Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid?

Summary The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0–4°C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid. A reappraisal.

The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0-4 degrees C) and at a pH of 2-2.5.     

On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0–4° C) and at a pH of 2–2.5.Paper presented at the 65th Session, Indian Science Congress, Ahmedabad, Jan. 3–7, 1978     

On the specificity of the alcoholic,acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The defects besetting the histochemical localization of ascorbic acid were removed in the modified method described here by the simultaneous fixation of the experimental material and its reaction with silver nitrate by the use of alcoholic, acidic silver nitrate reagent in the dark at 0–3°C for 24 hours or longer at pH 2–2.5.The fixatives like acetic acid and alcohol of the reagent ensure quick penetration of AgNO₃ for fixation of ascorbic acid in situ before sectioning. It has been experimentally established that none of the other reductants react with AgNO₃ at the pH and the temperature mentioned.The sections were devitaminized by treatment with 6–10% formaline for 3–4 hours to serve as a control.     

The Chemistry of the Silver Precipitation method Used for the Histochemical Localizgtion of Ascorbic Acid

The usefulness of reductive silver precipitation for the histo chemical localization of assorbic acid in mouse lung has been examined under standard conditions. The estimated stoichiometry of the reaction shows that only one quarter of the silver precipitated in fresh tissue by the silver nitrate reagent was due to ascorbic acid. Although no evidence was found that diffusion of ascorbic acid proceeded more rapidly than reductive silver precipitation in the tissue, diffusion artifacts at the cellular led would seem likely to occur. Thiosulphate was shown to be more effective than ammonia m removing un reacted silver from tissues treated with silver nitrate.     

Expression of senescence-enhanced genes in response to oxidative stress

Expression of the LSC54 gene, encoding a metallothionein protein, has been shown previously to increase during leaf senescence and cell death. Evidence is presented in this paper to indicate that the extent of LSC54 expression is related to levels of oxidative stress in the tissues. Treatment of Arabidopsis cotyledon and leaf tissues with the catalase inhibitor, 3-amino-1,2,4-triazole, or with silver nitrate result in the enhanced expression of LSC54. Combined treatments with quenchers of reactive oxygen species (ROS), such as ascorbate, tiron and benzoic acid indicated that this induced expression was due to increased levels of ROS. The expression of many other senescence-enhanced genes was also found to be inducible by the increase in ROS. Treatment of plant tissue with 3-amino-1,2,4-triazole, followed by silver nitrate, resulted in protection from the severe damage caused by the silver nitrate treatment and reduced expression of many of the genes examined. However, one gene, encoding a lipid hydroperoxide-dependent glutathione peroxidase, showed increased expression in the protected tissue, which may indicate a role for this enzyme in the protection of plant tissue from oxidative stress. ROS-enhanced expression of at least one of the genes investigated required the presence of the salicylic acid signalling pathway, which was not required for the expression of LSC54.     

Optical ascorbic acid sensor based on the fluorescence quenching of silver nanoparticles

A sensitive and selective fluorimetric sensor for the assay of ascorbic acid (AA) using silver nanoparticles as emission reagent was investigated. In this study, silver nanoparticles were prepared based on aqueous–gaseous phase reaction of silver nitrate solution and ammonia gas. The nanoparticles were water‐soluble, stable and had a narrow emission band. They were used as a fluorescence probe for the assay of ascorbic acid on its quenching effect on the emission of silver nanoparticles. The principal reason for quenching is likely to be a complexation between ascorbic acid and silver nanoparticles. The quenching mechanism was established by Stern–Volmer law. Under the optimum conditions, the quenched fluorescence intensity was linear with the concentration of ascorbic acid in the range of 4.1 × 10^?6 to 1.0 ×10^?4 m (r = 0.9985) with a detection limit of 1.0 × 10^?7 m . The RSD for repeatability of the sensor for the assay of ascorbic acid concentration of 3.0 × 10^?5 and 4.0 × 10^?6 m was found to be 1.5 and 1.3%, respectively. The proposed method was applied to the determination of ascorbic acid in vegetables and vitamin C tablets. Copyright © 2009 John Wiley & Sons, Ltd.     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Histochemical Differentiation of Chloride from Other Ions Precipitated by Silver Nitrate in Freeze-Substitution Fixation

The method is based on substitution fixation at —25° C of quickly frozen tissue with a 90% alcohol solution saturated with silver nitrate. The silver salts are photochemically reduced in the histological preparations. At this low temperature very little staining of the protein structure of the tissue takes place. Silver ions adsorbed by the tissue can be removed by treatment with a sodium nitrate solution. About 2/3 of the brown material in the histological preparations of cerebral cortex was due to the chloride in the tissue, 1/6 to the phosphate, 1/10 to an unidentified (probably organic) anion, and 1/20 to bicarbonate. When the alcoholic silver nitrate solution used for the fixation is acidified, or the sections are treated with nitric acid, the colored material consists of reduced silver chloride only. A comparison of the light absorption in histological preparations of cortex treated with neutral and with acid solutions supported the conclusion that about 2/3 of the colored material in the tissue is reduced silver chloride.     

