Anion-exchange High-performance Liquid Research Articles

Arsenic speciation analysis in cow's milk and plant-based imitation milks by HPLC-ICP-MS

Imitation milks based on vegetables have become relevant in recent years as a substitute for cow´s milk. Total and inorganic content of arsenic (As) determination in food is of utmost importance due to toxicological concerns. This study aimed to (1) optimize and validate a methodology for speciation analysis using anion exchange high performance liquid chromatography coupled to plasma mass spectrometry and (2) analyze the arsenic content in milk and imitation milks. Determination of As (III) and As (V) was done following a simple and innovative treatment, which consisted on the dilution of a small volume of sample in mobile phase and further filtration. Furthermore, acid microdigestion for total As determination with small amounts of acid and sample was optimized. The detection limits of the method ranged from 0.2 to 0.7 µg L−1. Regarding to arsenic content we found that imitation milks based on peanuts and nuts presented the lowest inorganic As content (between 1.7 and 1 µg L−1) whereas the highest values were found in imitations based on soy, rice and vetch (27.3, 13 and 8 µg L−1, respectively). The cow’s milk presented intermediate levels of inorganic As (4 µg L−1). Also, almond- and quinoa-based imitation milks showed inorganic As values below detection limits, so these imitation milks may be the safest to consume with respect to arsenic content.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

Soluble-protein-aggregate-assisted improvements in heat-induced gel properties: Effect of genipin-mediated crosslinks on egg white protein

Genipin, a covalent crosslinker, has attracted significant attention from the food industry. Herein, the color, heat-induced gelling properties, and soluble aggregate character of the genipin-treated egg white were investigated to clarify the impact of soluble protein aggregates on the gel properties of egg white protein.After genipin pretreatment, the egg white protein turned dark blue and retained this color after heat-induced gelling. The genipin-pretreated egg white protein gel exhibited up to 2.9-fold and 13% higher breaking strength and strain, respectively, compared to non-treated egg white gel. The gel-breaking strength and strain correlated well with the color, before and after gelling. Gel electrophoresis, size exclusion, and anion exchange high-performance liquid chromatography revealed that a soluble protein aggregate with a negatively charged surface formed during pretreatment. The genipin-treated egg white protein formed a finely structured gel with high breaking strength and strain at all tested NaCl concentrations, indicating that the enhanced negative charge of the surface did not contribute to improving the gel properties.The soluble protein aggregates formed via covalent crosslinking are important for improving gel properties. The proposed method is a novel environmentally friendly method for enhancing the gel properties of egg white protein.

Measurement of cholesterol levels of lipoprotein subclasses in human serum using anion-exchange high-performance liquid chromatography with a linear concentration gradient of sodium perchlorate.

Relationships between the subclasses of high-density lipoprotein (HDL) or low-density lipoprotein (LDL) and the risk of atherosclerotic cardiovascular disease have been studied, and using various methods, such as ultracentrifugation, electrophoresis, and nuclear magnetic resonance, for analysing lipoprotein subclasses. We established a method for HDL and LDL subclasses using anion-exchange high-performance liquid chromatography (AEX-HPLC) with a linear concentration gradient of sodium perchlorate (NaClO4). In the AEX-HPLC, the subclasses of HDL and LDL were separated, and detected using a post-column reactor with an enzymatic cholesterol reagent, that contained cholesterol esterase, cholesterol oxidase, and peroxidase as major ingredients. LDL subclasses were divided based on the absolute value of first-derivative chromatogram. Three HDL subclasses, HDL-P1, HDL-P2, and HDL-P3, and three LDL subclasses, LDL-P1, LDL-P2, and LDL-P3, were separated by AEX-HPLC, and detected in order. The major components of HDL-P2 and HDL-P3 were HDL3 and HDL2, respectively. The linearity was determined for each lipoprotein subclass. The coefficients of variation of cholesterol concentration of the subclasses for within-day assay (n = 10) and between-day assay (n = 10) ranged between 3.08-8.94% and 4.52-9.97%, respectively. Cholesterol levels in HDL-P1 of diabetic patients were positively correlated with oxidized LDL levels (r = 0.409, p = 0.002). Moreover, cholesterol levels in LDL-P2 and LDL-P3 were positively correlated with oxidized LDL levels (r = 0.393, p = 0.004 and r = 0.561, p < 0.001, respectively). AEX-HPLC may be highly suitable as an assay to clinically assess lipoprotein subclasses.

