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Honey is a complex mixture of carbohydrates, in which the monosaccharides glucose and fructose are the most abundant compounds. Currently, more than 20 oligosaccharides have been identified in different varieties of honey normally at quite low concentration. A method was developed and validated using high-performance anion-exchange chromatography coupled to a mass spectrometry detector to investigate the composition of carbohydrates in honey samples. The method was tested for linearity range, trueness, instrumental and method detection and quantification limits, repeatability, and reproducibility. It was applied to determine seven monosaccharides, eight disaccharides, four trisaccharides, and one tetrasaccharide in various honey samples. The present work describes the composition of sugars in unifloral, multifloral, and some honeydew honey, which were produced and collected by beekeepers in the Trentino Alto-Adige region. Statistical techniques have been used to establish a relationship based on levels of carbohydrates among different Italian honey. The results emphasize that mono- and oligosaccharide profiles can be useful to discriminate different honeys according to their floral characteristics and inter-annual variability.

To achieve the measurement reliability of monosaccharides used as diagnostic markers in clinical fields, it is essential to establish certified reference materials (CRMs). The purpose of this study is to develop a serum CRM by adopting high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as a new candidate reference measurement procedure for the measurement of glucose and galactose, common diagnostic markers of diabetes and galactosemia, respectively. Using various monosaccharides as internal standards, the accuracy of the HPAEC-PAD method was tested by measuring glucose CRM following treatment with three different deproteinization methods: ultrafiltration, protein precipitation by trichloroacetic acid (TCA), and protein precipitation by acetonitrile. Results showed that ultrafiltration and 5% TCA provided good accuracy with every tested monosaccharide as the internal standard. Accordingly, serum samples in this study were treated by ultrafiltration after adding 2-deoxy-d-glucose and arabinose, which were selected as internal standards for galactose and glucose, respectively. Both intra- and inter-day recovery tests showed good precision and accuracy within 2%. From the serum CRM batches prepared at two levels, 11 units were analyzed by exact-matched calibration methods, and the mass fractions of galactose and glucose were determined via HPAEC-PAD. The between-unit relative standard deviations were not more than 1.5%, showing homogeneity. The expanded uncertainties (%) of galactose and glucose for both levels were less than 3.6% and 2.3% at 95% confidence. The HPAEC-PAD method presented in this study can significantly improve the accuracy and precision of simultaneous monosaccharide analysis, allowing for the development of further serum CRMs for monosaccharides.

Background Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics. Results Epidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per-O-trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by 13C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes). Conclusions Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.

To sensitively determine 99Tc, a new method for internal quantification of its most common and stable species, [99Tc]TcO4-, was developed. Anion-exchange chromatography (IC) was coupled to inductively coupled plasma–mass spectrometry (ICP-MS) and equipped with an aerosol desolvation system to provide enhanced detection power. Due to a lack of commercial Tc standards, an isotope dilution-like approach using a Ru spike and called isobaric dilution analysis (IBDA) was used for internal quantification of 99Tc. This approach required knowledge of the sensitivities of 99Ru and 99Tc in ICP-MS. The latter was determined using an in-house prepared standard manufactured from decayed medical 99mTc-generator eluates. This standard was cleaned and preconcentrated using extraction chromatography with TEVA resin and quantified via total reflection X-ray fluorescence (TXRF) analysis. IC coupled to ICP-MS enabled to separate, detect and quantify [99Tc]TcO4- as most stable Tc species in complex environments, which was demonstrated in a proof of concept. We quantified this species in untreated and undiluted raw urine collected from a patient, who previously underwent scintigraphy with a 99mTc-tracer, and determined a concentration of 19.6 ± 0.5 ng L−1. The developed method has a high utility to characterize a range of Tc-based radiopharmaceuticals, to determine concentrations, purity, and degradation products in complex samples without the need to assess activity parameters of 99(m)Tc.

Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [→3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.

A novel peptidyl-lys metalloendopeptidase ( Tc -LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc -LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc- LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc -LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc -LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc -LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc -LysN’s activity up to ~100% and ~50%, respectively. Tc -LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc -LysN which was comparable to the sequence coverage of 90% by trypsin peptides. Key points • A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity • Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants • Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting

Brown seaweeds contain fucoidan, which has numerous biological activities. Here, the anti-fine-dust activity of fucoidan extracted from , an abundant brown seaweed from South Africa, was explored. Fourier transmittance infrared spectroscopy, high-performance anion-exchange chromatography with pulsed amperometric detection analysis of the monosaccharide content, and nuclear magnetic resonance were used for the structural characterization of the polysaccharides. The toll-like receptor (TLR)-mediated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were evaluated. The results revealed that purified leaf fucoidan fraction 7 (EMLF7), which contained the highest sulfate content, showed the best anti-inflammatory activity by attenuating the TLR-mediated NF-κB/MAPK protein expressions in the particulate matter-stimulated cells. This was solidified by the successful reduction of Prostaglandin E , NO, and pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β. The current findings confirm the anti-inflammatory activity of EMLF7, as well as the potential use of as a low-cost fucoidan source due to its abundance. This suggests its further application as a functional ingredient in consumer products.

