Osmium Reduction Research Articles

Reduction Mechanisms of Anticancer Osmium(VI) Complexes Revealed by Atomic Telemetry and Theoretical Calculations.

Resonant X-ray emission spectroscopy (RXES) has developed in the past decade as a powerful tool to probe the chemical state of a metal center and in situ study chemical reactions. We have used it to monitor spectral changes associated with the reduction of osmium(VI) nitrido complexes to the osmium(III) ammine state by the biologically relevant reducing agent, glutathione. RXES difference maps are consistent with the proposed DFT mechanism and the formation of two stable osmium(IV) intermediates, thereby supporting the overall pathway for the reduction of these high-valent anticancer metal complexes for which reduction by thiols within cells may be essential to the antiproliferative activity.

Osmium dimethyl sulfoxide complexes: Synthesis and properties of [H(dmso)2][OsIII(dmso)2Br4

The reaction of H2[OsBr6] with DMSO in ethanol solution resulted in DMSO complex [H(dmso-O)2][OsIII(dmso-S)2Br4] (1) described previously as an intermediate product in the reaction of K2[OsBr6] with DMSO and characterized by EAS and ESR spectra. The coordination of DMSO molecules was established by IR and 1H and 13C NMR spectroscopy. The oxidation state of osmium and trans arrangement of DMSO molecules in the anion were established by ESR. The behavior of complex 1 in solutions was studied by EAS, ESR, and mass-spectrometry: a displacement of Br− ions accompanied by the reduction of osmium to oxidation state +2 occurs in DMSO, a solvation with displacement of DMSO molecules is observed at the first stage in water and methanol (rate constants 2.3 × 10−4 and 1.7 × 10−3 s−1, respectively), the sequential substitution of DMSO molecules and osmium oxidation to form [OsIVBr6]2− ions takes place in 4 mol/L HBr.

Electrochemical oxidation and reduction of osmium and iridium complexes with fullerene C60

The electrochemical oxidation and reduction of a series of complexes of platinum-group metals (Os, Ir, and Rh) with fullerene C60 with common formula C60[MLn] (MLn is the metal and the metal-bonded ligands) in the tetrahydrofuran solution are studied using the method of cyclic voltammetry. The effect of metal and ligands on the variations in the redox properties of complexes and stability of anions, which are formed in the reduction, is considered. It is shown that all complexes studied are reduced more difficultly than free fullerene, and the reduced forms of metallofullerenes are less stable as compared with free fullerene anions.

Colorimetric Estimation of Melatonin in Pharmaceutical Formulations

Three simple and sensitive colorimetric methods (A–C) for the determination of melatonin in bulk samples and in pharmaceutical formulations are described. They are based on the formation of coloured species by reaction of ninhydrin with the drug (method A, λmax 397 nm) by oxidation of the indol moiety in melatonin with potassium persulphate (method B, λmax 450 nm) or by reduction of osmium (VIII) (method C, λmax 516 nm). Regression analysis of Beer-Lambert plots showed good correlations in concentration ranges between 0.8–14.2, 70.0–140.0 and 2.0–40.0 μg/mL for methods A, B and C, respectively. The molar absorptivity, Sandell sensitivity and detection limit were calculated. For more accurate analysis, Ringbom optimum concentration ranges were calculated. The validity of the proposed methods was tested by analysing pharmaceutical formulations containing melatonin. The relative standard deviations were ≤ 0.95% with recoveries 99.0–101.33%.

Synthesis and reduction of osmium(VI) nitrido complexes and x-ray crystal structure of tetra-n-butylammonium octachlorodinitrido(.mu.-pyrazine)diosmate(VI)

Synthesis and reduction of osmium(VI) nitrido complexes and x-ray crystal structure of tetra-n-butylammonium octachlorodinitrido(.mu.-pyrazine)diosmate(VI)

