Fourier transform infrared microspectroscopy analysis of ovarian cancerous tissues in paraffin and deparaffinized tissue samples.

Ovarian cancer is one of the deadliest cancers occurring in women. This is typically due to late diagnosis of the disease and difficult treatment. Infrared microspectroscopy is a complementary research method that can be helpful in the diagnosis of this disease, because it allows for the analysis of the tissues biomolecular composition. In this study, archival paraffin-embedded preparations of ovarian tissues, tumours and control, were used. However, the paraffin present in such specimens is a strong absorber of infrared radiation, which makes it impossible to reliably analyse the biomolecular composition of the sample. The solution to this problem is to deparaffinize the tissue before the analysis. However, the extend to which the paraffinization and deparaffinization processes influence the biomolecular composition of the tissues is unclear. Analysed tissues in the form of cores were placed in a paraffin micromatrix and FTIR measurements were performed. Then the samples were deparaffinized and the measurements were taken again. For both sets of samples (embedded in paraffin and deparaffinized) ratios of integrated peaks and massifs within the obtained spectra were calculated. The obtained ratios were compared for different types of diseased and healthy, control tissues. The Kruskal-Wallis test revealed statistically significant differences of the calculated ratios between most of the types of tissues. Random Forest models clearly showed that both samples in paraffin and deparaffinized retain enough information to classify the tissues reliably. The feature analysis revealed the most important feature for distinguishing between different types of samples, i.e. 1080 cm-1/1240 cm-1 ratio and lipid saturation for the samples embedded in paraffin and deparaffinized respectively. The study showed that the deparaffinization process leads to changes in the biomolecular composition of the analysed tissues. Despite this, classification of the tissues based on FTIR measurements remains possible.

The applicability of Fourier transform infrared microspectroscopy for correction against matrix effects in X-ray fluorescence microimaging of tissues.

X-ray fluorescence (XRF) and Fourier transform infrared (FTIR) microscopy techniques are now considered popular for rapid and label-free complementary spectrochemical analysis of chemical elements and molecular systems in biological specimens. The morphological heterogeneity but also the inhomogeneities associated with the thickness/density of biological samples demonstrate challenges for the quantitative XRF microimaging. Therefore, in the present work, we proposed for the first time the application of the total absorbance under the FTIR spectra as a mass surface correction procedure for two-dimensional (2D) XRF microimaging of tissues. We also evaluated the equivalence of the developed correction method based on total absorbance of FTIR spectra with the proposed approaches based on incoherent scattering of primary X-rays as well as on the membrane Si-Kα transmission signal, on the example of selected rat organ tissues. Thin cryo-sections taken from various organs of Wistar rats were deposited on silicon nitride membranes (Si3N4). The FTIR microscopy studies were performed to collect infrared absorption spectra, used then for the determination of total absorbance values in the selected areas of tissue samples. In turn, hard X-ray imaging based on synchrotron radiation allowed the determination of characteristic radiation intensities of the elements detectable from the tissue, as well as the characteristic radiation of the membrane Si and incoherently scattered X-ray photons (Compton scattering). The latter served as correction factors for the surface mass of the sample alongside the FTIR total absorbance. The qualitative and quantitative analyses showed a high agreement between the results of elemental surface mass correction using total absorbance under FTIR spectra of tissues with those obtained using surface mass correction factors determined directly from XRF spectra. Therefore, the proposed procedure is a good alternative in cases where the surface mass effect of the sample cannot be eliminated based on the information provided directly by the XRF spectrum, as in the case of using polymer films as sample support. We have also proposed a procedure for synchronizing SRXRF and FTIR images, not limited to visual inspection of imaging/mapping data, but also enabling quantitative analysis. We found that the total absorbance determined from FTIR spectra can be successfully used as a correction factor for eliminating the surface mass effect in XRF microimaging of thin freeze-fried tissues and therefore to obtain the surface mass-independent elemental quantities. The proposed approach for 2D-FTIR-XRF analysis can also be a powerful and versatile tool for fostering a correlation and co-localization analysis to search for common distribution patterns between molecular arrangements and chemical elements.

A combined X-ray fluorescence and infrared microspectroscopy study for new insights into elemental-biomolecular obesity-induced changes in rat brain structures.

