Carbohydrate Mixtures Research Articles

Separation of mixtures of carbohydrates by a supported liquid membrane containing methyl cholate as carrier

Separation of mixtures of carbohydrates by a supported liquid membrane containing methyl cholate as carrier

Cap and Capture−Release Techniques Applied to Solid-Phase Synthesis of Oligosaccharides

This paper reports a new strategy for oligosaccharide synthesis by combining solid-phase methods with cap and capture-release separation techniques, using the p-(5-(ethoxycarbonyl)pentyloxy)benzyl group (CPB) as a tag for the capture of desired oligosaccharides. After a complex carbohydrate mixture was obtained by solid-phase synthesis, the desired oligosaccharide containing a free carboxyl group derived from CPB was attached to an amino resin. The loaded resin was readily separated from side products by filtration and finally treated with acid to release the pure oligosaccharide product.

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed alpha-galactosidase, beta-galactosidase, and beta-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.

Measuring rates of biochemical flux in mice following a modified glucose tolerance test.

Insulin is an important regulator of glucose homeostasis, e.g. during a glucose challenge insulin stimulates glucose storage in the liver and the skeletal muscle. Since insulin acts on other processes, it is reasonable to expect that the syntheses of lipids and proteins are also stimulated. In cases of “insulin resistance” and Type 2 diabetes, impairments in insulin secretion/action lead to aberrant metabolic regulation. Understanding the extent to which specific pathways are affected by disease affect therapeutic options. We hypothesized that one could determine the rates of synthesis of (i) liver and muscle glycogen, (ii) fatty acids and cholesterol and (iii) proteins by administering 2H2O prior to a modified glucose tolerance test. Healthy C57BL/6J mice were fasted overnight prior to the study. On the morning of the study, mice were injected with 2H2O, randomized to receive an injection of saline (“fasted”) or a mixture of carbohydrate and essential amino acids (“fed”) and killed 3.5 h later. As compared to “fasted” mice, “fed” mice had an increase in the rates of syntheses of total liver glycogen, fatty acids, cholesterol and protein. Although further development and testing are needed, our approach may be useful in determining the metabolic response of carbohydrate, lipid and protein metabolism to a controlled challenge. Application of this method may help to resolve specific impairments in insulin action.

Copper-coated microsprayer interface for on-line sheathless capillary electrophoresis electrospray mass spectrometry of carbohydrates

A sturdy home-built sheathless CE/ESI-QTOF-MS system was developed and optimized for carbohydrate analysis. The interface and employed methodology provided a simple analytical solution to laborious CE/MS interfacing methods and to problems in characterization of complex carbohydrate mixtures that require high-resolution separation of the components. The CE/ESI interface, feasible in any MS laboratory, consists of a one-piece CE column having the CE terminus in-laboratory shaped as a microsprayer and coated with copper. The CE microsprayer was inserted into an in-house made stainless steel clenching device and the whole assembly was mounted onto a quadrupole TOF mass spectrometer. The analytical potential of the interface in terms of suitability, microsprayer performance, copper coat durability, ionization efficiency, spray stability, and sensitivity was tested first on a simple mixture of standard saccharides, which were separated, resolved, and detected with high separation efficiency. The approach was next assessed for the screening of a biological sample, a complex mixture of O-glycosylated sialylated amino acids from urine of a patient suffering from Schindler disease. Preliminary data allow this method to be considered as one of general applicability in structural glycobiology and glycomics and easy to be implemented for proteomic surveys as well.