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

Rapid freezing of freshly dissected or incubated neural lobes was explored as a means of obtaining ultrastructural preservation of the more natural state of this tissue. A quantitative assessment of the region of good fixation was made in order to determine the relative fractions occupied by axons, pituicytes and the extracellular space. The immunocytochemical distributions of neurophysins and the glycopeptide portion of the vasopressin precursor were evaluated using the immunogold technique in order to determine the relative numbers of oxytocin and vasopressin fibre types in the fixed region, and the subcellular localization of these antigens. The uncut surface of rat neural lobes was rapidly frozen against a highly polished copper plug and freeze-substituted in osmium-acetone either immediately after dissection (approximately 2 min), or after a 15 min incubation period in vitro in an oxygenated, balanced salt solution. Substituted neural lobes were prepared for either conventional electron microscopy, or for immunogold labelling of neurophysins and the glycopeptide precursor to vasopressin. Membranes, subcellular organelles and extracellular matrix were well preserved 10 microns deep to the contacted surface. The extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes accounted for about 30% of the area and axons 20-25%. Pituicytes occupied close to 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. There were no differences between neural lobes frozen immediately after dissection and those incubated for 15 min in any of these measures, suggesting minimal fluid redistribution. Gold particles were specifically localized over large (100-200 nm) dense core vesicles, and less frequently over multivesicular bodies and lysosomes. No etching of the plastic or reduction of osmium was necessary to achieve labelling. Specific labelling of one set of terminals and axons (about 80%) was observed with the monoclonal antibody previously shown to be specific for oxytocin-neurophysin, while in neighbouring sections the remaining 20% of the processes were labelled with the antiserum to the vasopressin precursor, or with non-specific antibodies to neurophysins. In conclusion, ultrarapid freezing preserves a large extracellular space in the neural lobe and provides for high resolution morphological and immunocytochemical studies of neurohypophysial structure.

Unbuffered osmium staining of cell organelles: alterations induced by cell injury.

We have studied the localization of osmium reduction products to investigate the functional state of organelles as well as organelle interrelationships during cell injury. In normal hepatocytes osmium deposits of variable intensity are seen in nuclear envelope, endoplasmic reticulum. Golgi cisternae and vesicles and lysosomes. Buffering of osmium with s- collidine (pH 7.4) prevents the deposition of osmium. Reversible (30 min) and irreversible (60 min) ischemia without reflow causes no change in the pattern of osmium deposition. Irreversible ischemia followed by reflow causes decreased staining of endoplasmic reticulum (ER) and redistribution of the osmium deposits through the cytoplasm. Reversibly injured pancreatic acinar cells in cultured explants manifest a similar loss of osmium staining in the endoplasmic reticulum cisternae. The administration of antimicrotubule drugs induces an accentuation of osmium staining in localized cisternal elements of hepatocytes. These heavily stained cisternae appear to give rise to the bounding membranes of drug-induced autophagic vacuoles. Cytoplasmic organelles sequestered inside the autophagic vacuoles acquire intense staining when they begin to undergo degradation. In homogenized liver tissue all the subcellular organelles show osmium deposits. The deposits are preferentially localized along the organelle membranes. In particular the dense deposits in the ER lumen are not seen in the subcellular fractions. Phospholipase A2 (3 units/mg protein) enhances the deposition of osmium in the lumen of microsomal vesicles, whereas the presence of detergent has no such effect. Addition of EDTA to the homogenizing medium enhances the ultrastructural preservation of the subcellular fractions but has little effect on the deposition of osmium. OsO4 deposition occurs at acid pH and the intensity and pattern of the stain can be modified in vivo and in vitro. Osmium tetroxide deposition is induced at sites of membrane transformation (autophagic vacuoles) and degradation (lysosomes). Calcium influx and phospholipase activation (ischemia, tissue homogenization, phospholipase addition) enhance osmium deposition and/or influence the localization of the staining pattern.

Extraction and spectrophotometric determination of osmium with 4-(2-pyridylazo)resorcinol

The formation and extraction of osmium complexes with 4-(2-pyridylazo)resorcinol and their application to the spectrophotometric determination of osmium were studied. The procedures for the fast and accurate determination of osmium, based on the reduction of osmium(VIII) to osmium(VI) by ethanol and the spectrophotometric measurement of the osmium(VI)-PAR complex in aqueous ethanol media or in the extract obtained with tetraphenylphosphonium chloride in chloroform, are described. The composition and nature of the complex species measured were determined by solution study and by characterization of the isolated solid compound.

