The use of cluster and discriminant analysis in the investigations of the role of trace metals in the pathogenesis of Parkinson’s disease

X-ray fluorescence microscopy was applied for two-dimensional elemental analysis of substantia nigra (SN) tissue. The samples representing Parkinson’s disease (PD) and control cases were examined at HASYLAB beamline L and at ESRF beamline ID22. Two-dimensional mapping of P, S, Cl, K, Ca, Fe, Cu, Zn, Se and Br was done with the spatial resolution of 15 and 5 μm. The masses per unit area of elements in neuromelanin reach nerve cells of SN were determined.The elemental data were processed using two multivariate techniques, namely cluster and discriminant analysis. The statistical methods were used for data reduction, both unsupervised and supervised classification as well as for the creation of a model that would simplify case identification based on the elemental analysis of SN tissue. The results of cluster analysis confirmed the statistical significance of the differences in elemental composition of PD and control SN nerve cells. Based on the results of discriminant analysis, the elements (P, Cl, Fe, Cu and Zn) that played the greatest role in the process of differentiation between neurons from examined groups were determined.     

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.     

X-ray fluorescence analysis of long-term changes in the levels and distributions of trace elements in the rat brain following mechanical injury

This paper describes the results of the application of X-ray fluorescence microscopy to the qualitative, topographic and quantitative elemental analysis of nervous tissue from rats with neocortical brain injury. The tissue samples were analyzed with a 15 μm beam defined by the size of the polycapillary focus. Raster scanning of the samples generated 2D cartographies, revealing the distributions of elements such as P, S, Cl, K, Ca, Fe, Cu, and Zn. Special emphasis was placed on the analysis of the areas neighboring the lesion site and the hippocampal formation tissue. The results obtained for rats with mechanical brain injuries were compared with those recorded for controls and animals with pilocarpine-induced seizures. There were no significant differences in the elemental compositions of gray and white matter between injured and uninjured brain hemispheres. A higher level of Ca was observed in the gray matter of both of the hemispheres in brains with neocortical injuries. A similar relation was noticed for Fe in the white matter. A comparative study of hippocampal formation tissue showed a statistically significant decrease in the mass per unit area of P in the dentate gyrus (DG) and the hilus (H) of DG for animals with brain lesions in comparison with the control group. Analogous relations were found for Cu in the DG and Zn in sector 3 of Ammon’s horn (CA3) and the DG. It is important to note that identical changes in the same areas were observed for animals with pilocarpine-induced seizures in our previous study.     

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.     

Elemental composition of the human atherosclerotic artery wall

The elemental composition of the human atherosclerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3 X 10 micron 2) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered.     

Regional distribution of potassium,calcium, and six trace elements in normal human brain

Eight elements (i.e. K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) were measured in 50 different regions of 12 normal human brains by particle-induced X-ray emission (PIXE) analysis. The dry weight concentrations of K, Fe, Cu, Zn, Se, and Rb were consistently higher for gray than for white matter areas. The K, Zn and Se concentrations for the regions of mixed composition and, to some extent, also the Rb concentrations, were intermediate between the gray and white matter values, and they tended to decrease with decreasing neuron density. The mean dry weight concentrations of K, Ca, Zn, Se, and Rb in the various brain regions were highly correlated with the mean wet-to-dry weight ratios of these regions. For Mn, Fe, and Cu, however, such a correlation was not observed, and these elements exhibited elevated levels in several structures of the basal ganglia. For K, Fe, and Se the concentrations seemed to change with age. A hierarchical cluster analysis indicated that the structures clustered into two large groups, one comprising gray and mixed matter regions, the other white and mixed matter areas. Brain structures involved in the same physiological function or morphologically similar regions often conglomerated in a single subcluster.     

Pixe analysis of reproductive fluids

An external 3.8-MeV proton beam was employed to induce X-rays in 100-mg pellets of human follicular fluids and in 4–8 mg pellets of spermatozoa. The elements Cl, K, Ca, Fe, Cu, Zn, and Br were quantitatively determined in follicular fluids, whereas the elements, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, and Zn were determined in the spermatozoa. Both samples had high interelement Spearman correlation coefficients. Correlations of elements in the samples were observed with several clinical parameters. The multielemental analysis of spermatozoa can be used to approximate quantities of trace elements inserted into the egg ooplasm at the time of fertilization. These elemental quantities appear to be in the femtogram (10^?15) range.     

Influence of the coat color on the trace elemental status measured by particle-induced x-ray emission in horse hair

The influence of hair color on the trace elemental status in horse's hair has been studied. A current analytical technique such as particle-induced X-ray emission (PIXE) used in this study has provided reliable, rapid, easy, and relatively inexpensive diagnostic methods. Twenty-eight elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) in mane hair were detected by the PIXE method. The gray hair contains significantly greter amounts of Cu, Ti, and Zn, and lower amounts of Br, Ca, Se, and Sr than those in other colored horse hairs (p<0.05). Those results measured in the horse's hair were similar to those found in human and dog hair. When interpreting a result, it should be kept in mind that hair color, especially gray hair, influences the concentrations of some elements in horse hair.     

