Biomedical subjects

F Marx

Publications and source records attributed to F Marx.

At least 37 records · Page 2Linked to original sources

PubMed1991

Homogeneous distribution of phosphofructokinase in the rat liver acinus: a quantitative histochemical study.

A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.

3,3'-Diaminobenzidine↗

PubMed1991

The effect of internal biliary drainage on bile pigment accumulation and acid phosphatase activity in human liver during obstructive jaundice.

The effect of internal drainage after biliary obstruction due to primary cholangiocarcinoma has been studied in seven human liver biopsies with respect to bile pigment accumulation and acid phosphatase activity. Enzyme activity was demonstrated at the light-microscopic level in unfixed cryostat sections using an incubation medium containing naphthol AS-BI phosphate as substrate, and hexazotised pararosaniline as simultaneous coupling agent, and at the ultrastructural level in fixed tissue blocks and chopped tissue sections using sodium beta-glycerophosphate as substrate and lead or cerium ions as capture reagent. Large amounts of bilirubin were found in cryostat sections of non-drained cholestatic livers, especially in pericentral areas. At these sites a high acid phosphatase activity was found. At the ultrastructural level, acid phosphatase activity was found only in the lysosomal compartment--possibly due to the procedure necessary for tissue processing. After internal biliary drainage, the amount of bilirubin diminished, with a concomitant decrease in acid phosphatase activity. The co-localization of accumulations of bile compounds and acid phosphatase activity indicates that lysosomes play a role in the breakdown of bile compounds.

Acid Phosphatase↗

PubMed1990

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy.

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.

Animals↗

PubMed1990

Quantitative changes in acid phosphatase, alkaline phosphatase and 5'-nucleotidase activity in rat liver after experimentally induced cholestasis.

Acid phosphatase, alkaline phosphatase and 5'-nucleotidase activities were analyzed cytophotometrically in cryostat sections of rat liver up to 8 weeks after ligation and transsection of the common bile duct. Ligation resulted in cholestasis and induced alterations in both localization and activity of the enzyme investigated. The cellular distribution but not the activity of acid phosphatase changed in liver parenchyma. In control liver, the final reaction product was localized as discrete granules in the bile canalicular region of hepatocytes. The final reaction product was precipitated more diffusely within the cytoplasm after induction of cholestasis, most probably due to increased fragility of lysosomal membranes. In control liver, alkaline phosphatase activity was low and localized in the bile canalicular plasma membranes only. The total parenchymal activity increased threefold after the induction of cholestasis and is considered to be a compensatory mechanism in order to enhance the excretion of bile salts from hepatocytes. 5'-Nucleotidase was present at the bile canalicular and sinusoidal surfaces of plasma membranes of hepatocytes in control liver; total activity in pericentral areas was significantly higher than in periportal areas. Induction of cholestasis resulted in higher total activity and redistribution of the activity over all three surfaces of the plasma membranes, whereas heterogeneity over the different zones of the acinus disappeared. The appearance of the enzyme at lateral plasma membranes is suggested to be related to the formation of new sites for bile salt transport out of the hepatocytes. With respect to all three enzymes studied, alterations of liver parenchymal cells due to a disturbed bile transport were already established during the first week of cholestasis.

5'-Nucleotidase↗

PubMed1990

Light microscopical detection of D-amino acid oxidase activity in unfixed cryostat sections of rat kidney and liver using the cerium-DAB-cobalt-H2O2 procedure and a semipermeable membrane.

The two-step method for the light microscopical detection of oxidase activity using cerium ions and a visualization procedure with diaminobenzidine and nickel or cobalt ions as introduced by Anger-müller and Fahimi, has been improved. Inactivation by fixation and leakage of D-amino acid oxidase molecules were avoided by the application of unfixed cryostat sections adhered to a semipermeable membrane separating the sections from a gelled incubation medium. Optimum activity in rat kidney and liver was obtained by increasing the cerium concentration from 3 to 30 mM and the Tris-maleate concentration from 100 to 200 mM in the incubation medium for the first step. D-proline was used as substrate and a concentration of 20 mM gave maximum activity. The second visualization step was performed in a medium consisting of DAB, cobalt and hydrogen peroxide. Final reaction product was found in a granular form in the basal part of the epithelial cells of the proximal tubules and in liver parenchymal cells. The activity in periportal areas was much higher than in pericentral areas. Cytophotometric analysis revealed that a 30 times higher activity could be detected when using the membrane technique in comparison with incubations in aqueous media. The specificity of the reaction was proven by a nearly complete inhibition of the reaction by beta-hydroxybutyrate.

