Novel Identification of Arsenolipids Using Chemical Derivatizations in Conjunction with RP-HPLC-ICPMS/ESMS

The identification of molecular structures of an arsenolipid is pivotal for its toxicological assessment and in understanding the arsenic cycling in the environment. However, the analysis of these compounds in a lipid matrix is an ongoing challenge. So far, only a few arsenolipids have been reported, including arsenic fatty acids (AsFAs) and arsenic hydrocarbons (AsHCs). By means of RP-HPLC-ICPMS/ESMS, we investigated Capelin oil (Mallotus villosus) for possible new species of arsenolipids. Twelve arsenolipids were identified in the fish oil including three AsFAs and seven AsHCs. Among the AsHCs, four that were identified had protonotated molecular masses of 305, 331, 347, and 359 and have not been reported before. In addition, the compounds with molecular formulas C₂₀H₄₄AsO⁺ and C₂₄H₄₄AsO⁺ were found in low concentrations and showed chromatographic properties and MS data consistent with cationic trimethylarsenio fatty alcohols. Derivatization by acetylation and thiolation coupled with accurate mass spectrometry was successfully used to establish the occurrence of this new class of arsenolipids as cationic trimethylarsenio fatty alcohols (TMAsFOH)

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

Concentrations of essential trace metals in the brain of animal species—A comparative study

The essential trace metals iron, zinc, and copper have a significant physiological role in healthy brain development and function. Especially zinc is important for neurogenesis, synaptogenesis, synaptic transmission and plasticity, and neurite outgrowth. Given the key role of trace metals in many cellular processes, it is important to maintain adequate levels in the brain. However, the physiological concentration of trace metals, and in particular zinc, in the human and animal brain is not well described so far. For example, little is known about the trace metal content of the brain of animals outside the class of mammals. Here, we report the concentration of iron, zinc, and copper in fresh brain tissue of different model-species of the phyla Chordata (vertebrates (mammals, fish)), Annelida, Arthropoda (insects), and Mollusca (snails), using inductively coupled plasma mass-spectrometry (ICP-MS). Our results show that the trace metals are present in the nervous system of all species and that significant differences can be detected between species of different phyla. We further show that a region-specific distribution of metals within the nervous system already exists in earthworms, hinting at a tightly controlled metal distribution. In line with this, the trace metal content of the brain of different species does not simply correlate with brain size. We conclude that although the functional consequences of the controlled metal homeostasis within the brain of many species remains elusive, trace metal biology may not only play an important role in the nervous system of mammals but across the whole animal kingdo

Fluorine Speciation Analysis Using Reverse Phase Liquid Chromatography Coupled Off-Line to Continuum Source Molecular Absorption Spectrometry (CS-MAS): Identification and Quantification of Novel Fluorinated Organic Compounds in Environmental and Biological Samples

Driven by increasing demand for the monitoring of industrial perfluorinated compounds (PFCs), the identification of novel fluorine containing compounds (FOCs) and the tracking of organofluorine drugs and their degradation products, there is a clear need for sensitive, fluorine-specific detection of unknown FOCs. Here we report the first ever direct fluorine-specific (speciation) method; capable of individually detecting untargeted FOCs in environmental and biological samples through the application of continuum source molecular absorption spectrometry (CS-MAS) using a commercial CS-AAS. Two model FOCs (2,4,6, trifluorobenzoic acid (TFBA) and 5-fluoroindol-5-carboxylic acid (FICA)) were used, achieving fluorine-specific detection across a range of 0.1 to 300 ng/mL fluorine, corresponding to a limit of detection of 4 pg F and 5.26 nM for both compounds. Both TFBA and FICA showed a similar response to CS-MAS detection, potentially enabling the quantification of fluorine content in novel FOCs without having molecular standards available. This paper also reports the use of reverse-phase high performance liquid chromatography (RP-HPLC) coupled off-line with CS-MAS for the identification of single organofluorines in a mixture of FOCs via fraction collection. The linear range of both FOCs was determined to be from 1 to 500 ng/mL. The limits of detection of those species were just above 1 ng/mL (100 pg) and can therefore compete with targeted analytical methods such as ESI-MS. Finally, as a proof of principle the analysis of a fluoride-containing groundwater sample from Ghana demonstrated that this method can be used in the detection of novel FOCs, with identification achieved through parallel ESI-MS. Coupled HPLC–CS-MAS/ESI-MS is the first analytical methodology capable of selectively detecting and identifying novel FOCs, making possible the quantification of all fluorine containing compounds in one sample. This is the necessary analytical requirement to perform <i>fluoronomics</i>

Selective chemical ablation of splenic red pulp macrophages recapitulates the renal iron accumulation phenotype in the absence of <i>Candida</i> infection.

<p><b>Panel A.</b> Immunohistochemical confirmation of selective ablation of splenic red pulp macrophages by clodronate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat.1003676-VanRooijen1" target="_blank">[41]</a> (100 µL of 5 mg/mL liposome-encapsulated clodronate per animal): for antibodies see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g005" target="_blank">Fig. 5D</a>. <b>Panel B.</b> LA-ICP MS mapping of ⁵⁶Fe distribution reveals medullary shift of renal iron in clodronate-treated mice. Normalised ⁵⁶Fe/¹³C ratios across kidney following 8 h clodronate treatment are presented (see panel <b>A</b>); for colour scale see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g002" target="_blank">Fig. 2</a>. In the bar chart (<b>B</b>, right) bars correspond to the percentage surface area with normalized ⁵⁶Fe/¹³C intensity≥2-fold (left, light coloured bar) and ≥3-fold above background (right, dark coloured bar): error bars, standard deviations from the mean; <i>n</i>, number of biological replicates. In all cases, the experiments represent <i>C. albicans</i> SC5314 infections in BALB/c mice. ‘Control’, experimental control with no primary antibodies. Size bars: 200 µm. Figure in <b>B</b> is representative of three biological replicates.</p

Systemic candidiasis disturbs host renal iron homeostasis and affects splenic function.

