High-pressure Anion-exchange Chromatography Research Articles

The disinfection by-products are in the air: Aerosol measurements in the urban area of Venice

Haloacetic acids (HAAs) are usually taken to be man-made, regularly determined in drinking water as disinfection by-products; yet they have been demonstrated as a probable photooxidation products from atmospheric halogenated hydrocarbons. Here we determined a total of fourteen HAAs, including for the first time iodinated-HAAs, in aerosol samples collected from April to June 2023 at Parco Bissuola (urbanized area of Venice) and Rio Novo (Venice island). The study provides a first insight and source identification about the anthropogenic or natural origin of HAAs. To fulfil this aim, we improved existing methods with a highly sensitive technique by using high pressure anion exchange chromatography coupled to a triple quadrupole mass spectrometer. Five HAAs have been found to be above the detection limits at both sites, and the major source should be attributed to a mixed contribution from atmospheric oxidation of both organic precursor, sea spray and a minor contribution of an external swimming pool located near the Parco Bissuola site. Considering the presence of iodinated HAAs and their cytotoxicity, the present study also provides a basis for the assessment of risks for humans.

Steering the Selectivity of Electrocatalytic Glucose Oxidation by the Pt Oxidation State.

Electrocatalytic glucose oxidation can produce high value chemicals, but selectivity needs to be improved. Here we elucidate the role of the Pt oxidation state on the activity and selectivity of electrocatalytic oxidation of glucose with a new analytical approach, using high-pressure liquid chromatography and high-pressure anion exchange chromatography. It was found that the type of oxidation, i.e. dehydrogenation of primary and secondary alcohol groups or oxygen transfer to aldehyde groups, strongly depends on the Pt oxidation state. Pt0 has a 7-fold higher activity for dehydrogenation reactions than for oxidation reactions, while PtOx is equally active for both reactions. Thus, Pt0 promotes glucose dialdehyde formation, while PtOx favors gluconate formation. The successive dehydrogenation of gluconate is achieved selectively at the primary alcohol group by Pt0, while PtOx also promotes the dehydrogenation of secondary alcohol groups, resulting in more complex reaction mixtures.

Airborne polar pesticides in rural and mountain sites of North-Eastern Italy: An emerging air quality issue

North-Eastern Italy and in particular Veneto Region, stands out as a major centre of agriculture and viticulture which has rapidly expanded in the last decade with high productivity indexes. In this context, assessing atmospheric pollution caused by crop spraying with pesticides in rural areas and their transport to high-altitude remote sites is crucial to provide a basis for understanding possible impacts on the environment and population health. We aim to improve existing methods with a highly sensitive technique by using high pressure anion exchange chromatography coupled to a triple quadrupole mass spectrometer. Thus, a total of fourteen polar pesticides were determined in aerosol samples collected from August to December 2021 at Roncade (Venetian plain) and Col Margherita Observatory (Dolomites). The observatory was chosen as the background site as it is representative of the surrounding alpine region. Some samples revealed a substantial amount of cyanuric acid mainly at Roncade (mean concentration of 10 ± 10 ng m−3), glyphosate and fosetyl-aluminium (0.1 ± 0.2 and 0.1 ± 0.1 ng m−3, respectively). Surprisingly, some pesticides have been also found at Col Margherita, a high mountain background site, with concentrations an order of magnitude lower than at Roncade. This is the first time that fourteen polar pesticides have been assessed in the aerosol phase of the Po’ Valley and detected at a high-altitude remote site, and consequently this study provides the first data on their occurrences in Italian aerosols. It represents a basis for the assessment of risks for humans.

Prebiotic Activity of Poly- and Oligosaccharides Obtained from Plantago major L. Leaves

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO− group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

Characterization of a new exopolysaccharide produced by Halorubrum sp. TBZ112 and evaluation of its anti-proliferative effect on gastric cancer cells.

