Indirect Measurement of Biologically Important Compounds by Means of X-Ray Microanalysis

 The main objective of our work was to investigate the possibility and usefulness of indirect methods in X-ray microanalysis for the quantification of biologically important compounds. Metallothionein-like proteins (MT-like proteins) from kidney and liver, rich in sulfur were chosen as an indicator of heavy metal presence in cells and their environment. Tissues from goldfish (Carassius auratus gibelio) were sampled after short and prolonged periods of exposure to Co⁺², CrO₄ ⁻², Pb⁺², Cu⁺² and control treatment and prepared for histochemical staining for peroxidated thiolate groups. Commonly used –S–S– bonds dye (Nitro Red) was replaced with iodine atoms and they were quantified at L line by means of X-ray microanalysis combined with SEM. After fish treatments with heavy metal solutions changes in MT-like proteins and in I atom contents were expected. There was statistically significant decrease in MT-like proteins level in kidney after lead treatment (Pb/C = 0.62). In liver a statistically significant increase in MT-like proteins concentration was observed after chromium, cobalt and lead ions treatment in comparison to control animals. The following ratios were noted: 3.04 for Cr/C, 2.18 for Co/C and 2.10 for Pb/C. Our finding indicates that the method of indirect measurement of MT-like proteins in fish and other animal tissues is possible. The concentration of iodine atoms is above their detection level by EDS and their changes are possible to identify. During histochemical procedures it is worth taking into account sample preparation methods which might disturb the quality and quantity of the analysed material.     

Co-Localization of Copper, Zinc and Lead with Calcium in Their Accumulation Sites in the Housefly’s Abdomen by Micro-PIXE

Our previous study showed that treatment of Musca domestica larvae with semi-lethal doses of Cu, Zn or Pb leads to their accumulation in the adult fly’s abdomen. Now we report that the distribution pattern of heavy metals is similar to that of Ca. The housefly’s larvae were reared on media contaminated with semi-lethal doses of Cu (800 ppm), Zn (2000 ppm) or Pb (10,000 ppm) and on control medium. Ten days after eclosion, adult houseflies were prepared for X-ray microanalysis. Analyses were performed on the whole freeze-dried abdomen specimens using a nuclear microprobe (PIXE and RBS) at iThemba LABS, South Africa. GeoPIXE II software was used to obtain quantitative elemental maps by the Dynamic Analysis method. On the basis of Cu, Zn and Pb accumulation sites, the concentrations of these elements were correlated with the concentration of Ca. There was a clear relationship between Ca and all heavy metals examined, indicating Ca co-localization in their deposition sites. In the control animals, Zn and Ca deposition sites were significantly correlated. Association of heavy metals with Ca in their accretion sites suggests the presence of metal-accumulating granules, the so-called “concretions” probably in the Malpighian tubules, midgut epithelium or in the fat body.     

PROZA and CALIBRATION CURVES for Quantitative X-Ray Microanalysis of Biological Samples

The main scope of our work was to investigate the usefulness of the PROZA correction method and CALIBRATION CURVE construction for quantitative X-ray microanalysis of biological soft, freeze-dried tissues. The test samples with a known elemental concentration of NaCl, MgCl₂, KH₂PO₄ and Na₂SO₄ were prepared on the basis of 20% porcine skin gelatine. A control sample containing 20% gelatine was used for sulphur subtraction. Dissolved gelatine solutions were cooled with liquid nitrogen and then cut in cryomicrotome into 16µm thick slices. The sections were lyophilised, coated with carbon and analysed by means of a scanning electron microscope combined with an energy dispersive (ED) spectrometer. The homogeneity of the prepared samples was verified using the Fishers test. Only homogenous samples were used for calibration. The significance of the (z) (PROZA) correction method for biological sample analysis was verified by comparing the prepared standard and entering it into the Voyager computer program memory with the remaining samples of a known elemental content. The differences between the standard and the samples were noted for all elements analysed. There was no sample matching the standard after the PROZA correction procedure. A high correlation r factor (above 0.99) for all analysed elements indicates that CALIBRATION CURVES construction could be suitable for quantitative X-ray microanalysis of biological samples.