The Effect of Formaldehyde on Tissue Lipids and on Histochemical Reactions for Carbonyl Groups

Histcchemical and chemical evidence indicates that formaldehyde combines with unsaturated lipids at the double bond. The resulting complex contains a free carbonyl group which probably originates from the formaldehyde. The reaction occurs over a wide pH range, and takes place in the absence of oxygen or moisture. The reaction product is visualized by the Schiff reagent, and by the Ashbel-Seligman procedure. In the plasmal procedure, when performed on formalin-treated material, the reaction has the same significance as the pseudo-plasmal reaction, i.e. it denotes the presence of double bonds. The Ashbel-Seligman technic seems to be more sensitive to this complex than the Schiff reagent and shows it more markedly than it does the true plasmals and the atmospherically oxidized unsaturated compounds.     

Minimization of a sodium dithionite-derived interference in nitrate reductase-methyl viologen reactions

The use of sodium dithionite as the reducing agent to produce the radical cation of methyl viologen cofactor for the nitrate reductase-catalyzed reduction of nitrate to nitrite causes a negative interference in the diazonium salt azo dye determination of nitrite. The interference results from bisulfite, a reaction product of dithionite, reacting with the diazonium salt formed from nitrite and sulfanilamide. Addition of formaldehyde before the diazotization reaction masks this interference but at low acidity introduces a nonlinearity in the nitrite calibration curve. This nonlinearity is due to a competing reaction of formaldehyde with sulfanilamide to yield a Schiff base and can be eliminated by increasing the HCL content of the sulfanilamide reagent.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Studies on the Quantitative Nature of the Feulgen Stain

Bacterial suspensions were stained with Schiff's reagent according to the procedure suggested in essence by Dondero et al. (1954). Cell suspensions, Schiff's reagent, supernatant fluids and stained cells were analyzed by a micro-Kjeldahl procedure in an effort to quantitate the Feulgen reaction. The concentration of the bacterial suspension, type of fixative, time of hydrolysis and pH of cells and dye were varied and the effects analyzed quantitatively. While the cells were often stained deeply as determined by visual observation, the quantity of dye nitrogen in the cells was not large enough to be measured with the procedure employed. Significant quantitative results were obtained consistently only when the pH of the Schiff's reagent was raised. Feulgen reactions with solutions of formaldehyde and with solutions of DNA were also analyzed quantitatively after removing the colored compounds with charcoal. The analyses indicated that the DNA solution and the formaldehyde solution reacted differently with the dye.     

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.     

A new enzymo-chemical method for simultaneous assay of methanol and formaldehyde

A new enzymo-chemical method for the simultaneous assay of methanol and formaldehyde in mixtures is described which exploits alcohol oxidase (AO) and aldehyde-selective reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme is used for methanol oxidation to formaldehyde and MBTH plays a double role: 1) at the first step of reaction, it forms a colorless azine adduct with pre-existing and enzymatically formed formaldehyde and masks it from oxidation by AO; 2) at the second step of reaction, non-enzymatic oxidation of azine product to cyanine dye occurs in the presence of ferric ions in acid medium. Pre-existing formaldehyde content is assayed by colorimetric reaction with MBTH without treating samples by AO, and methanol content is determined by a gain in a colored product due to methanol-oxidising reaction. Possibility of differential assay of methanol and formaldehyde by the proposed method has been proved for model solutions as well as for real samples of industrial waste and technical formaline. A threshold sensitivity of the assay method for both analytes is near 1 microM that responds to 30-32 ng analyte in 1 ml of reaction mixture and is 3.2-fold higher when compared to the chemical method with the use of permanganate and chromotropic acid. Linearity of the calibration curve is reliable (p < 0.0001) and standard deviation for parallel measurements for real samples does not exceed 7%. The proposed method, in contrast to the standard chemical approach, does not need the use of aggressive chemicals (concentrated sulfuric, phosphoric, chromotropic acids, permanganate), it is more simple in fulfillment and can be used for industrial wastes control and certification of formaline-contained stuffs.     

Peculiarities of cytochemical properties of cancer cells as revealed by study of deoxyribonucleoprotein susceptibility to Feulgen hydrolysis.

