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

1994 ◽  
Vol 42 (8) ◽  
pp. 1091-1096 ◽  
Author(s):  
W M Frederiks ◽  
K S Bosch ◽  
R J Van den Munckhof ◽  
C J Van Noorden
Keyword(s):  
Polyvinyl Alcohol ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Incubation Medium ◽  
Oxidase Activity ◽  
Specific Inhibitor ◽  
Semipermeable Membrane ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Xanthine Oxidase Activity

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.

scholarly journals A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.

1993 ◽  
Vol 41 (5) ◽  
pp. 667-670 ◽  
Author(s):  
W M Frederiks ◽  
F Marx
Keyword(s):  
Xanthine Oxidase ◽  
Oxidase Activity ◽  
Semipermeable Membrane ◽  
Myeloperoxidase Activity ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Xanthine Oxidase Activity ◽  
Membrane Technique ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

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

1991 ◽  
Vol 39 (1) ◽  
pp. 81-86 ◽  
Author(s):  
H R Patel ◽  
W M Frederiks ◽  
F Marx ◽  
A J Best ◽  
C J Van Noorden
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Specific Inhibitor ◽  
Physiological Role ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Specific Test ◽  
Final Reaction Product ◽  
Final Reaction

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.

Activity and stability of rat liver xanthine oxidase in the presence of pyridine

2011 ◽  
Vol 89 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Kaveh Amini ◽  
Mohammad-Hossein Sorouraddin ◽  
Mohammad-Reza Rashidi
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Enzyme Inactivation ◽  
Native Enzyme ◽  
Buffer Solution ◽  
Stability Parameters ◽  
Xanthine Oxidase Activity

In the present study, rat liver xanthine oxidase activity and its thermostability in the presence of pyridine were investigated. The activity of the enzyme was determined by following the formation of uric acid spectrophotometrically. The thermal stability of the enzyme was studied in the presence of 0.0%–2.0% of pyridine in Sorenson’s buffer. Thermal stability parameters (half-life, inactivation constant, and activation energies for enzyme inactivation), thermodynamic constants (ΔH*, ΔS*, and ΔG*) and the kinetic parameters (Km and Vmax), were determined in pyridine-free and pyridine-containing buffer solution. A dramatic reduction was observed in xanthine oxidase activity in the presence of pyridine. However, the pyridine-treated enzyme showed a marked enhancement in thermal stability compared with the native enzyme. The ΔG values for the enzyme activity in the presence of pyridine were found to be about 1.5-fold larger than that calculated for the native enzyme, indicating that the enzyme becomes kinetically more stable in the presence of pyridine. The Km value for xanthine oxidase in the presence of 0.5% pyridine increased by 4.8-fold compared with the enzyme in the pyridine-free buffer solution; however, there was 1.8-fold reduction in the Vmax value in the hydro-organic solution compared with the enzyme activity in the buffer solution. As the stability of enzymes is one of the most difficult problems in protein chemistry, this thermostability property of xanthine oxidase could be of great value in developing novel strategies to improve and expand its application in various areas.

Effects of hyperthermia on xanthine oxidase activity and glutathione levels in the perfused rat liver

1989 ◽  
Vol 4 (2) ◽  
pp. 119-125 ◽  
Author(s):  
Joseph L. Skibba ◽  
Anna Stadnicka ◽  
John H. Kalbfleisch ◽  
Robert H. Powers
Keyword(s):  
Xanthine Oxidase ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Perfused Rat Liver ◽  
Xanthine Oxidase Activity
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