Spatial distribution of trace metals and associated transport proteins during bacterial infection.

Innate immune systems alter the concentrations of trace elements in host niches in response to invading pathogens during infection. This work reports the interplay between d-block metal ions and their associated biomolecules using hyphenated elemental techniques to spatially quantify both elemental distributions and the abundance of specific transport proteins. Here, lung tissues were collected for analyses from naïve and Streptococcus pneumoniae-infected mice fed on a zinc-restricted or zinc-supplemented diet. Spatiotemporal distributions of manganese (55Mn), iron (56Fe), copper (63Cu), and zinc (66Zn) were determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry. The murine transport proteins ZIP8 and ZIP14, which are associated with zinc transport, were also imaged by incorporation of immunohistochemistry techniques into the analytical workflow. Collectively, this work demonstrates the potential of a single instrumental platform suitable for multiplex analyses of tissues and labelled antibodies to investigate complex elemental interactions at the host-pathogen interface. Further, these methods have the potential for broad application to investigations of biological pathways where concomitant measurement of elements and biomolecules is crucial to understand the basis of disease and aid in development of new therapeutic approaches.

Dietary zinc and the control of Streptococcus pneumoniae infection.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

The Iceman's Last Meal Consisted of Fat, Wild Meat, and Cereals.

The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [1], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-year-old European glacier mummy [2, 3]. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual.

Laser ablation-inductively coupled plasma-mass spectrometry imaging of white and gray matter iron distribution in Alzheimer's disease frontal cortex.

Iron deposition in the brain is a feature of normal aging, though in several neurodegenerative disorders, including Alzheimer's disease, the rate of iron accumulation is more advanced than in age-matched controls. Using laser ablation-inductively coupled plasma-mass spectrometry imaging we present here a pilot study that quantitatively assessed the iron content of white and gray matter in paraffin-embedded sections from the frontal cortex of Alzheimer's and control subjects. Using the phosphorus image as a confirmed proxy for the white/gray matter boundary, we found that increased intrusion of iron into gray matter occurs in the Alzheimer's brain compared to controls, which may be indicative of either a loss of iron homeostasis in this vulnerable brain region, or provide evidence of increased inflammatory processes as a response to chronic neurodegeneration. We also observed a trend of increasing iron within the white matter of the frontal cortex, potentially indicative of disrupted iron metabolism preceding loss of myelin integrity. Considering the known potential toxicity of excessive iron in the brain, our results provide supporting evidence for the continuous development of novel magnetic resonance imaging approaches for assessing white and gray matter iron accumulation in Alzheimer's disease.

Optimization of chemometric approaches for the extraction of isorhamnetin-3-O-rutinoside from Calendula officinalis L.

The application of Design of Experiments (DoE) to the determination of optimum conditions for an extraction process relies on the correct selection of mathematical models. The linear model is the one typically used; however, in some cases it does not always have superior performance, ignoring the real nature of the data and its appropriate descriptive model. In order to evaluate the extraction efficiency of isorhamnetin-3-O-rutinoside from flowers of Calendula officinalis L. a multivariate factorial analysis was used. Simulations were conducted using linear, quadratic, full cubic and special cubic models. A Simplex-Centroid design was chosen as it delivered greater precision with only minor errors versus other models tested. Analyses were performed by capillary zone electrophoresis using sodium tetraborate buffer (40mmolL(-1), pH 9.4) containing 10% methanol. The detection was linear over a range of 8.0-50.0mgL(-1) (r(2)=0.996), and the limits of detection (LOD) and quantification (LOQ) for isorhamnetin-3-O-rutinoside were 3.44mgL(-1) and 11.47mgL(-1), respectively. The full cubic model showed the best extraction results, with an error of 3.40% compared to analysis of variance, and a determination coefficient of 0.974. The difference between the responses at the reference point, calculated by the model, and the experimental response, varies around 2.72% for full cubic model. Comparison of the four models showed the full cubic model was the most appropriate one, allowing greater efficiency in the extraction of isorhamnetin-3-O-rutinoside. Selection of the model made it possible to obtain a 60% increase in sensitivity compared to the linear model.

Is early-life iron exposure critical in neurodegeneration?

The effects of iron deficiency are well documented, but relatively little is known about the long-term implications of iron overload during development. High levels of redox-active iron in the brain have been associated with neurodegenerative disorders, most notably Parkinson disease, yet a gradual increase in brain iron seems to be a feature of normal ageing. Increased brain iron levels might result from intake of infant formula that is excessively fortified with iron, thereby altering the trajectory of brain iron uptake and amplifying the risk of iron-associated neurodegeneration in later life. In this Perspectives article, we discuss the potential long-term implications of excessive iron intake in early life, propose the analysis of iron deposits in teeth as a method for retrospective determination of iron exposure during critical developmental windows, and call for evidence-based optimization of the chemical composition of infant dietary supplements.

