Metal localisation in gastropod shells: New insights from mass spectrometry techniques.

Gastropod shells are calcified structures made of several crystal layers. They grow throughout the lifecycle of mollusks by integrating some of the chemical elements present in their environment, including metals. This characteristic means mollusks can be useful bioindicators of metal exposure. The present study aimed to better understand the role of layer composition on metal accumulation. To that end, the gastropods Radix balthica were collected in a French river adjacent to a municipal wastewater treatment plant. Microchemical metal analyses in the different shell layers were performed by Femtosecond-Laser Ablation Inductively Coupled Plasma Mass Spectrometry (Fs-LA-ICP-MS) and analyses of the molecular environment of the metals were performed by Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS). Strontium, Ba and Mn were well distributed within the whole shell and the high concentrations of these elements were found to be related to the aragonite structure of the shell. Copper, Ni, Pb and Zn were mostly present at the outer surfaces of the shell where the organic constituents were more concentrated. The analysis of metal distribution in shell layers could improve our understanding of the relationships between metal exposure and accumulation in mollusks, therefore providing evidences of their use as powerful integrated bioindicator of metal contamination.

Quantification in bioimaging by LA-ICPMS - Evaluation of isotope dilution and standard addition enabled by micro-droplets.

This study explores quantitative bioimaging as enabled by laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS), designing standardization methods based on robotic micro-droplet dispensing. The potential of producing controlled and highly precise pL-volume droplets was exploited to establish on-tissue isotope dilution and standard addition. Both strategies eliminate matrix effects and offer high metrological order traceable to SI units. The absolute quantity was obtained for µm-sized regions of interest in tissue samples, as defined by the extension of the deposited pL-volume droplet. While the gold standard isotope dilution (ID) was restricted to the accurate quantification of a single element, i.d. platinum in different tissue samples (mouse liver, spleen and tumor tissue), multiplexed matrix-matched calibration was obtained by on-tissue standard addition by depositing a dilution series of certified multi-element standards. Here, the working range was determined by the heterogeneity of the tissue samples and the background levels of elements intrinsically present and/or artificially introduced during sample preparation. Both methods, ID and standard addition served as reference methods for validation of external calibration using gelatin-based micro-droplet standards. Given full ablation, these external standards revealed a high dynamic range together with an excellent repeatability. Where applicable, the cross-validation revealed consistent quantitative results for the three quantification approaches. The comparable sensitivity obtained for standard addition and external standardization, respectively expressed as slope of the calibration function, provided proof that gelatin-based micro-droplets could serve as matrix-matched calibrations. Therefore, gelatin micro-droplets offer a valid tool for multiplexed matrix-mimicking standardization at high-throughput.

Eyes and ears: A comparative approach linking the chemical composition of cod otoliths and eye lenses.

Fish eye lenses are a protein-based chronological recorder of microchemical constituents that are a potentially useful tool for interpretations of environmental, ecological and life-history experienced by fish. Here, we present the first study with data on the chemical composition of eye lenses from Baltic cod examined using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and compare these spatially resolved data to otoliths from the same fish also analysed by LA-ICPMS, measuring the isotopes 27 Al, 137 Ba, 43 Ca, 52 Cr, 65 Cu, 57 Fe, 39 K, 7 Li, 25 Mg, 55 Mn, 31 P, 208 Pb, 85 Rb, 45 Sc, 29 Si, 88 Sr, 47 Ti, 50 V, 149 Yb, 66 Zn and 90 Zr. Comparison of the variation in element concentrations between eye lenses and otoliths from the same individuals showed minor similarities, suggesting a different governance in the uptake processes. A strong overlap between the concentric growth rings in the eye lenses and the otolith Sr periodicity was observed, where each consecutive minima in the chemical profile with high accuracy correspond to the width of each lens ring. No comparable trends were seen between growth rings and all other elements measured from both lenses and otoliths. The characteristic rings observed in cod eye lenses do not seem to represent seasonal fluctuation nor are they found to be directly linked to age. With this research, we provide a baseline study identifying elements in corresponding eye lenses and otoliths that show potential for unravelling the environmental and biological conditions experienced by fish.

