D. Vandeputte

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney.

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.

Phosphorus, calcium and lead distribution in collagen in lead induced soft tissue calcification : an ultrastructural and X-ray microanalytical study

Repeated intraperitoneal injections of lead acetate in rats caused a calcification of the skin of the abdomen near the site of the injections. In the lead-induced calcifications, electron dense collagen bundles could be observed. On the surface of the collagen fibrils, needle-like crystals were visible. With energy-dispersive X-ray analysis, phosphorus, calcium and lead were detected in the electron dense collagen bundles. X-ray maps of the P-K alpha, Ca-K alpha, and Pb-L alpha plus Pb-L beta lines showed an equivalent distribution along the collagen fibrils for phosphorus and calcium. The occurrence of the most electron dense areas in the STEM-image was comparable to the lead distribution. A good correlation existed between the structural and the elemental images of the same area. Although the medicinal use of preparations containing lead is no longer recommended, some are still prescribed. From our results we can conclude that they should not be applied to injured or inflamed skin.

Localization of lead and fluoride in cultured tooth germs by laser microprobe mass analysis.

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl2 of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine.

Phosphorus, calcium and lead distribution in experimental calcification induced by lead acetate. An X-ray microanalytical study

Rats were in jected in t ra pe r i t onea l l y with lead acetate for several weeks. At autopsy we noted white deposits at the inner surface of the peritoneum and on in ternal organs. The skin near the area of the in jec t i ons was hard and seemed c a l c i f i e d . U l t ras t ruc tu ra l examination of the subcutaneous connective t issue revealed the presence of small electrondense deposits in the ex t race l l u l a r ground substance associated wi th apparently normal collagen f i b r i l s in some collagen bundles. In other collagen bundles the f i b r i l s were thickened and extremely electron dense. The energv d ispers ive X-Pay spectrum of a semi-thin sect ion of a sample f i xed in glutaraldehyde, dehydrated in alcohol and embedded in epoxy resin showed that the area near the electron dense collagen f i b r i l s contained large amounts of phosphorus, calcium and lead. Maps of the phosphorus K~ (2,02 keV), calcium K~(3.70 keV) and lead L=(10.56 keV) could be superimposed on the STEM image of the collagen f i b res and gave an essent ia l equivalent d i s t r i b u t i o n pattern for the three elements. Two mechanisms have been proposed for t h i s lead induced so f t t issue c a l i f i c a t i o n (1): a) lead may be i n i t i a l l y f i xed to a component of the collagen f i b res , thereby creat ing or exposing a binding s i t e for phosphorus and calcium on these f i b res , and b) p r e c i p i t a t i o n of lead wi th pyrophosphate, a powerfull i n h i b i t o r of c a l c i f i c a t i o n , may lead to spontaneous p rec ip i t a t i on of apa t i te .