Ia Determinants Research Articles

Characterization and Quantification of Arsenic Species in Foodstuffs of Plant Origin by HPLC/ICP-MS.

Arsenic is a well-known carcinogenic, mutagenic and toxic element and occurs in the environment both as inorganic arsenic (iAs) and organoarsenical compounds (oAsCs). Since the toxicity of arsenic compounds depends on their chemical form, the identification and determination of arsenic species are essential. Recently, the European Food Safety Authority, following the European Commission request, published a report on chronic dietary exposure to iAs and recommended the development and validation of analytical methods with adequate sensitivity and refined extraction procedures for this determination. Moreover, the authority called upon new arsenic speciation data for complex food matrices such as seaweeds, grains and grain-based products. Looking at this context, an optimized, sensitive and fast analytical method using high performance liquid chromatography followed by inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) was developed for the determination of iAs (sum of arsenite-AsIII and arsenate-AsV) and the most relevant oAsCs, arsenobetaine, dimethylarsinic acid and monomethylarsonic acid. The method was validated with satisfactory results in terms of linearity, sensitivity, selectivity, precision, recovery, uncertainty, ruggedness and matrix effect, and then successfully applied for the analysis of several matrices, i.e., processed and unprocessed cereal and cereal products, fruits, vegetables, legumes, seaweeds, nuts and seeds. The results obtained indicate that not only seaweed and rice matrices but also many cereals, legumes and plant-based foods for infants and young children contain significant concentrations of iAs and oAsCs. These findings contribute to the data collection necessary to assess the role of these matrices in the total arsenic exposure and if specific maximum limits have to be established.

Determination of inorganic arsenic in Argentinean rice by selective HGAAS: Analytical performance for paddy, brown and polished rice

Inorganic As (iAs) species require strict monitoring in foodstuff due to their toxicity. Rice is of particular concern, with recent legislations establishing limits for these species. In this work we develop a method based on selective hydride generation atomic absorption spectrometry (HGAAS) for the determination of iAs in different types of rice (polish, parboiled, brown and paddy rice). The multivariate design of experiments used for optimization showed interactions between different factors involved in hydride generation. Matrix effects were systematically studied, where the flow rate of the carrier gas proved to be a critical parameter for the minimization of matrix interferences. Nevertheless, matrix-matching was found to be necessary for accurate quantification of paddy rice samples. Comparison of the iAs concentrations obtained by HGAAS and HPLC-ICP-MS showed non-significant differences between the methods. Analysis of 86 rice samples from the province of Entre Ríos showed relatively low concentrations of iAs (<282 μg kg−1 for paddy rice and <120 μg kg−1 for polished rice), regardless of total As concentrations (which ranged from 63 μg kg−1 to 932 μg kg−1), suggesting that the rice analyzed tends to accumulate As in its organic forms.

Concentrations and health risks of inorganic arsenic and methylmercury in shellfish from typical coastal cities in China: A simultaneous analytical method study.

Shellfish consumption is an important pathway for coastal residents exposure to inorganic arsenic (iAs) and methylmercury (MeHg). An integrated method had been developed for simultaneous determination of iAs and MeHg in shellfish with microwave-assisted extraction and high performance liquid chromatography-inductively coupled plasma mass spectrometry identification. The limits of detection were 0.0520, 0.1003 and 0.0015 mg/kg (dry weight) for AsIII, AsV and MeHg, respectively. The proposed method was applied to evaluate the contents and health risks of iAs and MeHg in shellfish from the typical coastal cities (Lianyungang and Yantai) in China. The concentrations of iAs and MeHg in fresh shellfish were 0.10-1.69 mg/kg and 0.46-6.38 μg/kg, respectively. The target hazard quotient and target cancer risk values of iAs were the highest for the children and adults in Lianyungang, and were respectively larger than 1 and 1 × 10-4, indicating both non-carcinogenic and carcinogenic risks of iAs exposure were significant in this area.

