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.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography

Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.     

An automatic analyzer for the specific determination of amino sugars

The techniques previously employed for the extraction and determination of amino acids from different matrices are not necessarily optimal for the determination of the amino sugars. An analytical system is described which is a hybrid between the conventional amino acid analyzer and the liquid chromatographic system for the detection of reducing sugars. The major, naturally occurring amino sugars are separated in about 40 min, with sensitivites lying under the nanomole range, without interference from other co-extracted compounds such as amino acids and sugars. The reagent employed is noncorrosive and stable over long periods of time. The amino sugar analyzer can be readily constructed by simple modification of a conventional phenylketonuria or amino acid analyzer.     

Capillary electrophoresis for the determination of major amino acids and sugars in foliage: application to the nitrogen nutrition of Sclerophyllous species

Amino acids and sugars are probably the most commonly measured solutes in plant fluids and tissue extracts. Chromatographic techniques used for the measurement of such solutes require complex derivatization procedures, analysis times are long and separate analyses are required for sugars and amino acids. Two methods were developed for the analysis of underivatized sugars and amino acids by capillary electrophoresis (CE). Separation of a range of sugars and amino acids was achieved in under 30 min, with good reproducibility and linearity. In general, there was close agreement between amino acid analyses by CE and HPLC with post-column derivatization. An alternative, more rapid method was optimized for the common neutral sugars. Separation of a mixture of fructose, glucose, sucrose, and fucose (internal standard) was achieved in less than 5 min. How the source of N applied (nitrate or ammonium) and its concentration (8.0 or 0.5 mM) affects the amino acid and sugar composition of leaves from Banksia grandis Willd. and Hakea prostrata R. Br. was investigated. The amino acid pool of Banksia and Hakea were dominated by seven amino acids (aspartic acid, glutamic acid, asparagine, glutamine, serine, proline, and arginine). Of these, asparagaine and glutamine dominated at low N-supply, whereas at high N-supply the concentration of arginine increased and dominated amino-N. Plants grown with nitrate had a greater concentration of proline relative to plants with ammonium. In Banksia the concentration of amides was greatest and arginine least with a nitrate N-source, whereas in Hakea amides were least and arginine greatest with nitrate N-source. The concentration of sugars was greater in Banksia than Hakea and in both species at greater N-supply.     

Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.     

Gas chromatography of neutral and amino sugars in glycoproteins

A gas chromatographic method for determining the neutral and amino sugars commonly found in glycoproteins of animal origin is described. Following mild acid hydrolysis, the solution is neutralized with Dowex 1 HCO₃⁻, and the sugars are reduced to alditols in the cold with sodium borohydride. The solution is lyophilized, and the alditols are acetylated with acetic anhydride in pyridine. Monosaccharides can be determined on as little as 1 mg of a glycoprotein which contains 6% total carbohydrate.     

Analysis of neutral sugars as glycamines using an amino acid analyzer.

A new method of the analysis of neutral sugars was developed based on the separation of their corresponding glycamines. Sugars and ammonia were combined by means of the reduction with sodlum cyanoborohydride. The glycamines thus obtained were quantitatively analyzed by an automatic amino acid analyzer. Satisfactery results were obtained in the analyses of the constituent sugars of several polysaccharides and glycoconjugates.     

Quantitative analysis of total membrane-bound sugars and amino sugars as alditol acetates by combined thin-layer chromatography,gas-liquid chromatography,and radiogas chromatography

A sensitive method (0.4 μg of hexoses routinely detectable) for quantitative determinations of sugars and amino sugars in biological material, particularly in membranes, is described. The method consists of a combination of thin-layer chromatography (tlc), gas-liquid chromatography (glc), and radiogas chromatography (rgc), using a highly thermostable phase (Silar 10 c) for the analysis of the specific alditol acetates. In this method, the losses incurred during hydrolysis and preparation for glc are assessed by comparison with the specific recoveries of added radiolabeled internal standards.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Turnover of the amino sugars of erythrocyte and thrombocyte glycoproteins in rats maintained on different carbohydrate diets

The turnover of erythrocyte and platelet glycoprotein amino sugars in rats fed carbohydrates has been studied. Partial replacement of starch by sucrose results in an almost 3-fold increase in the half-life of erythrocyte glycoprotein amino sugars and in a 2-fold increase in that of platelets as early as 14 days after keeping the animals on carbohydrates. It is concluded that study of dynamic parameters of cell amino sugars can be used for evaluation of the role played by food in metabolic and adaptive reactions both in experimental animals and man.     

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gin, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

外源无机氮素形态对土壤氨基糖动态的影响

微生物生长对底物的可利用性存在不同的响应,外源氮素的形态可以显著影响微生物代谢过程,而土壤氨基糖作为微生物细胞壁残留物,其形成、分解和周转特征与外源碳氮供给密切相关,对土壤氨基糖的研究与同位素标记技术相结合,可以进一步反映微生物对底物的利用特征.本文以葡萄糖及15N标记的NH4+和NO3-为底物,利用气相色谱-质谱联机技术,通过测定氨基糖中同位素富集比例,跟踪新形成(标记)和原有(非标记)的土壤氨基糖的动态变化.结果表明:在培养过程中,15N标记的氨基糖含量显著增加,NH4+向氨基糖的转化显著高于NO3-,反映出微生物对NH4+的选择性利用.土壤中原有的氨基糖也发生了不同变化.其中,非标记氨基葡萄糖在N H4+为底物时,其含量有所增加,但在NO3-为底物时含量逐渐下降;非标记胞壁酸含量在2个处理中均不断下降,尤其以NO3-为底物时更为显著;非标记氨基半乳糖含量的增减幅度均小于20％.这种特异性变化表明,不同来源的微生物细胞壁残留物对土壤氮素周转和稳定的作用不同,真菌细胞壁残留物易于在土壤中积累,有利于土壤有机质的稳定,而细菌细胞壁残留物容易分解,在土壤有机质周转过程中起重要作用.     