Comparison of single pulse, multiple dosage, and 2D oversampling / deconvolution LA-ICPMS strategies for mapping of (ultra)low-concentration samples.

Elemental LA-ICPMS mapping in continuous scanning mode gathers the counts generated upon laser ablation in line scanning mode. Acquisition of counts can be performed for each single laser pulse separately or by summing the counts of multiple laser pulses. Conventionally, pixels in an LA-ICPMS map are associated with spot-resolved single laser pulses (zero-dimensional, 0D), but also sub-pixel convolution strategies are in use, associated with one-dimensional (1D) or two-dimensional (2D) overlapping laser shots, and where possible followed by deblurring. The imaging quality of several 0D, 1D, and 2D LA-ICPMS strategies were compared for mapping of (ultra)low-concentration samples, both via computational and experimental approaches. The data presented will be helpful to make the right decision about the best possible LA-ICPMS mapping strategy for the highest image quality.

Benthic foraminifera geochemistry as a monitoring tool for heavy metal and phosphorus pollution - A post fish-farm removal case study.

Measuring environmental contaminants in coastal areas is critical for monitoring and managing their impacts. Commonly used techniques involve repetitive field sampling, which provides a single moment in time during each effort. In this study, we examine the potential for using foraminifera in monitoring and risk assessment as recorders of bioavailable pollutants. Geochemical analysis of benthic foraminifera (Operculina ammonoides) shells sampled annually at a previous fish farm location showed extremely high levels of copper (Cu), zinc (Zn), and phosphorus (P) during the three years following the fish cages removal, with a general reduction afterwards. Levels of Cu/Ca were still more than 4-fold higher than background levels 10 years after the removal of the fish cages. Based on our finds, it is concluded that the geochemical analysis of recent benthic foraminifera shells can serve as a powerful monitoring tool of bioavailable contaminants in seawater. Additionally, the results highlight the need for heavy metal monitoring near marine aquaculture facilities and suggest that long-term effects extend spatially and temporally far beyond the original point source. Finally, we observed variations in micro-distribution of elements within the top 1-2 µm of the shells, where Cu/Ca, Zn/Ca and P/Ca are consistently higher in the chamber wall than in the septa. This observation is relevant for studies conducting single chamber analyses.

No more waste at the elemental analysis of airborne particulate matter on quartz fibre filters.

Particulate matter (PM) is the major environmental pollutant. Its elemental composition is routinely monitored. Inductively coupled plasma mass spectroscopy (ICPMS) is commonly applied after a PM sample has been digested by an acid during a microwave treatment. In this case, sample preparation procedure is laborious, sometimes incomplete and produces toxic waste. In this paper we show that direct sample introduction to ICPMS by laser ablation (LA-ICPMS) is of huge advantage. Minimal quantity of a sample is required for the analysis (<1 cm2) and no chemical waste is produced. The study focused on the most universal and widely used quartz fibre filter samples and we show that LA-ICPMS can be successfully applied for the determination of the elemental composition of such samples. Some effort is, however, still needed to develop an autosampler for the LA-ICPMS system and to provide commercial matrix-matched standards for this application to be implemented in environment laboratories worldwide.

Zinc distribution within breast cancer tissue of different intrinsic subtypes.

