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.

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.

Heart-cutting two-dimensional liquid chromatography combined with isotope ratio mass spectrometry for the determination of stable carbon isotope ratios of gluconic acid in honey.

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is used to analyze various types of samples, including foodstuffs, to determine their authenticity and trace their origin on the basis of their stable carbon isotope ratios (δ13C). However, multicomponent samples are difficult to analyze. For example, determining the δ13C values of the organic acids in honey is complicated by the presence of large amounts of carbohydrates. Herein, we present a heart-cutting two-dimensional LC/IRMS method for analysis of honey samples. In this method, the organic acids in the samples were first separated from the carbohydrates by a size-exclusion column, and then the organic acids were separated from each other by a reverse-phase column connected to the first column via a switching valve. By means of this method, the δ13C values for three organic acids in high-carbohydrate-content simulated honey samples could be determined with high accuracy and precision (≤0.3‰ and ≤0.1‰, respectively). In addition, the gluconic acid δ13C values for 25 honey samples were determined with high precision and found to range from -31.7 to -28.5‰ (mean: -30.0 ±â€¯0.7‰). These values shed some light on the mechanism of gluconic acid production. Taken together, our results suggest that this two-dimensional LC method has the potential to be more effective than one-dimensional LC for use in isotopic research.

Stable carbon isotope ratios for organic acids in commercial honey samples.

Stable carbon isotope ratios (δ13C) for glucose, fructose, disaccharides, trisaccharides, and organic acids in 116 commercial honey samples were measured by LC/IRMS. On the basis of EA/IRMS and LC/IRMS authenticity criteria, 39 of the samples were judged to have been adulterated. The δ13C values for organic acids from pure honey, reported here for the first time, ranged from -33.6 to -26.5‰. The mean Δδ13C (glucose-organic acids) value was +3.7 ±â€¯0.9‰. Glucose and organic acid δ13C values were strongly correlated (R = 0.71, P < 0.001). Gluconic acid, the predominant organic acid in honey, has been reported to be produced via decomposition of glucose by bee glucose-oxidase and certain Gluconobacter spp. This fact was confirmed by isotope analysis.

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.

Morphological and Functional Attenuation of Degeneration of Peripheral Neurons by Mesenchymal Stem Cell-Conditioned Medium in Spinocerebellar Ataxia Type 1-Knock-in Mice.

AIMS: Spinocerebellar ataxia type 1 (SCA1) is caused by the ataxin-1 protein (ATXN1) with an abnormally expanded polyglutamine tract and is characterized by progressive neurodegeneration. We previously showed that intrathecal injection of mesenchymal stem cells (MSCs) during the nonsymptomatic stage mitigates the degeneration of the peripheral nervous system (PNS) neurons in SCA1-knock-in (SCA1-KI) mice. We tested in this study whether the therapeutic effects of MSCs in SCA1-KI mice could be reproduced with MSC-releasing factor(s). METHODS: To test the effects of MSC-releasing factor(s), we used MSC-conditioned medium (MSC-CM). MSC-CM was intrathecally and/or intravenously injected into young SCA1-KI mice, and the therapeutic effects were assessed in the PNS at later ages using immunostaining, electrophysiology, and behavioral tests. RESULTS: MSC-CM attenuated the degeneration of axons and myelin of spinal motor neurons. Consequently, the injected SCA1-KI mice exhibited smaller reductions in nerve conduction velocity in spinal motor neurons and reduced motor incoordination than the untreated mice. CONCLUSIONS: These results suggest that factors released from MSC mitigate the morphological and functional abnormalities in the PNS that are observed in SCA1-KI mice in a paracrine manner.

Mesenchymal stem cells attenuate peripheral neuronal degeneration in spinocerebellar ataxia type 1 knockin mice.

Spinocerebellar ataxia type 1 (SCA1) is a devastating neurodegenerative disorder in which an abnormally expanded polyglutamine tract is inserted into causative ataxin-1 proteins. We have previously shown that SCA1 knockin (SCA1-KI) mice over 6 months of age exhibit a degeneration of motor neuron axons and their encasing myelin sheaths, as reported in SCA1 patients. We examined whether axon degeneration precedes myelin degeneration or vice versa in SCA1-KI mice and then attempted to mitigate motor neuron degeneration by intrathecally administering mesenchymal stem cells (MSCs). Temporal examination of the diameters of motor neuron axons and their myelin sheaths revealed a decrease in diameter of the axon but not of the myelin sheaths in SCA1-KI mice as early as 1 month of age, which suggests secondary degeneration of the myelin sheaths. We injected MSCs into the intrathecal space of SCA1-KI mice at 1 month of age, which resulted in a significant suppression of degeneration of both motor neuron axons and myelin sheaths, even 6 months after the MSC injection. Thus, MSCs effectively suppressed peripheral nervous system degeneration in SCA1-KI mice. It has not yet been clarified how clinically administered MSCs exhibit significant therapeutic effects in patients with SCA1. The morphological evidence presented in this current mouse study might explain the mechanisms that underlie the therapeutic effects of MSCs that are observed in patients with SCA1.

Mesenchymal stem cells as a potential therapeutic tool for spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a devastating progressive neurodegenerative disorder, for which no effective treatments have been developed. However, some studies have shown that an intracerebellar or intrathecal injection of mesenchymal stem cells (MSCs) was partially effective in some genetic mouse models of cerebellar ataxia such as SCA1 and Lurcher mutant. MSCs likely exert their therapeutic efficacy by secreting innate factors to induce neuronal growth and synaptic connection and reduce apoptosis. In this review, we introduce the therapeutic influence of MSCs on each mouse model for cerebellar ataxia and the possible mechanisms underlying the action of MSCs. We also introduce studies on the safety and effectiveness of umbilical cord MSCs for patients with SCA.

Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV-GFP mice, aged 8-12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

Impairment of spinal motor neurons in spinocerebellar ataxia type 1-knock-in mice.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of polyglutamine repeats in the Ataxin-1 protein. An accumulating body of cerebellar, histological and behavioral analyses has proven that SCA1-knock-in mice (in which the endogenous Atxn1 gene is replaced with mutant Atxn1 that has abnormally expanded 154 CAG repeats) work as a good tool, which resembles the central nervous system pathology of SCA1 patients. However, the peripheral nervous system pathology of the model mice has not been studied despite the fact that the clinical manifestation is also characterized by peripheral involvement. We show here that spinal motor neurons are degenerated in SCA1-knock-in mice. Histologically, some spinal motor neurons of the SCA1-knock-in mice have polyglutamine aggregates in their nuclei and also thinner and demyelinated axons. Electrophysiological examinations of the mice showed slower nerve conduction velocities in spinal motor neurons and lower amplitudes of muscle action potential, compared to wild-type mice. Consistently, the mice displayed decrease in rearing number and total rearing time. These results suggest that the knock-in mice serve as a definite model that reproduces peripheral involvement and are therefore useful for research on the peripheral nervous system pathology in SCA1 patients.