The objective of our research was to determine the brain changes at the molecular and elemental levels typical of early-stage obesity. Therefore a combined approach using Fourier transform infrared micro-spectroscopy (FTIR-MS) and synchrotron radiation induced X-ray fluorescence (SRXRF) was introduced to evaluate some brain macromolecular and elemental parameters in high-calorie diet (HCD)- induced obese rats (OB, n = 6) and in their lean counterparts (L, n = 6). A HCD was found to alter the lipid- and protein- related structure and elemental composition of the certain brain areas important for energy homeostasis. The increased lipid unsaturation in the frontal cortex and ventral tegmental area, the increased fatty acyl chain length in the lateral hypothalamus and substantia nigra as well as the decreased both protein α helix to protein ß- sheet ratio and the percentage fraction of ß-turns and ß-sheets in the nucleus accumbens were revealed in the OB group reflecting obesity-related brain biomolecular aberrations. In addition, the certain brain elements including P, K and Ca were found to differentiate the lean and obese groups at the best extent. We can conclude that HCD-induced obesity triggers lipid- and protein- related structural changes as well as elemental redistribution within various brain structures important for energy homeostasis. In addition, an approach applying combined X-ray and infrared spectroscopy was shown to be a reliable tool for identifying elemental-biomolecular rat brain changes for better understanding the interplay between the chemical and structural processes involved in appetite control.

Combined spectroscopic, biochemical and chemometric approach toward finding of biochemical markers of obesity.

BACKGROUND: Early-stage detection of subclinical obesity-driven systemic changes is a challenging area of medical diagnostics, where the most popular existing measures - such as body mass index - BMI - often fall short of providing a realistic estimate of adiposity and, therefore, of ongoing pathologies at the systemic, tissue and cellular level. In the quest for identifying new more robust diagnostic markers, whole-organ analysis of chemical elements is a promising approach for identifying candidate proxies of obesity status in the system. METHODS: Total Reflection X-ray fluorescence (TXRF) coupled with biochemical assays, chemometrics and statistical validation was used as a new integrated pipeline for marker identification in external ear samples of obese animals. The specimens were taken from obese animals fed a high calorie diet as well as from lean intact animals fed a standard diet. RESULTS: The most significant differences in the content of K, Fe, Br, and Rb between the studied groups of the animals were identified. However, with the methodology applied Rb was found the most robust biochemical discriminator of early-stage obesity effects, as validated by the logistic regression model. We observed no relationship between the levels of the elements consumed by the animals and their apparent content in the earlobe tissue samples. CONCLUSIONS: Our preliminary study confirms that obesity alters tissue trace metal metabolism and shows the proposed new approach as an accurate and reliable methodology for detecting tissue elemental obesity-related alterations. GENERAL SIGNIFICANCE: This result can be of practical significance for designing new point-of-care systems for obesity screening tests, taking advantage of direct/indirect Rb measurements.

The New Markers of Early Obesity-Related Organ and Metabolic Abnormalities.

The objective of our study was to identify new markers related to excessive body adiposity and its early consequences. For this purpose we determined serum FGF-19 and FGF-21 concentrations in obese rats, whose role in the pathogenesis of obesity is not yet established. In addition, a total reflection X-ray fluorescence technique was applied to determine the elemental chemistry of certain tissues affected by obesity. Next, the new biochemical and molecular parameters were correlated with well-known obesity-related markers of metabolic abnormalities. Our obese rats were characterized by increased calorie consumption and body adiposity, hypercholesterolemia, elevated levels of liver enzymes and FGF-21, while the level of FGF-19 was reduced. Strong relationships between new hormones and established metabolic parameters were observed. Furthermore, we demonstrated that obesity had the greatest effect on elemental composition in the adipose tissue and liver and that rubidium (Rb) had the highest importance in distinguishing the studied groups of animals. Tissue Rb strongly correlated with both well-known and new markers of obesity. In conclusion, we confirmed serum FGF-19 and FGF-21 as useful new markers of obesity-related metabolic alternations and we robustly propose Rb as a novel indicator of excessive body adiposity and its early consequences. However, further investigations are encouraged to address this clinical issue.

Repetitive transcranial direct current stimulation modulates the brain-gut-microbiome axis in obese rodents.

