Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Sialyl Lewis X Defines an Activated and Functional Regulatory T Cell Subpopulation in Mice.

Attempts have been made to elucidate the functional markers of regulatory T cells (Tregs), CD4+Foxp3+ T cells with an immunosuppressive function. Sialyl Lewis X (sLex), a tetrasaccharide Ag, is involved in leukocyte trafficking as selectin ligands and is a marker of highly differentiated Tregs in humans. However, the importance of sLex in murine Tregs remains unknown. In this study, we report that sLex defines the activated and functional subset of murine Tregs. The contact hypersensitivity model showed that murine Tregs strongly express sLex upon activation, accompanied by functional Treg marker elevation, such as Foxp3, CD25, CD103, CD39, and granzyme B. RNA sequencing analysis revealed sLex-positive (sLex+) Tregs expressed genes involved in Treg function at a higher level than sLex-negative (sLex-) Tregs. Using an in vitro suppression assay, we found that sLex+ Tregs could more efficiently suppress naive CD4+ T cell proliferation than sLex- Tregs. In the murine contact hypersensitivity elicitation model, the topical sLex+ Treg injection into the ears suppressed ear inflammation more efficiently than that of sLex- Tregs. Our results indicate that sLex could serve as a unique surface marker of activated and functional Tregs with immunosuppressive functions in mice.

Prevention of experimental autoimmune encephalomyelitis by targeting 6-sulfo sialyl Lewis X glycans involved in lymphocyte homing.

Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.

Apportioning Atmospheric Ammonia Sources across Spatial and Seasonal Scales by Their Isotopic Fingerprint.

Mitigating ammonia (NH3) emissions is a significant challenge, given its well-recognized role in the troposphere, contributing to secondary particle formation and impacting acid rain. The difficulty arises from the highly uncertain attribution of atmospheric NH3 to specific emission sources, especially when accounting for diverse environments and varying spatial and temporal scales. In this study, we established a refined δ15N fingerprint for eight emission sources, including three previously overlooked sources of potential importance. We applied this approach in a year-long case study conducted in urban and rural sites located only 40 km apart in the Shandong Peninsula, North China Plain. Our findings highlight that although atmospheric NH3 concentrations and seasonal trends exhibited similarities, their isotopic compositions revealed significant distinctions in the primary NH3 sources. In rural areas, although agriculture emerged as the dominant emission source (64.2 ± 19.5%), a previously underestimated household stove source also played a considerably greater role, particularly during cold seasons (36.5 ± 12.5%). In urban areas, industry and traffic (33.5 ± 15.6%) and, surprisingly, sewage treatment (27.7 ± 11.3%) associated with high population density were identified as the major contributors. Given the relatively short lifetime of atmospheric NH3, our findings highlight the significance of the isotope approach in offering a more comprehensive understanding of localized and seasonal influences of NH3 sources compared to emissions inventories. The refined isotopic fingerprint proves to be an effective tool in distinguishing source contributions across spatial and seasonal scales, thereby providing valuable insights for the development of emission mitigation policies aimed at addressing the increasing NH3 burden on the local atmosphere.

Long-Term Source Apportionment of Ammonium in PM2.5 at a Suburban and a Rural Site Using Stable Nitrogen Isotopes.

Ammonia gas (NH3) is an important alkaline air pollutant and a precursor to particulate matter, and its source has been thought to be agricultural, but in recent years, nonagricultural sources have been suspected. In this study, stable nitrogen isotope ratios of ammonium (δ15N-NH4+) in fine particulate matter (PM2.5) were measured at a suburban site and a rural site in Japan. Then, the long-term sources of NH4+ were identified using the δ15N-NH3 and an isotopic mixing model. The results showed that the averaged contribution from nonagricultural sources was 67% at the suburban site and 78% at the rural site. We also reanalyzed NH3 data collected at the same location. The result showed that the averaged contribution of nonagricultural sources to NH3 was 39%. This result is reasonable because bottom-up estimates are close to the contribution, and the NH3 emissions are affected by warm season activities in the rural site. It was first found that the sources vary greatly, depending on the gas and particles. Back-trajectory results suggested that PM2.5 measured at the rural site was derived from the Asian continent. We inferred that the NH4+ had been formed on the continent and that these particles thus represent transboundary pollution.