In the present study, we aimed to extract, purify, analyze monosaccharide composition of exopolysaccharide (EPS) produced by Halorubrum sp. TBZ112 (KCTC 4203 and IBRC-M 10773) and also to evaluate its possible antiproliferative activity against human gastric cancer (MKN-45) cell line and its biocompatibility effect on normal cells using human dermal fibroblast (HDF) cell line. Average molecular weight and monosaccharide composition were determined by high-pressure size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and high-pressure anion exchange chromatography (HPAEC), respectively. Fourier transform infrared (FTIR) spectroscopy was used for the partial characterization of the EPS. The EPS effect on the cell proliferation and viability of MKN-45 and HDF cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion, respectively. Strain TBZ112 excreted 480mg.l-1 of the EPS under optimal growth conditions. The EPS had a molecular weight of 5.052kDa and was a heteropolysaccharide containing ten moieties mainly composed of mannose (19.95%), glucosamine (15.55%), galacturonic acid (15.43%), arabinose (12.24%), and glucuronic acid (12.05%). No significant difference of the EPS treatments on the proliferation activity of MKN-45 and HDF cells were observed (P > 0.05). For the first time, the EPS from Halorubrum sp. TBZ112, an extremely halophilic archaeon related to Halorubrum genus, was isolated and chemically characterized. The EPS from Halorubrum sp. TBZ112 possesses a relatively low molecular weight and might be applied as a biocompatible compound. More investigations are needed to determine other biological activities of the EPS along with further details of its chemical structure.

Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.

Comparison of four glycosyl residue composition methods for effectiveness in detecting sugars from cell walls of dicot and grass tissues

BackgroundThe effective use of plant biomass for biofuel and bioproduct production requires a comprehensive glycosyl residue composition analysis to understand the different cell wall polysaccharides present in the different biomass sources. Here we compared four methods side-by-side for their ability to measure the neutral and acidic sugar composition of cell walls from herbaceous, grass, and woody model plants and bioenergy feedstocks.ResultsArabidopsis, Populus, rice, and switchgrass leaf cell walls, as well as cell walls from Populus wood, rice stems, and switchgrass tillers, were analyzed by (1) gas chromatography–mass spectrometry (GC–MS) of alditol acetates combined with a total uronic acid assay; (2) carbodiimide reduction of uronic acids followed by GC–MS of alditol acetates; (3) GC–MS of trimethylsilyl (TMS) derivatives; and (4) high-pressure, anion-exchange chromatography (HPAEC). All four methods gave comparable abundance ranking of the seven neutral sugars, and three of the methods were able to quantify unique acidic sugars. The TMS, HPAEC, and carbodiimide methods provided comparable quantitative results for the specific neutral and acidic sugar content of the biomass, with the TMS method providing slightly greater yield of specific acidic sugars and high total sugar yields. The alditol acetate method, while providing comparable information on the major neutral sugars, did not provide the requisite quantitative information on the specific acidic sugars in plant biomass. Thus, the alditol acetate method is the least informative of the four methods.ConclusionsThis work provides a side-by-side comparison of the efficacy of four different established glycosyl residue composition analysis methods in the analysis of the glycosyl residue composition of cell walls from both dicot (Arabidopsis and Populus) and grass (rice and switchgrass) species. Both primary wall-enriched leaf tissues and secondary wall-enriched wood/stem tissues were analyzed for mol% and mass yield of the non-cellulosic sugars. The TMS, HPAEC, and carbodiimide methods were shown to provide comparable quantitative data on the nine neutral and acidic sugars present in all plant cell walls.

RP-UHPLC-UV-ESI-MS/MS analysis of LPMO generated C4-oxidized gluco-oligosaccharides after non-reductive labeling with 2-aminobenzamide

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides.

A Survey of Commercially Available Isomaltooligosaccharide-Based Food Ingredients.

Isomaltooligosaccharides (IMOs) are included in many commercially available food products including protein/fiber bars, shakes, and other dietary supplements. Marketed as "high fiber," "prebiotic soluble fiber," and/or as a "low-calorie, low glycemic sweetener," IMO may be present in significant amounts, for example, more than 15 g/item or serving. Herein, high-pressure anion exchange chromatography with pulsed amperometric detection and high-pressure liquid chromatography with differential refractive index detection are used to compare 7 commercially available IMO-containing bulk food ingredients. The ingredients are typical of those produced either (a) via bacterial fermentation ("fermented" IMO or MIMO) of sucrose in the presence of a maltose acceptor mediated by a glucosyltransferase enzyme (dextransucrase), or (b) via transglycosylation of hydrolyzed starch with α-glucosidase ("industrial" IMO). Analysis of the results with respect to digestibility suggests that the potential glycemic impact of the ingredients and products containing "industrial" IMO may be inconsistent with the product labeling and/or certificates of analysis with respect to overall fiber content, prebiotic fiber content, and glycemic response and are thus inappropriate for diabetic patients and those on low-carbohydrate (for example, ketogenic) diets.

