A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier

A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cell is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used.     

A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier

Summary A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cells is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a "fixation" in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.     

A cytochemical method for measuring enzyme activity in individual preovulatory mouse oocytes

The activities of 6 enzymes involved in carbohydrate metabolism were determined quantitatively in preovulatory oocytes by cytochemical means per individual cell as well as biochemically in cell homogenates. Oocytes were incorporated in a polyacrylamide matrix for appropriate enzyme cytochemical staining. This incorporation preserves the morphology of the cells very well, and the enzymes keep their activity for a considerable period of time. This method could also be used to demonstrate more than one enzyme activity in the same cell. The results obtained by cytochemical means appeared to correlate very well with the biochemical data (P less than 0.005). Glucose 6-phosphate dehydrogenase, the key-enzyme in the pentose phosphate pathway, had very high activity in these preovulatory oocytes, but 6-phosphogluconate dehydrogenase activity was only about 2% of that of glucose 6-phosphate dehydrogenase. The activities of lactate dehydrogenase and to a lesser extent glucose phosphate isomerase and D-glyceraldehyde-3-phosphate dehydrogenase also appeared to be very high, while hexokinase showed a very low activity.     

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

Summary The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.     

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Summary A quantitative cytochemical assay for NAD⁺ kinase-like activity in the guinea-pig thyroid gland is described. The NADP⁺ produced by the activity of the kinase was used to drive the NADP⁺-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg²⁺ and NAD⁺) either alone or in combination. Kinase activity was dependent both on ATP and Mg²⁺, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.     

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes

Summary A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a fixation in 0.025% glutaraldehyde (under NADP⁺ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.     

A preliminary note on the hormonal induction of NADP-linked dehydrogenases in phytohaemagglutinin-stimulated human lymphocytes

Synopsis Human leucocytes were culturedin vitro with either phytohaemagglutin or 17 -oestradiol or both, and the enzyme activities of nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase were assayed. As a result, it was found that these enzymes were considerably induced by oestradiol in phytohaemagglutinin-stimulated lymphocytes, but not in non-stimulated cells. Electrophoretic studies on glucose-6-phosphate dehydrogenase showed that six molecular forms are demonstrable in normal and phytohaemagglutinin-stimulated leucocytes, and that one of the forms was specifically induced by oestradiol in the presence of phytohaemagglutinin.     

The effect of an anionic detergent on complex carbohydrates and enzyme activities in the epidermis of the catfish Heteropneustes fossilis (Bloch)

Summary The histochemistry of various oxidative enzymes and complex carbohydrates in the epidermis of the catfishHeteropneustes fossilis was investigated after exposure to sublethal concentrations of the detergent sodium alkulbenzenesulphonate.It was found that the detergent treatment was accompanied by a marked increase in the number of mucous cells which produce histochemically detectable amounts of acidic glycoproteins with a shift towards the production ofO-acetylated sialic acids. The activities of mitochondrial enzymes were lost in the superficial cell layers. In contrast the activities of glucose-6-phosphate and lactate dehydrogenase increased considerably. The rise in glucose-6-phosphate dehydrogenase was correlated with the metabolic requirements for the enhanced production of mucus under stress.The changes in both enzyme activities and in the chemical composition of mucus may provide a suitable experimental model for histochemical investigations of the effect of stress induced by pollulants on aquatic organisms.     

Studies on the phenazine methosulphate-tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. I. Localization artefacts caused by the escape of reduced co-enzyme during cytochemical reactions for NAD(P)+-dependent dehydrogenases

The correct localization of oxidative enzymes using cytochemical tetrazolium methods, in which low molecular weight electron carriers such as NAD(P)H and reduced phenazine methosulphate (PMSH) are used, can be endangered by the escape of the reduced intermediates before they react to form the insoluble formazan at the true enzyme-containing sites. To investigate this phenomenon, the glucose-6-phosphate dehydrogenase reaction was studied in fixed erythrocytes which, because of their microscopic dimensions, are well-suited for studying the loss of intermediates. A mixture of active and heat-inactivated fixed erythrocytes was incubated in a PMS-supplemented medium for glucose-6-phosphate dehydrogenase. The cytophotometric histograms showed that the final formazan precipitate was equally distributed over both active and inactivated cells. When bovine serum albumin was added to the medium, all the formazan was found to be bound to this protein and the erythrocytes remained essentially unstained. The false localization in this system could be explained by an unfavourable balance between the capture of electrons carried by NADPH within the erythrocyte and the diffusion of NADPH out of the erythrocyte. The rate constant of NADPH oxidation was determined, as was also the diffusion constant of NADPH in a protein matrix. Substituting the data obtained into formulae derived from the enzyme cytochemical localization theory of Holt & O'Sullivan (1958), it was calculated that the capture reaction was highly deficient and, theoretically, less than 1% of the total amount of formazan produced was localized within the erythrocyte which explains the false localization observed. The importance of these findings for the cytochemical demonstration of NAD(P)+-dependent dehydrogenases in cells and electropherograms is briefly discussed.     