An accurate characterization of the fine molecular structure of starch in terms of the chain length distribution (CLD) of amylopectin requires complete dispersion of the starch in a solvent, either buffer or water and the non-interference of this on the subsequent enzymatic debranching of starch with isoamylase (pretreatments before determining the CLD). In this study, the HYLON V and HYLON VII starches (commercial high-amylose corn starches) were selected, since high-amylose starches typically present difficulties in their dispersion and, therefore, the study of their fine structure can commonly present errors. The goal of this research was to study two water dispersion treatments (1. boiling water for 1 h and 2. autoclaving at 120 °C and 15 Psi for 15 min) to achieve a fast and reliable method of the fine structure of two high-amylose corn starches using high-performance size exclusion chromatography coupled with a refractive index detector (HPSEC-RID) and high-performance anion-exchange chromatography coupled a pulsed amperometric detector (HPAEC-PAD). The HPSEC-RID chromatograms of both starches showed better resolution using autoclaving than boiling water treatment. The two starches tested had higher amylose and degree of polymerization (DP) values utilizing the autoclaving than the boiling water treatment. However, no appreciable differences were observed in the CLD values employing HPAEC-PAD for either of the two water dispersion treatments. Lastly, the water dispersion treatment by autoclaving before enzymatic debranching is recommended to obtain a more accurate quantification of the amylose content of high-amylose starches.

The objective of this study was to determine and compare the sugar profile, distribution in fruits and leaves and sink-source relationship in three strawberry (‘Favette’, ‘Alba’ and ‘Clery’) and three blueberry cultivars (‘Bluecrop’, ‘Duke’ and ‘Nui’) grown in organic (OP) and integrated production systems (IP). Sugar analysis was done using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The results showed that monosaccharide glucose and fructose and disaccharide sucrose were the most important sugars in strawberry, while monosaccharide glucose, fructose, and galactose were the most important in blueberry. Source-sink relationship was different in strawberry compared to blueberry, having a much higher quantity of sugars in its fruits in relation to leaves. According to principal component analysis (PCA), galactose, arabinose, and melibiose were the most important sugars in separating the fruits of strawberries from blueberries, while panose, ribose, stachyose, galactose, maltose, rhamnose, and raffinose were the most important sugar component in leaves recognition. Galactitol, melibiose, and gentiobiose were the key sugars that split out strawberry fruits and leaves, while galactose, maltotriose, raffinose, fructose, and glucose divided blueberry fruits and leaves in two groups. PCA was difficult to distinguish between OP and IP, because the stress-specific responses of the studied plants were highly variable due to the different sensitivity levels and defense strategies of each cultivar, which directly affected the sugar distribution. Due to its high content of sugars, especially fructose, the strawberry cultivar ‘Clery’ and the blueberry cultivars ‘Bluecrop’ and ‘Nui’ could be singled out in this study as being the most suitable cultivars for OP.

This study aimed to characterize the structure of Chinese yam (Dioscoreae Rhizoma) polysaccharide (CYP) and to investigate its protective effect against H2O2-induced oxidative damage in IEC-6 cells. The chemical composition and structural characteristics of the samples were analyzed by chemical and instrumental methods, including high-performance gel permeation chromatography, high-performance anion-exchange chromatography (HPAEC), Fourier transformed infrared (FT-IR), ultraviolet (UV), and scanning electron microscopy (SEM). Antioxidant activity was evaluated by establishing a cellular model of oxidative damage. The molecular weight of CYP was 20.89 kDa. Analysis of the monosaccharide composition revealed that CYP was primarily comprised of galactose (Gal), glucose (Glu), and galacturonic acid (GalA), and the ratio between them was 28.57:11.28:37.59. Pretreatment with CYP was able to improve cell viability, superoxide dismutase (SOD) activity, and reduce intracellular reactive oxygen species (ROS) production and malondialdehyde (MDA) content after H2O2 injury. CYP also attenuated oxidative damage in cells through the mitogen-activated protein kinase (MAPK) signaling pathway. This study showed that CYP was an acidic heteropolysaccharide with a good protective effect against oxidative damage, and it thus has good prospects in food and biopharmaceutical industries.