Myroslav V. Zoriy

Determination of phosphorus and metals in human brain proteins after isolation by gel electrophoresis by laser ablation inductively coupled plasma source mass spectrometry

Phosphorus, sulfur, silicon and metal concentrations (Al, Cu and Zn) were determined in human brain proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after separation of protein mixtures by two dimensional (2-D) gel electrophoresis. The analysis of phosphorus, silicon and metals in single protein spots in the gel was performed with an optimized microanalytical method using a double-focusing sector field inductively coupled plasma mass spectrometer coupled to a commercial laser ablation system (LA-ICP-MS). Relative ion intensities for P, Si and metals with respect to sulfur in protein spots were determined by LA-ICP-MS. The detection limits for phosphorus and sulfur in protein spots with a silver staining procedure on the 2-D gels were compared with the Coomassie staining technique described previously.

Ultratrace determination of uranium and plutonium by nano-volume flow injection double-focusing sector field inductively coupled plasma mass spectrometry (nFI–ICP-SFMS)

A sensitive analytical procedure based on nano-volume flow injection (FI) and inductively coupled plasma double-focusing sector field mass spectrometry (ICP-SFMS) was developed for the ultratrace determination of uranium and plutonium. A 54-nl sample was injected by means of a nanovolume injector into a continuous flow of carrier liquid at 7 μl min−1 prior to ICP-SFMS. The absolute detection limits were 9.1 × 10−17 g (3.8 × 10−19 mol, ∼230000 238U atoms) and 1.5 × 10−17 g (6 × 10−20 mol, ∼38000 242Pu atoms) for uranium and plutonium, respectively. The method was validated for the determination of the uranium isotope ratios by the analysis of a certified isotope reference material (NIST U350). The analysis of a contaminated urine sample showed the enriched uranium to be the origin of contamination. Another application concerned the determination of plutonium at the subfemtomolar level in water with a detection limit down to the ag ml−1 range (6 × 10−18 g ml−1).

Metal Imaging on Surface of Micro- and Nanoelectronic Devices by Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Possibility to Measure at Nanometer Range

An analytical mass spectrometric method for the elemental analysis of nano-bioelectronic devices involved in bioengineering research was developed and applied for measurements of selected metals (Au, Ti, Pt, Cr, etc. ) on interdigitated electrode array chips (IDA-chip). An imaging laser ablation inductively coupled plasma mass spectrometric (LA-ICP-MS) procedure was used to map the elements of interest on the surface of the analyzed sample. The obtained images of metals were in a good agreement and corresponded to the micro- and nanofabricated metal electrode pattern. For the analysis at nanometer resolution scale a NF-LA-ICP-MS (NF-near-field) procedure was applied, which utilize thin Ag needle to enhance laser beam energy and improve spatial resolution of the method. The results show a ∼100× enhancement of analyte signal, when the needle was positioned in the “near-field region” to the sample surface and the laser shot was performed. In addition, mass spectrometric studies of reproducibly for five separated NF-LA shots in different places of analyzed sample yielded an RSD of the measurement of 16%.

Imaging of metals and metal-containing species in biological tissues and on gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS): A new analytical strategy for applications in life sciences*

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has become established as a very efficient and sensitive trace, ultratrace, and surface analytical technique in the life sciences. We have developed a new analytical imaging technique using LA-ICP-MS to study element distribution in biological tissues. Nowadays, LA imaging ICP-MS using double-focusing sector field (LA-ICP-SFMS) or quadrupole-based mass spectrometers (LA-ICP-QMS) can be applied as an exciting tool providing new information on the pathophysiology, pharmacology, and toxicology of elements of interest in biological systems. The quantitative determination of elements (e.g., Cu, Fe, Zn, Se, and others) in biological tissues is of growing interest especially in brain research (e.g., for studying neurodegenerative diseases such as Alzheimers or Parkinsons disease). LA-ICP-SFMS was employed to produce images of detailed regionally specific element distributions in thin tissue sections of different sizes (such as control human or rat brain tissues or tumor regions). In addition, imaging MS using LA-ICP-QMS was applied to study the uptake and transport of nutrient and toxic elements in plant tissues. Besides the quantitative imaging of essential and toxic elements in tissues, powerful analytical techniques are also required for the determination and characterization of phosphoproteins and metal-containing proteins within a large pool of proteins, after electrophoretic separation (e.g., blue native, BN and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) into 1D and 2D gels. LA-ICP-MS was used to detect metalloproteins in protein bands of 1D gels or protein spots separated after 2D gel electrophoresis (2D-GE). In addition to elemental determination by LA-ICP-MS, matrix-assisted laser desorption/ionization (MALDI)-MS was employed to identify metal-containing proteins. Recent progress will be discussed in applying LA-ICP-MS in the life sciences, including the imaging of thin slices of tissue and applications in proteome analysis in combination with MALDI-MS to investigate phosphoproteins and metal-containing proteins.

Element distribution is altered in a zone surrounding human glioblastoma multiforme.

