The tetrazolium-formazan system: design and histochemistry.

Our increasing knowledge about the chemistry and the correlations between chemical structure and histochemical properties of the tetrazolium/formazan system is resulting in: a better understanding of existing histochemical tetrazolium techniques; the selection of optimal tetrazolium salts for qualified use in histochemistry, cytochemistry and biochemistry; both qualitative and quantitative improvements in histochemical techniques for purposes demonstrating the activities of various dehydrogenating enzymes; an extended insight into the "state" of the tested biological object by means of tetrazolium indicators with special properties; and the combination of histochemical enzyme determination with further morphological techniques. This article has attempted to illustrate the progress in the use of the tetrazolium/formazan-system for histochemical purposes.     

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.     

A histochemical method for detecting xanthine oxidase activity in the liver

A histochemical method is described for localizing xanthine oxidase--the key enzyme in the purine catabolic pathway. The above method is based on the reduction of p-Nitroblue tetrazolium during hypoxanthine enzymatic oxidation, phenazine methosulfate being an intermediate electron acceptor. The patterns of the reaction product distribution suggest that the Kupffer cells and the sinusoidal endothelium possess the highest xanthine oxidase activity.     

The histochemical demonstration of phosphoglucose isomerase

Summary A technique for the histochemical demonstration of phosphoglucose isomerase, using an indirect tetrazolium method, is described. The enzyme is shown to be widely distributed, thus confirming biochemical findings. The wide distribution is of significance because phosphoglucose isomerase occupies a position of considerable importance in carbohydrate metabolism, particularly in the conversion of fructose to glucose by means of intermediate phosphates.     

A tetrazolium method for non-specific alkaline phosphatase

Summary A technique for the histochemical demonstration of non-specific alkaline phosphatase was developed using a medium containing indoxyl phosphate and a tetrazolium salt, Nitro B.T. The tetrazolium salt was reduced to diformazan by the hydrogen ions released by the formation of either indigo or indigo white by reaction of the enzyme on the indoxyl phosphate.The localization in the organs investigated was similar to that obtained by the standard azo dye and lead techniques.     

Histochemical study of superoxide dismutase in the ovary of the rat during the oestrous cycle

Superoxide dismutase, an enzyme which causes the dismutation of superoxide free radical anions to generate hydrogen peroxide, has been localized in the rat ovary. The negative-staining method was used to provide photo-induced reduction of nitro-blue tetrazolium in cryostat sections of rat ovaries for histochemical localization of superoxide dismutase. Superoxide dismutase was found in growing follicles, the membrane granulosa of Graafian follicles, ovulated follicles and blood vessels. It is suggested that superoxide dismutase may play a role in regulating follicular development, ovulation and luteal functions.     

THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.     

Histochemistry of the epimerases

Summary On the assumption that electron transfer is involved in the process of epimerization and by using tetrazolium salts as an indicator, a histochemical reaction for the demonstration of UDPGal-4-epimerase has been developed.By using UDPG or UDPGal as substrates it has been possible to ascertain the direction of the reaction catalysed by this enzyme in various tissues in normal physiological conditions.Biochemical tests support the concept that the histochemical reactions recorded were the result of UDPGal-4-epimerase activity.     

Quantitative microphotometric succinate dehydrogenase histochemistry in human nephron

A simple method for microphotometric evaluation of cryostat sections from human renal tissue routinely stained for succinate dehydrogenase activity by means of tetranitro-blue tetrazolium chloride is described and tested for validity. Manual absorbance measurement within single nephron segments from the same section allows to directly visualize the distribution pattern of this enzyme along the nephron. Photometric data can be expressed in relative enzyme activities by using the cortical collecting ducts within the same section as reference. This allows to compare measurements of different kidney sections stained by various incubation procedures. The agreement found between relative succinate dehydrogenase activities and recently published morphometric data on mitochondrial inner membranes along the rat nephron suggests that quantitative succinate dehydrogenase microphotometry is a useful histochemical tool for the assessment of renal mitochondrial cristae membranes.     

The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

Summary An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

The simultaneous demonstration of adrenergic fibres and enteric ganglion cells

Synopsis A method for the demonstration of adrenergic nerves and enteric neurons at the same time has been developed by combining the fluorescence histochemical technique for catecholamines and the histochemical technique for NADH:Nitro BT oxidoreductase.The method consists of a short incubation of the laminae from the wall of the intestine in an isotonic medium containing the substrate (NADH) and a tetrazolium salt (Nitro BT). After washing, the laminae are air dried, exposed to formaldehyde vapour and mounted.The adrenergic nerves in the myenteric plexus appear brightly fluorescent on excitation with u.v. light, whereas the neurons are heavily stained by deposits of formazan. Not all the neurons of the plexus are stained, but their morphology is well preserved. Differences in staining of the neurons reflect differences in penetration of the tetrazolium salt in the tissue and into the cells. The adrenergic axons do not establish exclusive connexions with individual neurons and some isolated neurons are not associated with any adrenergic fibres.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

Summary The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.     