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Arabino-oligosaccharides can be considered as potential prebiotics. Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60–360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of α1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower p I values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher p I values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

Variable hydration of small carbohydrates for predicting equilibrium properties in diluted and concentrated solutions

A physical–chemical model is proposed to describe the equilibrium properties of binary and multicomponent water–carbohydrate mixtures. The chemical part of the model accounts for the hydration equilibrium of carbohydrate with the formation of carbohydrate n-water molecules in a single stage process; n, the hydration number, and K, the hydration equilibrium constant, are the two independent parameters in this part. The physical part of the model is the UNIFAC group-contribution model as modified by Larsen et al. (I.E.C. Research, 26 (1987) 2274–2286). The original functional groups and the structural and interaction parameter values are maintained and introducing new groups such as pyranose rings, furanose rings and osidic bonds was not necessary. The model is compared to experimental data including water activity, osmotic coefficients, activity coefficients, freezing and boiling point temperatures and solubility for binary systems containing xylose, glucose, mannose, galactose, fructose, sucrose, maltose, lactose and trehalose and for food products containing sugars.

Interplay between the Maillard Reaction and Lipid Peroxidation in Biochemical Systems

The Maillard reaction and lipid peroxidation are two of the most important chemical reactions that take place in biochemical systems. Both include a whole network of different reactions in which an extraordinarily complex mixture of compounds is produced in very different amounts, with both positive and negative consequences. In addition, both reactions are intimately interrelated, and the products of each reaction influence the other. Furthermore, there are common intermediates and products in both pathways; these products are usually known as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Moreover, other AGE/ALEs are analogous and participate similarly in both amino acid degradation and amino phospholipid/protein polymerization by identical mechanisms. All these data suggest that the Maillard reaction and lipid peroxidation are so closely interrelated that both reactions should be considered simultaneously to understand the reaction mechanisms, kinetics, and products in the complex mixtures of carbohydrates, lipids, and proteins occurring in biochemical systems. In these systems, lipids and carbohydrates are competing in the chemical modification of amino phospholipids and proteins. Therefore, although there are significant differences between the Maillard reaction and lipid peroxidation, many aspects of both reactions can be better understood if they are included in only one general carbonyl pathway that can be initiated by both lipids and carbohydrates.

Fast electrophoresis in conventional capillaries by employing a rapid injection device and contactless conductivity detection

A purpose-made set-up featuring an automated fast injector allowed the easy optimization of the injected amount and the adjustment of the separation length of conventional capillaries from a minimum of 5 cm upward. It was found that a compromise in capillary length for separation efficiency and analysis time also has to take into account the injected amount, which in turn affects the sensitivity and hence the detection limit. The versatility of the system was demonstrated by the analysis of the major cations and anions in natural water samples in less than 1 min, the concurrent determination of a mixture of amino acids and carbohydrates in 160 s, and of three active ingredients in a pharmaceutical preparation in 40 s. Plate numbers were typically around 50,000 and detection limits down to 1 muM could be achieved.

Low-melting sugar–urea–salt mixtures as solvents for Diels–Alder reactions

Sweet solutions are obtained upon heating mixtures of simple carbohydrates, urea and inorganic salts to moderate temperatures, to give new chiral media for organic reactions.

Preoperative supplementation with a carbohydrate mixture decreases organ dysfunction-associated risk factors

Recently, both asymmetrical dimethylarginine and IL-6 have been suggested to be associated with the induction and severity of single and multiple organ dysfunction. The aims of the present study were to elucidate if these factors were increased in an ischemia reperfusion (IR) model and whether pre-operative carbohydrate supplementation can reduce the risk factors along with the IR injury. One group of male Wistar rats was fasted for 16 h (water ad libitum) prior to clamping the superior mesenteric artery (IR fasted n=14). A second group had ad libitum access to a carbohydrate solution prior to clamping (IR fasted CHO group n=11). Sham-fasted animals, which only received laparotomy and no clamping, served as controls (n=4). Plasma urea and ALAT activity were both increased in the IR fasted animals when compared to the sham rats (P=0.007 and P<0.02, respectively). Furthermore, it was shown that IR fasted rats had increased ADMA and IL-6 concentration in plasma when compared to sham animals (P<0.02). Moreover, the GSH level in lung was significantly decreased in the IR fasted animals (P=0.014). IR CHO supplemented showed no significant increase of ALAT activity and decrease of lung GSH. Furthermore, significantly lower plasma urea, ADMA and IL-6 concentration was seen in the IR CHO supplemented group when compared to the IR fasted rats (P=0.028, P<0.01 and P<0.02, respectively). The liver glycogen concentration in IR fasted rats was 48% of that IR rats supplemented the carbohydrate mixture. The present rat intestinal ischemia reperfusion model not only induces organ injury indicated by the classical parameters such as plasma urea and ALAT activity, but also increased plasma IL-6 and ADMA and decreased lung GSH concentration in IR fasted rats. Pre-operative supplementation with the carbohydrate mixture significantly lowered the plasma urea, IL-6 and ADMA concentrations and maintained lung GSH concentration. This indicates that pre-operative carbohydrate supplementation reduces post-operative organ injury.