Elemental composition of the human atherosclerotic artery wall

Summary The elemental composition of the human atheroselerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3×10 m²) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered.     

Correlation between 25 element contents in mane hair in riding horses and atrioventricular block

The influence of atrioventricular block (AV-block) on the trace elemental status in a horse hair was studied. The particle-induced X-ray emission (PIXE) method has provided a reliable, rapid, easy, and relatively inexpensive diagnostic method. Twenty-five elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Hg, K, Mg, Mn, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Ti, Y, and Zn) in mane hair and serum were measured by the PIXE method. A horse hair with first- and second-degree AV-block contained significantly greater amounts of Br, Ca, Sr, and Zn than those of horses without electrocardiographic abnormalities, whereas there was no significant differences in the elemental contents of the serum of the both groups. Those results in contents of a horse hair suggest that the evaluation of the degree of ionic imbalance by this method might be used to predict the susceptibility of a horse to heart disease much before symptoms appear.     

Pigmentation and temporal effects on trace elements in hair

Variations in trace element concentration in the head and facial hair of five individuals with ever-increasing amounts of white hair were examined. Hair was collected from the scalp, cheeks, and chin from one donor on a regular basis since 1984. Samples were separated into white and pigmented fractions, and analyzed at SLOWPOKE-Toronto by INAA for the short-lived, isotope-producing elements Br, Ca, Cl, Cu, I, Mg, Mn, Na, S, and Zn. Temporal concentration variations of these elements over time, and variations of the elemental concentrations in pigmented and white hair were established.

     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

Analysis of Trace Elements During Different Developmental Stages of Somatic Embryogenesis in Plantago ovata Forssk Using Energy Dispersive X-ray Fluorescence

Energy dispersive X-ray fluorescence (ED-XRF) technique has been used for the determination of trace element profile during different developmental stages of somatic embryogenic callus of an economically important medicinal plant, Plantago ovata Forssk. Somatic embryogenesis is a plant tissue culture-based technique, which is used for plant regeneration and crop improvement. In the present investigation, elemental content was analysed using ED-XRF technique during different developmental stages and also determine the effect of additives—casein hydrolysate and coconut water on the trace elemental profile of embryogenic callus tissue of P. ovata. Subsequent experiments showed significant alteration in the concentration of K, Ca, Mn, Fe, Zn, Cu, Br, and Sr in both the embryogenic and non-embryogenic callus. Higher K, Ca, Fe, Cu, and Zn accumulation was in embryogenic tissue stage compared to other stages, suggesting these elements are crucial for successful embryogenesis. The results suggest that this information could be useful for formulating a media for in vitro embryo induction of P. ovata.     

The association between content of the elements S,Cl, K,Fe, Cu,Zn and Br in normal and cirrhotic liver tissue from Danes and Greenlandic Inuit examined by dual hierarchical clustering analysis

ProjectMeta-analysis of previous studies evaluating associations between content of elements sulphur (S), chlorine (Cl), potassium (K), iron (Fe), copper (Cu), zinc (Zn) and bromine (Br) in normal and cirrhotic autopsy liver tissue samples.ProcedureNormal liver samples from 45 Greenlandic Inuit, median age 60 years and from 71 Danes, median age 61 years. Cirrhotic liver samples from 27 Danes, median age 71 years. Element content was measured using X-ray fluorescence spectrometry. Statistics: Dual hierarchical clustering analysis, creating a dual dendrogram, one clustering element contents according to calculated similarities, one clustering elements according to correlation coefficients between the element contents, both using Euclidian distance and Ward Procedure.ResultsOne dendrogram separated subjects in 7 clusters showing no differences in ethnicity, gender or age. The analysis discriminated between elements in normal and cirrhotic livers. The other dendrogram clustered elements in four clusters: sulphur and chlorine; copper and bromine; potassium and zinc; iron. There were significant correlations between the elements in normal liver samples: S was associated with Cl, K, Br and Zn; Cl with S and Br; K with S, Br and Zn; Cu with Br. Zn with S and K. Br with S, Cl, K and Cu. Fe did not show significant associations with any other element.ConclusionsIn contrast to simple statistical methods, which analyses content of elements separately one by one, dual hierarchical clustering analysis incorporates all elements at the same time and can be used to examine the linkage and interplay between multiple elements in tissue samples.     

Concentrations of Cd, Co, Cu, Fe, Mn, Rb, V, and Zn in formalin-fixed brain tissue in amyotrophic lateral sclerosis and parkinsonism-dementia complex of guam determined by high-resolution ICP-MS

Amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) are neurodegenerative disorders that occurred with extremely high frequency among the native population on Guam, especially in the 1950s and 1960s, but have substantially declined over the last half-century. The etiology of these diseases is unknown, but the most plausible hypothesis centers on imbalances in essential and toxic metals. We have determined the concentrations of Cd, Co, Cu, Fe, Mn, Rb, V, and Zn in formalin-fixed brain tissue collected during the period 1979–1983 from eight Guamanian patients with ALS, four with PDC, and five control subjects using high-resolution inductively coupled plasma-mass spectrometry. The concentrations of Cd are markedly and significantly elevated both in gray and white matter in ALS, but not in PDC patients. The concentrations of Zn are elevated for both patient groups, in both gray and white matter, but only the difference in gray matter for PDC is significant. For the other metals, no significant differences are found.     