Determination of selenium in serum in the presence of gadolinium with ICP-QQQ-MS.

Gadolinium (Gd)-based magnetic resonance imaging (MRI) contrasting agents interfere with the determination of selenium (Se) when analysed by single quadrupole inductively coupled plasma-mass spectrometry (ICP-MS). This paper demonstrates that an ICP-triple quadrupole-MS (ICP-QQQ-MS) with oxygen mass shift overcomes Gd(++) interference on Se(+) and mitigates typically encountered matrix and spectral based interferences. Normal human serum was diluted in a solution containing isopropanol, EDTA, NH4OH and Triton X-100. Samples were unspiked (control) serum; serum spiked with 0.127 µmol L(-1) Se or 127 µmol L(-1) Gd; and serum spiked with both 0.127 µmol L(-1) Se and 127 µmol L(-1) Gd. Consideration of collision/reaction gases and conditions for interference mitigation included helium (He); a 'low' and 'high' hydrogen (H2) flow, and oxygen (O2). The instrument tune for O2 was optimised for effective elimination of interferences via a mass shift reaction of Se(+) to SeO(+). The ICP-QQQ-MS was capable of detecting trace (>9.34 nmol L(-1)) levels of Se in serum in the presence of Gd in our simulated post-MRI serum sample. The multi-tune capabilities of the ICP-QQQ-MS may be adapted to eliminate other specific isobaric interferences that cause false positive results in other analyses where the analyte is confounded by doubly charged and/or polyatomic species.

Barium distributions in teeth reveal early-life dietary transitions in primates.

Early-life dietary transitions reflect fundamental aspects of primate evolution and are important determinants of health in contemporary human populations. Weaning is critical to developmental and reproductive rates; early weaning can have detrimental health effects but enables shorter inter-birth intervals, which influences population growth. Uncovering early-life dietary history in fossils is hampered by the absence of prospectively validated biomarkers that are not modified during fossilization. Here we show that large dietary shifts in early life manifest as compositional variations in dental tissues. Teeth from human children and captive macaques, with prospectively recorded diet histories, demonstrate that barium (Ba) distributions accurately reflect dietary transitions from the introduction of mother's milk through the weaning process. We also document dietary transitions in a Middle Palaeolithic juvenile Neanderthal, which shows a pattern of exclusive breastfeeding for seven months, followed by seven months of supplementation. After this point, Ba levels in enamel returned to baseline prenatal levels, indicating an abrupt cessation of breastfeeding at 1.2 years of age. Integration of Ba spatial distributions and histological mapping of tooth formation enables novel studies of the evolution of human life history, dietary ontogeny in wild primates, and human health investigations through accurate reconstructions of breastfeeding history.

Elemental bio-imaging of trace elements in teeth using laser ablation-inductively coupled plasma-mass spectrometry.

OBJECTIVES: In this study we present the application of a novel laboratory method that employs laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to construct two-dimensional maps of trace elements in teeth. METHODS: Teeth were sectioned longitudinally, embedded in resin and polished to a smooth surface. Data were generated by laser ablating the entire sectioned tooth surface. Elemental images were constructed using custom-built software. RESULTS: Quantified images of (66)Zn, (88)Sr, (111)Cd and (208)Pb, with a spatial resolution of 30 µm(2), were generated from three teeth. Concentrations were determined by single-point calibration against NIST SRM 1486 (bone meal). Zn and Sr concentrations were determined in the µg g(-1) range and Cd and Pb in the ng g(-1) range. Concentrations of Pb, Zn and Cd were higher in dentine particularly in regions adjacent the pulp. CONCLUSIONS: Elemental bio-imaging employing LA-ICP-MS is a novel method for constructing µm-scale maps of trace elements in teeth. This simple imaging method displays the heterogeneity of trace elements throughout the tooth structure that correspond to specific structural and developmental features of teeth. As a preliminary study, this work demonstrates the capabilities of LA-ICP-MS imaging in dental research.

Three-dimensional elemental bio-imaging of Fe, Zn, Cu, Mn and P in a 6-hydroxydopamine lesioned mouse brain.

Three dimensional maps of iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and phosphorous (P) in a 6-hydroxydopamine (6-OHDA) lesioned mouse brain were constructed employing a novel quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method known as elemental bio-imaging. The 3D maps were produced by ablating serial consecutive sections taken from the same animal. Each section was quantified against tissue standards resulting in a three dimensional map that represents the variation of trace element concentrations of the mouse brain in the area surrounding the substantia nigra (SN). Damage caused by the needle or the toxin did not alter the distribution of Zn, and Cu but significantly altered Fe in and around the SN and both Mn and Fe around the needle track. A 20% increase in nigral Fe concentration was observed within the lesioned hemisphere. This technique clearly shows the natural heterogeneous distributions of these elements throughout the brain and the perturbations that occur following trauma or intoxication. The method may applied to three-dimensional modelling of trace elements in a wide range of tissue samples.