Animals↗

PubMed1990

The effect of ischemia on glycogen phosphorylase activity in rat liver: a quantitative histochemical study.

The effects of ischemia in vitro for 0-60 min at 37 degrees C on glycogen phosphorylase activity in rat liver have been studied under different feeding conditions. Glycogen phosphorylase activity was demonstrated with a recently developed quantitative histochemical method using a semipermeable membrane and the PAS-reaction. The cytophotometrically measured glycogen phosphorylase activity in livers from 24 h-fasted rats was approximately five times the activity in livers from normally fed rats. The activity in periportal areas was about 1.5 times higher than the activity in pericentral areas in livers from starved rats, but more or less evenly distributed in livers from fed rats. Enzyme activity in pericentral areas of livers from 24 h-fasted rats started to decrease after 20 min of ischemia. After 50-60 min of ischemia, the activity was decreased to approximately 25% of the control activity. Livers from normally fed rats showed unchanged activity in periportal and pericentral areas after 10-60 min of ischemia. It has been assumed that the activation of the enzyme was disturbed by ischemia, possibly as a consequence of plasma membrane damage.

Animals↗

PubMed1989

Changes in acid phosphatase activity in rat liver after ischemia.

The effect of ischemia on the stability, i.e. the permeability of the lysosomal membrane of rat liver has been studied using quantitative histochemical analysis of acid phosphatase activity. Ischemia in vitro was performed for 0-240 min at 37 degrees C and ischemia in vivo for 60 min was followed by 1, 5, 24 and 48 h of reperfusion. Acid phosphatase activity was demonstrated in cryostat sections using naphthol AS-BI phosphoric acid as substrate and polyvinyl alcohol was added to the incubation medium to counteract diffusion phenomena. Ischemia in vitro up to 240 min did not affect the localization nor the total activity of acid phosphatase activity. After 60-min ischemia in vivo followed by 1-h reperfusion distinct areas showed decreased acid phosphatase activity. A further decrease in activity was observed after 5 h reperfusion. Final reaction product generated by acid phosphatase activity was rather diffusely distributed in border zones between normal and damaged tissue after 24 and 48 h of reperfusion following 60 min ischemia in vivo. It is concluded that not ischemia itself but rather reperfusion affects the stability of the lysosomal membrane due to the occurrence of oxygen-derived free radicals and/or imbalanced Ca2+ concentration. Restoration of the blood flow causes leakage of acid phosphatase from the lysosomes into the cytoplasm of liver parenchymal cells and from there to the blood.

Acid Phosphatase↗

PubMed1988

A quantitative histochemical study of 5'-nucleotidase activity in rat liver after ischaemia.

The lead salt method of Wachstein and Meisel15 has been applied using incubation media containing polyvinyl alcohol for the localization and quantification of 5'-nucleotidase (E.C.3.1.3.5) activity in cryostat sections from rat liver after ischaemia in vitro and ischaemia in vivo followed by different periods of re-perfusion. 5'-Nucleotidase activity at the bile canaliculi, especially in the pericentral areas, had already decreased after 60 min of ischaemia in vitro, although the total activity as measured densitometrically was not changed. After 120-240 min of ischaemia, a significant decrease of the total 5'-nucleotidase activity was found. At that stage, signs of irreversible cell damage were recognized. Short periods of re-perfusion (1 h) after ischaemia in vivo induced a decreased bile canalicular 5'-nucleotidase activity throughout the entire liver, but a restoration after longer periods of re-perfusion was observed (5, 24, and 48 h). Necrotic areas recognized by a decreased lactate dehydrogenase activity after all periods of re-perfusion showed decreased total 5'-nucleotidase activities. A correlation was observed between the decrease in bile canalicular 5'-nucleotidase activity and the disappearance of microvilli of the bile canaliculi. It is concluded that a decrease in the bile canalicular 5'-nucleotidase activity can be used as a very sensitive marker for ischaemic liver cell damage. Assessment of the irreversibility of the cell injury has to be determined using additional parameters such as a decreased lactate dehydrogenase activity.