Selective and fast screening method for inorganic arsenic in seaweed using hydride generation inductively coupled plasma mass spectrometry (HG-ICPMS)

A method for the determination of inorganic arsenic (iAs) without chromatography using hydride generation — inductively coupled plasma mass spectrometry (HG-ICPMS) is applied to 40 brown macroalgae samples (Fucus vesiculosus, Ascophyllum nodosum, Saccharina latissima). Determining iAs in seaweed matrices is a challenge and, in this study, using 2% HNO3 and 3% H2O2 extraction media the HG-ICPMS outperformed the most commonly used method of HPLC-ICPMS for the determination of iAs in the seaweed samples. There was a good comparison between the simple and fast HG-ICPMS and the more accurate but time-consuming HPLC-HG-ICPMS. Linear regression showed reasonable linearity (R2 of 0.82), with a slope slightly higher than 1 (1.15 ± 0.09) and intercept 0.03 ± 0.01 when comparing the concentrations of iAs determined with the two methods. Hence there is a minor overestimation of iAs with HG compared to the HPLC-HG, however, the method is well suited as a quick screening method. The iAs concentration was below 1% of total arsenic concentration for all the seaweed samples irrespective of using HG-ICPMS or HPLC-HG-ICPMS.

Determination and Uncertainty Analysis of Inorganic Arsenic in Husked Rice by Solid Phase Extraction and Atomic Absorption Spectrometry with Hydride Generation.

This study enables the selective determination of inorganic arsenic (iAs) with a low detection limit using an economical instrument [atomic absorption spectrometer with hydride generation (HG)] to meet the regulatory requirements as per European Commission (EC) and Codex guidelines. Dry rice samples (0.5 g) were diluted using 0.1 M HNO3-3% H2O2 and heated in a water bath (90 ± 2°C) for 60 min. Through this process, all the iAs is solubilized and oxidized to arsenate [As(V)]. The centrifuged extract was loaded onto a preconditioned and equilibrated strong anion-exchange SPE column (silica-based Strata SAX 500 mg/6 mL), followed by selective and sequential elution of As(V), enabling the selective quantification of iAs using atomic absorption spectrometry with HG. In-house validation showed a mean recovery of 94% and an LOQ of 0.025 mg/kg. The repeatability (HorRatr) and reproducibility (HorRatR) values were <2, meeting the performance criteria mandated by the EC. The combined standard measurement uncertainty by this method was less than the maximum standard measurement uncertainty; thus, the method can be considered for official control purposes. The method was applied for the determination of iAs in husked rice samples and has potential applications in other food commodities.

Inorganic Arsenic Determination in Food: A Review of Analytical Proposals and Quality Assessment Over the Last Six Years.

Here we review recent developments in analytical proposals for the assessment of inorganic arsenic (iAs) content in food products. Interest in the determination of iAs in products for human consumption such as food commodities, wine, and seaweed among others is fueled by the wide recognition of its toxic effects on humans, even at low concentrations. Currently, the need for robust and reliable analytical methods is recognized by various international safety and health agencies, and by organizations in charge of establishing acceptable tolerance levels of iAs in food. This review summarizes the state of the art of analytical methods while highlighting tools for the assessment of quality assessment of the results, such as the production and evaluation of certified reference materials (CRMs) and the availability of specific proficiency testing (PT) programmes. Because the number of studies dedicated to the subject of this review has increased considerably over recent years, the sources consulted and cited here are limited to those from 2010 to the end of 2015.

Simultaneous quantification of picfeltarraenins IA and IB in rat plasma by UPLC–MS/MS: Application to a pharmacokinetic study

A simple and rapid quantitative UPLC-MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C18 (50mm×2.1mm, 1.8μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m/z 785.4→639.5, m/z 815.5→669.5, and m/z 763.5→455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5-1150ng/mL and 13.0-1300ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats.

Determination of inorganic arsenic in rice by solid phase extraction and hydride generation atomic fluorescence spectrometry

Low-cost, simple methods are needed for accurate determination of iAs in food crops. Total arsenic (As) from rice was extracted and As5+ reduced to As3+. The combined As3+ was separated then quantified. This method appears suitable for general use due to its low cost.

Speciation without Chromatography Using Selective Hydride Generation: Inorganic Arsenic in Rice and Samples of Marine Origin

Because of the toxicity of inorganic arsenic (iAs), only iAs needs to be monitored in food and feedstuff. This demands the development of easy and quick analytical methods to screen large number of samples. This work focuses on hydride generation (HG) coupled with an ICPMS as an arsenic detector where the HG is added as a selective step to determine iAs in the gaseous phase while organically bound As remains in the solution. iAs forms volatile arsine species with high efficiency when treated with NaBH4 at acidic conditions, whereas most other organoarsenic compounds do not form any or only less volatile arsines. Additionally, using high concentrations of HCl further reduces the production of the less volatile arsines and iAs is almost exclusively formed, therefore enabling to measure iAs without a prior step of species separation using chromatography. Here, we coupled a commercially available HG system to an ICPMS and optimized for determination of iAs in rice and samples of marine origin using different acid concentrations, wet and dry plasma conditions, and different reaction gas modes. Comparing this method to conventional HPLC-ICPMS, no statistical difference in iAs concentration was found and comparable limits of detections were achieved using less than half the instrument time.