BACKGROUND: Complex interactions between the brain, gut and adipose tissue allow to recognize obesity as a neurometabolic disorder. The recent data have shown that gut microbiota can play a potential role in obesity development. Transcranial direct current stimulation (tDCS) is a safe and non-invasive technique to modulate the activity of cerebral cortex and other connected brain areas also in context of appetite control. The objective of this study was to evaluate the effects of repetitive anodal tDCS (AtDCS) of prefrontal cortex on feeding behavior, metabolic status and selected phyla of gut microbiota in rats with obesity induced by high-calorie diet (HCD). METHODS: 32 female Wistar rats were equally divided into 4 subgroups depending on diet effect (lean versus obese) and type of stimulation (active versus sham tDCS versus no stimulation). Feed intake, body weight, blood lipoproteins and leptin levels as well as Firmicutes and Bacteroidetes in intestines and stool were examined. RESULTS: HCD changed feeding behavior and metabolic parameters typically for obesity-related ranges and resulted in an abundance of Firmicutes at the expanse of Bacteroidetes in the large intestine and stool. AtDCS decreased appetite, body weight, and cholesterol levels. In addition, AtDCS reduced ratio of the average number of Firmicutes to average number of Bacteroidetes in all examined tissues. CONCLUSIONS: Repetitive AtDCS is not only effective for appetite restriction but can also modulate gut microbiome composition which demonstrates the existence of the brain-gut-microbiome axis and points at this technique as a promising complementary treatment for obesity. However, the effects should be further replicated in human studies.

Elemental Composition of Skeletal Muscle Fibres Studied with Synchrotron Radiation X-ray Fluorescence (SR-XRF).

Diseases of the muscle tissue, particularly those disorders which result from the pathology of individual muscle cells, are often called myopathies. The diversity of the content of individual cells is of interest with regard to their role in both biochemical mechanisms and the structure of muscle tissue itself. These studies focus on the preliminary analysis of the differences that may occur between diseased tissues and tissues that have been recognised as a reference group. To do so, 13 samples of biopsied human muscle tissues were studied: 3 diagnosed as dystrophies, 6 as (non-dystrophic) myopathy and 4 regarded as references. From these sets of muscle biopsies, 135 completely measured muscle fibres were separated altogether, which were subjected to investigations using synchrotron radiation X-ray fluorescence (SR-XRF). Muscle fibres were analysed in terms of the composition of elements such as Br, Ca, Cl, Cr, Cu, Fe, K, Mn, P, S and Zn. The performed statistical tests indicate that all three groups (dystrophies-D; myopathies-M; references-R) show statistically significant differences in their elemental compositions, and the greatest impact, according to the multivariate discriminate analysis (MDA), comes from elements such as Ca, Cu, K, Cl and S.

On 2D-FTIR-XRF microscopy - A step forward correlative tissue studies by infrared and hard X-ray radiation.

Correlative Fourier Transform Infra-Red (FTIR) and hard X-Ray Fluorescence (XRF) microscopy studies of thin biological samples have recently evolved as complementary methods for biochemical fingerprinting of animal/human tissues. These are seen particularly useful for tracking the mechanisms of neurological diseases, i.e., in Alzheimer/Parkinson disease, in the brain where mishandling of trace metals (Fe, Cu, Zn) seems to be often associated with ongoing damage to molecular components via, among others, oxidative/reductive stress neurotoxicity. Despite substantial progress in state-of-the-art detection and data analysis methods, combined FTIR-XRF experiments have never benefited from correlation and co-localization analysis of molecular moieties and chemical elements, respectively. We here propose for the first time a completely novel data analysis pipeline, utilizing the idea of 2D correlation spectrometry for brain tissue analysis. In this paper, we utilized combined benchtop FTIR - synchrotron XRF mapping experiments on thin brain samples mounted on polypropylene membranes. By implementing our recently developed Multiple Linear Regression Multi-Reference (MLR-MR) algorithm, along with advanced image processing, artifact-free 2D FTIR-XRF spectra could be obtained by mitigating the impact of spectral artifacts, such as Etalon fringes and mild scattering Mie-like signatures, in the FTIR data. We demonstrated that the method is a powerful tool for co-localizing and correlating molecular arrangements and chemical elements (and vice versa) using visually attractive 2D correlograms. Moreover, the methods' applicability for fostering the identification of distinct (biological) materials, involving chemical elements and molecular arrangements, is also shown. Taken together, the 2D FTIR-XRF method opens up for new measures for in-situ investigating hidden complex biochemical correlations, and yet unraveled mechanisms in a biological sample. This step seems crucial for developing new strategies for facilitating the research on the interaction of metals/nonmetals with organic components. This is particularly important for enhancing our understanding of the diseases associated with metal/nonmetal mishandling.

