scholarly journals DIFFERENTIAL DEMONSTRATION OF MUSCLE AND HEART TYPE LACTIC DEHYDROGENASE OF RAT MUSCLE AND KIDNEY

1967 ◽  
Vol 15 (1) ◽  
pp. 21-31 ◽  
Author(s):  
PAUL J. MCMILLAN
Keyword(s):  
Cytochrome Oxidase ◽  
Incubation Medium ◽  
Preliminary Report ◽  
Reaction Rate ◽  
Lactic Dehydrogenase ◽  
Histochemical Demonstration ◽  
Oxidase Inhibitor ◽  
Semipermeable Membrane ◽  
Phenazine Methosulfate ◽  
Product Diffusion

A method for the histochemical demonstration of lactic dehydrogenase (LDH) is presented. This method utilizes a semipermeable membrane to minimize enzyme diffusion and high tetrazole concentration with a controlled reaction rate to minimize product diffusion. It was demonstrated that phenazine methosulfate could be used to make the reaction independent of endogenous diaphorase providing either a cytochrome oxidase inhibitor or strict anaerobiosis is employed. Differentiation of cells containing predominantly H-LDH from cells containing predominantly M-LDH was achieved by the use of 4 molal urea or pyruvate in the incubation medium. A preliminary report of the staining patterns observed in rat gastrocnemius and kidney indicates the usefulness of the technique. A new avenue to the study of the distribution of the LDH subunits is thus opened.

scholarly journals Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

1978 ◽  
Vol 26 (3) ◽  
pp. 157-162 ◽  
Author(s):  
A C Frasch ◽  
M E Itoiz ◽  
R L Cabrini
Keyword(s):  
Cytochrome Oxidase ◽  
Incubation Medium ◽  
Histochemical Demonstration ◽  
Tongue Muscle ◽  
Tissue Sections ◽  
Direct Light ◽  
Fixed Concentration ◽  
Set Up ◽  
Cryostat Sections

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.

Measurement of Serum Lactic Dehydrogenase

1961 ◽  
Vol 7 (3) ◽  
pp. 265-274 ◽  
Author(s):  
H Alan Ells
Keyword(s):  
Present Method ◽  
Reaction Rate ◽  
Continuous Measurement ◽  
Colorimetric Method ◽  
Lactic Dehydrogenase ◽  
Phenazine Methosulfate ◽  
Ldh Activity ◽  
Serum Lactic Dehydrogenase ◽  
Method Agreement ◽  
Mediate Electron Transfer

Abstract A colorimetric method is described for the measurement of serum lactic dehydrogenase (LDH) activity. The method employs phenazine methosulfate to mediate electron transfer between reduced diphosphopyridine nucleotide and 2,6-dichloroindophenol, and allows continuous measurement of the reaction rate in the direction lactate to pyruvate. The LDH of a number of normal and pathologic sera was measured by the present method and by the ultraviolet spectrophotometric method. Agreement between the two methods is good.

A Study of the Histochemical Demonstration of Cytochrome Oxidase

1964 ◽  
Vol s3-105 (72) ◽  
pp. 497-502
Author(s):  
R. G. BUTCHER ◽  
J. V. DIENGDOH ◽  
J. CHAYEN
Keyword(s):  
Enzyme Activity ◽  
Cardiac Muscle ◽  
Cytochrome Oxidase ◽  
Histochemical Demonstration ◽  
Lugol’S Iodine ◽  
Liver And Kidney

Burstone's procedure for the histochemical demonstration of cytochrome oxidase has been studied and applied to sections prepared by freezing in hexane and cutting by the controlled-temperature freezing-sectioning technique. The method has been modified by the inclusion of a treatment with Lugol's iodine to yield a stronger colour, which is stable for at least several weeks and which localizes the enzyme activity more precisely. The variants of the reaction have been compared in their effect on cardiac muscle, liver, and kidney of the rat.

scholarly journals The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

1975 ◽  
Vol 23 (3) ◽  
pp. 216-234 ◽  
Author(s):  
G J Spector
Keyword(s):  
Hair Cell ◽  
Cytochrome Oxidase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Succinic Dehydrogenase ◽  
Lactic Dehydrogenase ◽  
Nicotinamide Adenine ◽  
Fixation Method ◽  
Inner Mitochondrial Membrane ◽  
Tetrazolium Chloride ◽  
Outer Compartment

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.

scholarly journals HISTOCHEMICAL DEMONSTRATION OF PHOSPHOLIPASE B (LYSOLECITHINASE) ACTIVITY IN RAT TISSUES

1966 ◽  
Vol 14 (12) ◽  
pp. 907-914 ◽  
Author(s):  
ATHOS OTTOLENGHI ◽  
JOHN P. PICKETT ◽  
WILLIAM B. GREENE
Keyword(s):  
Fatty Acid ◽  
Incubation Medium ◽  
Copper Ions ◽  
Isotonic Saline ◽  
Histochemical Demonstration ◽  
Interstitial Tissue ◽  
Rat Tissues ◽  
Positive Staining ◽  
Phospholipase B

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 µ frozen sections, fixed in cold calcium-formol and incubated at 37°C in Tris buffered medium at pH 6.6 containing 2.2 x 10–3 M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a blackbrown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60°C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent. diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.

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 Method for the Demonstration of Diphosphopyridine Nucleotide Diaphorase

1958 ◽  
Vol 4 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Marvin M. Nachlas ◽  
Donald G. Walker ◽  
Arnold M. Seligman
Keyword(s):  
Gastric Mucosa ◽  
Rat Kidney ◽  
Succinic Dehydrogenase ◽  
Lactic Dehydrogenase ◽  
Histochemical Demonstration ◽  
Cytochemical Localization ◽  
Dehydrogenase System ◽  
Special Distribution ◽  
Almost All ◽  
Mucous Lining

The present investigation concerning the histochemical demonstration of DPN diaphorase follows the development of a new reagent, Nitro-BT, which has already been used successfully for the cytochemical localization of the succinic dehydrogenase system. The most consistently favorable results were obtained with the lactate-lactic dehydrogenase system buffered at pH 7.4. Using sections of rat kidney and stomach, it was found that the intensity of stain was optimal after 15 minutes incubation at 37°C., conducted aerobically. By appropriate variations in the substrate mixture it was possible to selectively demonstrate the histochemical distribution of certain DPN-linked dehydrogenases in addition to DPN diaphorase. This was made possible by the special distribution of some of these dehydrogenases which distinguished them from one another. Of the dehydrogenases studied the distribution pattern of ß-hydroxybutyric dehydrogenase was the most singular. In the gastric mucosa ß-hydroxybutyric dehydrogenase was restricted to the cells of the mucous lining epithelium and the gland necks; and in the kidney the enzyme was limited to the cells of the proximal convoluted tubule and thick limbs of Henle's loop. In contrast, lactic dehydrogenase like DPN diaphorase was demonstrable in almost all cytologic elements of both the stomach and the kidney.

Sign in / Sign up

Export Citation Format

Share Document