A need for determination of arsenic species at low levels in cereal-based food and infant cereals. Validation of a method by IC–ICPMS

The present study arose from the need to determine inorganic arsenic (iAs) at low levels in cereal-based food. Validated methods with a low limit of detection (LOD) are required to analyse these kinds of food. An analytical method for the determination of iAs, methylarsonic acid (MA) and dimethylarsinic acid (DMA) in cereal-based food and infant cereals is reported. The method was optimised and validated to achieve low LODs. Ion chromatography-inductively coupled plasma mass spectrometry (IC–ICPMS) was used for arsenic speciation. The main quality parameters were established. To expand the applicability of the method, different cereal products were analysed: bread, biscuits, breakfast cereals, wheat flour, corn snacks, pasta and infant cereals. The total and inorganic arsenic content of 29 cereal-based food samples ranged between 3.7–35.6 and 3.1–26.0μgAskg−1, respectively. The present method could be considered a valuable tool for assessing inorganic arsenic contents in cereal-based foods.

Simultaneous Determination of Cryptotanshinone, Tanshinone IA and Tanshinone IIA in Three Processing Products of White Flower S. Miltiorrhiza by HPLC

A reversed-phase high performance liquid chromatographic method was established for the simultaneous determination of tanshinones in three processing products of white flower Radix salvia miltiorrhiza. Cryptotanshinone, tanshinone IA and tanshinone IIA were successfully separated on a Yilite C18 column (250 mm x 4.6 mm, 5 µm). The mobile phase was a mixture of methanol, water, tetrahydrofuran and glacial acetic acid (70:24:5:1, v/v/v/v), employing isocratic elution at a flow rate of 1.0 mL/min. Detection was 254 nm. Regression equations revealed good linear relationship between the peak areas of the compounds and their concentrations (correlation coefficients: 0.9994 for cryptotanshinone, 0.9996 for tanshinone I A and 0.9996 for tanshinone IIA). The recoveries were between 98.03 % and 103.1 %. The method is simple, accurate and effective and can be used to determine the contents of tanshinones in processing products of Salvia miltiorrhiza bge. f. alba, and the contents of three tanshinones presented the stir-frying processing＜the crude drugs＜the wine prosessing.

Simultaneous Determination of Escin Ia and Its Isomer Isoescin Ia by LC–MS–MS: Application to a Pharmacokinetic Study of Escin Ia in Rats

A rapid and sensitive LC–MS–MS method for the simultaneous determination of escin Ia and isoescin Ia in rat plasma, urine, feces and bile samples was developed and validated. Analytes and telmisartan [internal standard (IS)] were extracted by solid-phase extraction on C18 cartridges. Components in the extract were separated on an HC-C18 column (5 μm, 150 × 4.6 mm i.d.) using 10 mM ammonium acetate–methanol–acetonitrile (40:30:30, v/v/v) as the mobile phase. The method demonstrated good linearity from 5 ng mL−1 (LLOQ) to 1,500 ng mL−1 for both escin Ia and isoescin Ia. Intra- and inter-day precision measured as RSD was within ±15%. Recoveries and matrix effects of both escin Ia and isoescin Ia were satisfactory in all four matrices examined. The method was successfully applied to a pharmacokinetic study in Wistar rats after a single intravenous administration of escin Ia at the dose of 1.0 mg kg−1.

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Improvements in the prevention or control of rejection of the kidney and liver have been largely interchangeable (1, 2) and then applicable, with very little modification, to thoracic and other organs. However, the mechanism by which anti rejection treatment permits any of these grafts to be “accepted” has been an immunological enigma (3, 4). We have proposed recently that the exchange of migratory leukocytes between the transplant and the recipient with consequent long-term cellular chimerism in both is the basis for acceptance of all whole-organ allografts and xenografts (5). Although such chimerism was demonstrated only a few months ago, the observations have increased our insight into transplantation immunology and have encouraged the development of alternative therapeutic strategies (6).