Model-based correction algorithm for Fourier Transform infrared microscopy measurements of complex tissue-substrate systems.

Model-based algorithms have recently attracted much attention for data pre-processing in tissue mapping and imaging by Fourier transform infrared micro-spectroscopy (FTIR). Their versatility, robustness and computational performance enabled the improvement of spectral quality by mitigating the impact of scattering and fringing in FTIR spectra of chemically homogeneous biological systems. However, to date, no comprehensive algorithm has been optimized and automated for large-area FTIR imaging of histologically complex tissue samples. Herein, for the first time, we propose a unique, integrated and fully-automated Multiple Linear Regression Multi-Reference (MLR-MR) method for correcting linear baseline effects due to diffuse scattering, for compensating substrate thickness inhomogeneity and accounting for sample chemical heterogeneity in FTIR images. In particular, the algorithm uses multiple-reference spectra for histologically heterogeneous biological samples. The performance of the procedure was demonstrated for FTIR imaging of chemically complex rat brain frontal cortex tissue samples, mounted onto Ultralene® films. The proposed MLR-MR correction algorithm allows the efficient retrieval of "pure" absorbance spectra and greatly improves the histological fidelity of FTIR imaging data, as compared with the one-reference approach. In addition, the MLR-MR algorithm here presented opens up the possibility for extracting information on substrate thickness variability, thus enabling the indirect evaluation of its topography. As a whole, the MLR-MR procedure can be easily extended to more complex systems for which Mie scattering effects must also be eliminated.

Molecular and elemental effects underlying the biochemical action of transcranial direct current stimulation (tDCS) in appetite control.

Recent studies highlight that obesity may alter the electric activity in brain areas triggering appetite and craving. Transcranial direct current brain stimulation (tDCS) has recently emerged as a safe alternative for treating food addiction via modulating cortical excitability without any high-risk surgical procedure to be utilized. As for anodal-type tDCS (atDCS), we observe increased excitability and spontaneous firing of the cortical neurons, whilst for the cathodal-type tDCS (ctDCS) a significant decrease is induced. Unfortunately, for the method to be fully used in a clinical setting, its biochemical action mechanism must be precisely defined, although it is proposed that molecular remodelling processes play in concert with brain activity changes involving the ions of: Na, Cl, K and Ca. Herein, we proposed for the first time Fourier transform infrared (FTIR) and synchrotron X-ray fluorescence (SRXRF) microprobes for a combined molecular and elemental analysis in the brain areas implicated appetite control, upon experimental treatment by either atDCS or ctDCS. The study, although preliminary, shows that by stimulating the prefrontal cortex in the rats fed high-caloric nutrients, the feeding behavior can be significantly changed, resulting in significantly inhibited appetite. Both, atDCS and ctDCS produced significant molecular changes involving qualitative and structural properties of lipids, whereas atDCS was found with a somewhat more significant effect on protein secondary structure in all the brain areas investigated. Also, tDCS was reported to reduce surface masses of Na, Cl, K, and Ca in almost all brain areas investigated, although the atDCS deemed to have a stronger neuro-modulating effect. Taken together, one can report that tDCS is an effective treatment technique, and its action mechanism in the appetite control seems to involve a variety of lipid-, protein- and metal/non-metal-ion-driven biochemical changes, regardless the current polarization.

Combined brain Fe, Cu, Zn and neurometabolite analysis - a new methodology for unraveling the efficacy of transcranial direct current stimulation (tDCS) in appetite control.

