Use of laser microprobe mass analysis (LAMMA) for localizing multiple elements in soft and hard tissues

The potential of laser microprobe mass analysis (LAMMA) as a sensitive microanalytical technique was explored in applications relevant to nephrology. Aluminum and associated elements, such as iron, were localized in fresh tissue biopsies obtained from uremic patients treatment by chronic hemodialysis. The LAMMA was applied to serum, liver, bone, and parathyroid glands of such patients. In addition, we used LAMMA to evaluate the specificity and sensitivity of routine histochemistry, in particular on human bone sections stained by the aluminon method. The high, multielemental sensitivity and molecular microprobe potential of LAMMA established important advantages over other microchemical methods forin situ analysis at the micron level in histological sections.     

Laser Microprobe Mass Analysis (LAMMA) to Verify the Aluminon Staining of Bone

Triammonium aurin tricarboxylate (aluminon) has been used to localize aluminum in 2 μm sections of undecalcified, methyl methacrylate embedded bone obtained from patients with terminal chronic renal failure. Aluminum appeared in four cases as bright red lines at the mineralized-bone boundary. In two cases, however, purplish lines were found and one patient showed red as well as purplish lines. Laser microprobe mass analysis (LAMMA) identified aluminum at the location of the red lines and both aluminum and iron at the purplish lines. Furthermore, both iron and aluminum were found in histiocytic bone marrow cells, which showed brownish aluminon staining. It appears that when aluminum and iron occur together, aluminon staining may yield aberrant results. This study shows that LAMMA can be used for the identification of elements sought by histochemical methods and thus permits the evaluation of their staining effects.     

The application of laser microprobe mass analysis to the study of biological material

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1–3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.     

Cellular localization of quinolizidine alkaloids by laser desorption mass spectrometry (LAMMA 1000)

Stem sections of Lupinus polyphyllus and Cytisus scoparius have been analyzed for the distribution of quinolizidine alkaloids by laser desorption mass spectrometry, employing a LAMMA 1000 instrument. Sparteine and lupanine could be recorded and were found to be restricted to the epidermis and probably also to the neighbouring 1 or 2 subepidermal cell layers.Abbreviations QA quinolizidine alkaloids - GLC gas liquid chromatography - MS mass spectrometry     

Failure to verify the presence of pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase by gas chromatography/mass spectrometry

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.     

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.     

Cellular and subcellular localization of chromium in the crab Liocarcinus puber (Brachyrhyncha) by transmission electron microscopy,secondary ion mass spectrometry and electron microprobe analysis

The swimming crab Liocarcinus puber (Crustacea Brachyrhyncha) was exposed for 2 weeks to CrCl₃ (chromium occurs principally in the trivalent state in the natural environment). The gills, digestive gland and muscle were examined by several analytical techniques for cellular and subcellular localization of chromium. The techniques applied were secondary ion mass spectrometry (ion microscopy and ion microprobe analysis) associated with photon microscopy and X-ray spectrometry (electron microprobe analysis) together with transmission electron microscopy. The digestive gland was found to be free of chromium, whereas chromium was adsorbed onto the gill exoskeleton. The muscle was the only tissue with intracellular electron-dense precipitates with no surrounding membrane. The metal was detected in the heterophagic vacuoles of amoebocytes where it was associated with phosphorus and trapped in an unsoluble form. Mechanisms of chromium cellular and subcellular metabolism were compared between crabs and other aquatic organisms. L. puber does not appear to be a suitable bioindicator of chromium pollution because of molting and its low chromium bioaccumulation capability.     

Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining

This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.     

Physiological analysis of bone appetite (osteophagia)

The vegetation eaten by animals on large areas of several continents is deficient in phosphate and deleterious effects on physiology, particularly reproduction, ensue. Records on bone chewing behaviour by both pastoral andwild game animals extend over two centuries. In laboratory investigation of this apt behaviour it has been shown that the appetite for bones is innate and specific and cued predominantly by olfactory stimuli. It is suppressed by rapidly increasing the plasma phosphate concentration to normal but not influenced by increasing the phosphate concentration in cerebrospinal fluid. The central organization of this genetically programmed behaviour appears to differ from systems subserving thirst and sodium appetite.     

Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS)

The review describes methods available for analyzing mineralization of bone tissue in healing of fractures and implants in bone. The recent development of imaging MS, TOF-secondary ion MS (SIMS), enabling localization of hydroxyapatite (HA) in tissue samples will be presented in some detail. We strongly believe that imaging MS has the potential of becoming an important method for the studies of bone mineralization. Formation and mineralization of bone tissue with HA is a process controlled by cells, the osteoblasts, osteocytes, and osteoclasts. Formation, de novo, of bone in embryonic tissue takes place in extracellular areas within cell clusters that regulate the environment of the mineralization zone. The process of de novo formation of bone as in embryonic tissue is reactivated in adults for example during fracture healing, with or without the presence of bone implants. Thus, bone healing is one of few examples of scar-free healing of a differentiated tissue. Much of the interest of researchers in bone mineralization stems from a desire to influence the process of bone formation towards fast and endurable bone healing. There is also a wish to understand the pathogenesis of bone diseases, for example osteogenesis imperfecta, Turner's syndrome and osteoporosis.     