Off‐line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time‐of‐flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

AbstractImplementation and optimization of an off‐line capillary electrophoresis (CE)/(−)nanoESIchip‐quadrupole time‐of‐flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O‐glycosylated peptides from urine of a patient suffering from α‐N‐acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off‐line CE/MS analysis, could be positively addressed by the off‐line conjunction of CE with automated chip‐based ESI‐QTOF‐MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Effect of Water and Physical State on Near- and Mid-Infrared Partial Least Squares Calibrations for Multi-Component Carbohydrate Mixtures

The objective was to determine the effects of water and physical state on near- and mid-infrared calibrations for carbohydrate mixtures. Seventy-five mixtures of cellulose, starch, pectin, locust gum, glucose and xylitol were scanned in the near- and mid-infrared in three physical states (wet, simple mixtures of dry constituents and mixtures dried together). These final samples (mixtures dried together) were prepared by drying wet mixtures/solutions while under constant stirring to prevent separation of the insoluble polymers by settling, which occurs during normal oven drying. Final drying of the resulting solid mass was carried out in a vacuum oven at 60°C. The dried mass was then ground and scanned in the spectrometer. All dry samples were ground to a flour-like consistency using a A-10 Tekmar Analytical Mill to avoid effects due to gross differences in particle size. The wet samples were produced by adding sufficient water to result in samples of a putty like consistency. Spectra were taken on a Digilab FTS-65 Fourier transform spectrometer equipped with near- and mid-infrared benches by diffuse reflectance. Results demonstrated that the effects of water and physical state (matrix effects), seen previously with simple model systems, can be extrapolated to draw some general conclusions about real world calibrations, but that developing model systems to either predict the feasibility of developing a calibration or to determine the reason for the specific results of a calibration must be done with extreme care. For example, using simple mixtures of components to determine whether a calibration for the same components in a different physical condition (melted together, dried together etc.) might well lead to erroneous conclusions about its usefulness. Also, from the results, it would appear that factors (e.g. physical state) other than the mere presence of large amounts on water in samples can account for the decrease in calibration accuracy often seen in samples with high moisture contents as compared to their dried counterparts. Finally, the results seen using mid-infrared spectra indicate that mid-infrared diffuse reflectance spectroscopy might be feasible even with samples containing high percentages of water.

