Elemental Analysis of Renal Slices by Proton-Induced X-Ray Emission

Abstract

Methods for analyzing elements in biological samples are important for studying the effects of intoxication. Since its introduction in 1970 (1), proton-induced X-ray emission spectroscopy (PIXE) has been shown to be a powerful, multielemental analysis technique with high sensitivity (2). The PIXE technique involves bombarding a sample with protons which eject inner-shell electrons of the elements in the sample ; the subsequent decay of the induced electronic states results in the emission of X-rays whose energies are characteristic of the elements present in the sample and whose intensities are proportional to the concentrations of these elements. A liquid-nitrogen-cooled solid-state Si(Li) detector and a multichannel analyzer are used to determine the energies as well as the intensities of the emitted X-rays, and a computer program identifies the elements present and calculates their concentrations. Although the application of PIXE to biological samples is not new (3), and recent reviews have emphasized the usefulness of this technique (4-7), the optimization of PIXE for use in typical animal toxicity studies has not been described in the toxicology literature. Here we present the optimization of the instrumentation, sample preparation, and the application of PIXE to a disposition study of metals in renal tissue. The implementation of the PIXE technique has been limited mostly to physics and nuclear research laboratories because no commercial instrumentation is available. The proton beams for PIXE are generated by Van de Graaff accelerators that are not in high demand for applications in physics because much higher-energy ion beams are preferred in most nuclear physics research. The availability of high-energy accelerators has therefore made PIXE an attractive alternative to other conventional, multielemental analytical methods. The simultaneous quantification of a large number of elements in a sample makes PIXE an important analytical tool for use in diverse toxicological studies. Overview of PIXE Theory and Technique X-rays are produced as electrons from outer shells rapidly decay to replace inner-shell electrons ejected by high-energy protons. The energies of the X-ray emissions are characteristic of the elements, and the intensities of the emissions are proportional to the concentrations of the elements in the sample. The useful range of emitted X-ray energies for PIXE is 1-30 keV; the Si(Li) detector is capable of resolving X-ray emissions as closely spaced as 150 eV, and, as a consequence, 20-30 elements can be readily identified and determined simultaneously. An X-ray spectrum of a thin copper film,deposited on a carbon substrate is shown in Figure 1. The dominant features of the spectrum are the copper K. and K X-ray emission lines resulting from the ejection of the K (innermost) shell electrons of copper. The shape of the X-ray emission peaks in the spectrum is primarily Gaussian; the width of the peak is defined by the statistical distribution of the electrical pulses generated by X-ray photons in the...

Metal speciation in environmental and biological systems.

“…It has been estimated that the major fraction of metals in "solution" in aquatic systems are in reality adsorbed on solid surfaces of colloids or in particulate matter (11). There is general agreement that in the presence of realistic concentrations of anionic complexing agents and in the absence of strong complexing agents such as nitrilotriacetic acid (NTA) and fulvic acid, the major species of lead found in freshwater is the ion-association complex PbCO3 (12).…”

“…In the last 10 to 15 years there has been a renewed interest in the X-ray fluorescence method as a result of the availability of Van processing can be completely automated (11,12). Additionally, the proton beam can be focused and areas from 10 Pm2 to 100 pm2 can be analyzed for heavy elements (micro-PIXE) present at parts per million concentration levels.…”

Interaction of Metals during Their Uptake and Accumulation in Rabbit Renal Cortical Slices

“…Samples were placed in the freezer immediately after preparation until the day before the analysis, at which point they were placed in a desiccator and allowed to thaw and dry. The dried samples were then analyzed by PIXE, as described by Lowe et al (9). Briefly, the samples were placed in an evacuated chamber where they were subjected to a proton beam produced by a Van de Graaff generator.…”

“…By using a representative valid in vitro system, such as precision-cut renal cortical slices (8), various combinations of exposures can be more readily performed. In addition, proton-induced X-ray emission (PIXE) has proven to be a technique that allows for quantitation of multiple elements in these renal slices (9).…”

Use of the Caco-2 cell model to assess the relative lead-chelating ability of diasterioisomers of 2,3-dimercaptosuccinic acid.

“…This was accomplished by measuring the concentrations of lead, zinc, and other essential elements in cells exposed to lead solutions. The relative ability of the two forms of DMSA to mobilize lead, zinc, and other essential elements from inside the cells was assessed using macro-PIXE (22). This technique provides information on the concentrations of elements above atomic number 11 and is the only analytical method that can measure 20-30 elements nondestructively in a single small sample (<5 mg) with a detection limit of 1-5 ppm (dry weight).…”