Sulfated Glycans Recognized by S1 Monoclonal Antibody can Serve as a Diagnostic Marker for Malignant Pleural Mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a malignant neoplasm of the pleura caused by asbestos exposure. For diagnosis of MPM, immunohistochemistry using multiple markers is recommended to rule out differential diagnoses, such as pulmonary adenocarcinoma. However, the specificity of currently used markers is not fully satisfactory. We previously developed a monoclonal antibody named S1, which recognizes 6-sulfo sialyl Lewis x, an L-selectin ligand expressed on high endothelial venules. During the screening process, we discovered that this antibody stained normal pleural mesothelium. This finding prompted us to hypothesize that the epitope recognized by S1 might serve as a new diagnostic marker for MPM. METHODS: To test this hypothesis, we immunostained human MPM (n = 22) and lung adenocarcinoma (n = 25) tissues using S1 antibody. RESULTS: 77.3% of MPM were S1 positive, and if limited to epithelioid type, the positivity rate was 100%, while that of lung adenocarcinoma was only 36.0%. Statistical analysis revealed a significant difference in the S1 positivity rate between each disease. Furthermore, immunohistochemistry using a series of anti-carbohydrate antibodies combined with glycosidase digestion revealed the structure of sulfated glycans expressed in MPM to be 6-sulfo sialyl N-acetyllactosamine attached to core 2-branched O-glycans. CONCLUSION: We propose that the S1 glycoepitope could serve as a new diagnostic marker for MPM.

Cancer Malignancy Is Correlated with Upregulation of PCYT2-Mediated Glycerol Phosphate Modification of α-Dystroglycan.

The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on α-dystroglycan (α-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the α-DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as cytidine 5'-diphosphate-glycerol (CDP-Gro) synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.

Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans.

α-Dystroglycan (α-DG) is a glycoprotein specifically modified with O-mannosyl glycans bearing long polysaccharides, termed matriglycans, which comprise repeating units of glucuronic acid and xylose. The matriglycan is linked to the O-mannosyl glycan core through two ribitol phosphate units that can be replaced with glycerol phosphate (GroP) units synthesized by fukutin and fukutin-related protein that transfer GroP from CDP-Gro. Here, we found that forced expression of the bacterial CDP-Gro synthase, TagD, from Bacillus subtilis could result in the overproduction of CDP-Gro in human colon carcinoma HCT116 cells. Western blot and liquid chromatography-tandem mass spectrometry analyses indicated that α-DG prepared from the TagD-expressing HCT116 cells contained abundant GroP and lacked matriglycans. Using the GroP-containing recombinant α-DG-Fc, we developed a novel monoclonal antibody, termed DG2, that reacts with several truncated glycoforms of α-DG, including GroP-terminated glycoforms lacking matriglycans; we verified the reactivity of DG2 against various types of knockout cells deficient in the biosynthesis of matriglycans. Accordingly, forced expression of TagD in HCT116 cells resulted in the reduction of matriglycans and an increase in DG2 reactivity. Collectively, our results indicate that DG2 could serve as a useful tool to determine tissue distribution and function of α-DG lacking matriglycans under physiological and pathophysiological conditions.

Ion-exchange resin and denitrification pretreatment for determining δ15 N-NH4 + , δ15 N-NO3 - , and δ18 O-NO3 - values.