Thermal treatment affects aptamers’ structural profiles

Using anion-exchange high performance liquid chromatography under non-denaturing conditions, the conformational flexibility of adenosine-, ampicillin-, and quinine aptamers were studied. It was found that all three aptamers showed more than one species when not subjected to thermal anneal. Addition of ligand to untreated aptamers did not significantly change the structural distribution. Upon heating followed by slow cooling, however, all three aptamers were found to exist virtually solely in one structure, presumably the partial hairpin species. It was also found that sonication of quinine aptamer, but not adenosine and ampicillin aptamer, led to its elution off HPLC as virtually a single species. These changes in conformational distribution as a result of thermal anneal or sonication were further confirmed by UV/vis and circular dichroism spectroscopy, as well as melt curves. The findings provided basis for future optimization of aptamer selection and preparation, where thermal anneal can help optimize selection efficiency and improve the consistency in the interpretation of results.

Simultaneous speciation of chromate, molybdate and arsenate in lysimetric water from geotechnical composites installed in field lysimeters

Anion-exchange high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used for simultaneous speciation of chromate, molybdate and arsenate. The repeatability of measurement tested for multielemental standard solution of chromate, molybdate and arsenate (50 ng mL−1 of Cr, Mo and As, pH 12) was ± 0.9%, ± 4.9% and ± 4.1%, respectively. Limits of quantification (LOQs) were low (0.53 ng mL−1 for chromate and arsenate and 1.03 ng mL−1 for molybdate, expressed as elemental concentrations). A wide linear concentration range (from LOQs to 500 ng mL−1) was obtained. The performances of this method enabled simultaneous speciation analysis in samples of water from lysimeters, in which three geotechnical composites, made of recycled waste, were installed in parallel in compacted and uncompacted, 20 times less dense form. The release of toxic chemical species of elements into lysimetric waters from each composite was studied. The results revealed that the degree of compaction and the composition of composites both have a significant influence on leaching of chromate, molybdate and arsenate. The study proved that multielemental speciation analysis is fast and cost-effective method for investigations of environmental impacts of materials, made from recycled waste, and can be used in other similar applications.

Chemical Fruit Profiles of Different Raspberry Cultivars Grown in Specific Norwegian Agroclimatic Conditions

Raspberries are considered valuable fruits due to their high levels of nutrients and phytochemicals, which have many beneficial effects on humans. As many external factors affect the composition of these fruits (the type of cultivation, soil characteristics, ripeness, storage time and post-harvest technologies, cultivar/genotype, and climatic conditions), the goal of this study was to analyze different raspberry cultivars grown in Norway. Considering that Norway is a country with specific climatic conditions, as well as has a limited period of fruit vegetation, another important goal of this study was also to compare raspberries from different Norwegian areas, as well as different grown cultivars. Modern analytical techniques, such as high-performance anion-exchange liquid chromatography with pulsed amperometric detection (HPEAC-PAD), ultra-high-performance liquid chromatography with diode array detector coupled to triple quadrupole mass spectrometry (UHPLC-DAD MS/MS), and inductively coupled plasma–optical emission spectrometry (ICP-OES), provided a detailed examination of the raspberry extract samples. Based on their high levels of minerals (especially N, P, and K), organic acids (predominantly citric and malic acids), sugars (glucose, fructose, sucrose, and galactose), and polyphenols (ellagic acid, syringic acid, quercetin, and rutin), Norwegian raspberries could be considered fruits with increased health-beneficial compounds. The chemical composition of the studied cultivars depended on the locality of growth.

Identification of Recombinant Chimpanzee Adenovirus C68 Degradation Products Detected by AEX-HPLC.

Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.

The use of enriched stable isotopic tracers of 50Cr(VI) and 53Cr(III) in a study of Cr speciation in wine and beer

Wine and beer are beverages rich in polyphenols and other antioxidants that inhibit the existence of Cr(VI). To verify this hypothesis, the content of Cr(VI) in wine and beer samples was determined by applying a speciation analysis based on anion-exchange high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The total Cr concentrations in the wine and beer samples determined using ICP-MS ranged from 6 to 22 ng/mL Cr and 1.8–5 ng/mL Cr, respectively. The behaviour of Cr(III) and Cr(VI) was followed using enriched 50Cr(VI) and 53Cr(III) stable isotopic tracers added to the wine and beer samples. The results revealed that wine and beer do not contain toxic Cr(VI). In all the samples analysed, the concentration was below 0.06 ng/mL Cr(VI). It was also demonstrated that, due to the presence of antioxidants, the added isotopically enriched 50Cr(VI) was reduced to 50Cr(III). Our investigation refuted the statements of some researchers who reported that wine and beer contain toxic Cr(VI).

