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.     

Elemental profile of cerebrospinal fluid in patients with Parkinson''s disease

To ascertain the potential role of chemical elements (namely, Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Sb, Si, Sn, Sr, Tl, V, W, Zn and Zr) as markers in the Parkinson's disease (PD), the elemental concentration of cerebrospinal fluid (CSF) of 42 patients with PD and 20 age-matched controls was assessed. Analyses were performed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) and Sector Field Inductively Coupled Plasma Mass Spectrometry (SF-ICP-MS). Significantly lower levels of Co, Cr, Fe, Pb, Si and Sn were observed in the CSF of PD patients compared with those in controls, with a percentage of depletion up to 50% for Cr and Pb. No such variations were detected for all the other elements. Results suggested that Pb, Cr, Fe were the most suitable elements to distinguish between normality and PD. Different cut-off concentrations for these elements could be tentatively proposed as a predictive tool for the PD condition.     

Iron as catalyst for oxidative stress in the pathogenesis of Parkinson's disease?

The mechanisms leading to degeneration of melanized dopaminergic neurons in the brain stem, and particularly in the substantia nigra zona compacta (SNZC) in patients with Parkinson's disease (PD) are still unknown. Demonstration of increased iron Fe(III) in SNZC of PD brain has suggested that Fe-melanin interaction may contribute to oxidative neuronal damage. Energy dispersive X-ray electron microscopic analysis of the cellular distribution of trace elements revealed significant Fe-peaks, similar to those of a synthetic melanin-Fe(III) complex in intracytoplasmic electron-dense neuromelanin granules of SNZC neurons, with highest levels in a case of PD and Alzheimer's disease (AD). No Fe increase was found in Lewy bodies or in SN neurons of control specimens. The relevance of chemical reactions of dopamine (DA), 5-hydroxydopamine (5-OHDA), and 6-hydroxydopamine (6-OHDA) with Fe(III) and with dioxygen for the pathogenesis of PD was investigated. An initiating mechanism related to interaction between Fe and neuromelanin is suggested which results in accumulation of Fe(III) and a continuous production of cytotoxic species inducing a cascade of pathogenic reactions ultimately leading to neuronal death.     

Iron-Melanin Complex in Substantia Nigra of Parkinsonian Brains: An X-Ray Microanalysis

Using energy-dispersive x-ray analysis on an electron microscope working in the scanning transmission electron microscopy mode equipped with a microanalysis system, we studied the subcellular distribution of trace elements in neuromelanin-containing neurons of the substantia nigra zona compacta (SNZC) of three cases of idiopathic Parkinson's disease (PD) [one with Alzheimer's disease (AD)] and of three controls, in Lewy bodies of SNZC, and in synthetic dopamine-melanin chemically charged or uncharged with Fe. Weak but significant Fe peaks similar to those of a synthetic melanin-Fe3+ complex were seen only in intraneuronal highly electron-dense neuromelanin granules of SNZC cells of PD brains, with the highest levels in a case of PD plus AD, whereas a synthetic melanin-Fe2+ complex showed much lower iron peaks, indicating that neuromelanin has higher affinity for Fe3+ than for Fe2+. No detectable Fe was seen in nonmelanized cytoplasm of SNZC neurons and in the adjacent neuropil in both PD and controls, in Lewy bodies in SNZC neurons in PD, and in synthetic dopamine-melanin uncharged with iron. These findings, demonstrating for the first time a neuromelanin-iron complex in dopaminergic SNZC neurons in PD, support the assumption that an iron-melanin interaction contributes significantly to dopaminergic neurodegeneration in PD and PD plus AD.     

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.     