Recent data indicate that A(1) adenosine receptor (A(1)AR) density is increased in a zone surrounding human and experimental gliomas. On the contrary, tumor tissue and adjacent brain tissue show low to intermediate A(1)AR densities. In order to assess whether changes in A(1)AR expression are indicating further processes of a chemical reorganization of the peritumoral zone, we investigated element concentrations and distribution patterns of copper and zinc in six human glioblastoma multiforme (GBM) specimens by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Uranium and lead were used as external standards. Copper and zinc levels were increased in a peritumoral zone corresponding to the region of elevated A(1)AR density. They showed a lower density in the solid tumor in comparison to surrounding brain tissue, although the cellular density was higher within GBM. Our findings suggest that the immediate vicinity of GBM is characterized by increased levels of copper and zinc supporting the view that higher A(1)AR density surrounding GBM is not an isolated alteration of peritumoral tissue but an indicator of complex changes in the vicinity of infiltrative tumors. Further research is needed to explore the pathophysiological consequences of altered peritumoral element distribution.

Glia Protects Neurons against Extracellular Human Neuromelanin

Background: Neuromelanin-containing neurons of the substantia nigra are highly vulnerable to degenerate in Parkinson’s disease. Inhibition of the respiratory chain or formation of reactive oxygen species (ROS) by intracellular neuromelanin and triggering of inflammatory processes by extracellular neuromelanin emanating from melanized neurons after their demise are thought to be causally implicated in the high vulnerability of melanized neurons. Objective: We addressed the direct effect of purified neuromelanin on mitochondrial complex I activity, and its influence on ROS production and survival of primary mesencephalic neurons in the presence or absence of glia. Methods: Neuromelanin was isolated from midbrain of postmortem human brains. The content in iron and other elements was measured by inductively coupled mass spectrometry. The effect of neuromelanin on mitochondrial complex I activity was analyzed in post-nuclear extracts. Primary neuronal enriched and neuron-glia mixed cultures from midbrain were treated with different concentrations of neuromelanin. The generation of ROS was determined by fluorochrome detection. MAP2-positive and TH-positive neuronal viability was analyzed. Results: Neuromelanin did not affect complex I activity, but concentration-dependently increased ROS production in neurons and reduced the number of MAP2-positive and TH-positive cultured neurons. Glia protected neurons against the neuromelanin toxicity. Conclusion: Extracellular neuromelanin is detrimental to neurons implicating a mechanism of intracellular ROS production, but not complex I inhibition. ROS formation may be catalyzed by iron, which was sensitively identified in purified neuromelanin (3.3 mg/g). Importantly, we demonstrate that glial cells have the potential to mitigate the neurotoxic effect of neuromelanin.

Accreditation of testing laboratories in dosimetry: the use of a flexible scope at the Competent Incorporation Measuring Body Jülich

The accreditation of the Competent Incorporation Measuring Body at Jülich includes incorporation monitoring by means of direct measurements of the body activity as well as by means of indirect determination of the body activity by radiochemical analysis of excreta samples. In both testing areas, it proved to be very useful to have a flexible scope. In particular, the associated freedom in choosing testing procedures supports the continual improvement process of the laboratory. The modification of existing methods as well as the development and introduction of new procedures makes an immediate reaction to changed requirements feasible. At Jülich the use made out of the flexible scope included, e.g. the introduction of mathematical calibration in whole-body counting and the automation of sample preparation in radiochemical analysis. Advantages of the new procedures and modified methods include on the one hand the reduction of processing times, downtimes and hazard potentials on the other hand enhanced detection limits and improved cost-efficiency. In the result, it can be recommended to other qualified testing laboratories to go for a flexible scope.

No effect of digestate amendment on Cs-137 and Sr-90 translocation in lysimeter experiments

The soil-plant transfer of Cs-137 and Sr-90 in different crops was determined with respect to the present-day amendment practice of using digestate from biogas fermenters. The studies were performed using large lysimeters filled with undisturbed luvisol monoliths. In contrast to the conservative tracer, Br-, neither of the studied radionuclides showed a significant vertical translocation nor effect of the applied digestate amendment compared to a non-amended control was found. Furthermore, no significant plant uptake was measured for both nuclides in wheat or oat as indicated by the low transfer factors between soil-shoot for Cs-137 (TF 0.001-0.010) and for Sr-90 (0.10-0.51). The transfer into nutritionally relevant plant parts was even lower with transfer factors for soil-grain for Cs-137 (TF 0.000-0.001) and for Sr-90 (0.01-0.06). Hence, the amendment with biogas digestate is unfortunately not an option to further reduce plant uptake of these radionuclides in agricultural crops, but it does not increase plant uptake either.

Fractionation of uranium and other trace elements in soil samples collected in selected regions of the Republic of Kazakhstan

To determine the specific chemical forms of trace elements and to study of their mobility/availability in soil, samples collected in the Republic of Kazakhstan were analysed using the revised BCR protocol. A study was made with dependence of the mobility of different elements of interest on digestion procedures for collected soil samples and Standard Reference Material. The concentrations of the analysed elements (Sc, Mn, Co, Ni, Cu, Zn, Ga, Y, Cd, In, Tl, Pb, Th, U) were measured using ICP-MS in each sequential extraction step (exchangeable, reducible, oxidation and residual) and after a total digestion procedure.

Quality assurance of testing methods in incorporation monitoring at the officially recognised incorporation measurement office at Jülich, Germany.

The systematic quality assurance (QA) and control of testing methods in incorporation monitoring consists of continual measures for internal QA and additional measures such as external laboratory controls. This includes among other aspects accuracy, precision and descriptions of the methods as well as the representation and timely availability of analytic results of measurements and internal dose assessment. At the officially recognised incorporation measurement office at Jülich, QA is performed for direct measurements (whole-body counter), indirect measurements with radiochemical testing methods of excretion samples and internal dose assessment.