O0070 IN-VITRO BIOAVAILABILITY OF GLUCOSE FROM INFANT CEREALS

Introduction: Dietary carbohydrates are digested and absorbed at different rates and amounts depending on the food sources and their origins. Rapidly and highly elevated blood glucose levels postprandially may be detrimental to health (type 2 diabetes, obesity). Diets rich in slowly digested carbohydrates may protect against chronic diseases. Altering the quality of carbohydrates in diets of young children may exert important health implications. This study determines the quantity of glucose released from carbohydrate mixtures, expressed as the available glucose portion (AG). Distinction is made between rapidly (RAG) and slowly available glucose portions (SAG). Methods: The in-vitro dynamic-digestive-dialysis-model (1) was adapted to measure glucose availability from cereals. 9.5 g of carbohydrate mixture was added to 100 ml milk. Sample A contains rice flour (31.7%), tapioca (31.1%), sucrose and maize starch (13.5%). Sample B is composed of rice flour (37.5%), maltodextrose, tapioca (28.8%) and maize starch, no sucrose is added. Sample C is made of wheat flour (95.2 %), rice flour (1.1 %), rye flour, maize starch (1.1%) and sucrose. Samples were treated to determine free soluble glucose (FSG = free Glc + Glc from sucrose) and total glucose (TG). Glucose concentrations were analysed spectrophotometrically. Total starch (TS) was calculated by multiplying TG by factor 0.9. Results: The method yields CVs of 0.73 % intra-assay and 16.5 % inter-assay for measurement of the AG. Sample A contains high FSG (148 ±3 mg/g), which corresponds to the high RAG fraction (34.8 ±7.5 mg/g). SAG and total AG are 94.9 mg/g (±15.3) and 130 mg/g (±17). TG and TS are 0.86 g/g (±0.06) and 0.64 g/g (±0.0). Sample B contains low FSG (4.9 ±1.2 mg/g) and low RAG (15.9 ± 4.8mg/g) due to the lack of added sugars. SAG is relatively high (80.6 ±16.3 mg/g) compared to the total amount of AG (96.5 ±19.6 mg/g). TG and TS are 1.00 g/g (±0.03) and 0.9 g/g (±0.00). FSG of sample C is 65.8 mg/g (± 0.3) with relatively low RAG (15.6 ± 3.7 mg/g). SAG is also low (45.7 ± 12.6 mg/g) due to the relatively low total AG of the high cereal mixture (61.2 ± 13.0 mg/g). TG and TS are 0.81 g/g (±0.07) and 0.67 g/g (± 0.00). Conclusion: Carbohydrate mixtures of different composition (quality and quantity) exert different effects on the amount of available glucose thereby influencing the portions of rapid or slowly available glucose. Added sugars increase the rapid available glucose portion, whereas dietary fibres may decrease overall glucose availability with special attention to the lowering effects on the rapid available glucose fraction.

Glucose fermentation in the presence of linoleic, oleic and stearic acids by a mixed culture

AbstractLong Chain Fatty Acid (LCFA) mixtures containing linoleic, oleic and stearic acids plus carbohydrates are found in a variety of effluents arising from fried food manufacture and milk processing. Accumulation of Volatile Fatty acids (VFAs) due to the presence of LCFAs may impair the operation of an anaerobic system treating effluents containing a mixture of triglycerides and carbohydrates. In this study, the effects of linoleic (C18:2), oleic (C18:1), and stearic (C18:0) acids on glucose fermentation were investigated at 21 °C using a culture acclimated to glucose. In cultures receiving ≥300 mg dm−3 LCFAs, residual amounts of glucose remained after approximately 8 h and none was detected after 24 h. Acetate degradation was inhibited in the presence of 300 or more mg dm−3 linoleic acid (LA), oleic acid (OA), or stearic acid (SA) with more acetate accumulation observed in cultures receiving LA. In comparison to the controls, similar amounts of propionate accumulation were observed in cultures receiving ≤100 mg dm−3 of each LCFA. However, in cultures receiving ≥300 mg dm−3 LCFAs, more propionate accumulated with complete removal observed within 20 days for only those cultures receiving oleic or stearic acids. Butyrate accumulation was observed only in cultures receiving ≥300 mg dm−3 LA and none was detected after 10 days of incubation. Copyright © 2004 Society of Chemical Industry

Optimizing Storage Stability of Lyophilized Recombinant Human Interleukin-11 with Disaccharide/Hydroxyethyl Starch Mixtures