Assessing Plasma Levels of Selenium,Copper, Iron and Zinc in Patients of Parkinson’s Disease

Trace elements have been recognized to play an important role in the development of Parkinson’s disease (PD). However, it is difficult to precisely identify the relationship between these elements and the progression of PD because of an insufficient number of patients. In this study, quantifications of selenium (Se), copper (Cu), iron (Fe) and zinc (Zn) by atomic absorption spectrophotometry were performed in plasma from 238 PD patients and 302 controls recruited from eastern China, which is so far the largest cohort of PD patients and controls for measuring plasma levels of these elements. We found that plasma Se and Fe concentrations were significantly increased whereas Cu and Zn concentrations decreased in PD patients as compared with controls. Meanwhile, these four elements displayed differential changes with regard to age. Linear and logistic regression analyses revealed that both Fe and Zn were negatively correlated with age in PD patients. Association analysis suggests that lower plasma Se and Fe levels may reduce the risk for PD, whereas lower plasma Zn is probably a PD risk factor. Finally, a model was generated to predict PD patients based on the plasma concentrations of these four trace elements as well as other features such as sex and age, which achieved an accuracy of 80.97±1.34% using 10-fold cross-validation. In summary, our data provide new insights into the roles of Se, Cu, Fe and Zn in PD progression.     

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.     

Hepatic distribution of iron, copper, zinc and cadmium-containing proteins in normal and iron overload mice

Subcellular distribution of metal-containing proteins of Fe, Cu, Zn and Cd were determined in the liver samples of iron overload mice by size exclusion high performance liquid chromatography with on-line coupling to UV and inductively coupled plasma mass spectrometry. Collision cell techniques was used to remove polyatomic interferences for some elements, such as Fe. Comparative molecular weight (MW) information of the elemental fraction was obtained within a retention time of 40 min. Fe was present only in high-MW (HMW) protein; Cu, Zn and Cd were found in different MW proteins. It was also observed that these four elements studied showed predominant association with HMW fractions. Moreover, compared with the normal group, we found that the contents of these elements except Cu significantly increased and the distribution of some elements like Cd changed in iron overload mouse liver. It means that excessive iron accumulation in vivo may affect the metabolism of other element such as Zn and Cd.     

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.     

Estimation of Trace Elements in Mace (Myristica fragrans Houtt) and Their Effect on Uterine Cervix Cancer Induced by Methylcholanthrene

In the present work, trace elemental analysis of mace (Myristica fragrans Houtt) was carried out by the atomic absorption spectrometry technique. The concentrations of various elements analyzed in this medicine were ranked in decreasing order: selenium (Se)?>?zinc (Zn)?>?magnesium (Mg)?>?iron (Fe)?>?calcium (Ca)?>?manganese (Mn)?>?lead (Pb). The concentrations of Mg, Zn, Fe, Mn, Ca, and Se were significantly decreased in serum of methylcholanthrene tumor models (P?<?0.001) compared with the control and mace groups. It is consistent with the result of tumor incidence. These trace elements could be directly or indirectly responsible for the antitumor activity of mace. The inorganic elements in this folk remedy can partly account for the antitumor.     

Distribution of elements in rabbit liver lobule

The elemental composition of rabbit liver was determined by the PIXE and micro-PIXE methods. The mean concentrations of P, S, Cl, K, Fe, Cu, Zn, and Rb measured by both methods were similar. The latter method also allowed for localization of elements within lobule territory. It has been found that some elements are more prevalent in the veins (Cl, Fe) and others in the liver parenchyma (P, Cu, Zn). Moreover, Zn showed the characteristic intralobular distribution. Some methodological aspects of microbeam application to biological materials were also discussed.     

Correlation of Trace Elemental Profiles in Blood Samples of Indian Patients with Leukoplakia and Oral Submucous Fibrosis

In order to ascertain possible correlation between alterations in trace elemental profile and the progression or regression of two most common potentially malignant disorders affecting oral cavity, namely oral submucous fibrosis and leukoplakia, blood from 60 patients from each group of patients as well from 30 healthy individuals was analyzed for elemental profiling employing EDXRF technique. Out of the 16 detected elements (K, Si, Ca, V, Cr, Ni, Mn, Fe, Cu, Zn, Se, Br, Rb, Sr, Co, and Pb), Mn, Fe, Zn, Br, and Co showed remarkable alteration in their profile in both leukoplakia and oral submucous fibrosis patients with respect to the normal healthy individuals. While Zn, Br, and Fe reflected similar changes--showing gross depletion in both the diseased groups, Mn and Co depicted inverse pattern of alterations in their concentrations in the two types of precancerous disorders when compared to the control subjects.     

