Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium.

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.

Effects of ischaemia and reperfusion on NADH coenzyme Q reductase activity in rat liver.

NADH coenzyme Q reductase (EC 1.6.5.3) has been suggested in the literature to be inactivated by ischaemia. In the present study, NADH coenzyme Q reductase activity was localized in unfixed cryostat sections of ischaemic rat livers and quantified using image analysis. In vitro ischaemia was induced by storage of rat liver fragments for 30, 60, and 120 min at 37 degrees C. In vivo ischaemia was provoked by clamping the afferent vessels of median and left lateral liver lobes for 60 min followed by 30, 60 and 180 min of reperfusion. NADH coenzyme Q reductase activity was demonstrated with the tetrazolium salt method in the presence of polyvinyl alcohol. Final reaction product was found in liver parenchymal cells and its distribution was homogeneous within liver lobules. Only low amounts of final reaction product were formed when the incubation was performed in the absence of the substrate NADH. A non-linear relation was found between the absorbance and incubation time when the reaction was performed in the presence of NADH. Therefore, the initial velocity was taken as the true rate of enzyme activity. A linear relationship was found for the initial velocity and section thickness up to 6 microm followed by a levelling off. Electron microscopically, NADH coenzyme Q reductase activity was localized at the outer and inner membranes of mitochondria. In vitro ischaemia up to 120 min did not affect NADH coenzyme Q reductase activity. At 30 min reperfusion after in vivo ischaemia for 60 min enzyme activity was slightly decreased in certain foci which also showed diminished lactate dehydrogenase activity. A further decrease of enzyme activities in foci was observed at 180 min reperfusion after ischaemia. It is concluded that NADH coenzyme Q reductase activity is not sensitive to ischaemia. Furthermore, it is likely that the enzyme leaks from liver parenchymal cells into the circulation during reperfusion after ischaemia.

In situ detection of constitutive superoxide anion production in granules of mast cells.

3,3'-Diaminobenzidine, in the presence of manganese and cobalt ions, was applied for the detection of superoxide anions in unfixed cryostat sections of rat oesophagus, trachea, skin and intact mesenterium. In all connective tissues, a blue final reaction product was found in a granular form in mast cells. The amount of final reaction product formed after incubation with diaminobenzidine and cobalt ions was increased by the addition of manganese ions. Electron microscopical analysis revealed that the electron-dense final reaction product was exclusively present in the granules of mast cells and on elastin fibres. It was found that the constitutive spontaneous formation of final reaction product in mast cells was enzymatic and dependent on the presence of oxygen in the medium. Of all the enzyme inhibitors and free radical scavengers tested, only azide strongly reduced the amount of final reaction product. It was concluded that the reaction was partly caused by peroxidase activity, but that superoxide anions are also constitutively and spontaneously produced in mast cell granules. The exact enzymatic source could not be established. Whether this property of mast cell granules plays an antimicrobial role in connective tissues can only be speculated.

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine.

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3'-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co(2+)-containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB-cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehyde oxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB-cobalt complex.

Localization of superoxide dismutase activity in rat tissues.

Superoxide anion radicals have been implicated in a variety of pathological processes. Under physiological conditions, superoxide dismutase (SOD) is effectively able to disproportionate superoxide anions into hydrogen peroxide and dioxygen. Until now, no techniques have been available to localize SOD activity within tissues. In the present study, SOD activity was detected in different rat tissues using a thin film of xanthine oxidase between the glass slide and the unfixed cryostat section and a medium containing hypoxanthine as a source of electrons for the production of superoxide anions. The incubation medium also contained cerium ions to precipitate the hydrogen peroxide product and polyvinyl alcohol to prevent leakage of soluble and/or loosely bound enzymes from the sections into the incubation medium. The cerium perhydroxides that are formed were visualized for the light microscope in a second step using an incubation medium consisting of 3,3'-diaminobenzidine, cobalt ions, and hydrogen peroxide, which results in oxidation of the diaminobenzidine to the final insoluble blue reaction product. By this methodology, high enzyme activity was found not only in endothelial cells of liver and kidney but also in hepatocytes of liver, myocytes of heart, smooth and striated cells of muscle, acinar cells of pancreas, epithelial cells of kidney ducts, and epithelial cells of the small intestine and colon. These findings were largely in agreement with immunohistochemical data obtained using antibodies against the Cu/Zn- and Mn-SODs. However, high activity was also detected extra-cellularly at the surface of epithelia of trachea, esophagus, small intestine, and colon and at the extracellular matrices, cartilage, and connective tissues. We conclude from these latter data that the activity of the extracellular form of the dismutase is localized. The present method allows the analysis of all three types of known SOD activity (Cu/Zn, Mn, and extracellular) in different tissues and cell compartments.