Demonstration and Biochemical Characterisation of Rat Brain NADPH-Dependent Diaphorase

An enzyme responsible for the NADPH-dependent reduction of nitroblue tetrazolium HCl (NBT) has been isolated from rat brain. Although other tetrazolium salts could be utilised, NBT was the preferred substrate, and the enzyme had an absolute requirement for NADPH. An in vitro assay was developed and used to determine the kinetic constants: Km NBT = 17.3 microM; Km NADPH = 1.9 microM, Vmax = 30.8 mumol product produced/min/mg protein. Substrate inhibition by NADPH was observed in some instances. Brain subcellular fractionation indicated highest enzyme activities in the microsomal fraction. Activity was present in all brain regions and in a variety of peripheral tissues. Relative molecular mass determinations of the native enzyme yielded an Mr = 170-180,000. It seems likely that the enzyme activity described in this study relates directly to the histochemical demonstration of brain NADPH-diaphorase-positive neurons. As yet, the natural substrate for the enzyme is unknown. However, the isolation and purification of NADPH-dependent diaphorase may be anticipated to assist in the elucidation of its function in the brain, and in the special characteristics of those neurons that contain the enzyme in abundance.     

The effect of superoxide dismutase on histochemical detection of dehydrogenases on zymograms using sorbitol dehydrogenase as an example

Sorbitol dehydrogenases from the cytoplasm of plant, animal, and microbial cells was used to study the effect of superoxide dismutase on histochemical exposure of dehydrogenases after their electrophoretic separation. In some organisms, both enzymes are localized in the same region, which finally leads to the formation of hydrasine tetrazolium, a soluble colourless compound, but not of diformazan, an insoluble stained derivative of tetrazolium nitroblue. The experimental conditions for histochemical exposure of the enzymes in gels are discussed.     

Histochemical Demonstration of Endogenous Dehydrogenase Systems by Supravital Perfusion

Endogenous dehydrogenase activity is demonstrated in fresh, intact organs by supravital perfusion with a tetrazolium solution. The animal is first injected intravenously with 1.5 mg Heparin/100 gm body weight. It is then anesthetized and a fine polyethylene cannula (PE50, Intramedic) is inserted into a major artery and secured with a ligature. An initial perfusion with warm (37°C) M/20 phosphate buffer (pH 7.6) to remove the blood from the tissues is followed by a 10 min perfusion with the same kind of buffer to which has been added 0.25% neotetrazolium chloride (Dajac Laboratories). The tetrazolium solution is delivered to the tissue at the rate of 1 ml/minute. A final perfusion with 10% formalin in warm phosphate buffer (pH 7.6) flushes and fixes the tissues. Frozen sections can then be cut and mounted in glycerol jelly. Fine, colored formazan crystals are deposited at the sites of enzyme activity. The method is simple and yields excellent histochemical preparations.     

Succinic dehydrogenase activity in liver,kidney and brain of rat

Summary A method is described for histochemical quantification of the activity of succinic dehydrogenase in various tissues of rat by means of Nitroblue tetrazolium. This method can be used for comparison of enzyme activities; the activities calculated correspond to values obtained by biochemical methods. The necessity to quantify the nothing dehydrogenase is established as well as the amount of half-formazan.Accepted as doctoral dissertation by the Faculty of Medicine, Westfälische Wilhelms-University Münster     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Quantitative aspects of the histochemical tetrazolium salt reaction on monoamine oxidase activity in rat liver

Summary The tetrazolium method for the histochemical detection of monoamine oxidase (MAO) activity in rat liver cryostat sections has been tested for its specificity and its possible use in quantification. The tetrazolium salt tetranitro blue tetrazolium is recommended for the localization of MAO activity, rather than nitro blue tetrazolium or BPST [2-(2-benzothiazolyl)-3(4-phthalhydrazidyl)-5-styryl-tetrazolium]. Hardly any formazan was produced in the absence of the substrate tryptamine and Marsilid, a specific inhibitor of MAO activity, prevented formazan production almost completely. A linear relationship between the integrated absorbance measured with a microdensitometer and either the incubation period or section thickness was obtained. We conclude that the method described in this paper can be used for the quantitative analysis of MAO activity in tissue sections of rat liver. MAO activity was found to be 20–25% higher in the periportal zone of rat liver than in the perivenous zone.