RATIONALE: There has never been a highly sensitive method for simultaneously measuring the δ15 N and δ18 O values of nitrate ions (NO3 - ) and the δ15 N values of ammonium ions (NH4 + ) in particulate matter using denitrifying bacteria. In this study, we explored a method that combines use of an anion-exchange resin and denitrifying bacteria to make such measurements. METHODS: The δ15 N-NH4 + values of samples obtained using the hypobromite and denitrifying bacteria method were measured by isotope ratio mass spectrometry. Tests (effect of flow rate, breakthrough, and acid concentration) were conducted to verify the removal of NO3 - using an AG1-X8 anion-exchange resin for NH4 + measurements and the enrichment of NO3 - . For aerosol samples, the optimized method was used to measure the δ15 N-NO3 - , δ18 O-NO3 - , and δ15 N-NH4 + values of atmospheric particulate matter (PM2.5 , aerodynamic diameter < 2.5 µm). RESULTS: The δ15 N-NO3 - and δ18 O-NO3 - values measured following extraction with 1-6 mol/L HCl, at sample flow rates of 1-2 mL/min, with total anion amounts of less than 2.2 mmol, and in concentration tests were found to be in very close agreement with reagent values. The precisions and the accuracies of the δ15 N-NH4 + and δ15 N-NO3 - values were in all cases less than 1‰. In addition, the accuracies for the δ18 O-NO3 - values were less than 1.4‰ and generally acceptable. The δ15 N-NH4 + , δ15 N-NO3 - , and δ18 O-NO3 - values in six PM2.5 samples were similar to those reported in previous studies. CONCLUSIONS: Our proposed method for removing anions using AG1-X8 resin, for isotopic analysis using denitrifying bacteria, and for concentrating samples containing low concentrations of NO3 - will make it possible to perform high-precision and accurate analyses easily and inexpensively. These methods are applicable not only to aerosols, but also to samples from diverse locations such as rivers, oceans, and Antarctica.

Carbon isotope ratio of organic acids in sake and wine by solid-phase extraction combined with LC/IRMS.

We developed an analytical procedure for determining the δ13C values of organic acids in sake and wine using solid-phase extraction combined with liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). First, the solid-phase extraction (SPE) procedure was performed and various tests were conducted to extract organic acids from alcoholic beverages using the simulated sake sample. Under the optimal SPE procedure, high recovery rates (96-118%) and good accuracies (≤ 0.7‰) were thus achieved for the simulated sake and wine samples. Next, we determined the δ13C of organic acid (tartaric acid, malic acid, lactic acid, succinic acid) in 9 sake and 11 wine samples. Finally, the δ13C values of lactic acid in nine sake samples suggested that lactic acid had been added during the brewing process. The high correlation between the δ13C values of tartaric acid and malic acid in 11 wine samples was consistent with their common source, grapes. This analytical method may help to identify when organic acids have been added to sake and wine and to elucidate the process of organic acid production therein. Graphical abstract.

Therapeutic Effects of an Anti-sialyl Lewis X Antibody in a Murine Model of Allergic Asthma.

Asthma is an allergic disease that causes severe infiltration of leukocytes into the lungs. Leukocyte infiltration is mediated by the binding of sialyl Lewis X (sLex) glycans present on the leukocytes to E-and P-selectins present on the endothelial cells at the sites of inflammation. Here, we found that mouse eosinophils express sLex glycans, and their infiltration into the lungs and proliferation in the bone marrow were significantly suppressed by an anti-sLex monoclonal antibody (mAb) F2 in a murine model of ovalbumin-induced asthma. The percentage of eosinophils in the bronchoalveolar lavage fluid and bone marrow and serum IgE levels decreased significantly in the F2-administered mice. Levels of T helper type 2 (Th2) cytokines and chemokines, involved in IgE class switching and eosinophil proliferation and recruitment, were also decreased in the F2-administered mice. An ex vivo cell rolling assay revealed that sLex glycans mediate the rolling of mouse eosinophils on P-selectin-expressing cells. These results indicate that the mAb F2 exerts therapeutic effects in a murine model of allergen-induced asthma, suggesting that sLex carbohydrate antigen could serve as a novel therapeutic target for allergic asthma.