PURPOSE: To show feasibility of laser ablation inductively coupled mass spectrometry (LA-ICPMS) for analysis of zinc content and concentration in breast cancer tissue and to correlate this with validated prognostic and predictive markers, i.e. histological grading and expression of steroid receptors (estrogen receptor, ER; progesterone receptor, PR) and human epidermal growth-factor receptor 2 (Her2). METHODS: 28 samples of human invasive ductal breast cancer tissue were subclassified into groups of four different intrinsic subtypes according to the expression of ER, PR and Her2 by immunohistological staining and then analyzed for zinc content and distribution by LA-ICPMS applying a calibration technique based on spiked polyacrylamide gels. A correlation of zinc concentration with histological grading and molecular subtypes was analyzed. RESULTS: Consistent with results of a pilot-study LA-ICPMS was feasible to show zinc accumulation in cancerous tissue, even more adjacent healthy stroma was with proportional increase of zinc. Zinc levels were most elevated in triple-positive (TPBC) and in triple-negative (TNB) breast cancers. CONCLUSION: LA-ICPMS was feasible to confirm a connection between zinc and grade of malignancy; furthermore, focusing on a correlation of zinc and intrinsic breast cancer subtypes, LA-ICPMS depicted an upwards trend of zinc for "high-risk-cancers" with highest levels in Her2-positive and in triple-negative (TNBC) disease. The currently uncommon alliance of clinicians and analytical chemists in basic research is most promising to exploit the full potential of diagnostic accuracy in the efforts to solve the enigma of breast cancer initiation and course of disease.

Geochemistry of large benthic foraminifera Amphisorus hemprichii as a high-resolution proxy for lead pollution in coastal environments.

Anthropogenic lead (Pb) contamination resulting from the rapid growth of industrialization in coastal environments poses significant challenges. In this study, we report a novel approach utilising the large benthic foraminifera Amphisorus hemprichii as a biogeochemical archive for monitoring Pb pollution in tropical to warm-temperate coastal waters. Live juvenile specimens of A. hemprichii were cultured in the laboratory for 16 weeks with a range of seawater Pb concentrations. Lead uptake in both newly grown and pre-existing chambers of individual specimens was characterised using the microanalytical technique, Laser ablation-ICP mass spectrometry. We found that Pb concentration in the tests of cultured foraminifera in the laboratory is proportional to seawater [Pb] with the lead partition coefficient (KDPb) of 8.37 ± 0.3. This calibration together with a new biomineralisation model now enables A. hemprichii to be utilised as a naturally occurring bio-archive to quantitatively monitor anthropogenic Pb pollution in coastal waters.

Does trace element composition of bivalve shells record utra-high frequency environmental variations?

Saint-Pierre and Miquelon (SPM) is a small archipelago where instrumental measures based on water column velocity and temperature profiles compiled comprehensive evidence for strong near-diurnal (25.8h) current and bottom temperature oscillations (up to 11.5 °C) which is possibly the largest ever observed - at any frequency - on a stratified mid-latitude continental shelf. The main objective of our study was to identify if Placopecten magellanicus can record on its shell these high frequency environmental variations. To this end, we have tried to identify proxies for water temperature and food availability through development of a new ultra-high resolution LA-ICPMS analyses method capable of resolving shell surface elemental composition with a 10 µm resolution. This method was applied on two shell fragments, both representing the third year of growth and 2015 annual growth period, respectively coming from two environmentally contrasted sites, more (30 m depth) or less (10 m depth) affected by high frequency thermal oscillations. Our results strongly suggest a relationship between phytoplankton biomass and barium incorporation into P. magellanicus shells at both sites. Even if P. magellanicus might present a physiological control of magnesium incorporation, the shape of the two Mg/Ca profiles seems to illustrate that temperature also exerts a control on magnesium incorporation in P. magellanicus shells from SPM. While U/Ca and Mg/Ca profiles show a strong positive correlation for 30 m site shell, suggesting that uranium incorporation in P. magellanicus shell is at least partially temperature dependent. The absence of such correlation for 10 m site shell suggests differences in uranium environmental availability or in P. magellanicus biomineralization between these two sites. The resolution of this new analytical method raises questions about such data interpretation related to P. magellanicus growth dynamics and physiology or individual scale based environmental measurements.

A unifying model for the accretion of chondrules and matrix.