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Effect of cyclosporine A on the regulation of Ia antigen keratinocytes expression

Since many skin diseases characterized by positive Ia keratinocytes show improvement with cyclosporine therapy, the purpose of this study was to determine whether cyclosporine A (CyA) alters the expression of Ia keratinocytes. Nude mice were injected with normal mouse serum (NMS) to induce keratinocyte expression of the Ia antigen. The injected mice were then divided into four groups: one was treated with oral CyA; the second was treated topically with CyA twice a day; the third was treated topically with olive oil; and the fourth was injected with nude mouse serum. The third and fourth groups served as Ia positive and Ia negative controls, respectively. The mice were treated during the first 10 days after the injections. On Day 10, epidermal sheets were analyzed for Ia expression. Analysis was made by an indirect immunoperoxidase staining method using monoclonal antibodies specific for Ia determinants. Quantitation of the number of Langerhans cells was analyzed on epidermal sheets using immunodiagnostic reagents, anti-MHC-Ia, and surface ectoenzyme, ATPase. A significant reduction of Ia-positive keratinocytes was noted in the oral CyA group vs topical and olive oil groups (64.9 ± 29.9% vs 20.1 ± 18.7%, respectively, P < 0.01). In a second set of experiments mice were injected with NMS, but treatment was started only on Day 10 after injections, for 10 days. The results showed that CyA failed to down-regulate Ia expression. Topical and systemic CyA did not modify Langerhans cell population. The present study showed that systemic administration of CyA significantly reduced Ia induction by keratinocytes of nude mice that were injected with NMS.

Adoptive immunotherapy is suppressed in C57BL/6J AND B6.C‐H‐2bm12 mice following recognition of congenic class II MHC antigen determinants

In previous reports, we have shown that adoptive transfer of tumor-sensitized T (immune) cells to tumor-bearing mice that have received a prior injection of cyclophosphamide (CY) results in the induction of permanent tumor regression in syngeneic strains. It has also been shown that adoptive immunotherapy results in an increased expression of class II MHC antigens (Ia) by macrophages at the tumor site and in the peritoneal cavity and is associated with expansion of CD4+ and CD8+ T cells at the site of tumor regression. In this report, we use the Ia mutant strain, B6.C-H-2bm12, and congenic C57BL/6J (B6) mice to determine the relative importance of Ia expression in regulating amplification of immune responses following adoptive immunotherapy and to test the hypothesis that recognition of congenic Ia determinants will result in the induction of suppressor mechanisms that down-regulate active immunity. The data indicated that the adoptive transfer of immune congenic T cells (B6 immune cells into CY-treated tumor-bearing bm12 mice and vice-versa) down-regulated active immunity, while the transfer of syngeneic immune cells resulted in permanent tumor regression. By using radiation-chimeric mice, it was shown that down-regulation was associated with incompatibility of the transferred immune T cells and bone-marrow-derived cells (putatively expressing the Ia haplotype of donor-derived macrophages) and the appearance of long-lived splenic suppressor cells. Suppression per se was shown to be induced in response to the Ia difference between the two strains and not in response to the MCA/76-9 sarcoma, which appears to be one of the few tumors that can induce active immunity in both the syngeneic and congenic strains without obvious subsequent down-regulation by suppression.

ChemInform Abstract: Strong Interaction Between an Aliphatic Carbon‐Hydrogen Bond and a Metal Atom: The Structure of (Diethylbis(1‐pyrazolyl)borato)allyldicarbonylmolybdenum(II).

AbstractThe single‐crystal X‐ray structure determination of (Ia) (space group P1, Z=2) shows a very short Mo to H distance, suggesting a strong interaction between an aliphatic C‐H bond and the Mo atom.

Cell surface molecules involved in early events in T-cell mitogenic stimulation by staphylococcal enterotoxins.