Trace elements in human parotid saliva

Although various proteins and some electrolytes have been measured in human saliva, little systematic data about the major and minor elemental components of this body fluid have been obtained. In order to obtain such data, concentrations of C, Na, P, Cl, K, Ca, Sc, Cr, Fe, Co, Zn, Se, Br, Rb, Sb, I, and Cs in human parotid saliva were measured by instrumental nuclear methods. The data obtained confirmed the relative lack of Zn in saliva of patients with hypogeusia (decreased taste acuity) and suggested that concentrations of Na, Cl, Br, and Ca followed the order: normals > hypogeusia > hyposmia (decreased smell acuity). To compare concentrations of elements in saliva with those in blood and urine, absolute concentrations were normalized to that of Na through the use of a concept called an enrichment factor. On this basis, parotid saliva is relatively depleted in Se, Zn, and Fe and enriched for most other elements relative to blood plasma indicating that the fluid is not simply a transudate of blood plasma. Using this same technique, saliva composition was found more similar to urine than blood plasma, being relatively depleted in Se, Cs, and Co, being enriched in I, Br, and Cr and having about the same relative concentrations of P, Cl, Zn, Fe, Ca, K, and Rb. As the total body concentrations of many of the enriched elements in saliva are extremely small, their enrichment in saliva suggests special roles for these elements in the oral cavity. Because of its accessibility, ease of collection, and interaction with some body constituents, saliva represents a useful, albeit neglected, tool in the diagnosis of some physiological and pathological changes in body function and in understanding important aspects of trace metal metabolism.     

A search for trace elements in some human intracranial tumors by instrumental neutron activation analysis

A investigation was undertaken to measure the presence of trace elements in some intracranial tumors using the instrumental neutron activation analysis technique. The following 20 minor and trace elements were investigated: Na, Mg, Al, P, Cl, K, Ca, Cr, Mn, Fe, Co, Cu, Zn, As, Se, Br, Rb, Sb, I, and Cs. Our results are compared with other trace element analyses in human brain tissue.     

NAA-SLR and ICP-AES Application in the Assessment of Mass Fraction of 19 Chemical Elements in Pediatric and Young Adult Prostate Glands

The effect of age on the mass fraction of 19 chemical elements in the intact prostate of 50 apparently healthy 0–30-year-old males was investigated by neutron activation analysis with high-resolution spectrometry of short-lived radionuclides and inductively coupled plasma atomic emission spectrometry. Mean values (M ± standard error of the mean) for mass fraction (in milligrams per kilogram, on dry weight basis) of chemical elements were as follows: Al, 77?±?17; B, 1.31?±?0.29; Ba, 4.0?±?1.2; Br, 37.7?±?4.3; Ca, 1,536?±?189; Cl, 13,414?±?949; Cu, 12.3?±?2.1; Fe, 132?±?11; K, 11,547?±?468; Li, 0.064?±?0.009; Mg, 922?±?89; Mn, 1.88?±?0.16; Na, 9,834?±?411; P, 6,741?±?335; S, 8,034?±?251; Si, 199?±?34; Sr, 1.40?±?0.19; and Zn, 277?±?33. The upper limit of mean mass fraction of V was ≤0.24. This work revealed that there is significant tendency for the mass fractions of Ca, K, Mg, and Zn in the prostate tissue of healthy individuals to increase with age from the time of birth up to 30 years. It means that Ca, K, Mg, and Zn mass fractions in prostate tissue are the androgen-dependent parameters. Our finding of a positive correlation between the prostatic Zn and Ca, K, Mg, P, and S mass fractions indicates that there is a special relationship of Zn with some main electrolytes (Ca, K, and Mg) and with P- and S-containing compounds in the prostate. It was shown also that high levels of Al, B, Ba, Br, Cl, Li, Na, and Sr mass fraction in prostate tissue do not indicate a direct involvement of these elements in the reproductive function of the prostate.     

江苏海岸带陆生盐土植物矿质元素含量的特点及生物循环

陆生盐土植物在生长过程中吸收积累了大量的Cl和Na;从海向陆随着土壤和植被的生态演替,植物中Cl和Na的浓度逐渐降低;N与Cl、Na有相似的水平分布规律;植物种类是影响元素吸收积累的主要因素。在盐地碱蓬中N、P、K、Ca、Mg、Na、Cl、Mn和Zn的含量均是生长前期较高,随着其生长老化逐渐降低,大穗结缕草、白茅与盐地碱蓬相比,Ca的含量前期低后期高,Na、Mn、Cu和Zn的季节变化不明显。参加盐地碱蓬系统生物循环的元素中,Cl和Na的比例最大,在大穗结缕草和白茅生态系统中比例较小;由于白茅被收割利用,一些元素从此生态系统中流失。     

Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na⁺,K⁺-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.