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

Frederiks, Wilma M.; Bosch, K. S.; Munckhof, R. J. M. Van Den

doi:10.1007/bf01464338

Histochem Cell Biol

1995

DOI: 10.1007/bf01464338

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

Wilma M. Frederiks

K. S. Bosch²,

R. J. M. Van Den Munckhof

Abstract: 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… Show more

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Cited by 4 publications

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“…Free radicals directly react with DAB, forming an insoluble DAB polymer in a reaction catalyzed by the presence of Mn . This technique has been successfully used to identify ROS-producing sites (13)(14)(15)(16).…”

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confidence: 99%

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

Remans

Oosterhout

Smeets

et al. 2005

Arthritis & Rheumatism

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Objective. To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium.Methods. Cytochemical procedures based on the 3,3-diaminobenzidine (DAB)-Mn 2؉ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanatelabeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2,7-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested.Results. Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H 2 O 2 , since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H 2 O 2 , diphenyleneiodonium (an inhibitor of NADPH oxidase), N G -monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production.Conclusion. Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H 2 O 2 , although the oxidase(s) involved in its generation remains to be determined.

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mentioning

confidence: 99%

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

Remans

Oosterhout

Smeets

et al. 2005

Arthritis & Rheumatism

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

Frederiks

Bosch

1996

Hepatology

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Xanthine oxidoreductase is a rate-limiting enzyme of the Activity of xanthine oxidoreductase (total xanthine catabolism of purines (reviewed by Kooij 1 ). The enzyme exists dehydrogenase plus xanthine oxidase) and xanthine oxiin two functionally distinct forms: an oxidized nicotinamide dase was determined cytophotometrically in periportal adenine dinucleotide-dependent dehydrogenase form (Dand pericentral areas of livers of rats under various form; E.C. 1.1.1.204) that produces reduced nicotinamide ad-(patho)physiological conditions that are known to affect enine dinucleotide and urate and an oxygen-dependent oxithe content of reduced glutathione. For this purpose, dase form (O-form; E.C. 1.2.3.2) that produces superoxide rats were either normally fed or fasted for 24 hours, anion radicals and/or hydrogen peroxide and urate. Converfasted for 24 hours, and treated with diethylmaleate that sion of xanthine dehydrogenase into xanthine oxidase may depleted glutathione or treated by in vivo ischemia for occur reversibly via oxidation of sulfhydryl groups and irre-2 hours in the livers. Xanthine oxidoreductase activity versibly by proteolysis. 2-7 These findings were mainly based was shown histochemically with the use of a tetrazolium on biochemical studies performed on homogenates. It was salt procedure, and xanthine oxidase activity was localassumed for a long time that different pathological conditions ized with a cerium-diaminobenzidine-cobalt-hydrogen such as ischemia lead to in vivo conversion of the D-form into peroxide technique in unfixed cryostat sections of the the O-form. 8-13 However, Kooij et al. 14 showed recently that livers. Cytophotometric measurements showed that toconversion of the D-form into the O-form did not occur to any tal xanthine oxidoreductase activity was decreased after significant extent in vivo in livers from fed or fasted rats fasting and ischemia, whereas only ischemia caused reeither or not after in vivo ischemia. However, conversion ocduced xanthine oxidase activity. Moreover, the percentcurred rapidly in vitro in the supernatants of homogenates age of xanthine oxidase of total xanthine oxidoreductase of these livers, and then the conversion rate was increased activity was constant in both periportal and pericentral by fasting and in vivo ischemia. When homogenization proceareas at the level of approximately 4% in normally fed dures were performed under strictly controlled conditions, to and 24-hour fasted and diethylmaleate-treated rats.avoid in vitro conversion of the D-form into the O-form, a Ischemia reduced this percentage in both areas of the constant percentage of 15%-20% oxidase activity of total xanliver to 2%. It was concluded that the amount of endogethine oxidoreductase activity in livers of rats was found under nous reduced glutathione did not affect the percentage all conditions studied. To test the hypothesis that conversion of xanthine oxidase. The low percentage of xanthine oxiof the D-form into the O-form does not occur in vivo to any dase as determined in the pre...

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In situ heterogeneity of peroxisomal oxidase activities: An update

Munchof

1996

Histochem J

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Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of the in situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidases D-amino acid oxidase, L-alpha-hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.

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

Cited by 4 publications

References 18 publications

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

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

In situ heterogeneity of peroxisomal oxidase activities: An update

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