Chaperone-mediated secretion switching from early to middle substrates in the type III secretion system encoded by Salmonella pathogenicity island 2.

The bacterial type III secretion system (T3SS) delivers virulence proteins, called effectors, into eukaryotic cells. T3SS comprises a transmembrane secretion apparatus and a complex network of specialized chaperones that target protein substrates to this secretion apparatus. However, the regulation of secretion switching from early (needle and inner rod) to middle (tip/filament and translocators) substrates is incompletely understood. Here, we investigated chaperone-mediated secretion switching from early to middle substrates in the T3SS encoded by Salmonella pathogenicity island 2 (SPI2), essential for systemic infection. Our findings revealed that the protein encoded by ssaH regulates the secretion of an inner rod and early substrate, SsaI. Structural modeling revealed that SsaH is structurally similar to class III chaperones, known to associate with proteins in various pathogenic bacteria. The SPI2 protein SsaE was identified as a class V chaperone homolog and partner of SsaH. A pulldown analysis disclosed that SsaH and SsaE form a heterodimer, which interacted with another early substrate, the needle protein SsaG. Moreover, SsaE also helped stabilize SsaH and a middle substrate, SseB. We also found that SsaE regulates cellular SsaH levels to translocate the early substrates SsaG and SsaI and then promotes the translocation of SseB by stabilizing it. In summary, our results indicate that the class III chaperone SsaH facilitates SsaI secretion, and a heterodimer of SsaH and the type V chaperone SsaE then switches secretion to SsaG. This is the first report of a chaperone system that regulates both early and middle substrates during substrate switching for T3SS assembly.

Analysis of malto-oligosaccharides and related metabolites in rice endosperm during development.

MAIN CONCLUSION: Linear glucans with degree of polymerization of up to 23 were detected in rice endosperm at the very early developmental stage of endosperm and considered to play an important role in the de novo synthesis of branched glucans. Little is known concerning the contribution of malto-oligosaccharides (MOS) and longer linear glucans to the starch biosynthesis in cereal endosperm. In the present study, the changes in the amount of major metabolic intermediates including MOS and linear glucans with a degree of polymerization (DP) of ≤ 9 and ≥ 10, respectively, in rice endosperm were measured during the development. Significant amounts of linear glucans of at least DP23 were present in the endosperm at 3 and 5 days after pollination (DAP), whereas most MOS of DP up to 8 were detected in the endosperm throughout the development up to 20 DAP. It was also found that a significant amount of simple sugars such as sucrose, glucose, and fructose, and organic acids such as malic acid, citric acid, and succinic acid were present in the developing endosperm. Although the levels of metabolites are not directly related to the extent of the metabolic flux, the present results suggest that MOS and linear glucans as well as these sugars and organic acids are involved in starch biosynthesis of rice endosperm. It is thought that linear glucans might play a role in starch biosynthesis in rice endosperm, presumably as the precursor for the subsequent synthesis of branched glucans involved in the initiation process that is possibly active in the endosperm at the very early developmental stage.

Rapid immunosurveillance by recirculating lymphocytes in the rat intestine: critical role of unsulfated sialyl-Lewis X on high endothelial venules of the Peyer's patches.