Elemental fingerprint as a cerebrospinal fluid biomarker for the diagnosis of Parkinson's disease

The diagnosis of Parkinson's disease (PD) still lacks objective diagnostic markers independent of clinical criteria. Cerebrospinal fluid (CSF) samples from 36 PD and 42 age‐matched control patients were subjected to inductively coupled plasma‐sector field mass spectrometry and a total of 28 different elements were quantified. Different machine learning algorithms were applied to the dataset to identify a discriminating set of elements yielding a novel biomarker signature. Using 19 stably detected elements, the extreme gradient tree boosting model showed the best performance in the discrimination of PD and control patients with high specificity and sensitivity (78.6% and 83.3%, respectively), re‐classifying the training data to 100%. The 10 times 10‐fold cross‐validation yielded a good area under the receiver operating characteristic curve of 0.83. Arsenic, magnesium, and selenium all showed significantly higher mean CSF levels in the PD group compared to the control group (p  = 0.01, p  = 0.04, and p  = 0.03). Reducing the number of elements to a discriminating minimum, we identified an elemental cluster (Se, Fe, As, Ni, Mg, Sr), which most importantly contributed to the sample discrimination. Selenium was identified as the element with the highest impact within this cluster directly followed by iron. After prospective validation, this elemental fingerprint in the CSF could have the potential to be used as independent biomarker for the diagnosis of PD. Next to their value as a biomarker, these data also argue for a prominent role of these highly discriminating six elements in the pathogenesis of PD.

     

Distribution of trace elements in normal and tumor-bearing mice using the multitracer technique

A multitracer solution obtained from the nuclear reaction of selenium with 25-MeV/nucleon ⁴⁰Ar ions was orally administered to normal and tumor-bearing Balb/c male mice. After 96 h, the mice were sacrificed and the elemental distribution was determined in various tissues, organs, and blood. The uptake of Na, Rb, Ga, Sc, V, Cr, Mn, Co, Fe, Zn, Y, Zr, Tc, Ru, Ag, and In in normal and, except for zinc, in tumor-bearing mice was simultaneously detected. Most elements were distributed in about the same manner in the skin and liver of animals in both groups. The distribution of Rb, Ga, V, Cr, Tc, and In showed little or no significant differences between the two study groups. The distribution of Na, Mn, Fe, Ag, Sc, and Co showed significant differences between normal and tumor-bearing mice. In the blood, spleen, and kidney of the normal mice, there was good absorption of Na, Mn, Fe, Ag, Co, and Zn. In the heart, these elements were well absorbed, except for Na and Mn.     

Trace element distribution in the hair of some sickle cell anemia patients and controls

Hair samples of some young sickle cell anemia (SCA) and control patients in Nigeria were analyzed for 12 elements, viz, Se, Hg, Cr, Fe, Zn, Co, Cu, Br, As, Sb, Na, and Sc, using Instrumental Neutron Activation Analysis (INAA).

With the exception of Cu, which was found to be significantly higher in the hair of SCA patients (at the 0.05 level of the t-test), there were generally no significant differences in elemental concentrations within the two groups. A preliminary study of the elemental contents of the fingernails of the same subjects showed a higher abundance of most of the elements in nail than in hair. These preliminary results were compared with similar studies from some other parts of the world.

