Trace element determinations using a 15-keV synchrotron X-ray microprobe.

At the Synchrotron Radiation Source (SRS), Daresbury, U.K., a synchrotron microprobe was constructed, in order to create an instrument capable of analyzing at the ppm or in favorite cases sub-ppm level with a lateral resolution of 10 x 15 microns2. In order to span a wide range of elements to be analyzed, a beam energy of 15 keV was chosen. Focusing and monochromation of the white beam was done in one single step with a high-precision ellipsoidally concave curved Si(111) crystal. Sufficient flux of X-rays in a narrow energy band is available in the spot to measure trace elements at the femtogram level. Measurements on standard materials, reference standard materials, and biological samples showed the lower relative minimum detection limits and higher sensitivity for the higher Z-elements obtainable with micro-SXRF (synchrotron X-ray fluorescence) as compared with microtechniques using ion accelerators. Moreover, the much lower energy deposited in the specimen represents a major argument to prefer X-rays to ions for the analysis of radiation-sensitive samples.

Biomedical applications of proton induced X-ray emission.

Apart from studies on aerosols, the majority of applications of proton induced X-ray emission (PIXE) with a normal beam or a microprobe (micro-PIXE) is found in biology and medicine. Two aspects of broad beam PIXE are often decisive for the choice of this analytical technique. Compared to other techniques capable of analysis down beyond the ppm level, PIXE can be carried out with a very small amount of material and minute fractions of the composite samples, even in the scale of micrometers and quite often with minimal sample preparation, which are important requirements for biomedical investigations. Secondly, the speed of the total analysis opens the possibility to analyze large numbers of samples in a reasonable time, which is often necessary in biomedical studies in order to obtain sufficiently significant correlations between trace element concentrations and biomedical phenomena. Few, if any, techniques can compete with micro-PIXE; quantitative trace element analysis on a micrometer scale still represent a challenging problem. The electron microprobe normally lacks the sensitivity while the laser induced techniques suffer as yet from quantification problems. This paper describes recent developments especially in micro-PIXE in biomedical research.

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus. A microbeam PIXE study. Particle Induced X-Ray Emission.

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus was investigated using PIXE (Particle Induced X-Ray Emission) spectroscopy. Using the proton microbeam (60 X 60 microns), 2 mm line-scans were made on hippocampal sections and the data were expressed as absolute zinc concentrations. Values of 55 and 136 ppm (dry weight) were found for the mean background zinc level and the maximum mossy fiber zinc level, respectively, in animals fed a control diet containing 50 ppm zinc. Treatment of these animals with dithizone caused about 50% reduction in the maximum mossy fiber zinc level. Feeding a zinc-deficient diet for 28 days did not cause a decrease in the mossy fiber zinc level, however, feeding the zinc-deficient diet for 90 days reduced the maximum mossy fiber zinc level by about 30%. The results are discussed in relation to the behavioral abnormalities that have been observed in zinc-deficient animals.

Incorporation routes of elements into human hair; implications for hair analysis used for monitoring.

A study has been made on the incorporation of some trace elements and minerals into human hair by measuring their distributions across hair diameters. For this study the Particle Induced X-ray Emission (PIXE) technique using the proton microbeam of the Vrije Universiteit of Amsterdam has been applied. Results of measurements on single hair strands at different positions in the root and outside the skin provide evidence that the elements not only can enter the cortex via the matrix but also via the hair root sheaths. Moreover, there are indications that the distribution of an endogenous element is not always homogeneously spread over a cross section of the hair. Endogenous Fe and Pb seem to be peaked on the periphery of the hair by natural means, while the elements S,Zn and Cu of the samples measured are found to be distributed homogeneously across the hair diameter. The consequences for hair analysis in use for monitoring the trace elements status of an individual are discussed.

Copper and zinc metabolism in aspartylglycosaminuria and Salla disease.

Changes in the metabolism of copper and zinc are described in aspartylglycosaminuria (AGU) patients. AGU patients had significantly reduced serum zinc concentrations. However, hair zinc levels were normal, and hyperzincuria could not be demonstrated. The copper content in the hair of AGU patients was highly elevated. Serum copper and ceruloplasmin concentrations were within normal range. In AGU, small-molecular-weight glycoasparagine storage products accumulate in tissues and are excreted in urine in large amounts. They may interfere with the transport mechanisms of trace elements, and thus alter their distribution and availability for tissues. The changes in copper and zinc levels may contribute to the pathogenesis of some of the clinical signs of AGU and Salla disease.

Disturbed metabolism of copper and zinc in aspartylglycosaminuria: possible involvement with connective tissue changes.

In aspartylglycosaminuria (AGU), a lysosomal storage disorder of glycoprotein degradation, there are some abnormalities in collagen and proteoglycan metabolism. Because of earlier observations suggesting a disturbance of copper balance, the metabolism of copper and zinc was studied in more detail to find out if possible trace metal disturbances could be correlated with connective tissue disorder. Highly elevated copper concentrations in the hair and significantly reduced zinc levels in serum and urine were detected in AGU patients indicating a disturbance of trace element balance. However, the patients had normal serum copper levels, and the concentrations of zinc and copper in cultured fibroblasts did not differ from those of control cells. Normal lysyl oxidase activities in cell culture indicate that collagen cross-link formation is not affected. The changes in copper and zinc balance are probably secondary to the basic enzyme deficiency, and may contribute to the development of the clinical signs and symptoms of AGU although the mechanisms involved are not yet understood.