The so far unique role of our Solar System in the universe regarding its capacity for life raises fundamental questions about its formation history relative to exoplanetary systems. Central in this research is the accretion of asteroids and planets from a gas-rich circumstellar disk and the final distribution of their mass around the Sun. The key building blocks of the planets may be represented by chondrules, the main constituents of chondritic meteorites, which in turn are primitive fragments of planetary bodies. Chondrule formation mechanisms, as well as their subsequent storage and transport in the disk, are still poorly understood, and their origin and evolution can be probed through their link (i.e., complementary or noncomplementary) to fine-grained dust (matrix) that accreted together with chondrules. Here, we investigate the apparent chondrule-matrix complementarity by analyzing major, minor, and trace element compositions of chondrules and matrix in altered and relatively unaltered CV, CM, and CR (Vigarano-type, Mighei-type, and Renazzo-type) chondrites. We show that matrices of the most unaltered CM and CV chondrites are overall CI-like (Ivuna-type) (similar to solar composition) and do not reflect any volatile enrichment or elemental patterns complementary to chondrules, the exception being their Fe/Mg ratios. We propose to unify these contradictory data by invoking a chondrule formation model in which CI-like dust accreted to so-called armored chondrules, which are ubiquitous in many chondrites. Metal rims expelled during chondrule formation, but still attached to their host chondrule, interacted with the accreted matrix, thereby enriching the matrix in siderophile elements and generating an apparent complementarity.

Fourier-Transform Infrared Imaging Spectroscopy and Laser Ablation -ICPMS New Vistas for Biochemical Analyses of Ischemic Stroke in Rat Brain.

Objective: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Understanding the post-stroke biochemical and molecular changes are critical for patient survival and stroke management. The aim of this work was to investigate the photo-thrombotic ischemic stroke in male rats with particular focus on biochemical and elemental changes in the primary stroke lesion in the somatosensory cortex and surrounding areas, including the corpus callosum. Materials and Methods: FT-IR imaging spectroscopy and LA-ICPMS techniques examined stroke brain samples, which were compared with standard immunohistochemistry studies. Results: The FTIR results revealed that in the lesioned gray matter the relative distribution of lipid, lipid acyl and protein contents decreased significantly. Also at this locus, there was a significant increase in aggregated protein as detected by high-levels Aß1-42. Areas close to the stroke focus experienced decrease in the lipid and lipid acyl contents associated with an increase in lipid ester, olefin, and methyl bio-contents with a novel finding of Aß1-42 in the PL-GM and L-WM. Elemental analyses realized major changes in the different brain structures that may underscore functionality. Conclusion: In conclusion, FTIR bio-spectroscopy is a non-destructive, rapid, and a refined technique to characterize oxidative stress markers associated with lipid degradation and protein denaturation not characterized by routine approaches. This technique may expedite research into stroke and offer new approaches for neurodegenerative disorders. The results suggest that a good therapeutic strategy should include a mechanism that provides protective effect from brain swelling (edema) and neurotoxicity by scavenging the lipid peroxidation end products.

Highly-sensitive open-cell LA-ICPMS approaches for the quantification of rare earth elements in natural carbonates at parts-per-billion levels.

This work presents a high-sensitivity approach to quantify ultra-trace concentrations of rare earth elements (REEs) in speleothem carbonates using open-cell laser ablation-sector field-inductively coupled plasma mass spectrometry (open-cell LA-SF-ICPMS). Specifically, open-cell LA in combination with a gas exchange device enabled sampling of large-scale carbonate specimens in an ambient environment. The use of a "jet" vacuum interface and the addition of small amounts of N2 gas allowed for a 20-40 fold sensitivity enhancement compared to the conventional interface configuration. Mass load effects, quantification capabilities and detection power were investigated in analyses of reference materials using various combinations of spot sizes and laser repetition rates. From a 160 µm diameter circular laser spot and 10 Hz ablation frequency, limits of detection were in the low or sub-ng g-1 range for REEs. Little dependence of Ca normalized sensitivity factors on the amount of material introduced into the plasma was observed. Relative deviations of quantified concentrations from USGS MACS-3 preferred values were smaller than 12%. The analytical approach enabled the determination of REE concentration profiles at the single digit ng g-1 level. Application to a 15-cm piece stalagmite collected from East Timor revealed at least two abrupt elevations in light rare earth elements (LREEs) within a scanning distance of 8 mm. These anomaly regions extended over a distance of ≈200 µm and showed LREE abundances elevated by at least one order of magnitude. This high-resolution open-cell LA-SF-ICPMS method has the potential to be applied in micro-domain analyses of other natural carbonates, such as travertine, tufa, and flowstones. This is promising for a better understanding of earth and environmental sciences.