Limb bone proportions and body mass of the cave bear (Ursus spelaeus)

The European cave bear evolved during the Middle Pleistocene and adapted to mountain environments. Earlier workers have described the cave bear as a robust bear. In this study the cave bears limb bone morphology is compared to the limb bone morphology of extant bears. Body mass estimates for the cave bear are made both based on different limb bone characters and based on dental and cranial characters. The shafts are wider in the cave bear limb bones than in the extant bear limb bones, and consequently the shaft widths give higher weight estimates to the cave bear than the other dimensions. The widened shafts are suggested to be a special adaptation (of presently unknown significance) rather than an indicator of an increased body mass.     

Electrolyte analysis of biological fluids with the electron microprobe

When a method of ultramicrochemical analysis is needed for nanoliter samples, conventional methods will not suffice. Electron microprobe analysis, however, is accurate, the sample preparation is simple, and the measurement can be executed rapidly.The probe also has a great advantage in that it is extremely versatile; its possibilities are limited only by the imagination of the user.     

Silver staining analysis on spermatozoa of Nucella lapillus (Gastropoda,Prosobranchia)

Summary

Sperm of Nucella lapillus was studied by electron microscopy, including the application of a cytochemical silver method. Using silver impregnation a dense precipitation of Ag granules in spermatocyte II nucleoli was seen over the fibrillar component and a slight one in the granular component. On longitudinal sections of the spermatozoon the results demonstrate that argyrophilic proteins are located in the external limiting zone of the acrosome in the anterior portion of the nucleus between the cytoplasmic and the nuclear membranes, in the posterior end of the nucleus and in the terminal portion of the middle region. These data indicate an affinity for silver in areas of the cytoplasm containing microtubules and in zones of transition.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in “doublets” as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

Cytochemistry and X-ray microprobe analysis of the midgut of Tomocerus minor lubbock (Insecta,Collembola) with special reference to the physiological significance of the mineral concretions

Summary Histochemical and cytochemical analyses have been made on the mineral concretions within the midgut cells of Tomocerus minor. The classical histochemical methods are not specific and precise enough and have been supplemented with cytochemical techniques on ultrathin sections. The most interesting of these was the K-pyroantimonate technique combined with glutaraldehyde-osmium fixation. This technique shows the distribution of cations such as Ca⁺⁺, K⁺, Mg⁺⁺ and Na⁺ on the concentric layers of the concretions. Chloride ions can be detected by means of the silver lactate technique. The action of calcium chelators such as E.D.T.A. shows an important distribution of calcium ions in the concretions. The spectra obtained by electron probe microanalysis from areas of fresh, dried and carbon coated midguts as well as from carbon coated semithin or ultrathin sections reveal the presence of Ca, K, Mg, S, Cl and P principally. Other elements such as aluminium, silicon and manganese have also been detected. Iron is not always present. The chemical and X-ray analytical investigations indicate that the midgut concretions are mainly built up of calcium, potassium, magnesium and sodium phosphates, perhaps associated with chlorides and carbonates. An organic matrix formed by polysaccharides seems to join the different mineral layers. These concretions may be formed within the vesicles of rough endoplasmic reticulum. The midgut cells are highly differentiated and very active in transport. Extensive basal infoldings and apical microvilli as well as lateral membranes are a site of small cationic deposits. The possible pathway of ion transport in the cell and the physiological significance of the concretions are discussed. The principal function of these concretions seems to be the maintenance of the mineral balance and to trap foreign and excess ions.

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Résumé L'analyse chimique des sphérocristaux de l'intestin moyen de Tomocerus minor a été réalisée. Les méthodes histochimiques courantes manquant souvent de spécificité et de sensibilité ont été complétées avec des méthodes cytochimiques sur coupes ultrafines. La plus intéressante a été la technique du pyroantimonate de K montrant la distribution des cations Ca⁺⁺, K⁺, Mg⁺⁺, Na⁺ sur les couches concentriques des sphérocristaux. La technique au lactate d'argent permet de déceler les ions Cl^-. L'action d'agents chélateurs du Ca tels l'E.D.TA. montre une importante distribution du calcium dans les sphérocristaux. L'analyse spectrographique d'étalements de mésentérons séches, carbonés et de coupes semi-fines ou ultrafines carbonées montre la présence de Ca, K, Mg, S, Cl, P, Na. D'autres éléments tels l'Al et le Si ont pu être détectés. Le Fe n'est pas toujours présent. Les sphérocristaux semblent formés essentiellement de phosphates de calcium, de potassium, de magnésium, de sodium associés peut-être à des chlorures ou des carbonates. Une matrice organique constituée essentiellement par des polysaccharides semble lier les différentes couches minérales. Ces sphérocristaux prennent naissance à l'intérieur des vésicules de l'ergastoplasme. Les cellules de l'intestin moyen sont très différenciées et sont le siège de nombreux transports actifs. Les replis basaux de la membrane plasmique, les microvillosités apicales, de même que les membranes latérales sont le siège de dépôts de cations. Le transport des ions dans les cellules ainsi que le rôle physiologique des sphérocristaux sont discutés. Le maintien de la balance hydrique ainsi que le piégeage d'ions étrangers ou en surplus semblent être la principale fonction des sphérocristaux.

     

Electron microprobe X-ray microanalysis of diseased coconut (Cocos nucifera) roots

Summary Electron microprobe X-ray analysis of root (wilt) diseased and healthy coconut roots were scanned for the deposition of metal ions to implicate the involvement of these metal toxicity in the root (wilt) disease of coconut. The results indicated that a high concentration of Al, Mn, Cu and Co ions localised in the disease roots compared to healthy palms. The chemical analysis of tissue samples and soils also confirmed the results of XMA.