Chromatin of human squamous carcinoma cells reacts more intensively to short (1-2 min) acid hydrolysis in the Feulgen reaction and is, after such treatment, more intensively stained by Schiff reagent than chromatin of normal cells of the same origin. To reveal this difference in chromatin properties the use of a fluorescence variant of the Feulgen reaction is necessary because nuclei-binding of Schiff reagent after such short hydrolysis is so weak that the amount of the stain bound by means of absorption technique is hardly possible. The use of increased sensitivity of cancer cells chromatin to acid hydrolysis for cancer cytodiagnosis is suggested, especially for the diagnosis of so called diploid cancers for which detection on the basis of deoxyribonucleic acid content determination is impossible.     

Colorimetric determination of acetaldehyde in the presence of formaldehyde

A procedure is described that enables use of the p-phenylphenol color reaction to determine acetaldehyde in the presence of formaldehyde. The sample is first treated with an acidic 2,4-pentanedione reagent, which selectively removes formaldehyde. The method is applicable to blochemical reactions using tissue preparations.     

Reactions of aldehydes with unsaturated fatty acids during histological fixation

Synopsis Formaldehyde reacts with unsaturated fatty acids in tissues during histological fixation. For example, the reaction of formaldehyde with oleic acid gives rise to compounds with the structures shown below. The structures of the major reaction products have been confirmed, but those of the minor products have not been conclusively demonstrated. Esterified unsaturated fatty acids react more slowly, but the reaction is otherwise similar. Acrolein has been found to react in a similar fashion, but the reaction is far more complex.The occurrence of such compounds in tissues partly explains the loss of lipids during fixation, and raises some interesting possibilities regarding new fixatives and new histochemical reactions. The experimental work described in this paper was performed in the Departments of Pathology and Applied Biology, University of Cambridge.     

Studies in lipid histochemistry

Summary The effect of bromination (carried out especially with potassium tribromide and with bromine water) on various chemical groups in lipids and nonlipidic substances in tissue sections and in spot tests was investigated. Bromination blocks rapidly and effectively the reaction of double bonds with osmium tetroxide in unsaturated fatty acids, in phosphoglycerides, and nonpolar lipids, whereas in sphingolipids some residual staining persists even when the blocking period is prolonged. Similar results were obtained in the case of the UV-Schiff reaction. The vinylether bond in plasmalogens is blocked completely but not so rapidly.Besides double bonds bromination blocks the pseudoplasmal reaction in unsaturated lipids and the reaction of higher fatty aldehydes with Schiffs reagent and with dinitrophenyl hydrazine. Moreover it oxidizes the hydroxy group of cholesterol to a ketoderivative and in the case of ceramide mono- and dihexosides increases the intensity of the PAS reaction.The influence of bromination on various protein groups and the extraction of some loosely bound mucosubstances were demonstrated. The practical implication of these observations in applied lipid histochemistry is discussed.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

Studies of the mechanism of the periodic acid-Schiff histochemical reaction for glycogen using infrared spectroscopy and model chemical compounds.

It has been proposed in the literature that Schiffs reagent reacts with aldehydes to form one of the following types of compounds: alkylsulfonic acids, N-sulfinic acid derivatives, or Schiff bases. Model compounds whose structures are consistent with those proposed in the literature have been synthesized and subjected to infrared analysis. Also, actual products of Schiff reagent reactions with various aldehydes have been isolated and examined using infrared spectroscopy. Comparison of the spectra of the model compounds with those of Schiff-aldehyde reaction products yielded the following conclusions: 1. The reaction of simple organic aldehydes with Schiff's reagent produces an alkylsulfonate-type reaction product. 2. The reaction of periodate-oxidized glycogen with Schiff's reagent probably involves the formation of an alklsulfonate-type compound. 3. The product of the Schiff-aldehyde reaction exists as neither an N-sulfinic acid nor a Schiff base derivative of the fuchsin molecule.     

Comparative Spectroscopy of Basic Fuchsin, Schiff Reagent, and a Formalin-Schiff Derivative in Relation to Dye Structure and Staining

Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO₃H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl^- or SO₃H^- to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so reduced shows the fomation-NH₃ groups. Infrared (IR) spectra of basic fuchsin, Schiff crystals, and a crystalline formalin-Schiff reaction product support these observations and indicate that the final colored product is a methylsulfonic acid derivative of basic fuchsin. Identical IR spectra were obtained for two types of crystals derived from Schiff reagents indicating that both are the same chemically, although only one became colored on exposure to air. When these crystals were redissolved and SO₂ added, a Schiff reagent of appropriate pH was produced. Since it is derived from a crystalline product, this type of reagent should be useful in histochemical studies     

Histochemical studies of the mechanism of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure

Summary Histochemical investigations of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure were carried out on tissues containing carbohydrate macromolecules known to produce on periodate oxidation, only sialic acid monoaldehydes or hexosedialdehydes or mixtures of the two. The results indicated that the PAPS reaction is a generalized phenomenon independent of the hydrazine or hydrazide used, the nature of the Schiff reagent or the presence of anionic groups. It is proposed that phenylhydrazine condenses with periodate-engendered sialic acid monoaldehydes to yield the corresponding phenylhydrazone and with periodate-engendered dialdehydes to yield the corresponding morpholine or azido derivatives. Subsequent Schiff treatment results in the reversal of the blockade of sialic acid monoaldehydes but not of the dialdehydes, thus leading to selective Schiff staining of sialic acid residues.     