5'-Nucleotidase↗

PubMed1988

Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle.

Creatine kinase (EC 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP. The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues. In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.

Animals↗

PubMed1988

A quantitative histochemical study of 5'-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol.

5'-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein-Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5 mM AMP, 10 mM magnesium chloride, 7.2 mM lead nitrate, 0.1 M Tris-maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5'-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8 micron. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4 mM in pericentral zones and 0.8 mM in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.

5'-Nucleotidase↗

PubMed1987

Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study.

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.

5'-Nucleotidase↗

PubMed1987

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction.

Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction. Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60-120 mM. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section. p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (KM) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of the a form only in periportal areas and of the a and b forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4-16 min). A linear relationship was also found with section thickness (4-10 micron). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity. It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.

Animals↗

PubMed1987

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

Extrahepatic 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.

Animals↗

PubMed1987

Changes in cytoplasmic and mitochondrial enzymes in rat liver after ischemia followed by reperfusion.

The behavior of cytoplasmic and mitochondrial enzymes has been studied in rat liver at 1, 5, and 24 hr after 60 min of ischemia using histochemical methods. This period of ischemia resulted 24 h after ischemia in liver cell necrosis in about 15% of the volume of the ischemic liver lobes. As early as after 1 hr reperfusion lactate dehydrogenase (LDH, cytoplasm) activity decreased in a certain proportion of the liver parenchymal cells, whereas glutamate dehydrogenase (GDH, mitochondrial matrix) activity started to decrease after 5 hr reperfusion; the activities of mitochondrial membrane enzymes, monoamine oxidase and succinate dehydrogenase, did not decrease before 24 hr of reperfusion. It has been concluded that the early decrease in LDH activity is caused by leakage into the blood and reflects reversible damage; when this decrease is accompanied by a decrease in GDH activity irreversible liver cell damage is assumed. Diminished activity of mitochondrial membrane enzymes, due to leakage and denaturation, is observed when real necrosis can be assessed.

Animals↗

PubMed1987

Demonstration of creatine kinase in myocardial and skeletal muscle using the semipermeable membrane technique.

The histochemical method as described by Sjövall (1967) for the detection of creatine kinase activity in myocardium and skeletal muscle has been modified by the introduction of a semipermeable membrane method. With this technique a semipermeable membrane is interposed between incubating solution and tissue sections preventing diffusion of the enzyme into the medium during incubation. A very high enzyme activity was demonstrated along myofibrils and probably also in mitochondria. Addition of ZnCl2 or 2,4-dinitrofluorobenzene to the incubation medium prevented the production of formazan, confirming the specificity of the staining reaction. Creatine kinase activity was observed in both white (type II) and red (type I) skeletal muscle fibres without notable difference in level of activity.

Adenosine Diphosphate↗

PubMed1987

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.

Acid Phosphatase↗

PubMed1987

Diurnal variation in glycogen phosphorylase activity in rat liver. A quantitative histochemical study.

The diurnal variations of the glycogen content and of glycogen phosphorylase activity in periportal and pericentral areas of rat liver parenchyma have been analyzed in periodic acid Schiff (PAS)-stained cryostat sections using quantitative microdensitometry. Glycogen content and phosphorylase activity were always higher in periportal areas than in pericentral areas throughout the daily cycle. The glycogen content was highest at the end of the active period during darkness and lowest at the end of the resting period. Phosphorylase activity appeared to be inversely correlated with the glycogen content in both areas. It is concluded that the glycogen content is regulated by phosphorylase activity, which may be due to local cAMP concentration.

Animals↗