Discrepancies between histological and physical methods for trace element mapping in the rat brain.

This paper contains a comparison between four experimental methods for the detection of trace metals in biological tissues, e.g. autoradiography, dithizone-, Timm-staining and microPIXE (Particle Induced X-ray Emission). As research object the hippocampus of the rat was chosen and mainly the Zn-distribution in the fascia dentata has been examined. The comparison was judged on four criteria: spatial resolution, specificity, quantification and influence of the protein binding of the metal on the outcome of each method. Some additional Timm and microPIXE experiments have been performed to compare the metal concentrations for both normal and Zn-deficient rats. The Zn/Cu ratio for both kinds of rats were calculated and some striking differences in the concentrations were obtained. The Zn/Cu ratio for a normal rat was found to be about 40% higher than for a deficient one.

Mechanism of action of the copper(I) complex of 2,9-dimethyl-1,10-phenanthroline on Mycoplasma gallisepticum.

Evidence was found that the inhibitory action of Cu(DMP)2NO3, the copper(I) complex of 2,9-dimethyl-1,10-phenanthroline (DMP), on Mycoplasma gallisepticum is a consequence of the ultimate toxicity of copper, and not that of the ligand, DMP. From uptake studies with radiolabeled 67Cu and [14C]DMP, we concluded that significantly more copper than DMP is bound to the mycoplasmal cell. It appeared that dissociation of Cu(DMP)2+ occurred shortly after interaction with the cell membrane. Copper was transported across the cytoplasmic membrane. A strong dependence of copper uptake on the incubation medium was observed in the absence of DMP. The main function of the ligand DMP appeared to be as a vehicle for the transport of copper from nontoxic copper-medium complexes to membrane-buried cellular ligands.

Mode of action of the copper(I) complex of 2,9-dimethyl-1,10-phenanthroline on Mycoplasma gallisepticum.

Various physiological important activities of Mycoplasma gallisepticum were inhibited by the copper(I) complex of 2,9-dimethyl-1,10-phenanthroline [Cu(DMP)2NO3]. The energy-yielding metabolism was inhibited because the conversion of pyruvate into lactate was found to be blocked by Cu(DMP)2NO3, indicating a selective inhibition of lactate dehydrogenase. Also, the production rate of acetate and the rate of oxygen uptake by whole cells of M. gallisepticum appeared to be strongly decreased. Experiments with crude cell extracts showed an inhibition of reduced nicotinamide adenine dinucleotide (NADH) oxidase by Cu(DMP)2NO3 and an even stronger inhibition of NADH oxidase and lactate dehydrogenase by CuSO4. No preferential inhibition of adenosine 5'-triphosphatase and pyruvate kinase was found. Investigations on the influence of Cu(DMP)2NO3 on deoxyribonucleic acid, ribonucleic acid, and protein synthesis with growing cells of M. gallisepticum showed a selective inhibition of the incorporation of [14C]thymidine into deoxyribonucleic acid. Cu(DMP)2NO3 induced a decrease in the total amount of accessible sulfhydryl groups of whole cells of M. gallisepticum, indicating that the observed diverse toxicity of Cu(DMP)2NO3 may be associated with the interaction of copper ions with protein sulfhydryl groups.

Mode of action of copper complexes of some 2,2'-bipyridyl analogs on Paracoccus denitrificans.

Copper complexes of 2,2'-bipyridyl and related compounds and CuSO4 inhibited the growth of paracoccus denitrificans. The copper(I) complex of 2,9-dimethyl-1,10-phenanthroline [Cu(DMP)2NO3] showed the highest activity, whereas the copper(II) complex of 1,10-phenanthroline and CuSO4 inhibited the growth to a lesser extent. The uncomplexed ligands (1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline) showed little activity, but in the presence of noninhibitory amounts of CuSO4 this activity increased markedly. Copper ions therefore proved to be essential for the growth-inhibitor effect. The extent of inhibition appeared to be strongly dependent on the initial cell density and on the growth medium. No selective inhibition of deoxyribonucleic acid, ribonucleic acid, or protein synthesis was observed with Cu(DMP)2NO3. Respiratory electron transport of P. denitrificans appeared to be strongly inhibited by Cu(DMP)2NO3 and to a somewhat lesser extent by CuSO4. Both aerobic and anaerobic respirations were inhibited to the same extent, and from the cytochrome redox kinetics it is concluded that the site of this inhibition in the respiratory electron transport chain must be located before cytochrome b. Cu(DMP)2NO3 did not significantly influence the H+/O ratio with whole cells of P. denitrificans, suggesting that the efficiency of oxidative phosphorylation is not affected by CU(DMP)2NO3. Growing cultures of P. denitrificans showed a decrease in intracellular potassium ion content in the presence of increasing amounts of Cu(DMP)2NO3. It is concluded that interference with the cytoplasmic membrane, resulting in inhibition of respiratory electron transport, probably constitutes the main mode of action of copper complexes of 2,2'-bipyridyl analogs on P. denitrificans.