Effect of UV-light on formation of fluorescent products of lipid peroxidation

The rate of fluorescent product formation during the peroxidation of polyunsaturated linolenic acid or egg phosphatidylethanolamine and also during the oxidation of linolenic acid together with a phenylalanine and synthetic phosphatidylethanolamine 1,5--3 times more intensive after previous UV-irradiation of the unsaturated fatty acid. Schiff bases are fluorescent products in amine containing systems which are produced in the reaction of the malonaldehyde with amines. It is possible that fluorochromes produced during the only unsaturated acid oxidation are the result of the radical recombination. Accumulation of the oxidated products determined by TBA-reactive substances does not inevitably correlate with the fluorescent intensity in explored systems.     

Periodic acid-Schiff reaction combined with quantitative autoradiography of 3H-thymidine- or 85S-sulfate-Labeled epithelial cells of colon

Summary The influence of periodic acid-Schiff staining on grain counts was examined using autoradiography of guinea pig colon labeled with either ³H-thymidine or ³⁵S-sulfate. Prestaining decreased the grain count, an effect due to the Schiff reagent but not to periodic acid. Poststaining altered the grains which were partly or completely lost, an effect due to periodic acid but not to the Schiff reagent. The suggested procedure is to pretreat the sections with periodic acid, process for autoradiography, and poststain with the Schiff reagent. No silver grain is then lost.     

The apparent failure of sodium borohydride reduction to block further PAS reactivity in rat epithelial mucins

Synopsis Sections of rat small intestine were oxidized with 1% periodic acid for periods of 1, 2, 5, 10 and 30 min and were subsequently either (a) stained with Schiff reagent, or (b) reduced with sodium borohydride and then treated with either Schiff reagent alone or or by the standard PAS procedure. It was found that whereas sodium borohydride reduction abolished all Schiff staining, initial periods of oxidation in excess of 10 min were necessary to abolish any subsequent PAS reactivity. The theoretical and practical significance of these data is discussed in relation to the recent publication of Bayliss & Adams (1976).     

Unexpected isomeric equilibrium in pyridoxamine Schiff bases

Pyridoxamine is a vitamin B₆ derivative involved in biological reactions such as transamination, and can also act as inhibitor in protein glycation. In both cases, it has been reported that Schiff base formation between pyridoxamine and carbonyl compounds is the main step. Nevertheless, few studies on the Schiff base formation have been reported to date. In this work, we conduct a comparative study of the reaction of pyridoxamine and 4-picolylamin (a pyridoxamine analog) with various carbonyl compounds including propanal, formaldehyde and pyruvic acid. Based on the results, 4-picolylamin forms a Schiff base as end-product of its reactions with propanal and pyruvic acid, but a carbinolamine with formaldehyde. On the other hand, pyridoxamine forms a Schiff base with the three reagents, but the end-product is in equilibrium with its hemiaminal form, which results from the attack of the phenolate ion of the pyridine ring on the imine carbon. This isomeric equilibrium should be considered in studying reactions involving amine derivatives of vitamin B₆.     

Supravital Staining of Mitochondria with Amethyst Violet

Schiff reagent may be made by simply adding 1 g. fuchsin, 1.9 g. sodium metabisulfite to 100 ml. 0.15 N hydrochloric acid, shaking at intervals or mechanically for 2 hours, decolorizing with charcoal and filtering.

To obtain perfectly colorless Schiff reagent, it is necessary that fresh activated charcoal be used. If the final product is not water white, a fresh sample of activated charcoal should be obtained, and the product retreated and refiltered.

Schiff reagent precipitates a white crystalline substance after varying periods of cold storage, apparently a polymer which can be partly depolymerized by boiling and reconstituted into a Schiff reagent. The reconstituted reagent rather promptly precipitates again. Hence reconstitution is impractical and preparation of fresh batches at intervals of 2-3 weeks is recommended.