Naive lymphocytes systemically recirculate for immunosurveillance inspecting foreign antigens and pathogens in the body. Trafficking behavior such as the migration pathway and transit time within the gastrointestinal tract, however, remains to be elucidated. Rat thoracic duct lymphocytes (TDLs) were transferred to a congeneic host that had undergone mesenteric lymphadenectomy. The migration pathway was investigated using newly developed four-color immunohistochemistry and immunofluorescence. Donor TDLs showed rapid transition in gut tissues from which they emerged in mesenteric lymph around 4 h after intravenous injection. Immunohistochemistry showed that donor TDLs predominantly transmigrated across high endothelial venules (HEVs) at the interfollicular area of the Peyer's patches (PPs), then exited into the LYVE-1+ efferent lymphatics, that were close to the venules. The rapid recirculation depended largely on the local expression of unsulfated sialyl-Lewis X on these venules where putative dendritic cells (DCs) were associated underneath. Recruited naive T cells briefly made contact with resident DCs before exiting to the lymphatics in the steady state. In some transplant settings, however, the T cells retained contact with DCs and were sensitized and differentiated into activated T cells. In conclusion, we directly demonstrated that lymphocyte recirculation within the gut is a very rapid process. The interfollicular area of PPs functions as a strategically central site for rapid immunosurveillance where HEVs, efferent lymphatics and resident DCs converge. PPs can, however, generate alloreactive T cells, leading to exacerbation of graft-versus-host disease or gut allograft rejection.

Endothelial heparan sulfate controls chemokine presentation in recruitment of lymphocytes and dendritic cells to lymph nodes.

Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased because of loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.

Nitrogen Isotope Fractionation from Ammonia Gas to Ammonium in Particulate Ammonium Chloride.

The nitrogen stable isotope ratios of NH3 gas (δ15N-NH3(g)) and particulate NH4+ (δ15N-NH4+(p)) have been used to determine the sources and elucidate the environmental dynamics of NH3(g) and NH4+(p). Some studies have determined the sources from δ15N-NH4+(p) in the ambient environment by using the nitrogen isotope fractionation value (Δδ15N). Studies of Δδ15N are limited and estimated the Δδ15N by theoretical calculation or laboratory experiments in closed systems. Here, we produced actual submicron-sized particles and exposed them to NH3(g) in a dynamic chamber under various experimental conditions of temperature and NH3(g) turnover rate. Δδ15N was lower when temperature was higher. For low turnover rates (about 0.9 per day), the Δδ15N was 31.6 ± 2.0‰ (mean ± 1SD), almost equal to the Δδ15N from theoretical calculations and laboratory tests in closed systems. Also, the relationship between Δδ15N, the temperature, and the turnover rate can be described in the equation form. This relationship can help to explain the discrepancies between Δδ15N values in closed and open systems. Our results could help to clarify the sources and mechanisms of particle formation.

Forced Expression of CXCL10 Prevents Liver Metastasis of Colon Carcinoma Cells by the Recruitment of Natural Killer Cells.

CXC chemokine ligand 10 (CXCL10) is a CXC chemokine family protein that transmits signals by binding to its specific receptor, CXCR3. CXCL10 is also known as an interferon-γ-inducible chemokine involved in various biological phenomena, including chemotaxis of natural killer (NK) cells and cytotoxic T lymphocytes, that suppress tumor growth and inhibition of angiogenesis. In this study, we examined the effects of forced expression of CXCL10 in a murine colon carcinoma cell line (CT26) on growth and metastasis in syngeneic mice. We first established CT26 cells that were stably expressing murine CXCL10 (CT26/CXCL10) and compared their growth with their parental CT26 cells in vitro and in vivo. The in vitro growth of the CT26/CXCL10 and CT26 cells was comparable, whereas the in vivo growth of the CT26/CXCL10 cells in the skin was strongly suppressed. Liver metastasis of the CT26/CXCL10 cells was also significantly suppressed after intra-splenic implantation. Removal of NK cells by the administration of anti-asialo GM1 antibody canceled the suppression of subcutaneous growth and liver metastasis of CT26/CXCL10 cells. Immunofluorescence clearly showed that abundant NKp46-positive NK cells were recruited into the liver metastatic lesions of the CT26/CXCL10 cells, consistent with specific NK cell migration towards the culture supernatant from the CT26/CXCL10 cells in the chemotaxis assay using transwells. These findings indicate that CXCL10 prevents in vivo growth and metastasis of colon carcinoma cells by recruiting NK cells, suggesting that forced expression of CXCL10 in the colon tumors by gene delivery should lead to a favorable clinical outcome.