Advances on the Determination of the Astatine Pourbaix Diagram: Predomination of AtO(OH)2 (-) over At(-) in Basic Conditions.

It is generally assumed that astatide (At(-) ) is the predominant astatine species in basic aqueous media. This assumption is questioned in non-complexing and non-reductive aqueous solutions by means of high-pressure anion-exchange chromatography. Contrary to what is usually believed, astatide is found to be a minor species at pH=11. A different species, which also bears a single negative charge, becomes predominant when the pH is increased beyond 7. Using competition experiments, an equilibrium constant value of 10(-6.9) has been determined for the formation of this species from AtO(OH) with the exchange of one proton. The identification of this species, AtO(OH)2 (-) , is achieved through relativistic quantum mechanical calculations, which rule out the significant formation of the AtO2 (-) species, while leading to a hydrolysis constant of AtO(OH) in excellent agreement with experiment when the AtO(OH)2 (-) species is considered. Beyond the completion of the Pourbaix diagram of astatine, this new information is of interest for the development of (211) At radiolabeling protocols.

Sugars in Antarctic aerosol

The processes and transformations occurring in the Antarctic aerosol during atmospheric transport were described using selected sugars as source tracers.Monosaccharides (arabinose, fructose, galactose, glucose, mannose, ribose, xylose), disaccharides (sucrose, lactose, maltose, lactulose), alcohol-sugars (erythritol, mannitol, ribitol, sorbitol, xylitol, maltitol, galactitol) and anhydrosugars (levoglucosan, mannosan and galactosan) were measured in the Antarctic aerosol collected during four different sampling campaigns.For quantification, a sensitive high-pressure anion exchange chromatography was coupled with a single quadrupole mass spectrometer. The method was validated, showing good accuracy and low method quantification limits.This study describes the first determination of sugars in the Antarctic aerosol. The total mean concentration of sugars in the aerosol collected at the “Mario Zucchelli” coastal station was 140 pg m−3; as for the aerosol collected over the Antarctic plateau during two consecutive sampling campaigns, the concentration amounted to 440 and 438 pg m−3.The study of particle-size distribution allowed us to identify the natural emission from spores or from sea-spray as the main sources of sugars in the coastal area. The enrichment of sugars in the fine fraction of the aerosol collected on the Antarctic plateau is due to the degradation of particles during long-range atmospheric transport. The composition of sugars in the coarse fraction was also investigated in the aerosol collected during the oceanographic cruise.

A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

A novel α-L-arabinofuranosidase of family 43 glycoside hydrolase (Ct43Araf) from Clostridium thermocellum.

The study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding α-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50°C. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg−1 and 5.0 Umg−1, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of α-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both p-nitrophenol-α-L-arabinofuranoside and p-nitrophenol-α-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% β-sheets, 49% random coils but only 3% α-helices.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants.Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume.We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape.SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit.

Changes in carbohydrate metabolism during grape berry development play a central role in shaping the final composition of the fruit. The present work aimed to identify metabolic switches during grape development and to provide insights into the timing of developmental regulation of carbohydrate metabolism. Metabolites from central carbon metabolism were measured using high-pressure anion-exchange chromatography coupled to tandem mass spectrometry and enzymatic assays during the development of grape berries from either field-grown vines or fruiting cuttings grown in the greenhouse. Principal component analysis readily discriminated the various stages of berry development, with similar trajectories for field-grown and greenhouse samples. This showed that each stage of fruit development had a characteristic metabolic profile and provided compelling evidence that the fruit-bearing cuttings are a useful model system to investigate regulation of central carbon metabolism in grape berry. The metabolites measured showed tight coordination within their respective pathways, clustering into sugars and sugar-phosphate metabolism, glycolysis, and the tricarboxylic acid cycle. In addition, there was a pronounced shift in metabolism around veraison, characterized by rapidly increasing sugar levels and decreasing organic acids. In contrast, glycolytic intermediates and sugar phosphates declined before veraison but remained fairly stable post-veraison. In summary, these detailed and comprehensive metabolite analyses revealed the timing of important switches in primary carbohydrate metabolism, which could be related to transcriptional and developmental changes within the berry to achieve an integrated understanding of grape berry development. The results are discussed in a meta-analysis comparing metabolic changes in climacteric versus non-climacteric fleshy fruits.