Engineering immunomodulatory nanoplatforms from commensal bacteria-derived polysaccharide A.

Capsular zwitterionic polysaccharides (CZPs), typically found on the surfaces of commensal gut bacteria, are important immunomodulatory molecules due to their ability to produce a T cell dependent immune response upon processing by antigen presenting cells (APCs). Their immunological activity makes them potentially useful for generating material constructs that are applicable for the treatment of diseases, or as vaccines. Herein, we explored synthetic strategies to generate immunologically active polymer-carbohydrate conjugates and nanomaterials of the CZP, Polysaccharide A (PSA) derived from Bacteroides fragilis. Initially, we addressed the purification of PSA, which is critical for the realization of materials applicable for biomedical purposes. Anion exchange high performance liquid chromatography in the presence of a surfactant (CHAPS) enabled the isolation of pure PSA. Through modification of purified PSA with azide groups, we demonstrated that polymers or antigens could be incorporated with PSA via click chemistry reactions to generate conjugates that can be fabricated into nanoparticles. By conjugation of PSA with a DBCO end functionalized polyphosphoester polymer with hydrophobic pendant terminal alkyne groups, an amphiphilic conjugate was obtained which formed nanoparticles of about 100 nm in aqueous solution. Moreover, terminal alkyne groups could be modified with charged thiol molecules (amine/carboxylate) via thiol-yne radical chemistry to generate conjugates, which could be incorporated into nanoparticles via electrostatic interactions building onto a charged nanoparticle template. The conjugates and nanoparticles exhibited immunological activity as assessed by the toll-like receptor 2 (TLR2) activation assay and positive cytokine production (IL-10) following their co-incubation with APCs and T cells. Summarily, this work plainly demonstrates chemical biology strategies for fabricating immunomodulatory nanomaterials from commensal microorganisms that can potentially be novel vaccines or immunotherapeutics.

A rapid method for extraction, purification and structure analysis of chondroitin sulfate from six marine tissues

Abstract This study was designed to establish a strategy for the extraction, purification, and structure analysis of chondroitin sulfate (CS) in milligram amounts. Crude acidic polysaccharides were extracted from six kinds of marine animals by enzymatic hydrolysis and hexadecylpyridinium chloride precipitation and purified by Q Sepharose Fast Flow strong anion exchange column. The purification of each crude polysaccharide was completed within 1 h. The structure of the polysaccharides, i.e. their chemical characterization, functional group, molecular weight and monosaccharide composition, were analyzed by colorimetry, nuclear magnetic resonace and high-performance liquid chromatogrpahy, respectively. All polysaccharides were identified as CS. The oligosaccharide profile produced by enzyme hydrolysis of polysaccharides was determined by strong anion-exchange high-performance liquid chromatorgraphy. This method can be widely applied to the extraction and characterization of chondroitin sulfate from unknown raw materials, screening high-quality sources of functional polysaccharides, and laying a good foundation for the following study of the structure–function relationship of polysaccharides.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified.Graphical abstract.

Clinical Significance of Intermediate-Density Lipoprotein Cholesterol Determination as a Predictor for Coronary Heart Disease Risk in Middle-Aged Men.