     

Serum trace element levels and the complexity of inter-element relations in patients with Parkinson's disease

Trace elements have been postulated to play a role in Parkinson's disease (PD). In order to elucidate whether changes in the serum levels of trace elements reflect the progression of PD, we assessed serum levels of 12 elements (Na, K, Fe, Al, Cu, Zn, Ca, Mg, Mn, Si, P and S) in early PD, severe PD and normal subjects, using inductively coupled plasma atomic emission spectrometry. The concentrations in μmol/ml, the relative mole percentage distribution and inter-element relations were computed. Statistical analysis of these data showed a definite pattern of variation among certain elements in early and severe PD compared to controls. In both early and severe PD serum, Al and S concentrations were significantly decreased (p<0.05) compared to the controls. Fe (p<0.01) and Zn (p<0.05) concentrations were significantly lower in severe PD, while K, Mg, Cu (p<0.01) and P (p<0.05) concentrations were higher in early and severe PD compared to the controls. The data revealed an imbalance in the inter-element relations in both early and severe PD serum compared to controls, as shown by the direct and inverse correlations. These results suggest a disturbance in the element homeostasis during the progression of PD.     

A Comparison of Washing Methods for Hair Mineral Analysis: Internal Versus External Effects

A major difficulty in hair elemental (mineral) analysis for biomonitoring is adequate understanding of the effectiveness of washing procedures. A review of washing protocols used in hair analysis publications showed little consensus with regard to solvents and surfactants used, washing times, and number of washing stages. Two washing approaches were subsequently used to compare their influence on internal and external surface elemental signals determined with time-of-flight secondary ion mass spectrometry. Na, K, Ca, Mg, and Fe were assessed with regard to their relative signal compared to carbon. Both washing methods had similar effect. All elements except for Fe appear to be removed from the surface of the hair as well as from inside the hair. Only the internal Fe content changed with washing and could indicate that external surface bound Fe may not be removed with most washing procedures. It is shown that washing procedures can have a significant effect on reducing the internal elemental signal levels in hair.     

Changes in the elemental content of rat heart as a result of the fixation in formalin analyzed by synchrotron radiation X-ray fluorescent analysis

The aim of the study was to investigate possible changes in the elemental content of small biological tissue samples (with low weight) during the short period of fixation in formalin. Therefore, the effect of the fixation time (4 h to 6 days) was studied on the elemental content of the tissue. Synchrotron radiation X-ray fluorescent analysis (SRXRF) technique was used for the determination of the elemental concentrations. The levels of Cl, K, Ca, Br, Rb, and Sr in the samples fixed for 4 h (and longer) show significant differences in comparison with levels in fresh samples, because these ions have a high mobility and capacity for moving through the cell membrane. The content of Zn, Se, Fe, and Cu in the samples fixed by formalin solution during 4 h do not show significant differences compared with fresh samples. The ions of these elements can form complexes with the large protein molecules in the tissue and can hardly be removed from it.     

Distributions of arsenic and essential elements in pinna of arsenic hyperaccumulator Pteris vittata L.

The distributions of arsenic and 6 essential elements in the pinna of As hyperaccumulator, Pteris vittata L., were studied using synchrotron radiation X-ray fluorescence (SRXRF). Significant correlation between the distribution and mobility of the elements revealed that SRXRF study on the elemental distribution was feasible to inspect the transportations of elements in plants. The distribution of As in the pinna showed that As had great abilities to be transported in xylem vessels and from xylem to mesophyll. The distribution of K, one of the most mobile elements in plants, was similar to that of As, whereas the distributions of Fe and Ca with less mobility in plants were almost opposite to that of As in the pinna.     

Distributions of arsenic and essential elements in pinna ofarsenic hyperaccumulator Pteris vittata L.

The distributions of arsenic and 6 essential elements in the pinna of As hyperac-cumulator, Pteris vittata L., were studied using synchrotron radiation X-ray fluorescence (SRXRF). Significant correlation between the distribution and mobility of the elements revealed that SRXRF study on the elemental distribution was feasible to inspect the transportations of elements in plants. The distribution of As in the pinna showed that As had great abilities to be transported in xylem vessels and from xylem to mesophyll. The distribution of K, one of the most mobile elements in plants, was similar to that of As, whereas the distributions of Fe and Ca with less mobility in plants were almost opposite to that of As in the pinna.