We tested the mitogenic response to staphylococcal enterotoxin (SE) type A and SE type B in spleen cells from five strains of mice and found consistent and significant differences among the strains. We chose to study the mitogenic responses of two of these strains, C58BL/6J and BALB/cJ, in greater detail. We investigated the effects of specific monoclonal antibodies to cell surface determinants on SE-induced mitogenesis. Monoclonal antibodies against Ia (class II major histocompatibility complex) determinants blocked SE-induced mitogenesis. Both I-A and I-E molecules can participate in the stimulation, and in BALB/cJ mice which express both types of class II molecules both must be blocked to prevent mitogenesis. Mitogenesis was not inhibited by monoclonal antibodies specific for class I major histocompatibility complex antigens or monoclonal antibodies specific for Mac-1, Lyt-1, or Lyt-2 cell surface proteins. Monoclonal antibodies specific for the T-cell surface antigens L3T4 and T3 also substantially inhibited SE-induced mitogenesis. This implicates participation of the T-cell antigen receptor complex in stimulation induced by the SEs. Elimination of L3T4+ helper-inducer T cells abolished the mitogenic response of spleen cells to SE. Reconstitution of L3T4-depleted spleen cells with L3T4+ T cells showed that the level of the mitogenic response was directly proportional to the number of L3T4+ cells added. Elimination of Lyt-2+ cells resulted in a 50% decrease in the response to SEs. These results indicate that L3T4+ T cells are required for the mitogenic response to SE, but both L3T4+ and Lyt 2+ T cells participate in SE-induced mitogenesis. Our results suggest that both Ia and the T-cell antigenic receptor complex are involved in SE-induced mitogenesis.

Activation of Lyt-2+ (CD8+) and L3T4+ (CD4+) T cell subsets by anti-receptor antibody.

The mAb F23.1, specific for V beta 8-related determinants on the TCR, was used to study the requirements for TCR cross-linking and for accessory cells (AC) in the induction of proliferation or IL-2 responsiveness in L3T4+ (CD4+) and Lyt-2+ (CD8+) T cells. T cells were exposed in vitro to soluble native F23.1 antibody, to heteroconjugates composed of the Fab fragments of F23.1 linked to Fab fragments of antibodies specific for Ia determinants on AC, or to F23.1 immobilized on an insoluble matrix. Soluble F23.1 antibody-induced proliferation in naive T cells only in the presence of both AC and exogenous IL-2, and these responses were confined to Lyt-2+ T cells. In contrast, heteroconjugates capable of crosslinking F23.1+ TCR to AC surface Ia determinants were capable of inducing proliferation in both L3T4+ and Lyt-2+ T cells in the absence of added lymphokine. Moreover, binding to and presumably multi-valent crosslinking of the TCR by immobilized F23.1 was sufficient to induce proliferation in both Lyt-2+ and L3T4+ T cells in the absence of AC or exogenous IL-2. Further, it was found that the conditions necessary for T cell growth factor secretion paralleled closely those required for induction of T cell proliferation in the absence of added lymphokine, suggesting that production of endogenous lymphokine might be the limiting process for triggering of T cell proliferation. Taken together, these findings suggest that under optimal conditions of TCR cross-linking, TCR occupancy and cross-linking is sufficient to deliver all of the signals necessary to initiate proliferation in naive populations of both L3T4+ and Lyt-2+ T cells. However, when conditions for TCR signaling are suboptimal, as may be the case for normal Ag-mediated stimulation, a role for second signals delivered by AC or exogenous lymphokines can become critical for T cell activation.

Murine Macrophage Cell Line AP284 Presents Antigen to Cloned MT4 +, Lyt-2 − T Cells in vitro and in vivo

A murine macrophage cell line AP284 that appeared to be mature in phenotype was isolated. After repeated cloning, the cell line expressed the markers Mac-1, Mac-2, Mac-3, 2.4G2, F4/80 as well as Ia antigens. Moreover, it was positive for the enzymes nonspecific esterase and acid phosphatase, negative for alkaline phosphatase and was able to phagocytize latex beads. We studied whether this cell line was able to present antigen to cloned MT4 +, Lyt-2 − T cells specific for methylated bovine serum albumin (mBSA) or ovalbumin (OVA). The in vitro proliferative response of the cloned T cells specific for mBSA or OVA was found to be effectively supported by AP284. This proliferation could be blocked by monoclonal antibodies against Ia determinants. AP284 also effectively presented antigen in vivo as was shown in a foot swelling assay measuring delayed type hypersensitivity (DTH) to mBSA caused by specific cloned T cells with the helper phenotype. This offers a unique model system for studying the process of antigen presentation in which both the antigen presenting cells and the T cells are monoclonal.