Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.     

Oxidative enzymes in the development of Fasciola hepatica L. IV. The activity of oxidases and dehydrogenases in redia.

The object of the study was to investigate the occurrence and localization of oxidative enzymes in the redia -- the third larval stage of Fasciola hepatica L. The author detected cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases (diaphorases), as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde phosphate, glucose-6-phosphate, 6-phosphogluconate, beta-hydroxybutyrate, L-glutamate, and alcohol dehydrogenases. The presence and localization of the enzymes in various periods of development of the redia were detected with histochemical methods. Out of the studied oxidases and dehydrogenases only cytochrome oxidase was found to be absent from the stages of young rediae. It was ascertained that the redia uses all three paths of release of energy i.e. the glycolytic, Krebs, and pentose cycles, glycolysis being presumably the principal mode of energy production.     

Factors influencing the histochemical demonstration of coenzyme-dependent dehydrogenases and diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, beta-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases

Summary A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but -OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results.     

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.     

Factors Influencing the Histochemical Demonstration of Coenzyme-Dependent Dehydrogenases and Diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, β-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

Dehydrogenase enzyme histochemistry on freezedried or fixed resin-embedded tissue

Summary A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4°C or –20°C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at –20°C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.     

Oxidative enzymes in the development of Fasciola hepatica L. III. The activities of oxidases and dehydrogenases in the sporocyst.

The object of the study was the investigation of the occurrence and distribution of some oxidative enzymes in the sporocyst of Fasciola hepatica L. The samples were examined for the presence of cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases, as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde-3-phosphate, glucose-6-phosphate, beta-hydroxybutyrate, L-glutamate and alcohol dehydrogenases. All of them save cytochrome oxidase were found to occur in the sporocyst. The presence and localization of these enzymes were examined by histochemical methods in various stages of development of the sporocyst. These investigations permitted it to be established that glycolytic processes are the principal way of release of energy for all developmental groups of this larva. Moreover, the functions of the tricarboxyl acid and pentose-phosphate cycles were detected and found to play a less important part in processes of energy production in the sporocyst. In addition, the functioning and metabolism of each larval organ in various stages of its development were discussed in so far as was possible on the basis of the analysis of the above-mentioned oxidative enzymes.     

Brain enzyme histochemistry following stabilization by microwave irradiation

Summary The activities of various enzymes present in brain homogenates were assayed biochemically (a) with no pretreatment, (b) following a standard microwave treatment in saline and (c) after a standard microwave treatment in formalin. All enzyme activity was lost after the microwave — formalin in treatment. Following microwave — saline treatment, the activities of alkaline phosphatase, 5-nucleotidase, isocitrate and succinate dehydrogenases were reduced. In contrast, the activities of lactate and malate dehydrogenases were unchanged, and that of acetylcholinesterase apparently increased.Analogous outcomes were seen following attempted histochemical demonstrations of these enzymes. Thus satisfactory histochemical demonstration of all enzymes was achieved (except with alkaline phosphatase, lactate and malate dehydrogenases) following the microwave-saline pretreatment. Since acid phosphatase, catalase and peroxidase were also successfully demonstrated, it seems that microwave-saline pretreatments permit both retention of sufficient enzyme activity for histochemical demonstration to occur and retention of sufficient structural integrity for critical morphological investigations. Since the failure to stain the sites of lactate and malate dehydrogenases is not due to microwave inactivation of these enzymes, their demonstration may be possible by varying the staining procedures.     

The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases

Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.     

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.     

Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat

Summary The segmentation of the proximal tubules of the male rat kidney was studied by means of enzyme histochemical reactions. Soluble oxidoreductases (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, NAD- and NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase) were demonstrated using methods which reduce enzyme diffusion (incubating in presence of polyvinyl alcohol) and eliminate interference from tissue tetrazolium reductases. Less soluble or insoluble enzymes (glucose 6-phosphatase, -hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases) were demonstrated by incubation in conventional watery media.Segmental differences were observed in respect to all enzymes studied, and most reactions clearly visualized the three segments known to exist from ultrastructural as well as previous histochemical studies: The pars convoluta includes the first (P₁) and most of the second (P₂) segment. The transition to the third segment (P₃) is in the beginning of the pars recta. Also these reactions revealed a difference between the first part of the P₃, which runs through the cortex in the medullary rays, and the terminal part transversing the outer stripe of the medulla. In most instances intensity of reaction decreased in the last portion of the P₃.A number of the enzymes studied were mainly or solely localized to the P₃ (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenases, -hydroxybutyrate dehydrogenase, 3-hydroxysteroid dehydrogenase, decarboxylating malate dehydrogenase and uridine diphosphate glucose dehydrogenase). Some possible functional implications of the findings are discussed.Supported by grants from Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — Mr. Kaj L. Pedersen is thanked for valuable photographic assistance.     

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.     