Differential microbial uptake of dissolved amino acids and amino sugars in surface waters of the Atlantic Ocean

Nitrogen bioavailability is considered to limit the productivityof oceanic oligotrophic gyres, the largest biomes on Earth.In order to assess the microbial requirement for small organicnitrogen molecules in these and other waters, the microbialuptake rates of amino acids (leucine, methionine and tyrosine)and amino sugars (glucosamine and N-acetyl-glucosamine) as wellas glucose were compared using a bioassay technique of radiotracerdilution. The bioassays were carried out on four mid-Atlanticmeridional transects spanning a latitudinal range from 60°Nto 42°S. The mean concentrations of both bioavailable N-acetyl-glucosamineand glucose in the gyres were 1 nM, four times higher than themean leucine concentration. Despite its lower concentration,the mean turnover time of leucine in the gyres of 15 h was 90and 9 times shorter than the turnover time of N-acetyl-glucosamineand glucose, respectively. In addition, among amino acids, leucinewas taken up in the gyres at a rate of 1.5 times faster thanmethionine and 2.5 times faster than tyrosine. Hence, oceanicbacterioplankton as a community showed a clear preference foramino acids, particularly leucine, compared with amino sugars.The preferential uptake of amino acids to sugars challengesthe concept of microbial nitrogen or carbon limitation in theopen ocean.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Phloem loading and unloading of sugars and amino acids

In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino nitrogen (N) compounds in a bulk flow of solution, is propelled though the phloem by osmotically generated hydrostatic pressure differences between source (net nutrient export) and sink (net nutrient import) ends of phloem paths. Source loading and sink unloading of sugars, amino N compounds and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. A symplasmic component is characteristic of most loading and unloading pathways which, in some circumstances, may be interrupted by an apoplasmic step. Raffinose series sugars appear to be loaded symplasmically. However, sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes of their sieve element/companion cell (se/cc) complexes. Sucrose transporters, with complementary kinetic properties, are conceived to function as membrane transporter complexes that respond to alterations in source/sink balance. In contrast, symplasmic unloading is common for many sink types. Intervention of an apoplasmic step, distal from importing phloem, is reserved for special situations. Effluxers that release sucrose and amino acids to the surrounding apoplasm in phloem loading and unloading are yet to be cloned. The physiological behaviour of effluxers is consistent with facilitated membrane transport that can be energy coupled. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered along with mechanisms regulating symplasmic transport. The latter is hypothesized to exert significant control over phloem unloading and, in some circumstances, phloem loading.     

A transfer of carbon atoms from fatty acids to sugars and amino acids in yellow lupine (Lupinus luteus L.) seedlings

The metabolism of 14C-acetate was investigated during the in vitro germination of yellow lupine seeds. Carbon atoms (14C) from the C-2 position of acetate were incorporated mainly into amino acids: aspartate, glutamate, and glutamine and into sugars: glucose, sucrose, and fructose. In contrast to this, 14C from the C-1 position of acetate was released mainly as 14CO2. Incorporation of 1-14C and 2-14C from acetate into amino acids and sugars in seedling axes was more intense when sucrose was added to the medium. However, in cotyledons where lipids are converted to carbohydrates, this process was inhibited by exogenous sucrose. Since acetate is the product of fatty acid beta-oxidation, our results indicate that, at least in lupine, seed storage lipids can be converted not only to sucrose, but mainly to amino acids. Inhibitory effects of sucrose on the incorporation of 14C from acetate into amino acids and sugars in cotyledons of lupine seedlings may be explained as the effect of regulation of the glyoxylate cycle by sugars.     

Faster accumulation and greater contribution of glomalin to the soil organic carbon pool than amino sugars do under tropical coastal forest restoration

Microbial metabolic products play a vital role in maintaining ecosystem multifunctionality, such as soil physical structure and soil organic carbon (SOC) preservation. Afforestation is an effective strategy to restore degraded land. Glomalin-related soil proteins (GRSP) and amino sugars are regarded as stable microbial-derived C, and their distribution within soil aggregates affects soil structure stability and SOC sequestration. However, the information about how afforestation affects the microbial contribution to SOC pools within aggregates is poorly understood. We assessed the accumulation and contribution of GRSP and amino sugars within soil aggregates along a restoration chronosequence (Bare land, Eucalyptus exserta plantation, native species mixed forest, and native forest) in tropical coastal terraces. Amino sugars and GRSP concentrations increased, whereas their contributions to the SOC pool decreased along the restoration chronosequence. Although microaggregates harbored greater microbial abundances, amino sugars and GRSP concentrations were not significantly affected by aggregate sizes. Interestingly, the contributions of amino sugars and GRSP to SOC pools decreased with decreasing aggregate size which might be associated with increased accumulation of plant-derived C. However, the relative change rate of GRSP was consistently greater in all restoration chronosequences than that of amino sugars. The accumulation of GRSP and amino sugars in SOC pools was closely associated with the dynamics of soil fertility and the microbial community. Our findings suggest that GRSP accumulates faster and contributes more to SOC pools during restoration than amino sugars did which was greatly affected by aggregate sizes. Afforestation substantially enhanced soil quality with native forest comprising species sequestering more SOC than the monoculture plantation did. Such information is invaluable for improving our mechanistic understanding of microbial control over SOC preservation during degraded ecosystem restoration. Our findings also show that plantations using arbuscular mycorrhizal plants can be an effective practice to sequester more soil carbon during restoration.