Schistosoma mansoni: Cytochemistry and morphology of the gastrodermal Golgi apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.

Investigations of changes in the oxidation state of the catalyst during the OsO4-catalysed decomposition of hydrogen peroxide

Abstract By using different methods, such as spectrophotometry, potentiometric titration, polarography and extraction, it was found that at pH 8.5 osmium (VIII) is reduced by hydrogen peroxide to osmium(VI) to various extents. At pH 10.6, where the rate of the OsO4-catalysed decomposition of hydrogen peroxide reaches its maximum, the concentration ratio of osmium (VIII) and osmium (VI) was found to be nearly one. This favours the explanation that the maximum rate of hydrogen peroxide decomposition is found at the pH where the rate of reduction of osmium (VIII) by hydrogen peroxide just becomes equal to the rate of oxidation of osmium (VI) by hydrogen peroxide.

A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by x-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle.

A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed. X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO4 and CuRuO4. Therefore, and based on indexation methods, ZnOsO4 was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice. In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time. According to the microanalysis results, the zinc osmate has been associated to Ca2+ high affinity sites since Zn2+ is either replacing Ca2+ and/or displacing it by having a higher affinity for Ca2+ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca2+ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca2+. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation. It is likely that OsO4 alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO4). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.

Reduction of osmium(VIII) to osmium(II) by reaction with an olefin and EDTA. Formation of an O S II—EDTA complex

The complex Na 6H 4[O S II (EDTA) 3] is formed by reaction between osmium tetroxide, an olefin and EDTA. The same complex is produced by reaction between potassium osmate and EDTA. The EDTA analogue, trans-1,2-diaminocyclohexane-N,N,N′,N′- tetraacetate, forms a similar complex.

Phosphatase activities and osmium reduction in cell organelles ofMicrasterias americana

Organelles in resting and growing cells ofMicrasterias americana were examined using electron microscopy after cytochemical procedures for four kinds of phosphatases, acid phosphatase (ACPase), alkaline phosphatase (ALPase), thiamine pyrophosphatase (TPPase), and inosine diphosphatase (IDPase), and osmium tetroxide reduction. Special attention was paid to activities in the Golgi apparatus.

Ruthenium labeling of micropinocytotic activity in the rat visceral yolk-sac placenta

Visceral yolk-sac placentas of rats were autopsied from 12-, 17-, and 22-day pregnancies. The membrane is known to transport proteins from maternal to fetal systems only during the last of these 3 stages. Protein absorption previously has been shown to be initiated by micropinocytosis at the apices of the epithelial cells which cover the membrane. The pits and vesicles which are formed by this process are fuzzy-coated, or alveolate. In the present study, the yolk-sac membrane from each selected stage was treated with ruthenium red which binds at the site of micropinocytotic vesicle formation and which catalyzes osmium reduction during subsequent processing of the tissue. By this method, the micropinocytotic process was observed in detail. Morphologically, the alveolate vesicles come into close relationship with a subapical system of microvesicles and canaliculi, a system which, in turn, communicates with large storage vacuoles. By the ruthenium method, relative micropinocytotic activities at different gestational ages also were determined. The 12-day membrane appeared micropinocytotically very active. At the 17-day stage, on the other hand, the membrane was relatively quiescent. At 22 days, the placental membrane exhibited a degree of activity about like that at 12 days. Consequently, at the gestational ages which were examined, no correlation was observed between the rate of micropinocytotic uptake of protein and known protein transport rates.

Enterochromaffin properties of granular cells in the heart of the snails Helix aspersa and Strophocheilus oblongus

1. 1. Specific granular cells were found in the heart, mantle and subesophageal ganglia of the snails Helix aspersa and Strophocheilus oblongus. 2. 2. Histochemical methods for the demonstration of enterochromaffin granules gave positive results in these cells. 3. 3. Differential osmium reduction by nerves in these hearts showed two kinds of fibres: osmiophilic and non-osmiophilic nerves and gives histological evidence of a double innervation (inhibitory and stimulatory fibres) to the snail heart. 4. 4. Osmiophilic endings are reported in the ventricle and auricle and are shown to end very close to the granular cells. 5. 5. No neurosecretory-staining material could be traced along nerves in these hearts. 6. 6. One large bipolar neuron is described in these hearts. 7. 7. Histological preparations of the heart of Strophocheilus oblongus that had long been stimulated through its cardiac nerve show a release of the granular content in the cells and disclose stages of a secretory cycle. It is suggested that these cells are glandular tissue and perform a hormonal function.