Online wet oxidation/isotope ratio mass spectrometry method for determination of stable carbon isotope ratios of water-soluble organic carbon in particulate matter.

RATIONALE: Water-soluble organic carbon (WSOC) is formed by oxidation of organic compounds in particulate matter (PM) and accounts for 25-80% of the organic carbon in PM. Stable carbon isotope ratio (δ13 C) analysis is widely used to identify the sources of PM, but determining the δ13 C values of WSOC is complicated and requires a time-consuming pretreatment process. METHODS: We have developed an online wet oxidation/isotope ratio mass spectrometry method with a reduced pretreatment time. We have measured the δ13 C values of WSOC by using this method. RESULTS: The method showed high accuracy (0.1‰) and precision (0.1‰) for levoglucosan, and the limit of detection was sufficiently low for WSOC analysis. Using this method, we determined δ13 C values of WSOC in PM2.5 samples collected in Japan during the period from July to November 2017 and found that the values ranged from -26.5‰ to -25.0‰ (average, -25.8‰). CONCLUSIONS: Our simple, low-blank method could be used for rapid quantitative analysis of the δ13 C values of WSOC in PM2.5 . We propose that this online method be used as a standard method for δ13 C analysis of WSOC.

Determination of carbon isotope ratios for honey samples by means of a liquid chromatography/isotope ratio mass spectrometry system coupled with a post-column pump.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been used to authenticate and trace products such as honey, wine, and lemon juice, and compounds such as caffeine and pesticides. However, LC/IRMS has several disadvantages, including the high cost of the CO2 membrane and blocking by solidified sodium persulfate. Here, we developed an improved system for determining carbon isotope ratios using LC/IRMS. METHODS: The main improvement was the use of a post-column pump. Using the improved system, we determined δ13 C values for glucose with high accuracy and precision (0.1‰ and 0.1‰, respectively; n = 3). The glucose, fructose, disaccharide, trisaccharide, and organic acid constituents of honey samples were analyzed using LC/IRMS. RESULTS: The δ13 C values for glucose, fructose, disaccharides, trisaccharides, and organic acids ranged from -27.0 to -24.2‰, -26.8 to -24.0‰, -28.8 to -24.0‰, -27.8 to -22.8‰, and - 30.6 to -27.4‰, respectively. The analysis time was a third to a half of that required for analysis by previously reported methods. CONCLUSIONS: The column flow rate could be arbitrarily adjusted with the post-column pump. We applied the improved method to 26 commercial honey samples. Our results can be expected to be useful for other researchers who use LC/IRMS.

Classification of nine malathion emulsion samples by using carbon isotope ratios and the ratio of organic solvents.

The compound specific isotope analysis is nowadays an important and powerful tool in geochemical, environmental and forensics field. On November 2013, Aqli Foods Corporation in Japan dealt with complaints about stench from frozen foods produced. Subsequently, very high concentrations of organophosphorus pesticide as malathion, ethylbenzene and xylene were detected in recovered frozen foods. In particular case, we present the method to measure the stable carbon isotope ratio (δ13C) of nine malathion emulsion pesticides using gas chromatography/isotope ratio mass spectrometry (GC/IRMS) to identify the source. The δ13C values of malathion ranged from -30.6‰ to -29.5‰. Because malathion used in all malathion emulsions sold in Japan is imported from the same overseas company, Cheminova, Denmark. The δ13C values of ethylbenzene ranged from -28.2‰ to -20.8‰ and those of m,p-xylene from -28.7‰ to -25.2‰. The differences in the δ13C values may be because of the material itself and chemical processing. We also determined the ratio of ethylbenzene to m,p-xylene and finally categorized the nine malathion samples into five groups on the basis of this ratio and the δ13C values of ethylbenzene and m,p-xylene. The results of isotopic fractionation during volatilization (refrigerate, room temperature and incubator) was negligible small.