Reduction of silver nitrate by cystoliths ofFicus elastica

Summary The cystoliths ofFicus elastica turn dark when a 2 per cent solution of silver nitrate is applied to the leaf sections. The cystoliths appear as if they reduce the silver ions and impregnate themselves with metallic silver. The reducing activity is, however, accounted for by ascorbic acid, and not by the cystoliths in themselves. The darkening reaction takes place in the presence of ascorbic acid either natively contained in fresh leaves or exogenously replenished after killing of the leaf sections. The cystoliths can react, however, only when they maintain the calcareous cortex intact. The reaction no longer occurs even in the presence of ascorbic acid when the cystoliths have been deprived of the cortex by exposure to Na₂-EDTA.     

An alcohol-soluble Schiff's reagent: a histochemical application of the complex between Schiff's reagent and phosphotungstic acid.

A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

An alcohol-soluble Schiff's reagent: A histochemical application of the complex between Schiff's reagent and phosphotungstic acid

Summary A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent sued as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

THE CYTOCHEMICAL LOCALIZATION OF ASCORBIC ACID IN ROOT TIP CELLS

The intracellular distribution of ascorbic acid was studied in frozen-dried root tips of Allium cepa and Vicia faba by the silver nitrate procedure. The sites of the ascorbic acid as indicated by the deposited silver appear as spherical (0.2 to 0.6 µ in diameter) cytoplasmic particles. The site appears to have small amounts of lipides and to be rich in ribonucleic acid. These particles are concluded to be submicroscopic in size and associated, in the elongating cell, with the cell surface. In the meristematic cells they appear fewer in number and are distributed throughout the cytoplasm.     

Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs

Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues.     

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.     

AN ELECTRON MICROSCOPE STUDY OF SILVER NITRATE REDUCTION IN LEAF CELLS

Brown , W. V., H. Mollenhauer , and C. Johnson . (U. Texas, Austin.) An electron microscope study of silver nitrate reduction in leaf cells. Amer. Jour. Bot. 49(1): 57–63. Illus. 1962.—As reported earlier in many studies, AgNO₃ is reduced quickly by the living chloroplasts of angiosperms. Electron microscope study has resolved the conflict of opinions concerning the exact location of the silver particles. Reduction of AgNO₃, as indicated by location of silver particles, occurs within the chloroplasts but not within the grana or pure stroma; it appears to be associated with the intergranal (also called stroma) lamellae. Silver particles are formed also at the surfaces of the cell wall, both in the middle lamella and at the inner surface, and also within plasmodesmata. It is concluded that chlorophyll is probably not involved directly in the reduction. There is some slight support for the popular hypothesis that ascorbic acid may be the chief reducing agent.     

Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress,hepatic stellate cell activation,cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs

Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β₁, TNF-α and α₁(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β₁, TNF-α and α₁ (I) collagen in hepatic tissues.     

Periodic-Acid-Foot Stain for Connective Tissue

A marked increase in reticular argyrophilia may be obtained in the Foot ammoniated silver carbonate technic by interposing a strong periodic acid oxidation, 4% aqueous for 2 hours at 25-27°C., prior to silvering. Sections so oxidized before the silver bath show a histological picture of connective tissue that is stronger than that given by the original technic. Stroma of lymphoid tissues (but not other types) is further intensified by brief (5-10 sec.) passage through aqueous 1.5% uranium nitrate after oxidation but before silver impregnation. The specific action of periodic acid (cleavage of the 1,2-glycol linkage to produce aldehyde radicals) strengthens the premise that the carbonyl radical plays an important part in the phenomenon of connective tissue argyrophilia.     

A New Technique for Localization of Cellulase in situ Using Silver Nitrate

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.     

The location of silver in frog epidermis after treatment by ranvier's method,and possible implication of the flask cells in transport

Summary Frog skin, bladder wall, and sciatic nerve were treated by Ranvier's silver nitrate method and subsequently fixed and sectioned for electron microscopy. In the epidermis of the skin, more silver is found deposited in the flask cells than elsewhere, especially as a sub-apical plaque in the neck of the flask, which appears after the skin has been flooded with silver nitrate for 5 minutes. Mitochondria rich cells in the bladder also accumulate more silver than the surrounding epithelial cells, but do not show such a distinct sub-apical plaque. In myelinated nerve fibres treated similarly, silver accumulates in the paranodal regions of the axon, and outside the axon at the node. It is suggested that silver may accumulate near a site of ion transport, due to structural specialisations not visible by standard electron microscope techniques, and that the flask cells may therefore be implicated in transport in the frog skin.