Oxidation of ascorbic acid by osmium(VIII)

Reduction of osmium(VIII) by ascorbic acid has been studied in the presence of hydrochloric acid. The reaction is found to be directly proportional to the concentration of ascorbic acid and osmium(VIII) and to the reciprocal of the hydrogen ion concentration. In the proposed mechanism, the reacting species have been shown to be perperosmic acid and ascorbate ion; this was supported by the negligible effect of the ionic strength. Influence of temperature was studied in the range 25–40 °C, and the energy and entropy of activation were calculated as 14.1 ± 0.2 kcal mole−1 and −8.6 ± 0.1 e.u., respectively. Osmium(IV) was found to be a product of the reaction.

Osmic Impregnation of Amphibian and Gastropod Photoreceptors

Friend and Murray found that long osmification of mouse epididymis revealed localized osmium reduction in epithelial cells; in this instance, in only the outer cisternae and vesicles of the Golgi apparatus. Although many substances reduce osmium, aldehydes and compounds with multiple double bonds are particularly active. Knowing that retinaldehyde (vitamin A aldehyde) is a highly unsaturated aldehyde we applied their technique to eyes of tadpoles of two amphibians (Xenopus laevis and Hyla regilla) and to adult eyes of one pulmonate snail (Helix aspersa). Eyes were extirpated, immersed in unbuffered 2% aqueous osmium tetroxide for two or three days at 40°C, and prepared for electron microscopy by rapid dehydration with ethanol, embedment in Epon, and examination of ultrathin sections without further staining.

OSMIUM IMPREGNATION OF THE GOLGI APPARATUS.

AbstractThe Golgi complex consists of a heterogeneous assemblage of small vesicles, larger vacuoles and characteristic packets of flattened cisternae morphologically distinct from other membranous organelles of the cytoplasm. It is polarized with respect to its position in the cell and also shows recognizable polarity in the arrangement of structural components within the complex. This polarity holds for cells in which the Golgi complex seems to have quite different functions, such as in the epithelial cells of mouse epididymis and of Brunner's gland, and plasma cells in the lamina propria of the intestine examined in this study. The present investigation employs a classical method for impregnation of the Golgi apparatus with the objective of more accurately localizing with the electron microscope the sites of osmium reduction within this heterogeneous organelle and of comparing the distribution of these sites with morphological indications of polarization within the Golgi lamellar systems. The osmium impregnation procedure generally used in this study consisted of fixing the tissues in 1.3% osmium tetroxide buffered with s‐collidine followed by osmication in 2% aqueous osmium tetroxide for 40 hours at 40°C. After osmication the three cell types studied consistently exhibited a selective localization of osmium in the cisternae and vesicles on one side of the Golgi complex. The observations that only the outer vesicles and cisternae contain osmium while other cytologically similar elements under the same conditions do not, emphasizes the heterogeneous, bipolar nature of the Golgi apparatus. The substances responsible for osmium reduction are quite dependent on the local environment provided are apparently formed during the prolonged phase of the exposure of the tissue to osmium and are most likely localized at the inner face of the membrane of the outer cisternae and vesicles.

THE COMPARATIVE ROLES OF OXYGEN AND INHIBITORS IN THE PASSIVATION OF IRON. IV. OSMIUM(VIII) OXIDE

Cathodic polarization of iron electrodes after passivation in osmium(VIII) oxide was used as a means of determining the relative contributions of oxygen and the passivating inhibitor to the total cathodic process. It was found that the rate of reduction of osmium(VIII) oxide greatly exceeds that of oxygen. The reduction product, Os(OH)4, was shown to accelerate the reduction processes, as was found previously with reduction of the pertechnetate ion. A possible mechanism for this action is suggested. The results of the four papers in the series are summarized and discussed in their relation to theories of passivation.