalous larvae of the bonefish (Albula sp.), which were in the early stages of metamorphosis, using both associative and dissociative conditions in the presence of protease inhibitors. The procedure was rapid and resulted in an extraction efficiency of 75% (associative) and 85-90% (dissociative). The majority of co-extracted protein could be effectively separated from the PGs by utilizing either Sepharose CL-2B or CL-6B gel chromatography. Sepharose CL-2B chromatography of extracted PGs after treatment with bacterial keratan sulfate-endo-β-galactosidase (keratanase) showed that most of the high molecular weight (M r) carbohydrate was degraded. Free keratan sulfate (KS) chains were prepared from whole-larva extracts (which also contain small amounts of chondroitin sulfate) by both chondroitinase ABC treatment and ethanol fractionation. Sepharose CL-6B chromatography under dissociative conditions showed that larval KS chains were much larger (M r∼55,000) than those from cornea. These chains tended to aggregate when chromatographed under associative conditions. Larval KS was degraded by keratanase and resistant to chondroitinase, ABC and testicular hyaluronidase. Differences were also noted in the oligosaccharides produced by keratanase treatment of the two preparations. However, biochemical composition of larval and corneal KS was similar.

Carbon-13 nuclear magnetic resonance spectroscopy of some edible mushrooms.

In this paper, carbon-13 nuclear magnetic resonance (13C-NMR) spectra of some edible mushrooms, Grifola frondosa, Lentinus edodes, Flammulina velutipes, Pholiota nameko, Agaricus bisporus, Lyophyllum cinerascens, Lyophyllum aggregatum, and Peziza vesiculosa, as aqueous suspensions were measured and the glucan compositions in these mushrooms were compared. The 13C-NMR spectrum of each native mushroom gave signals attributable to both low and high molecular weight carbohydrates. Low molecular weight carbohydrates were different in each mushroom. As high moelcular weight carbohydrates, all the mushrooms belonging to Basidiomycotina (G. frondosa, L. edodes, F. velutipes, P. nameko, A. bisporus, L. cinerascens, and L. aggregatum) showed β-linked glucan signals. G. frondosa, and P. nameko also showed α-glucan signals. After removal of low molecular weight substances and α-glucans by refluxing with 80% ethanol, dialysis or amylase digestion, all of the spectra were quite similar and showed representative signals at 104, 85, 77, 76, 74, 70, 69, 62 ppm. On the other hand, though P. vesiculosa, a fungus belonging to Ascomycotina, contains antitumor β-glucans, this mushroom showed strong signals attributable to α-glucans and only weak signals attributable to β-glucans. After treatment of G. frondosa with periodate and borohybride, most of the β-glucan signals disappeared. These results suggest that (1) 13C-NMR spectroscopy is applicable to chemotaxonomical examination of fungi, (2) all of the mushrooms belonging to Basidiomycotina contain quite similar glucans, (3) β-signals in these spectra are attributable to periodate-sensitive linkages.

Mineral Complexing Properties of Food Carbohydrates

AbstractThe complexing of minerals by both low and high molecular weight carbohydrates is now well established and can be demonstrated by, for example, depression of conductivity, alteration of specific rotation, redox potential or techniques such as ligand exchange chroma‐tography. It is likely that mineral complexes will exist in food systems and, in mixtures of sugars such as glucose syrups, a variety of complexes will be present. Recent interest in the hydrogenated carbohydrates shows that these too complex minerals, sometimes better than their unhydrogenated counterparts. The complexing of Cu2+ by hydrogenated glucose syrups, for example, increases as degree of polymerisation decreases and iron salts are also complexed by various simple carbohydrate molecules. Mineral complexing has many consequences for the taste, nutritional value and technological quality of foods. The taste of iron salts is masked for example, by complexing with carbohydrates at high pH, whereas the opposite occurs at low pH. Vitamin C is protected from Cu2+ catalyzed oxidation when the cation is complexed with various carbohydrates or their hydrogenated derivatives. Such results have implications for the design, formulation and storage of food.

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.

Changes in the composition and carbohydrate constituents of okra ( Abelmoschus esculentus, Linn.) with age

Changes which occurred in the proximate composition, concentrations of free sugars, high molecular weight carbohydrates (water-soluble polysaccharides, starch hemicellulose and cellulose) and lignin in four varieties of okra when harvested at different ages have been studied. Samples were collected from the field at 1, 4, 7 and 10 days after flowering. Crude protein, which initially ranged from 19·9 to 24·7 g/100 g DM, decreased with age whilst crude fibre, 12·5–16·7 g/100 g DM, increased with age. Ether extract varied between 1·60 and 2·19 g/100 g DM whilst ash was 8·00–8·59 g/100 g DM. Available carbohydrates (starch + sugars) ranged from 11·2 to 13·1 g/100 g DM whilst unavailable carbohydrates increased from 33·9–42·0 g/100 g DM to 51·9–59·7 g/100 g DM. Overall varietal differences were apparent for ethanol-soluble sugars and the structural carbohydrates while differences among the means due to age were significant ( P < 0·05).

VARIATION IN THE BIOCHEMICAL COMPOSITION OF ACID EXTRACTS FROM BARLEYS OF CONTRASTING MALTING QUALITY

Acid extracts from barleys of poor malting quality were characterized by high viscosity caused by enhanced concentrations of high molecular weight carbohydrates of which more than 80 per cent was β-glucan. Up to 67 per cent of the β-glucan in these barleys was soluble in the acid extracting solution. In contrast, only about 25 per cent of the β-glucan in good malting barleys was soluble, resulting in extracts having low viscosity. No evidence was found for autolysis in any of the extracts, and extracts from good malting varieties were characterized by high concentrations of fructose and sucrose. Protein concentration in the extracts varied little, although the protein content of the barleys ranged from 9·1 per cent to 15·4 per cent. Soluble protein has no effect on extract viscosity or the gel filtration properties of the carbohydrates. Some quantitative variation in acid-soluble and salt-soluble proteins was seen after electrophoresis on polyacrylamide.