Background: Not only low-density lipoprotein (LDL) cholesterol but also non-high-density lipoprotein cholesterol (non-HDL-C), very low-density lipoprotein (VLDL) cholesterol (VLDL-C), and intermediate-density lipoprotein (IDL) cholesterol (IDL-C) are reported to be significant risk markers for coronary heart disease (CHD). We reported the relevance of IDL-C to Framingham risk score (F-score), but the present study addressed the relevance of IDL-C to Suita score (S-score), a risk score for coronary heart disease (CHD) developed for the Japanese individuals in addition to F-score.Methods: The cholesterol levels of lipoproteins, including triglyceride (TG)-rich lipoproteins (IDL and VLDL), were measured by an anion exchange high-performance liquid chromatography (AEX-HPLC). This study enrolled 476 men, aged mean 51 years and free of CHD and stroke.Results: Non-HDL-C, IDL-C, and VLDL-C significantly correlated with F-score and S-score. In the multiple stepwise regression analysis, IDL-C as well as body mass index (BMI) significantly correlated with both F-score and S-score in both the total subjects and the subjects without drug therapy. The multivariate logistic analysis with the model composed of BMI and IDL-C as the predictor variables demonstrated that 1 SD increase in IDL-C was an independent predictor for 10-year CHD risk >10% of F-score (OR 1.534, 95% CI 1.266–1.859, p < 0001) and that of S-score (OR 1.372, 95% CI 1.130–1.667, p = 0.0014) in the total subjects. Even in the subjects without the drug therapy, the increased IDL-C, as well as BMI, were significant predictors for 10-year CHD risk >10% of S-score as well as F-score.Conclusion: These results suggest the significant relevance of the increased IDL-C for CHD risk scores in middle-aged men free of CHD and stroke. Further investigations are needed in women and elderly subjects.

A fit-for-purpose copper speciation method for the determination of exchangeable copper relevant to Wilson’s disease

Exchangeable copper (CuEXC), mainly comprised copper (Cu) bound to albumin, has been proposed as a specific marker of Cu overload in Wilson's disease (WD). To the author's knowledge, there are no methods capable of determining reliably CuEXC to meet the requirements and challenges faced by a clinical trial. The present work describes a novel speciation strategy for the determination of the main Cu-species in human serum by anion-exchange high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A label-free protein quantification approach was conducted where the concentration of Cu associated to the protein fraction was based on its relative peak area distribution and the total Cu concentration in the sample. Such a methodology was characterized in terms of selectivity, sensitivity, precision, and robustness. Due to the lack of speciated Cu-reference materials, protein recovery was assessed by comparison with that of species-specific (SS) isotope dilution (ID). For this, a double SS HPLC-ICP-IDMS method for Cu-albumin was developed and presented here for the first time. Three human sera (two frozen LGC8211 and ERM®-DA250a, and the lyophilised Seronorm™ Human) were analyzed using both the relative and ID quantification methods. The validated relative approach, with relative expanded uncertainties (k = 2) between 5.7 and 10.1% for Cu-albumin concentrations ranging from 112 to 455 μg kg-1 Cu, was found to be able to discriminate between healthy and WD populations in terms of Cu-albumin content. Also, using such methodology, underestimation of CuEXC by the classical EDTA/ultrafiltration method was demonstrated. The methodology developed in this work will be invaluable for quality control assessment and WD drug monitoring. This work describes a Cu-protein quantification approach for the determination of exchangeable Cu relevant to Wilson's Disease.

Development and Validation of an Anion Exchange High-Performance Liquid Chromatography Method for Analysis of Empty Capsids and Capsids Encapsidating Genetic Material in a Purified Preparation of Recombinant Adeno-Associated Virus Serotype 5.

The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the laboratory to the clinic. The active deployment of these analytical technologies for process and product characterization has helped define critical quality attributes and improve the quality of the clinical grade material. In this article, we report an anion exchange high-performance liquid chromatography (AEX-HPLC) method for relative and as well as absolute quantification of empty capsids (EC) and capsids encapsidating genetic material (CG) in purified preparations of adeno-associated virus (AAV) using serotype 5 as a model. The selection of optimal chromatographic buffer composition and step-gradient elution protocol offered baseline separation of EC and CG in the form of two peaks, as validated with the respective reference standards. The native amino acid fluorescence-based detection offered excellent linearity with a correlation coefficient of 0.9983 over two-log dilutions of the sample. The limit of detection and limit of quantification values associated with the total AAV5 capsid assay are 3.1E + 09 and 9.5E + 09, respectively. AEX-HPLC showed method comparability with the analytical ultracentrifugation (AUC) method for determination of relative proportions of EC and CG, supporting the reported HPLC method as an easy-to-access alternative to AUC with operational simplicity. Moreover, rapid and easy adaptation of this method to AAV8 material also demonstrated the robustness of the proposed approach.

Comparison of cholesterol levels among lipoprotein fractions separated by anion-exchange high-performance liquid chromatography in periparturient Holstein-Friesian dairy cows.

