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

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 degrees 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.     

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.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

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 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.     

Decrease of nitrate biosynthesis in scorbutic mutant rats unable to synthesize ascorbic acid

The effect of ascorbic acid deficiency on the urinary excretion of nitrate was investigated using a mutant strain of rats (osteogenic disorder syndrome rats; ODS rats) unable to synthesize ascorbic acid. The amount of urinary nitrate excreted by ODS rats with or without ascorbic acid supplementation were measured before and after the intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). Urinary nitrate excretion increased markedly after LPS injection. Urinary nitrate excretion by ODS rats not supplied with ascorbic acid was significantly less than that of those supplied with ascorbic acid both before and after LPS injection. These results show that ascorbic acid enhances both LPS-stimulated and constitutive nitrate production in vivo.     

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.     

Mechanism of Prophylaxis by Silver Compounds against Infection of Burns

To clarify tthe mechanism by which local application of silver compounds protects burns against infection, an ion-specific electrode was used to measùre the concentration of silver ions in solutions. By this method it was shown that in burn dressings silver ions were reduced to a very low level by precipitation as silver chloride. The antibacterial effect was found to depend on the availability of silver ions from solution in contact with precipitate. Between 10^-5 and 10^-6 molar silver nitrate solution in water was rapidly bactericidal. The minimal amount of silver nitrate causing inhibition of respiration of skin in tissue culture was about 25 times the minimal concentration of silver nitrate that inhibited growth of Pseudomonas aeruginosa.     

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.     

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.     

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     

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.     

The Bielschowsky Staining Technic a Study of the Factors Influencing Its Specificity for Nerve FIBERS

By comparing results obtained with adult mammalian tissue from introducing variables into each separate step in block-staining by the Bielschowsky silver method, the following conclusions were reached:

1. No specific means for inhibiting the staining of connective tissue and still permitting complete staining of nerve fibers was found, but the avoidance of overstaining was very helpful toward such differentiation.
2. Overstaining could be corrected by reducing the concentration of the silver nitrate bath or by adding an excess of ammonia to the ammoniated silver bath.
3. Staining of fine fibers was favored by adding acetic acid to the formaldehyde used for fixation or by adding pyridin to the silver nitrate bath.
4. Addition of protein-precipitating organic acids (trichloracetic or sulfosalicylic) to the fixative was disadvantageous.
5. Prolonged fixation favored an increase in intensity of the stain. Four days' time was sufficient.
6. Extraction of lipids with ammoniated alcohol gave results similar to those obtained after extraction with pyridin, but the stain was lighter.
7. Ammoniated silver carbonate without excess ammonia had an action similar to ammoniated silver hydroxide with excess ammonia.
8. An excess of ammonia in the ammoniated silver solution (Ag 0.1 N) was tolerated, without apparent impairment of nerve-fiber staining, up to 6 M NH₃, altho the use of more than 3 M excess (2 cc. concentrated ammonia water added to 100 cc. of balanced ammoniated silver hydroxide solution) seemed unnecessary.
9. Impregnation with 1.7% (0.1 N) silver nitrate solution was quite satisfactory and variations in the concentrations of this bath suggested that the practical limits of concentrations that would be generally satisfactory lay between 0.3 and 3.0%.
10. The writers' experiences agreed with Agduhr's relative to the advantage of washing in 2.5% acetic acid between the ammoniated silver bath and formaldehyde reduction.

     

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.     

Simultaneous determination of the amounts of metallic and "reducible" silver in histologic specimens.

Acids and weak complexing agents (pK less than 8) are not able to remove, without leaving a residue, silver bound to biological tissues by ionic or complex bonds ("reducible" silver), whereas, strong complexing agents (pK greater than 8) can also partially or completely dissolve metallic silver formed under the influence of reducing groups in the tissue. For this reason, the chemical nature of the silver contained in tissue sections, be it metallic or reducible, must not be determined on the basis of solubility tests; moreover, the amount of neither of the two above fractions can be determined by removing the other with any kind of washing. Using radioactive impregnating baths, radioactive silver bound to the tissue as reducible silver can be replaced in a quantitative manner with inactive silver ions by means of a one-hour incubation in 1% inactive silver nitrate dissolved in 10% acetic acid, but the radioactive silver existing in reduced (atomic) state will be left unaffected. Consequently, radioactivity remaining in the tissue after the above treatment represents metallic silver. The amount of reducible silver can be calculated by subtracting that of the metallic silver from the total silver content of the sections.     

Decreased ascorbic acid levels and brown core development in pears (Pyrus communis L. cv. Conference)

Changes in ascorbic acid content are measured in the cortex tissue of Conference pears stored at various compositions of carbon dioxide and oxygen. Enhanced carbon dioxide levels cause ascorbic acid concentrations to decline. Soon after ascorbic acid has declined below a certain value, browning of the core tissue can be observed. Reducing carbon dioxide levels before this value is reached causes ascorbic acid levels to increase again and prevents browning to a great extent. In preliminary experiments with a photoacoustic laser-based detection system, it was shown that pears that show browning produce ethane, which is most likely a result of membrane peroxidation. Storage conditions, ascorbic acid levels and browning in pears are discussed in relation to diffusion characteristics, energy metabolism and energy maintenance levels of the fruit.     

Histochemical studies of uric acid in some insects. I. Storage in the fat body of Periplaneta americana and the action of the symbiotic bacteria

An improved histochemical method for uric acid consists in precipitation as silver magnesium urate combined with fixation of the tissues in formol/glutaraldehyde followed by argentaffin reaction with silver nitrate buffered with "tris" to pH 9.5. This reveals urates both in solid deposits and in solution in the tissues. Polyphenols concerned in sclerotin formation also react. In Periplaneta, uric acid synthesized in the trophocytes is carried by intracellular and intercellular channels to form the intercellular deposits of solid spheres. The symbiotic bacteria in the mycetocytes in contact with the deposits appear to metabolize the uric acid and they disperse and eliminate the deposits.     

Linking radiosilver to monoclonal antibodies reduced by ascorbic acia

Radiosilver-111 and Radiogold-199 were proposed by us (1) as suitable isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111T _1/2=7.45 d and Au-199T _1/2=3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dialysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1∶1, 1∶2, 1∶5, 1∶10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver efflux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The comparative results with the two techniques are described.     

Simultaneous high-performance liquid chromatographic determination of a glucuronyl prodrug of doxorubicin, doxorubicin and its metabolites in human lung tissue

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer–acetonitrile–methanol–tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86–99%) and a linear calibration range of 20 ng/g–80 μg/g for doxorubicin and 1–600 μg/g for HMR 1826.