The proportion of xanthine oxidase activity of total xanthine oxidoreductase activity in situ remains constant in rat liver under various (patho)physiological conditions.

Activity of xanthine oxidoreductase (total xanthine dehydrogenase plus xanthine oxidase) and xanthine oxidase was determined cytophotometrically in periportal and pericentral areas of livers of rats under various (patho)physiological conditions that are known to affect the content of reduced glutathione. For this purpose, rats were either normally fed or fasted for 24 hours, fasted for 24 hours, and treated with diethylmaleate that depleted glutathione or treated by in vivo ischemia for 2 hours in the livers. Xanthine oxidoreductase activity was shown histochemically with the use of a tetrazolium salt procedure, and xanthine oxidase activity was localized with a cerium-diaminobenzidine-cobalt-hydrogen peroxide technique in unfixed cryostat sections of the livers. Cytophotometric measurements showed that total xanthine oxidoreductase activity was decreased after fasting and ischemia, whereas only ischemia caused reduced xanthine oxidase activity. Moreover, the percentage of xanthine oxidase of total xanthine oxidoreductase activity was constant in both periportal and pericentral areas at the level of approximately 4% in normally fed and 24-hour fasted and diethylmaleate-treated rats. Ischemia reduced this percentage in both areas of the liver to 2%. It was concluded that the amount of endogenous reduced glutathione did not affect the percentage of xanthine oxidase. The low percentage of xanthine oxidase as determined in the present in situ histochemical study indicates that in vivo the percentage oxidase in rat liver is lower than is assumed on the basis of biochemical assays in liver homogenates even after strictly controlled homogenization procedures. Apparently, conversion of xanthine dehydrogenase into xanthine oxidase may occur in vitro to yield percentages of xanthine oxidase of 10%-20% as are reported in the literature. The latter increase in the percentage of xanthine oxidase may be caused by changes in the local environment of the enzymes, which is left completely intact in histochemical assays. The finding of this low percentage of xanthine oxidase further stresses that the main function of xanthine oxidoreductase in the liver is not the production of superoxide anion radicals and/or hydrogen peroxide but rather the metabolism of xanthine to uric acid, which can act as a potent antioxidant.

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions.

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19,000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the polyDAB-cobalt complex as final reaction product of oxidase reactions was established to be 140,000.

Demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure.

In the present study a technique was developed to demonstrate 5'-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4 degrees C rather than at 37 degrees C. The optimized incubation medium contained 300 mM Tris-maleate buffer, pH 7.2, 5 mM adenosine monophosphate as substrate, 30 mM cerium chloride as capturing agent for liberated phosphate, 10 mM magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5'-nucleotidase activity were obtained when incubations were performed at 37 degrees C and 4 degrees C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37 degrees C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8 micrograms. 5'-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37 degrees C than at 4 degrees C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37 degrees C, in comparison with that at 4 degrees C, were the appearance of multi-lamellar structures at bile canaliculi at 37 degrees C. We conclude that the present method is valid for the demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver.

The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light- and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mM diaminobenzidine, 44 mM hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 microns. Catalase in rat liver showed a Km value of 2.0 mM for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mM. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.

Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver.

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

The role of xanthine oxidase in ischemia/reperfusion damage of rat liver.

Oxygen radicals have been proposed to be involved in the induction of liver cell damage during reperfusion after ischemia. The role of xanthine oxidase in this process and the potential of the antioxidant system have been studied in a model of in vivo ischemia of rat liver followed by 1 h reperfusion by the use of enzyme histochemistry. Based on decreased lactate dehydrogenase activity in certain areas of liver parenchyma, cell damage could already be detected at 1 h reperfusion after ischemia. Incubations performed on serial sections showed that the same areas contained decreased activities of xanthine oxidoreductase, xanthine oxidase, catalase and glucose-6-phosphate dehydrogenase. Some individual cells in the undamaged liver parenchyma expressed a very high glucose-6-phosphate dehydrogenase, which suggests that these cells have a good defence against oxidative stress. It is concluded that oxygen radicals derived from xanthine oxidase do not play a decisive role in the induction of cell damage immediately at reperfusion after ischemia. However, it cannot be excluded that xanthine oxidase present in the blood stream can give rise to the development of additional damage later on.