PURIFICATION AND CHARACTERIZATION OF SOY COTYLEDON β-GLUCOSIDASE

ABSTRACT β-Glucosidase F42 of soy cotyledons was purified by ammonium sulfate fractionation, ion-exchange chromatography (CM-Sephadex-C-50, Sigma, St. Louis, MO) and gel filtration (Sephadex G-100, Sigma). The enzyme was purified 111.8-fold relative to its concentration in the crude extract. It had an apparent molecular mass of 53 kDa in gel filtration experiments and produced a 33-kDa band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that it is dimeric. The purified β-glucosidase F42 was characterized as a glycoprotein after the identification of fucose, galactosamine and glucosamine by high-pressure anion-exchange chromatography–pulsed amperometric detector. Its highest activity was observed at pH 5.0 and 45C, and it was stable for up to 4 days at 25C. The Km of the enzyme was 0.12 mM p-nitrophenyl-β-d-glucopyranoside. β-Glucosidase F42 showed specificity for different substrates, and its activity was inhibited by 1 mM HgCl2, 10 mM glucono-δ-lactone or 150 mM glucose and increased by 10 mM MnCl2. PRACTICAL APPLICATIONS β-Glucosidase is an enzyme that hydrolyzes β-glucosidic bonds to liberate glucose and hydrolyzes isoflavones to release aglycones. Soy aglycones have been broadly investigated because of their biological activity in the prevention and treatment of some chronic diseases. Soy β-glucosidase can be used in the food industry to alter soy isoflavones for the production of functional foods that are rich in aglycone isoflavones. Therefore, it was an established method of purification of the enzyme that has great biotechnological potential.

Is DTPA a good competing chelating agent for Th(IV) in human serum and suitable in targeted alpha therapy?

The interaction between thorium and human serum components was studied using difference ultraviolet spectroscopy (DUS), ultrafiltration and high-pressure-anion exchange chromatography (HPAEC) with external inductively conducted plasma mass spectrometry (ICP-MS) analysis. Experimental data are compared with modelling results based on the law of mass action. Human serum transferrin (HSTF) interacts strongly with Th(IV), forming a ternary complex including two synergistic carbonate anions. This complex governs Th(IV) speciation under blood serum conditions. Considering the generally used Langmuir-type model, values of 1033.5 and 1032.5 were obtained for strong and weak sites, respectively. We showed that trace amounts of diethylene triamine pentaacetic acid (DTPA) cannot complex Th(IV) in the blood serum at equilibrium. Unexpectedly this effect is not related to the competition with HSTF but is due to the strong competition with major divalent metal ions for DTPA. However, Th–DTPA complex was shown to be stable for a few hours when it is formed before addition in the biological medium; this is related to the high kinetic stability of the complex. This makes DTPA a potential chelating agent for synthesis of 226Th-labelled biomolecules for application in targeted alpha therapy.

Characterization and in vitro immunomodulatory screening of fructo-oligosaccharides of Asparagus racemosus Willd.

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC–FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2→1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7–8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2–50μg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9±4.4% at 5.6μg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8±1.2% at 25μg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the ‘Rasayana’ properties of this plant.

Enzymatic Modification of a Model Homogalacturonan with the Thermally Tolerant Pectin Methylesterase from Citrus: 1. Nanostructural Characterization, Enzyme Mode of Action, and Effect of pH

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).

Green liquor extraction of hemicelluloses from southern pine in an Integrated Forest Biorefinery

Loblolly Pine chips were extracted with fresh green liquor solutions containing sufficient alkalinity to approximately neutralize the acids released upon treatment of the chip-liquor mass at elevated temperatures of 170, 180 and 190 °C for various times. The components of green liquor extracts were then quantified using high pressure anion exchange chromatography and UV spectrophotometer. The time–temperature effect on the extracted yields including the wood weight loss of wood chips after extraction could be well described by the H-factor approach. Sugar components were all found in their polymeric forms in the liquor. About 2.5–3% of wood mass was extracted as total sugars at an H-factor of about 4000. In general, mannose showed lower extraction than xylose. With increasing chemical charge, arabinoglucuronoxylan and galactoglucomannan extracts decreased, whereas dissolved lignin increased. Cellulose showed a minimum dependence on H-factor and chemical charge. The total sugar showed an inverse proportionality to the final liquor pH that rapidly dropped from alkali to the neutral in the very early stages of extraction and leveled off slowly reaching the acid region. Significant difference between total organic yield and the weight loss provides a possible evidence for the presence of significant amount of low molecular weight dissolved organics that were not accounted for.