Determination of changes in the concentration and distribution of elements within olive drupes (cv. Leccino) from Se biofortified plants, using laser ablation inductively coupled plasma mass spectrometry.

BACKGROUND: Biofortification of food crops has been used to increase the intake of Se in the human diet, even though this may change the concentration of other elements and modify the nutritional properties of the enriched food. Selenium biofortification programs should include routine assessment of the overall mineral composition of enriched plants. RESULTS: Laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) was used for the assessment of mineral composition of table olives. Olive trees were fertilized with sodium selenate before flowering. At harvest, the edible parts of drupes proved to be significantly enriched in Se, delivering 6.1 µg g-1 (39% of the RDA for five olives). Such enrichment was followed by significant changes in the concentrations of B, Mg, K, Cr, Mn, Fe and Cu in edible parts, which are discussed for their impact on food quality. CONCLUSION: The biofortification of olive plants has allowed the enrichment of fruits with selenium. Enrichment with selenium has caused an increase in the concentration of other elements, which can change the nutritional quality of the drupes. The analytical technique used well as a valuable tool for routinely determining the chemical composition of all fruit parts. © 2018 Society of Chemical Industry.

Discrimination of habitat use between two sympatric species of mullets, Mugil curema and Mugil liza (Mugiliformes: Mugilidae) in the rio Tramandaí Estuary, determined by otolith chemistry

Two sympatric species of marine mullets, Mugil curema and M. liza, use the rio Tramandaí Estuary as nursing grounds. When two closely related species are sympatric, various mechanisms may permit their coexistence, including spatial or temporal segregation that results in the divergent use of the resources for which they compete. To investigate the spatial segregation, we used otolith chemistry inferred through laser ablation-inductively coupled plasma mass spectrometry. Our results indicate that in the rio Tramandaí Estuary, M. curema is associated with high salinity waters and can be classified as a Marine Migrant in the Marine Estuarine-opportunist subcategory. Mugil liza is associated with lower salinity and can be classified as a Marine Migrant in the Estuarine Dependent subcategory. The intra-specific variation in estuarine habitat use indicates that the migratory behaviors in mullets are far more complex than previously known.(AU)

Duas espécies de tainha simpátricas, Mugil curema e M. liza, usam o estuário do Rio Tramandaí como zona de berçário. Quando espécies do mesmo gênero são simpátricas, vários mecanismos podem permitir sua coexistência, incluindo segregação espacial ou temporal que resultam no uso distinto dos recursos pelos quais elas competem. Para investigar a segregação espacial nós usamos a análise de elementos químicos em otólitos inferidos por espectrometria de massas através de plasma indutivamente acoplado com amostras extraídas a laser (LA-ICPMS). Mugil curema está associada a águas de alta salinidade podendo ser classificada como Marinho Migrante sub-categoria Marinha estuarina-oportunista. Mugil liza está associada a águas menos salinas, devendo ser classificada como Marinha Migrante sub-categoria Estuarina Dependente. A variação intraespecífica no uso do habitat estuarino indica um comportamento migratório mais complexo que o previsto em tainhas.

Corrigendum: Identification of Cerebral Metal Ion Imbalance in the Brain of Ageing Octodon degus.

[This corrects the article on p. 66 in vol. 9, PMID: 28405187.].

Identification of Cerebral Metal Ion Imbalance in the Brain of Aging Octodon degus.