Changes in lipoprotein profiles occur in dairy cows during the periparturient period and in cows with transition cow disease. Here, the lipoprotein profiles of Holstein–Friesian dairy cows during the periparturient period were obtained by anion-exchange, high-performance liquid chromatography to evaluate the usefulness of lipoprotein profile evaluation during the periparturient period and in cows with fatty liver and milk fever. Lipoprotein levels (including total and high- (HDL-C) and low-density lipoprotein (LDL-C) cholesterol) in 10 healthy cows were low 4 weeks prepartum, with the lowest values at calving or within 1 week of calving; the values increased at 8 weeks postpartum. The lipoprotein levels were measured in 16 cows diagnosed with fatty liver (n=10) or milk fever (n=6) and compared to 10 healthy dairy cows. A significant difference was observed in HDL-C between healthy cows (at calving and 1 week postpartum), and the fatty liver and milk fever cows. Cows with fatty liver and milk fever had a lower mean HDL-C than the 10 healthy dairy cows at calving and 1 week postpartum. HDL-C might be a good indicator of energy balance for differentiating healthy cows from those with transition cow disease.

Verification of Low-Density Lipoprotein Cholesterol Levels Measured by Anion-Exchange High-Performance Liquid Chromatography in Comparison with Beta Quantification Reference Measurement Procedure.

A new lipoprotein testing method based on anion-exchange HPLC (AEX-HPLC) was recently established. We verified the accuracy of LDL-C levels, a primary therapeutic target for the prevention of cardiovascular disease (CVD), measured by AEX-HPLC comparing with LDL-C levels measured by beta quantification-reference measurement procedure (BQ-RMP), homogenous assays, and calculation methods. We compared LDL-C levels measured by AEX-HPLC (adLDL-Ch: LDL-Ch and IDL-Ch) and BQ-RMP using blood samples from 52 volunteers. AdLDL-Ch levels were also compared with those measurements by homogeneous assays and calculation methods (Friedewald equation, Martin equation, and Sampson equation) using blood samples from 411 participants with dyslipidemia and/or type 2 diabetes. The precision and accuracy of adLDL-Ch were verified by BQ-RMP. The mean percentage bias [bias (%)] for LDL-C was 1.2%, and the correlation was y = 0.990x + 3.361 (r = 0.990). These results met the acceptable range of accuracy prescribed by the National Cholesterol Education Program. Additionally, adLDL-Ch levels were correlated with LDL-C levels measured by the 2 homogeneous assays (r > 0.967) and the calculation methods (r > 0.939), in serum samples from patients with hypertriglyceridemia. AEX-HPLC is a reliable method for measuring LDL-C levels for CVD risk in daily clinical laboratory analyses.

Arsenic speciation in fish on the market

Arsenic speciation in fish samples on the market was analyzed using gradient anion exchange high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICPMS) detection. Freeze-dried samples were extracted by methanol and water mixture (8:2v/v) with a high-speed Soxhlet system; the concentration of arsenobetaine (AsB), dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), arsenate (As(V)) and arsenite (As(Ⅲ)) in the extract were then determined using HPLC-ICPMS. The method for determining arsenobetaine concentration was confirmed using standard reference material (SRM) of BCR 627 (tuna fish) and DORM-2 (dogfish muscle); recovery rates were 95 and 90%, respectively. The arsenic compounds in 60 market-ready fish muscle samples were investigated. The predominant arsenic compound found in samples was AsB. Arsenate was detected in low concentrations (ranged in 0.02-0.34 mg/kg As for fresh weight), whereas DMAA, MMAA and arsenite content were undetectable. Average AsB content in cephalopods, small fish and large fish were 5.42, 1.57 and 1.54 mg/kg As (fresh weight), respectively. The weekly intake of As was calculated based on the consumption of fish by Taiwanese residents. The calculated results demonstrated that the intake is 30.6 μg/kg (total As) body weight/week, higher than the acceptable weekly intake of 15 μg/kg body weight/week for inorganic arsenic that was suggested by WHO. However, around 87% of As in fish muscle was AsB. AsB is non-toxic and non-carcinogenic to humans, and is rapidly excreted after ingestion. Therefore, intake of fish muscle is low risk based on investigation results.

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC.

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing 'high-energy' diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing ‘high-energy’ diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.