A quantitative histochemical study of xanthine oxidase activity in rat liver using the cerium capture method in the presence of polyvinyl alcohol.

A recently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (w/v) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfixed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificity of the reaction was proven by the generation of only small amounts of final reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 microns). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity.

NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection.

Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum lipid peroxidation was observed when the pro-oxidant system contained 0.2 mM NADPH, 1 mM ADP and 15 microM FeCl2. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 microM prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher than 1 mM inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH 7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37 degrees C with the pro-oxidant system. A linear relationship was found between the thickness of the sections (up to 20 microns) and the amount of lipid peroxidation products. The addition of scavengers of O2-. (superoxide dismutase), hydrogen peroxide (catalase) and OH. (mannitol) to the first step medium did not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver.

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5'-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 microns in thickness. For all the enzymes that could be detected, the 6 microns:3 microns ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.

Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells.

Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5'-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.

Histochemical detection of glycogen phosphorylase activity as parameter for early ischemic damage in rat heart.

In the present study we have investigated whether enzyme histochemical parameters can be applied to detect early ischemic damage in rat heart after ischemia without restoration of the blood flow. Ischemia was induced by incubating heart fragments for 0, 10, 20, 30, 60, 120 and 240 min at 37 degrees C. The activity and localization of the following enzymes was studied in unfixed cryostat sections using quantitative histochemical methods: lactate dehydrogenase, creatine kinase, succinate dehydrogenase, phosphofructokinase, acid phosphatase, 5'-nucleotidase and glycogen phosphorylase. Moreover, the ultrastructure of the tissue was studied with special attention to the appearance of flocculent densities in mitochondria, which can be seen as a sign of irreversible cell damage. It was shown that glycogen phosphorylase activity in rat heart decreased after short periods (30 min) of in vitro ischemia, whereas all other enzymes studied were not decreased up to 240 min, with the exception of lactate dehydrogenase and phosphofructokinase activities which were diminished only at 240 and 120 min of ischemia, respectively. Some reaction product was found after incubating for 5'-nucleotidase activity in the absence of substrate, indicating the presence of endogenous substrate(s). This endogenous substrate disappeared from the myocytes after 20 min of ischemia. It is assumed that AMP and/or other phosphate-containing compounds play an essential role in the activation of glycogen phosphorylase. Significant reduction of glycogen phosphorylase activity is correlated with the irreversible stage of damage of myocytes as judged from the ultrastructure.

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver.

The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25 degrees C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5'-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25 degrees C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.

Protective effect of silymarin on rat liver injury induced by ischemia.

The effect of silymarin on liver cell damage induced by ischemia was studied in rats fasted for 24 h. In the first series of experiments in vitro ischemia was induced by storing tissue blocks in closed vials at 37 degrees C for 15, 30, 45 and 60 min. Cell injury was detected by the cytophotometrical measurement of glycogen phosphorylase activity in unfixed cryostat sections demonstrated by a modified histochemical procedure. In the second series of experiments in vivo ischemia was provoked by clamping the afferent vessels to the median and left lateral lobes of the liver for 60 min, followed by removal of the clamp and reperfusion. The extent of cell damage was determined by measuring the ALAT and ASAT activities in serum at 1, 3, 6 and 24 h after ischemia and by quantifying the extent of necrosis in the liver after 24 h reperfusion by measuring the unstained areas in cryostat sections incubated for lactate dehydrogenase activity. Silymarin (100 mg/kg b.w.) was administered intravenously at 5 min before both the induction of ischemia and the restoration of blood flow (in vivo ischemia) and at 1 h and at 5 min before sacrifice (in vitro ischemia). Controls received an equal amount of saline. The serum amino-transferase activities after 24 h reperfusion were significantly reduced in the silymarin-treated group (n = 10); ALAT 293 +/- 193 U/L, ASAT 343 +/- 229 U/L compared with the control group (n = 7): ALAT 1238 +/- 743 U/L, ASAT 948 +/- 541 U/L (p < 0.03), and the extent of necrosis decreased from 25.6 +/- 16.0% ( n = 7) to 7.8 +/- 8.3% (n = 10) (p < 0.01) after treatment with silymarin.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme.

A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mM inosine, 0.47 mM methoxyphenazine methosulphate and 1 mM Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section wa allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mM p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.