Elemental bio-imaging of thorium, uranium, and plutonium in tissues from occupationally exposed former nuclear workers.

Internal exposure from naturally occurring radionuclides (including the inhaled long-lived actinides (232)Th and (238)U) is a component of the ubiquitous background radiation dose (National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States; NCRP Report No. 160; NCRP: Bethesda, MD, 2009). It is of interest to compare the concentration distribution of these natural alpha-emitters in the lungs and respiratory lymph nodes with those resulting from occupational exposure, including exposure to anthropogenic plutonium and depleted and enriched uranium. This study examines the application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantifying and visualizing the mass distribution of uranium and thorium isotopes from both occupational and natural background exposure in human respiratory tissues and, for the first time, extends this application to the direct imaging of plutonium isotopes. Sections of lymphatic and lung tissues taken from deceased former nuclear workers with a known history of occupational exposure to specific actinide elements (uranium, plutonium, or americium) were analyzed by LA-ICPMS. Using a previously developed LA-ICPMS protocol for elemental bio-imaging of trace elements in human tissue and a new software tool, we generated images of thorium ((232)Th), uranium ((235)U and (238)U), and plutonium ((239)Pu and (240)Pu) mass distributions in sections of tissue. We used a laboratory-produced matrix-matched standard to quantify the (232)Th, (235)U, and (238)U concentrations. The plutonium isotopes (239)Pu and (240)Pu were detected by LA-ICPMS in 65 mum diameter localized regions of both a paratracheal lymph node and a sample of lung tissue from a person who was occupationally exposed to refractory plutonium (plutonium dioxide). The average (overall) (239)Pu concentration in the lymph node was 39.2 ng/g, measured by high purity germanium (HPGe) gamma-spectrometry (Lynch, T. P.; Tolmachev, S. Y.; James, A. C. Radiat. Prot. Dosim. 2009, 134, 94-101). Localized mass concentrations of thorium ((232)Th) and uranium ((238)U) in lymph node tissue from a person not occupationally exposed to these elements (chronic natural background inhalation exposure) ranged up to 400 and 375 ng/g, respectively. In lung samples of occupationally nonexposed to thorium and uranium workers, (232)Th and (238)U concentrations ranged up to 200 and 170 ng/g, respectively. In a person occupationally exposed to air-oxidized uranium metal (Adley, F. E.; Gill, W. E.; Scott, R. H. Study of atmospheric contaminiation in the melt plant buiding. HW-23352(Rev.); United States Atomic Energy Commission: Oakridge, TN, 1952, p 1-97), the maximum (235)U and (238)U isotopic mass concentrations in a lymph node, measured at higher resolution (with a 30 mum laser spot diameter), were 70 and 8500 ng/g, respectively. The ratio of these simultaneously measured mass concentrations signifies natural uranium. The current technique was not sufficiently sensitive, even with a 65 mum laser spot diameter, to detect (241)Am (at an overall tissue concentration of 0.024 ng/g, i.e., 3 Bq/g).

Elemental bio-imaging of melanoma in lymph node biopsies.

The spatial distribution of trace elements in human lymph nodes partially infiltrated by melanoma cells was determined by elemental bio-imaging. Imaging of (31)P within the nodal capsule and normal lymph node tissue showed a clear demarcation of the tumour boundary, with a significant decrease in relative (31)P concentration within the tumour. The location of the tumour boundary was confirmed by haematoxylin and eosin staining of serial sections and observation by light microscopy. Further enhancement of the tumour boundary was achieved by imaging the (31)P/(34)S ratio. (31)P/(66)Zn ratio images showed a decreasing ratio beyond the tumour boundary that extended into peritumour normal lymph node tissue.

Elemental bio-imaging of calcium phosphate crystal deposits in knee samples from arthritic patients.

Laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) was employed to image deposits of calcium phosphate based crystals in knee cartilage and synovial fluid from arthritic patients. A reaction/collision cell containing hydrogen minimised plasma interferences on calcium and also improved the image quality without significant sensitivity reduction. Areas of high calcium and phosphorus intensities consistent with crystal deposits were observed for both the cartilage and synovial fluid samples. These areas were also characterised by high magnesium and strontium intensities. Distribution patterns of other elements such as copper and sulfur did not correlate with the crystal deposits. Filtered and non-filtered solutions of calcium phosphate crystals grown in synthetic synovial fluid were also imaged as further evidence of crystal deposits. The crystal deposits were detected in the unfiltered solution, and were absent from the filtered solutions.