Breakdown of mucin and plant polysaccharides in the human colon

To obtain an estimate of the extent to which complex carbohydrates are degraded by bacteria in the human colon, aqueous extracts of colon contents from four human subjects were separated into high and low molecular weight fractions by chromatography on a Sephadex G-100 column. The composition of these fractions was compared with the composition of similar fractions from ileal contents, i.e., from material entering the colon. In all four subjects, high molecular weight carbohydrate concentrations were lower in the colon than in the ileum, indicating that breakdown of complex carbohydrate occurs in the colon. The high molecular weight carbohydrate fraction contained sugars characteristic of plant polysaccharides (arabinose, xylose, mannose, rhamnose) as well as sugars characteristic of mucin (fucose, hexosamines, sialic acids). Concentrations of most of these sugars were uniformly lower in the colon than in the ileum. Since high molecular weight protein concentrations were lower in the colon than in the ileum of two of the four subjects tested, some degradation of protein may also occur in the colon.

Chemical analysis of the phenol-water-extractable materials from Anabaena flos-aquae

The high molecular-weight carbohydrate substances extracted in the aqueous and phenol phases by phenol-water extraction of Anabaena flos-aquae A-37 were found to be polysaccharides without lipid attached.

Production and control of extracellular enzymes in Micrococcus sodonensis.

Micrococcus sodonensis has been shown to produce several extracellular enzymes: an alkaline phosphatase, at least two forms of phosphodiesterase, a 5′-nucleotidase, and an alkaline proteinase. The quantitative release of these enzymes into the culture medium during logarithmic growth under all the various culture conditions tested indicates that these enzymes are truly extracellular in nature. Inorganic phosphate repressed the production of the alkaline phosphatase in synthetic as well as in complex media, whereas, the repression of the production of active diesterase and 5′-nucleotidase by inorganic phosphate was partly reversed by the addition of supplemental organic nutrients to the culture medium. Proteinase production was independent of the culture conditions used. A mutant strain of M. sodonensis with an altered production of diesterase was obtained; the other extracellular enzymes were unaffected. These results suggest that the extracellular enzymes of M. sodonensis are not produced in a pleiotropic fashion since the level of one of the enzymes can be changed without affecting a corresponding change in the levels of the other enzymes. An extracellular high molecular weight carbohydrate fraction was shown to be produced by M. sodonensis in synthetic medium. The fraction was also shown to contain glycoprotein.

A study of the breakdown of carbohydrate by redox-generated free radicals.

Abstract Extensive autoxidative degradation of low and high molecular weight carbohydrates was achieved using an electrolysis cell and iron-EDTA as reductant at neutral pH. As ascertained by chemical methods this free-radical induced degradation of sugars was in each case of the order of 20% after a period of 10 h. The method should be useful for studying the nature of breakdown products derived from the hydroxyl free-radical initiated decomposition of sugars.

ACTIVATION OF GLYCOPROTEIN BIOSYNTHESIS BY TESTESTERONE PROPIONATE ON MOUSE EXORBITAL GLANDS.

The authors have studied the effects of testosterone propionate on the lacrimal gland of the mouse. Repeated injections of the hormone have determined some structural and functional modifications of the gland. The former were characterized by cytoplasmic vacuoles; the latter by an increase of glycoprotein material. The androgen used seemed to stimulate the synthesis of high molecular weight carbohydrates in a tissue that per se is mainly protein secreting.

PATTERNS OF OXIDATIVE ASSIMILATION IN STRAINS OF PSEUDOMONAS AND ACHROMOBACTER.

Tomlinson, Geraldine A. (University of British Columbia, Vancouver, B.C., Canada) and J. J. R. Campbell. Patterns of oxidative assimilation in strains of Pseudomonas and Achromobacter. J. Bacteriol. 86:434-444. 1963.-Oxidative assimilation of glucose-U-C(14) in the absence of added nitrogen was studied by use of washed-cell suspensions of Pseudomonas aeruginosa, P. fluorescens, Achromobacter strain B81, and Achromobacter viscosus (Alcaligenes viscolactis). The suggestion that oxidative assimilation in these organisms is the reincorporation of endogenously produced ammonia by way of alpha-ketoglutarate is tenable. Each of the four organisms accumulated intermediate compounds which acted as pacemakers for the oxidation of glucose. This phenomenon, partly because it ensured the availability of additional ammonia, undoubtedly increased the degree of oxidative assimilation. Products accumulating in the supernatant fluids during glucose oxidation were alpha-ketoglutarate, pyruvate, gluconate, a low molecular weight carbohydrate, and dicarboxylic acids. No two bacteria formed the same products. Assimilation of radioactivity into the cells, which accounted for 12 to 26% of the available C(14), continued as long as an oxidizable substrate was present, and was paralleled by uptake of endogenously produced ammonia. During the early stages of glucose oxidation, compounds of the cold trichloroacetic acid-soluble pool constituted a major portion of the total radioactivity of the cells. The lipid fractions of P. aeruginosa and Achromobacter B81 were also of high relative activity during this time. The labeling of the nucleic acid fractions of all four bacteria increased with time, more radioactivity being found in fractions from the two Achromobacter species than in those from the pseudomonads. At the completion of the experiment, the largest percentage of incorporated radioactivity was present in the protein fractions. One of the organisms, Achromobacter B81, synthesized a high molecular weight carbohydrate material.