Obesity is a chronic, multifactorial origin disease that has recently become one of the most frequent lifestyle disorders. Unfortunately, current obesity treatments seem to be ineffective. At present, transcranial direct current brain stimulation (tDCS) represents a promising novel treatment methodology that seems to be efficient, well-tolerated and safe for a patient. Unfortunately, the biochemical action of tDCS remains unknown, which prevents its widespread use in the clinical arena, although neurobiochemical changes in brain signaling and metal metabolism are frequently reported. Therefore, our research aimed at exploring the biochemical response to tDCS in situ, in the brain areas triggering feeding behavior in obese animals. The objective was to propose a novel neurochemical (serotoninergic and dopaminergic signaling) and trace metal analysis of Fe, Cu and Zn. In doing so, we used energy-dispersive X-ray fluorescence (EDXRF) and high-performance liquid chromatography (HPLC). Anodal-type stimulation (atDCS) of the right frontal cortex was utilized to down-regulate food intake and body weight gain in obese rats. EDXRF was coupled with the external standard method in order to quantify the chemical elements within appetite-triggering brain areas. Major dopamine metabolites were assessed in the brains, based on the HPLC assay utilizing the external standard assay. Our study confirms that elemental analysis by EDXRF and brain metabolite assay by HPLC can be considered as a useful tool for the in situ investigation of the interplay between neurochemical and Fe/Cu/Zn metabolism in the brain upon atDCS. With this methodology, an increase in both Cu and Zn in the satiety center of the stimulated group could be reported. In turn, the most significant neurochemical changes involved dopaminergic and serotoninergic signaling in the brain reward system.

Sources and fate of microplastics in marine and beach sediments of the Southern Baltic Sea-a preliminary study.

Microplastics' (particles size ≤5 mm) sources and fate in marine bottom and beach sediments of the brackish are strongly polluted Baltic Sea have been investigated. Microplastics were extracted using sodium chloride (1.2 g cm-3). Their qualitative identification was conducted using micro-Fourier-transform infrared spectroscopy (µFT-IR). Concentration of microplastics varied from 25 particles kg-1 d.w. at the open sea beach to 53 particles kg-1 d.w. at beaches of strongly urbanized bay. In bottom sediments, microplastics concentration was visibly lower compared to beach sediments (0-27 particles kg-1 d.w.) and decreased from the shore to the open, deep-sea regions. The most frequent microplastics dimensions ranged from 0.1 to 2.0 mm, and transparent fibers were predominant. Polyester, which is a popular fabrics component, was the most common type of microplastic in both marine bottom (50%) and beach sediments (27%). Additionally, poly(vinyl acetate) used in shipbuilding as well as poly(ethylene-propylene) used for packaging were numerous in marine bottom (25% of all polymers) and beach sediments (18% of all polymers). Polymer density seems to be an important factor influencing microplastics circulation. Low density plastic debris probably recirculates between beach sediments and seawater in a greater extent than higher density debris. Therefore, their deposition is potentially limited and physical degradation is favored. Consequently, low density microplastics concentration may be underestimated using current methods due to too small size of the debris. This influences also the findings of qualitative research of microplastics which provide the basis for conclusions about the sources of microplastics in the marine environment.

FTIR imaging of the molecular burden around Aß deposits in an early-stage 3-Tg-APP-PSP1-TAU mouse model of Alzheimer's disease.

Alzheimer's disease is one of the major causes of dementia in the elderly. The disease is caused by the misfolding of water soluble alpha-helical proteins, which leads to the accumulation of ß-sheets in the form of amyloid plaques, which can subsequently affect surrounding tissue areas by oxidative stress neurotoxicity. The aim of the present study was to design a novel methodology to analyze the extent to the neuronal burden around protein-rich Aß plaques suspected to affect molecular components by oxidative stress induced by inflammatory states. To do so, sagittal brain tissue sections from triple transgenic APPxPSP1xTAU mice were used to carry high magnification FTIR-FPA bench-top chemical imaging. The study used the combination of chemometric procedures involving spectral curve fitting and image processing to study the molecular changes occurring around the plaques. The study shows the performance of the approach by demonstrating its usefulness to co-localize molecular changes to different areas around the plaques. The results, although very preliminary, point to the strong interplay between the distance from the plaque and co-accumulation of molecular components indicative of inflammatory states.

Synchrotron radiation based X-ray fluorescence shows changes in the elemental composition of the human substantia nigra in aged brains.

