Arnold Kooij

On the nature of the ‘nothing dehydrogenase’ reaction

SummaryThe biochemical mechanism underlying the ‘nothing dehydrogenase’ reaction during the histochemical demonstration of dehydrogenases using tetranitro BT as the final electron acceptor has been investigated in unfixed, frozen rat liver sections. The reaction is stronger with NAD+ than either with NADP+ or in the absence of coenzyme. As much as 50% of the reaction is due to lactate dehydrogenase converting endogenous lactate and is largely inhibited by pyruvate. No NAD+-dependent alcohol dehydrogenase activity was detected at pH 7.45, the pH used for the incubations. The coenzyme-independent activity may be caused by SH-groups present in proteins and compounds like glutathione and cysteine and can be inhibited byN-ethylmaleimide andp-chloromercuribenzoic acid. It was also found that the ‘nothing dehydrogenase’ reaction mainly occurs during the first few minutes of incubation, levelling off quickly to a slow rate. When studying the kinetics of dehydrogenase reactions with tetrazolium salts, it should be realized that the ‘nothing dehydrogenase’ reaction, which as a whole is nonlinear with time, can interfere seriously with the dehydrogenase reaction to be analysed and may yield initial reaction rates that are too high. The findings of the present study reveal the nature of the reactions used for detection of necrosis in tissues with tetrazolium salts.

A re-evaluation of the tissue distribution and physiology of xanthine oxidoreductase

SummaryXanthine oxidoreductase is an enzyme which has the unusual property that it can exist in a dehydrogenase form which uses NAD+ and an oxidase form which uses oxygen as electron acceptor. Both forms have a high affinity for hypoxanthine and xanthine as substrates. In addition, conversion of one form to the other may occur under different conditions. The exact function of the enzyme is still unknown but it seems to play a role in purine catabolism, detoxification of xenobiotics and antioxidant capacity by producing urate. The oxidase form produces reactive oxygen species and, therefore, the enzyme is thought to be involved in various pathological processes such as tissue injury due to ischaemia followed by reperfusion, but its role is still a matter of debate. The present review summarizes information that has become available about the enzyme. Interpretations of contradictory findings are presented in order to reduce confusion that still exists with respect to the role of this enzyme in physiology and pathology.

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.

High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells. A relation between localization and function?

SummaryThe localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells from skin, vagina, uterus, penis, liver, oral and nasal cavities, tongue, esophagus, fore-stomach and small intestine. In addition activity was demonstrated in sinusoidal cells of liver and adrenal cortex, endothelial cells in various organs and connective tissue fibroblasts. Xanthine oxidoreductase produces urate which is a scavenger of oxygenderived radicals. Because the enzyme is found in epithelial and endothelial cells which are subject to relatively high oxidant stress, it is postulated that in these cells xanthine oxidoreductase is involved in the antioxidant enzyme defense system. In addition, a possible role for the enzyme in proliferation and differentiation processes is discussed.

Distribution of xanthine oxidoreductase activity in human tissues — a histochemical and biochemical study

SummaryLocalization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

SummaryThe effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5′-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.

Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.