STUDIES OF CYTOCHEMICAL LOCALIZATION OF SPECIFIC DEHYDROGENASES IN FROZEN,DISEASED TISSUES OF PINUS

Intracellular activity of individual dehydrogenases in frozen tissues of Pinus monticola and Cronartium ribicola was demonstrated by supplying a specific substrate and the appropriate pyridine-nucleotide-linked coenzyme. Freezing broke cell permeability barriers releasing endogenous coenzymes and substrates which had produced nonspecific enzymatic reduction of nitro blue tetrazolium by miscellaneous dehydrogenases throughout fresh tissues. Freezing enhanced specificity by accentuating the differences between control and treatment sections. Succinic, ethanol, glutamic, α-glycerophosphate, isocitric, lactic, malic, glucose-6-phosphate, and 6-phos-phogluconate dehydrogenases and NAD and NADP diaphorases were localized within cells of the blister rust fungus and its western white pine host. NAD- and NADP-linked forms of glutamic, isocitric, and malic dehydrogenases were also detected. The distribution and activity of the enzymes are described for cell types of host and pathogen. β-Hydroxybutyric and pyruvic dehydrogenases were not detected. Calcium and magnesium (5 × 10⁻³ m final conen) and zinc (1.5 × 10⁻⁵ m final concn) had little or no effect on localization. Amytal increased reduction by 6-phosphogluconate, glutamic, and ethanol dehydrogenases while azide depressed the reaction for the last enzyme. Cyanide augmented diformazan formation with succinate. Transhydrogenase was eliminated as a likely contributor to spurious localization in these frozen tissues. Enzymatically produced diformazan appeared on the surface of lipid droplets in cells of both organisms in fresh and frozen sections. The use and interpretation of data from frozen and fresh tissues in tetrazolium cytochemistry are discussed.     

Changes in enzyme histochemical profiles of identified spinal motoneurons of the European eel,Anguilla anguilla, following cordotomy

Summary The enzyme histochemical profiles of glucose-6-phosphate dehydrogenase (a marker of synthetic performance), succinate dehydrogenase (an indicator of oxidative metabolism), and NADH tetrazolium reductase (a marker of overall neuronal activity) were determined for identified white muscle motoneurons in six control and six cordotomized eels. Images were digitized and mean integrated absorbances obtained using appropriate hardware and software. For motoneurons caudal to the transection site there was a significant decrease in the mean absorbance value for NADH tetrazolium reductases which declines from 0.28 a.u. (arbitrary units) in control animals to 0.23 a.u. in cordotomized animals. However, no significant changes were detected in the activities of glucose-6-phosphate and succinate dehydrogenases. The cross-sectional area of the motoneuronal cell body was not affected by cordotomy. The decrease by around 20% in overall neuronal activity, as expressed by NADH tetrazolium reductase activity, might be expected from the decline in body motility that follows cordotomy. Changes in SDH and G6PDH activities would also be expected to follow this surgery, but none were seen, perhaps because they are compensated for by changes in neuronal metabolism that result from deafferentation.     

A Cytochemical Study of the Dehydrogenases of Mitochondria and Mitochondrial Particulates by a Monotetrazolium-Cobalt Chelation Method

In one of the current histochemical methods for dehydrogenases and diaphorases the final product is a metal-formazan dye derived from reduction of an N-thiazolyl-substituted tetrazolium. Sites of enzymic activity consistently appear as intramitochondrial dots 0.2 to 0.3 µ in diameter. When applied to active particles from disrupted mitochondria (Keilin-Hartree preparation, electron transport particle, Cooper-Lehninger particle) the individual particles appear as black dots 0.1 to 0.3 µ in diameter. It is clear that formazan is deposited progressively upon the particles and the results suggest that the latter may be spatially arranged in mitochondria so that areas of activity are separated by quiescent regions.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Cytochemical method for detecting dehydrogenase activity in the leukocytes of native blood smears

A cytochemical method of detection of dehydrogenases in blood leucocytes is proposed. Native smears are dried up in the air to be incubated at 37 degrees C in gel-containing medium composed of polyvinyl alcohol, sucrose, a corresponding substrate, cofactors and inhibitors of cytochrome oxidases activity. Using corresponding media, activities of succinate, malate, glutamate, lactate-, alpha-glycerophosphate, alcohol, beta-oxybutyrate and glucoso-5-phosphate dehydrogenases were revealed. Half-reduced diformazan providing diffuse rosy staining of cells was removed after the incubation, and the incubation medium was washed out by rinsing the smears in 60% acetone solution. As a result monochromatic micropreparations may be received. Finally, smears are fixed in formalin. The above method provides a reduced loss of enzymes, preserves a good cell morphology and eliminates non-dehydrogenase effects of tetrazolium reduction into formazan.     

A colorimetric assay to quantify dehydrogenase activity in crude cell lysates

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 degrees C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.     

The Cytochemical Localization of Oxidative Enzymes : II. Pyridine Nucleotide-Linked Dehydrogenases

Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.