Human brain aging is considered to be the leading risk factor for a variety of neurodegenerative alterations. In particular, it is thought that the human substantia nigra might play a pivotal role in age-associated dopamine depletion which could be responsible for neuronal demise and subsequent emergence of different neurological alterations. A plethora of neurochemical redox- and non-redox-driven mechanisms is mainly associated with modifications in the elemental composition of both neuromelanin-pigmented neurons and extraneuronal spaces in the human substantia nigra pars compacta (SNpc). An age-associated variation in the content of Fe, Cu, Zn and Ca has recently received great interest in neurology, as these elements are implicated in different biochemical mechanisms underlying malicious neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. Interestingly, to the best our knowledge, there is lack of a comprehensive study on age-associated variation in the elemental composition of the human SNpc. In that respect, the aim of the present study was to make a preliminary attempt to unravel some of the age-associated mechanisms responsible for the metabolism of some redox-active and redox-inactive elements in the elderly. To do so, substantia nigra tissue specimens, drawn from 37 individuals who deceased without any signs of neurodegeneration, were subjected to spectroscopic studies using synchrotron radiation based X-ray fluorescence. Both neuromelanin-pigmented neurons and extraneuronal areas were studied. It appears that in the neurons, Fe tends to decrease, whilst Cu, Zn and Ca were found to accumulate as an individual gets older.

Peripheral vagus nerve stimulation significantly affects lipid composition and protein secondary structure within dopamine-related brain regions in rats.

Recent immunohistochemical studies point to the dorsal motor nucleus of the vagus nerve as the point of departure of initial changes which are related to the gradual pathological developments in the dopaminergic system. In the light of current investigations, it is likely that biochemical changes within the peripheral nervous system may influence the physiology of the dopaminergic system, suggesting a putative role for it in the development of neurodegenerative disorders. By using Fourier transform infrared microspectroscopy, coupled with statistical analysis, we examined the effect of chronic, unilateral electrical vagus nerve stimulation on changes in lipid composition and in protein secondary structure within dopamine-related brain structures in rats. It was found that the chronic vagal nerve stimulation strongly affects the chain length of fatty acids within the ventral tegmental area, nucleus accumbens, substantia nigra, striatum, dorsal motor nucleus of vagus and the motor cortex. In particular, the level of lipid unsaturation was found significantly increasing in the ventral tegmental area, substantia nigra and motor cortex as a result of vagal nerve stimulation. When it comes to changes in protein secondary structure, we could see that the mesolimbic, mesocortical and nigrostriatal dopaminergic pathways are particularly affected by vagus nerve stimulation. This is due to the co-occurrence of statistically significant changes in the content of non-ordered structure components, alpha helices, beta sheets, and the total area of Amide I. Macromolecular changes caused by peripheral vagus nerve stimulation may highlight a potential connection between the gastrointestinal system and the central nervous system in rat during the development of neurodegenerative disorders.

Variability of protein and lipid composition of human subtantia nigra in aging: Fourier transform infrared microspectroscopy study.

There is growing evidence that a variety of biochemical processes that underlie the most frequent neurodegenerative diseases may have much in common with those connected with natural aging. It was shown that they involve, among others, lipid peroxidation and/or generation of insoluble in water protein deposits (i.e. alpha-synuclein and/or beta amyloid). Therefore, it is likely that the analysis of changes in both lipid and protein composition may be interesting in the light of any potential pathologies occurring within the dopaminergic system during physiological aging. Thereby, this paper presents a methodology for the analysis of age-related changes in a lipid and protein composition within human subtantia nigra tissue by means of Fourier transform infrared microspectroscopy (FTIRM). Particularly, the changes in the lipid saturation, unsaturation as well as in the protein secondary structure were examined. The studies were carried out on samples from 35 individuals who died without any signs of neurologic dysfunctions. Our results show that the level of lipid saturation increases inside the subtantia nigra tissue with age, though the total content of lipid decreases with age of individuals. Moreover, the statistically significant decrease in the protein content within neuron bodies was observed. Interestingly, it is presented that the content of the anti-parallel beta sheets for neuron bodies decreases from seventh to eighth decades of life and subsequently markedly increases from eighth to ninth decades of life, whilst, as regards extraneuronal spaces, the opposite trends are reported i.e. increase from the seventh to eighth decades, and subsequent decrease in the ninth decade of life. These observations, though preliminary, shed the light on a putative contribution of various pathological lipid- and protein-related processes underlying senescence, suggesting a "biochemical link" between the aetiology of the most common neurodegenerative diseases and physiological aging.