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.     

Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowman's glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture: I. Quantitative changes in intracellular enzyme activities following a single dose of carcinogen.

The effects of exposure of adult rat hepatocytes to chemical carcinogens have been studied using a short-term maintenance culture system. Scanning microdensitometry was used to quantitate the observed changes in enzyme activity. The dose-response curves showed a biphasic response for all 4 enzymes studied (glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH oxidase and gamma-glutamyl transpeptidase) there being decreased enzyme activities at the higher dose levels used, possibly indicating cytotoxicity. The enhancement of enzyme activity at low dose levels was due to generalised increases occurring in every cell, rather than to selection of a cell species particularly high in enzyme activity. A culture period of 24 h was necessary for the complete adaptation of the cells to the culture environment as evidenced by the response of intracellular glucose-6-phosphate dehydrogenase activity to carcinogen treatment. These findings are discussed in relation to previously reported in vivo studies.     

Cytoplasmic NADP-linked dehydrogenase activities in avian tissues

Cytoplasmic activities of NADP-linked malic enzyme (E.C. 1.1.1.40), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and NADP-linked isocitrate dehydrogenase (E.C. 1.1.1.42) were determined in tissues of selected avian species, and compared with those in mammals. Malic enzyme was generally more active in avian liver and kidney than in the corresponding mammalian tissues. Hepatic activities as high as 200 units/g wet wt and 100 units/g wet wt were recorded in the Nectariniidae and the Ploceidae respectively. Glucose-6-phosphate dehydrogenase was generally less active in avian tissues than malic enzyme. In passerine birds activities were very low indeed, and in most cases spectrophotometrically undetectable. Malic enzyme and glucose-6-phosphate dehydrogenase were highly active in the adipose tissue of mammals but were inactive in the adipose tissue of birds. Marked increases in hepatic malic enzyme and glucose-6-phosphate dehydrogenase activities were associated in birds with premigratory fattening. Activities of isocitrate dehydrogenase were comparable in the corresponding avian and mammalian tissues, including adipose tissue.     

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r(2)=0.857), between catalase and glucose-6-phosphate dehydrogenase activities (r(2)=0.924) and between protein carbonyl levels and glucose-6-phosphate dehydrogenase activity (r(2)=0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1-fold higher level of carbonyl proteins than that of wild-type strain. Our data suggest that in vivo catalases may protect glucose-6-phosphate dehydrogenase against oxidative inactivation.     

In situ determination of different dehydrogenase activity profiles in the linings of odontogenic keratocysts and radicular cysts

Summary The levels of succinate, lactate, glutamate, glycerophosphate and glucose-6-phosphate dehydrogenases within the linings of keratinizing and non-keratinizing odontogenic cysts were investigated using static end-point and continuously monitored Nitroblue Tetrazolium-based histochemical methods. The use of TV image analysis for quantification of formazan final reaction products was validated by demonstrating significant relationships between the integrated absorbance at 585 nm and the amount of formazan in, and thickness of, gelatin films containing reduced tetrazolium salt (r=1.0,p<0.001). Absorbance readings of stained sections gave mean coefficients of variation of 1.8±0.9% between day of measurement, and of 5.65±1.32% between serial sections. End-point assays indicated that the linings of odontogenic keratocysts contained higher levels of glucose-6-phosphate dehydrogenases (p<0.0002) and lower levels of lactate dehydrogenase (p<0.002) than those of radicular cysts. Succinate, glutamate and glycerophosphate dehydrogenase activities were similar in both cyst types. Results from continously monitored assays, performed for glucose-6-phosphate and succinate dehydrogenases, demonstrated linear reaction rates over the first 2.75 min of reaction. The calculated enzyme activities from continuous assays were between 1.49 and 3.49 times higher than those determined from end-point assays and confirmed that levels of glucose-6-phosphate dehydrogenase were significantly higher in the linings of odontogenic keratocysts than those of radicular cysts (p<0.004). By contrast, succinate dehydrogenase activity was significantly higher in radicular cyst linings (p<0.03). These results highlight the benefits of an approach toin situ determination of enzyme activity using image analysis and continous monitoring methodologies. Overall, the high level of glucose-6-phosphate dehydrogenase found in keratocyst linings is consistent with their clinical behaviour and higher level of proliferation and synthetic activity whereas the level of lactate dehydrogenase in radicular cysts probably reflects the presence of local tissue damage within these inflammatory lesions.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum-free medium

A serum-free, hormone-supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum-supplemented media. To quantitatively compare cell behavior in SFHM with serum-supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+-linked sn-glycerol-3-phosphate dehydrogenase, malate dehydrogenase, NAD+-linked isocitrate dehydrogenase, NADH-cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum-supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: 1) many enzyme activities that may represent dedifferentiation are elevated by serum; 2) NAD-linked glycerol-3-phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; 3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; 4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 micrograms/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.