G. N. Jonges

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

SummaryThe indoxyl—tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100mm Tris-HCl buffer, pH 9.0, 0.2–1.0mm substrate, 0.32mm 1-methoxyphenazine methosulphate, 10mm MgCl2, 5mm sodium azide and 1mm tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1–2mm and 5mm respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 µm and therefore, the reaction obeyed the Beer—Lambert law. Variation of the substrate concentration yielded aKM of 0.05mm for aqueous media and aKM of 0.55mm for polyvinyl alcohol-containing media. The inhibition with tetramisole appeared to be competitive withKi = 0.07mm for aqueous media andKi = 0.7mm for polyvinyl alcohol-containing media. These values indicate that the indoxyl—tetranitro BT method is considerably more sensitive than any metal salt or diazonium salt method developed so far. It is concluded that the optimized method described here is a specific, sensitive and valid quantitative histochemical method for the demonstration of alkaline phosphatase activity.

Analysis of enzyme reactions in situ

SummaryEstimations of metabolic rates in cells and tissues and their regulation on the basis of kinetic properties of enzymes in diluted solutions may not be applicable to intact living cells or tissues. Enzymes often behave differently in living cells because of the high cellular protein content that can lead to homologous and heterologous associations of protein molecules. These associations often change the kinetics of enzymes as part of post-translational regulation mechanisms. An overview is given of these interactions between enzyme molecules or between enzyme molecules and structural elements in the cell, such as the cytoskeleton. Biochemical and histochemical methods are discussed that have been developed for in vivo and in situ analyses of enzyme reactions, particularly for the study of effects of molecular interactions. Quantitative (histochemical) analysis of local enzyme reactions or fluxes of metabolites has become increasingly important. At present, it is possible to calculate local concentrations of substrates in cells or tissue compartments and to express local kinetic parameters in units that are directly comparable with those obtained by biochemical assays of enzymes in suspensions. In situ analysis of the activities of a number of enzymes have revealed variations in their kinetic properties (Km and Vmax) in different tissue compartments. This stresses the importance of in vivo or in situ analyses of cellular metabolism. Finally, histochemical determinations of enzyme activity in parallel with immunohistochemistry for the detection of the total number of enzyme molecules and in situ hybridization of its messenger RNA allow the analysis of regulation mechanisms at all levels between transcription of the gene and post-translational activity modulation.

In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

SummaryThe reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37°C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37°C revealed that the highest specific activity of G6PDH was present in the intermediate area (Vmax=7.79±1.76 µmol H2 cm−3min−1) and of PGDH in the pericentral and intermediate areas (Vmax=17.19±1.73 µmol H2 cm−3 min−1). In periportal and pericentral areas,Vmax values for G6PDH activity were 4.48±1.03 µmol H2 cm−3 min−1 and 3.47±0.78 µmol H2 cm−3 min−1, respectively. PGDH activity in periportal areas showed aVmax of 10.84±0.33 µmol H2 cm−3 min−1. Variation of the substrate concentration for G6PDH activity yielded similarKm values of 0.17±0.07mm, 0.15±0.13mm and 0.22±0.11mm in periportal, pericentral and intermediate areas, respectively.KM values of 0.87±0.12mm in periportal and of 1.36±0.10mm in pericentral and intermediate areas were found for PGDH activity. The significant difference betweenKM values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed betweenKM andVmax values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take placein vivo.

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method.

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.

Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis

SummaryExtrahepatic cholestasis induced by ligation and transsection of the common bile duct caused a change in the parenchyma/stroma relationship in rat liver. Two weeks after ligation, the periportal zones of the parenchyma were progressively invaded by expanding bile ductules with surrounding connective tissue diverging from the portal areas. Parenchymal disarray developed and small clumps of hepatocytes or isolated hepatocytes were scattered within the expanded portal areas. These cells showed normal activity of lactate, succinate and glutamate dehydrogenase and may, therefore, be considered to be functionally active. After cholestasis the remainder of the liver parenchyma showed adaptational changes with respect to glucose homeostasis, as demonstrated by histochemical means. Glycogen stores disappeared completely whereas glycogen phosphorylase activity increased about ten fold. The increased glycogen phosphorylase activity and glycogen depletion indicate a greater glycogenolytic capacity in liver parenchyma after bile duct ligation to maintain as far as possible a normal plasma glucose concentration. The parenchymal distribution pattern of glucose-6-phosphatase activity did not change significantly after bile duct ligation. The isolated hepatocytes within the expanded portal tracts showed a high activity of this enzyme whereas the pericentral parenchyma was only moderately active. The distribution patterns of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in the liver parenchyma were also largely unchanged after bile duct ligation, but the histochemical reaction for glucose-6-phosphate dehydrogenase activity demonstrated infiltration of the remainder of the parenchyma by non-parenchymal cells, possibly Küpffer cells and leucocytes as part of an inflammatory reaction. Under normal conditions the mitochondrial enzymes succinate and glutamate dehydrogenase show an opposite heterogenous distribution pattern in liver parenchyma. Following cholestasis both enzymes became uniformly distributed. The underlying regulatory mechanism for these different changes in distribution patterns of enzyme activities is not yet understood.

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.

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique.

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.

Effects of partial hepatectomy, phenobarbital and 3-methylcholanthrene on kinetic parameters of glucose-6-phosphate and phosphogluconate dehydrogenase in situ in periportal, intermediate and pericentral zones of rat liver lobules

Glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) are heterogeneously distributed in liver lobules of female rats. The maximum activity of both enzymes is approximately twice higher in intermediate and pericentral zones than in periportal zones. Enzyme activities and their distribution patterns were manipulated by partial hepatectomy and treatment with phenobarbital (PB) or 3-methylcholanthrene (3-MC). Vmax values of G6PDH for glucose-6-phosphate decreased mainly in intermediate and pericentral zones after partial hepatectomy, whereas they increased after PB treatment. Vmax values of PGDH for phosphogluconate decreased after partial hepatectomy in both zones, whereas other treatments did not have any effect. The affinity of G6PDH for glucose-6-phosphate was similar in all zones and it was decreased 2-3 fold by PB and 3-MC treatment. The affinity of PGDH for phosphogluconate was 1.4-2.3 times lower in intermediate and pericentral zones than in periportal zones of all livers tested and was not affected by treatment. From these data it can be concluded that not only the maximum activity of enzymes may differ in periportal, intermediate and pericentral zones of the liver lobule but also the affinity of enzymes for their substrates. The implication of these findings is that metabolic flux rates as they occur in vivo in these different metabolic compartments may be significantly different from predictions on the basis of maximum enzyme activities as detected immunohistochemically, microchemically or cytophotometrically.

The histochemical G6PDH reaction but not the LDH reaction with neotetrazolium is suitable for the oxygen sensitivity test to detect cancer cells

We used the oxygen sensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium (NT) reduction to discriminate cancer cells from normal cells. Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in oxygen instead of nitrogen. Competition for reductive equivalents between NT and oxygen via superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating NAD-dependent lactate dehydrogenase (LDH) activity instead of NADP-dependent G6PDH activity in normal rat liver and colon, in human colon carcinoma, and in experimentally induced metastases of colon carcinoma in rat livers. Reactions for both enzymes were determined cytophotometrically in an atmosphere of pure oxygen or nitrogen. G6PDH acted as described previously, showing distinct activity in cancer cells but strongly reduced activity in normal cells after incubation in oxygen, but this was not the case with LDH because formazan was also generated in normal tissue in oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of oxygen compared with nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon carcinoma and in colon carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon carcinoma and metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and oxygen via SOD, because NADPH generated by G6PDH and NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically NADPH-converting cellular systems such as NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous NADPH as substrate for these systems revealed oxygen sensitivity. Involvement of NADPH-dependent lipid peroxidation in the oxygen sensitivity test is discussed.

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor β superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.