The oxidation states and chemical environments of iron and zinc as potential indicators of brain tumour malignancy grade - preliminary results.

Despite the enormous advances in medicine, brain tumours are still among the lesser-known types of tumours and carry the worst prognoses. Transition metals are believed to play an essential role in carcinogenesis. The aim of this study was to determine differences in the average oxidation state and trends in the changes in the chemical environment of iron and zinc contained in healthy and neoplastic tissues of the human brain. For this purpose, X-ray Absorption Spectroscopy was used, which enables the study of disordered matter. The samples were taken intraoperatively and then immediately frozen to slow down chemical processes. Sixteen tumour samples with various malignancy grades were studied as well as one control sample. For each sample four to eight spectra were recorded, with a shift between them not greater than 0.2 eV. In all of the samples, iron occurred in compounds with both Fe(2+) and Fe(3+). However, the ratio of Fe(ii) to Fe(iii) content in the tissue visibly increased with the tumour malignancy grade. The change in the oxidation state of iron did not correlate with the hypoxia level of the tissues. Analysis of EXAFS spectra of zinc atoms showed that the chemical environment of zinc atoms differed with the tumour malignancy grade. Additionally, cryogenic conditions were found to produce positive results in studies of biological samples, whose form under such conditions is close to their native state, without preparation-caused artefacts.

Elemental micro-imaging and quantification of human substantia nigra using synchrotron radiation based x-ray fluorescence--in relation to Parkinson's disease.

Synchrotron radiation based x-ray fluorescence (SRXRF) was applied to the quantitative evaluation of elemental changes in substantia nigra pars compacta (SNc) in Parkinson's disease (PD) in the framework of a study on the role of chemical elements in the pathophysiology of PD. The analysis was carried out for dopaminergic nerve cells and extraneuronal spaces. The mass fractions of P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Rb were determined. The application of standard samples developed especially for the determination of elemental mass fractions in thin tissue sections using the SRXRF technique is presented. Two-dimensional maps of elemental distribution show that the location of nerve cells in SNc sections is precisely visualized by the high levels of most elements. It was found that statistically significant differences between control and PD neurons are observed for S (p = 0.04), Cl (p = 0.02), Ca (p = 0.08), Fe (p = 0.04) and Zn (p = 0.04). The mass fractions of P (p = 0.08), S (p = 0.07), Cl (p = 0.04), Zn (p = 0.08) and Rb (p = 0.08) in areas outside the nerve cell bodies differed significantly between PD and control groups. A clear cluster separation between the PD nerve cells and neurons representing the control group was noticed. It was found that Cl, Fe, Ca and Zn are the most significant elements in the general discrimination between PD nerve cells and the control. The comparison between the extraneuronal spaces showed that Cl, Fe and Cu differentiate the PD and control group the most. The evident contribution of chemical elements to the pathophysiology of PD was shown.

The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats.

Recent studies of Parkinson's disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental micro-imaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats.

First step toward the "fingerprinting" of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis.

Synchrotron-radiation-based X-ray fluorescence was applied to the elemental microimaging of neoplastic tissues in cases of various types of brain tumors. The following cases were studied: glioblastoma multiforme, gemistocytic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ganglioglioma, fibrillary astrocytoma, and atypical transitional meningioma. Apart from neoplastic tissue, the analysis included areas of tissue apparently without malignant infiltration. The masses per unit area of P, S, Cl, K, Ca, Fe, Cu, Zn, Br, and Rb were used to construct a diagnostic classifier for brain tumors using multiple discriminant analysis. It was found that S, Cl, Cu, Fe, K, Br, and Zn are the most significant elements in the general discrimination of tumor type. The highest similarity in elemental composition was between atypical transitional meningioma and fibrillary astrocytoma. The smallest differentiation was between glioblastoma multiforme and oligodendroglioma. The mean percentage of correct classifications, estimated according to the a posteriori probabilities procedure, was 99.9%, whereas the mean prediction ability of 87.6% was achieved for ten new cases excluded previously from the model construction. The results showed that multiple discriminant analysis based on elemental composition of tissue may be a potentially valuable method assisting differentiation and/or classification of brain tumors.