<p><b>Panels A–C.</b> Immunohistochemical detection of iron homeostasis associated proteins in kidneys of healthy and infected animals reveals correlation with renal iron loading. Ferritin, HO-1, HO-2, and the transferrin receptor (TfR) all increase in infected kidneys in comparison to healthy controls (<b>A</b>) Ferritin is distributed outside areas occupied by <i>C. albicans</i> (<b>A</b>, arrowheads). In healthy tissue (<b>B, left</b>), ferritin content is low and mainly cortical, while increased and mainly medullary in animals with advanced candidiasis (<b>B, right</b>). HO-1 is concentrated in rings encompassing fungal lesions (<b>A,</b> arrowheads) and is produced by cell type(s) other than the Ly-6G or F4/80-producing immune cells (<b>C</b>). <b>Panel D</b>. Immunohistochemical analyses of murine spleens. Blue areas correspond to white pulp (dotted lines), embedded in the red pulp. Red pulp macrophages stain selectively with anti-HO-1 and anti-F4/80 antibodies: the stain for HO-1 is depleted in tissues from infected animals (arrows) (<b>D</b>). In all cases, the experiments represent <i>C. albicans</i> SC5314 infections in BALB/c mice. ‘Pas_h’, sections processed for histology; ‘c’, kidney cortex; ‘m’, medulla; ‘control’, experimental control with no primary antibodies. Size bars: panels <b>A</b> and <b>D</b>, 500 µm; <b>B</b>, 1000 µm; <b>C</b>, 200 µm.</p

Spatial distributions of HBA and haem mirror ⁵⁶Fe distributions in healthy and infected kidneys.

<p>Tissue sections were analysed with MALDI IMS to map the distributions of HBA and haem B (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#s4" target="_blank">Materials and Methods</a>, and Supporting Material). Similarly to iron distribution (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g002" target="_blank">Fig. 2</a>), in the healthy tissue (top panels) both HBA and haem are distributed to the cortex, whereas in kidneys obtained from animals with advanced infection (bottom panels) they are medullary. Transverse kidney sections sequential to those presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g002" target="_blank">Fig. 2A</a> (‘Healthy’) and 2C (‘Infected’), respectively, are shown. The images are representative of at least three biological replicates.</p

Systemic candidiasis affects host iron homeostasis. A.

<p>Immunohistochemical detection of hepcidin in kidneys of healthy (right) and infected (left) animals. <b>B.</b> Perls staining of non-haem iron in livers of healthy and infected animals. The intensity of the blue stain is proportional to the amount of hepatic non-haem iron. <b>C.</b> Immunohistochemical analysis of Kupffer cells in mouse liver with anti-HO1 antibodies. The red arrowheads indicate hepatic Kupffer cells stained dark brown with anti-HO-1 antibodies. 1C1& 1C3 represent negative controls lacking the primary antibody. <b>D.</b> Perls staining of non-haem iron deposits in spleens of healthy and infected animals. The blue arrowheads highlight splenic non-haem iron deposits. <i>C. albicans</i> SC5314 infections were conducted in BALB/c mice and all images are representative of at least three separate biological replicates. Size bars correspond to 100 µm. Experimental details can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#s4" target="_blank">Materials and Methods</a>.</p

<i>C. albicans</i> infection is accompanied by dramatic changes in the renal iron landscape.

<p>LA-ICP MS mapping of iron distribution in transverse mouse kidney sections. Normalised ⁵⁶Fe/¹³C ratios are presented, with the colour scale indicating fold increases in signal intensities relative to background. As the infection progresses, iron loading increases and the iron becomes redistributed from the cortex of healthy kidneys (<b>A</b>), to the medulla in intermediate (<b>B</b>) and advanced infections (<b>C</b>). Histology insets (<b>D</b>) are representative of healthy, intermediate and advanced infections, and correspond to the tissues imaged in panels <b>A</b>–<b>C</b>. The pie charts in (<b>D</b>) present the percentage total tissue area with a given ⁵⁶Fe/¹³C intensity and the colour scale represents increments of 0.5-fold intensity changes from background (black) to 8-fold increase (white). In the bar chart (<b>D</b>) bars correspond to the percentage surface area with normalized ⁵⁶Fe/¹³C intensity ≥2-fold (left, light coloured bars) and ≥3-fold above background (right, dark coloured bars): error bars, standard deviations from the mean; <i>n</i>, number of biological replicates. The effects observed with the virulent <i>C albicans</i> isolate SC5314 (epidemiological clade 1) are replicated with a different clinical isolate AM2003/0191(clade 2) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat.1003676-MacCallum2" target="_blank">[30]</a> (panels <b>E</b> and <b>F</b>, bottom). Infections with <i>C. albicans</i> SC5314 stimulate significant immune infiltrates (<b>F</b>, top, dotted line), while AM2003/0191 elicits minimal immune infiltrates (<b>F</b>, bottom, solid line).</p

Flow chart of individuals identified as having received SSG treatment between 2006 and 2010.

<p>Additional outcomes: One patient had a still birth during antimonial treatment. One patient was identified with PKDL at the time of study.</p