The accumulation of redox-active transition metals in the brain and metal dyshomeostasis are thought to be associated with the etiology and pathogenesis of several neurodegenerative diseases, and Alzheimer's disease (AD) in particular. As well, distinct biometal imaging and role of metal uptake transporters are central to understanding AD pathogenesis and aging but remain elusive, due inappropriate detection methods. We therefore hypothesized that Octodon degus develop neuropathological abnormalities in the distribution of redox active biometals, and this effect may be due to alterations in the expression of lysosomal protein, major Fe/Cu transporters, and selected Zn transporters (ZnTs and ZIPs). Herein, we report the distribution profile of biometals in the aged brain of the endemic Chilean rodent O. degus-a natural model to investigate the role of metals on the onset and progression of AD. Using laser ablation inductively coupled plasma mass spectrometry, our quantitative images of biometals (Fe, Ca, Zn, Cu, and Al) appear significantly elevated in the aged O. degus and show an age-dependent rise. The metals Fe, Ca, Zn, and Cu were specifically enriched in the cortex and hippocampus, which are the regions where amyloid plaques, tau phosphorylation and glial alterations are most commonly reported, whilst Al was enriched in the hippocampus alone. Using whole brain extracts, age-related deregulation of metal trafficking pathways was also observed in O. degus. More specifically, we observed impaired lysosomal function, demonstrated by increased cathepsin D protein expression. An age-related reduction in the expression of subunit B2 of V-ATPase, and significant increases in amyloid beta peptide 42 (Aß42), and the metal transporter ATP13a2 were also observed. Although the protein expression levels of the zinc transporters, ZnT (1,3,4,6, and 7), and ZIP7,8 and ZIP14 increased in the brain of aged O. degus, ZnT10, decreased. Although no significant age-related change was observed for the major iron/copper regulator IRP2, we did find a significant increase in the expression of DMT1, a major transporter of divalent metal species, 5'-aminolevulinate synthase 2 (ALAS2), and the proto-oncogene, FOS. Collectively, our data indicate that transition metals may be enriched with age in the brains of O. degus, and metal dyshomeostasis in specific brain regions is age-related.

Metal and complementary molecular bioimaging in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, affecting over 27 million people worldwide. AD represents a complex neurological disorder which is best understood as the consequence of a number of interconnected genetic and lifestyle variables, which culminate in multiple changes to brain structure and function. These can be observed on a gross anatomical level in brain atrophy, microscopically in extracellular amyloid plaque and neurofibrillary tangle formation, and at a functional level as alterations of metabolic activity. At a molecular level, metal dyshomeostasis is frequently observed in AD due to anomalous binding of metals such as Iron (Fe), Copper (Cu), and Zinc (Zn), or impaired regulation of redox-active metals which can induce the formation of cytotoxic reactive oxygen species and neuronal damage. Metal chelators have been administered therapeutically in transgenic mice models for AD and in clinical human AD studies, with positive outcomes. As a result, neuroimaging of metals in a variety of intact brain cells and tissues is emerging as an important tool for increasing our understanding of the role of metal dysregulation in AD. Several imaging techniques have been used to study the cerebral metallo-architecture in biological specimens to obtain spatially resolved data on chemical elements present in a sample. Hyperspectral techniques, such as particle-induced X-ray emission (PIXE), energy dispersive X-ray spectroscopy (EDS), X-ray fluorescence microscopy (XFM), synchrotron X-ray fluorescence (SXRF), secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled mass spectrometry (LA-ICPMS) can reveal relative intensities and even semi-quantitative concentrations of a large set of elements with differing spatial resolution and detection sensitivities. Other mass spectrometric and spectroscopy imaging techniques such as laser ablation electrospray ionization mass spectrometry (LA ESI-MS), MALDI imaging mass spectrometry (MALDI-IMS), and Fourier transform infrared spectroscopy (FTIR) can be used to correlate changes in elemental distribution with the underlying pathology in AD brain specimens. Taken together, these techniques provide new techniques to probe the pathobiology of AD and pave the way for identifying new therapeutic targets. The current review aims to discuss the advantages and challenges of using these emerging elemental and molecular imaging techniques, and highlight clinical achievements in AD research using bioimaging techniques.