Optimal storage stability of a protein in a dry formulation depends on the storage temperature relative to the glass transition temperature (Tg) of the dried formulation and the structure of the dried protein. We tested the hypothesis that optimizing both protein structure and Tg—by freeze-drying recombinant human interleukin-11 (rhIL-11) with mixtures of disaccharides and hydroxyethyl starch (HES)—would result in increased storage stability compared with the protein lyophilized with either disaccharide or hydroxyethyl starch alone. The secondary structure of the protein in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures by infrared spectroscopy. After rehydration, aggregation was monitored by size exclusion chromatography. Oxidation levels and cleavage products were quantified by reversed-phase chromatography. For the formulation with HES alone, which has a relatively high Tg, storage stability of rhIL-11 was poor, because HES failed to inhibit lyophilization-induced unfolding. The sugar formulations inhibited unfolding, and had intermediate Tg values and storage stabilities. Addition of hydroxyethyl starch to sucrose or trehalose increased Tg without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization. Optimal storage stability of lyophilized rhIL-11 was achieved by using a mixture of disaccharide and polymeric carbohydrates. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition.

Water dynamics in samples of ceramide tetrasaccharide (Gg4Cer) vesicles and GM1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (2H-NMR). GM1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10(-10) s, typical of water molecules near to the polar groups. Pure GM1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from -18 to 32 degrees C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to GM1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into GM1 micelles substantially increases the mobility of water molecules compared with that found in pure GM1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid-lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.

Acute tryptophan depletion induced by a gelatin-based mixture impairs object memory but not affective behavior and spatial learning in the rat

One manner to study the role of serotonin (5-HT) in behavioral functions is through nutritional manipulation of its precursor tryptophan (TRP). By means of the method of acute TRP depletion, plasma TRP levels can be reduced in a reversible way in both humans and rats. In the present study a TRP-free protein–carbohydrate mixture was used to investigate the behavioral effects of lowering TRP and 5-HT concentrations in adult male rats. These animals were tested in models of anxiety (open field, home cage emergence test), depression (forced swimming test) and cognition (object recognition test and Morris water escape test). The TRP-free protein–carbohydrate mixture substantially reduced the ratio TRP/ΣLNAA within 2 and 4 h by 75 and 60%, respectively. It was found that 4 h after administration, the treatment did not affect anxiety-related behavior nor did it cause depressive-like behavior. Also, no treatment effect was found on spatial learning performance in a Morris water escape test. On the other hand, performance in an object recognition test was clearly impaired after TRP depletion. Taken together, these data suggest that acute lowered central 5-HT levels are not associated with changes in affective behavior (i.e. anxiety and depression), but do impair object memory in adult rats.

Acute tryptophan and serotonin depletion using an optimized tryptophan-free protein–carbohydrate mixture in the adult rat

In contrast to humans, a tryptophan (TRP)-free amino acid (AA) mixture only leads to moderate depletion in plasma TRP levels in adult rats. In this study we evaluated the effects of an acute administration of a TRP-free protein–carbohydrate nutritional mixture in adult male Wistar rats. Plasma amino acid levels were examined at 2 and 4 h starting after the first administration. Furthermore, the concentrations of amino acid, serotonin (5-HT), dopamine (DA) and their metabolite (5-hydroxyindolacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC), respectively) were measured within the striatum, hippocampus and cortex. In the TRP depleted animals, the TRP/ΣLNAA ratio (LNAA: large neutral amino acids) was substantial decreased at 2 and 4 h after the first administration of the oral administration (by 71 and 78%, respectively). Four hours after treatment central TRP and 5-HT concentrations were decreased by 50%. Both peripheral and central TRP levels returned to basal values in the group treated with the nutritional mixture supplemented with TRP. Surprisingly, tyrosine levels were also reduced after oral administration of the protein–carbohydrate mixture without affecting central DA concentrations. In conclusion, the TRP-free protein–carbohydrate nutritional mixture appears to be an efficient tool to substantially reduce plasma and central TRP levels in adult rat.