• Analytical Science and Technology
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• v.36 no.1
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• pp.44-52
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• 2023

This protocol clarifies a simple and precise method for measuring the activity of xanthine oxidase (XO) enzyme inhibitor. XO enzyme, which accelerates oxidative stress-related disorders through its capacity to generate hydrogen peroxide and superoxide anion radicals (O₂^•-), has been found to be inhibited by several plant extracts. Enzyme samples were incubated with a suitable buffer containing adequate amounts of xanthine as a substrate to determine XO activity. The method depends on direct measurements of uric acid and hydrogen peroxide production to test XO with and without interference. The CUPRAC reagent (Cu(Nc)₂²⁺) was used to inhibit enzyme reaction after incubation was complete. The generated urate and peroxide reduced the Cu(II)-neocuproine complex (Cu(Nc)₂²⁺) to a brightly colored Cu(I)-neocuproine complex (Cu(Nc)₂⁺), which was assessed with a spectrophotometer at 450 nm. XO activity was found to be directly related to the increased absorbance of the colored Cu(I)-neocuproine complex (Cu(Nc)₂⁺). To eliminate catalase enzyme interference, the proposed method used sodium azide and was validated against XO activity using the UV method in matched samples with t-test analysis. The proposed assay can determine XO activity with high precision, as indicated by the correlation coefficient (R² = 0.9935) from comparison with the reference protocol.

• Journal of Nutrition and Health
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• v.33 no.7
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• pp.739-744
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• 2000

The purpose of this study was to elucidate protective effect of Fructus Schsandrae(FS) water extract against xanthine oxidase/hypoxanthine(XO/HX)-induced cardiotoxicity in myocardial cells this experiment was performed. Cardiotoxicity of XO/HX was examined by MTT(MTT [3-(4,5-dimethylthiazol-2-yl)-2.5,-diphenyl tetrazolium bromide) assay. XO/HX induced the decrease of cell viability. Also XO/HX induced the increase of LDH activity and the decrease of beating rate on cultured myocardial cells in a dose-dependent manner. To investigate cardioprotective effect of FS water extract cultures were preincubated with FS water extract for 3 hours. Cultures were then exposed to XO/HX for 72 hours. FS water extract have an efficacy in decreaasing LDH activity and increasing heart beating rate on cultured myocardial cells damaged by XO/HX. From the results it is suggested that XO/HX may show toxic effect in cultured myocardial cells derived from neonatal mouse and FS water extract is effective in the prevention of XO/HX-induced cardiotoxicity.

• The Journal of Korean Medicine
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• v.20 no.4
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• pp.3-10
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• 2000

In order to elucidate the toxic mechanism of neurotoxical damage and neuroprotective effect of Jangwon-hwan(Zhuangyuan-wan) water extract, this experiment was performed. Neurotoxic effects of xanthine oxidase/hypoxanthine(XO/HX) were examined by MTT and NR assay, neuroprotective effects of Jangwon-hwan(Zhuangyuan-wan) water extract were examined by neurofilament enzymeimmuno assay(EIA). XO/HX induced an increase in cell viability, and a decrease in the amount of neurofilament on cultured mouse cerebral cortical neurons in dose-dependent manner. In neuroprotective effect of herb medicine, Jangwon-hwan(Zhuangyuan-wan) water extract increased the amount of neurofilament on cultured mouse cerebral cortical neurons damaged by XO/HX. From the results, it is suggested that XO/HX showed toxic effect in cultured mouse cerebral cortical Neurons and Jangwon-hwan(Zhuangyuan-wan) water extract is very effective in the prevention of neurotoxicity induced by XO/HX.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.4
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• pp.739-744
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• 1998

To evaluate an effect of ethanol pretreatment on the liver xanthine oxidase(XO) activity, 0.25ml of xylene(50% in olive oil) per 100g body weight was daily given four days to the rats at 2hrs after aministration of ethanol each day, while each control group(ethanol, xylene, olive oli) was treated as the same dose described as above. The animals were sacrificed at 24hrs after last injection. Xylene-treated rats showed the more decreased activity of liver XO compared to the control. But the pretreatment of ethanol to the xylene-treated rats enhanced the liver XO activity. Furthermore, the xylene-treated rats led to more increased Vmax value in liver XO compared to the only xylene-treated rats. On the other hadn, hepatic aldehyde dehydrogenase activity was more decreased in xylene-treated rats pretreated with ethanol than in xylene-treated rats. And the intermediated xylene metabolites, methyl benzylalcohol or aldehyde inhibited the XO activity "in vitro". In conclusion, the phenomenon that pretreatment of ethanol to the xylene-treated rats led to the enhancement of liver XO activity, may be due to an influence of acetaldehyde.taldehyde.

• Biomedical Science Letters
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• v.6 no.1
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• pp.37-43
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• 2000

To investigate an impact of skin occlusion on the dermal xanthine oxidase (XO) activity, the dorsal part in rats was covered with closed petri dish-shaped chamber, 46 mm in diameter and 10 mm in height, which was made of glass. The crack between top of chamber and skin was sealed by an adhesive agent. After 5 days, the quantity of sweat accumulation was about 400 mg, whereas after 10 days that was decreased about to 21 mg. The 5 days skin occlusion showed the more increased activity of dermal XO compared with the control, and the increased ratio of enzyme activity to the control was higher than that of 10 days skin occlusion, with the increase being associated with sweat accumulation in chamber. Furthermore, the V$_{max}$ of dermal XO in 5 days skin occlusion was higher than that in the control. In conclusion, it may be hypothesized that the XO system may play an role for defence mechanism in dermal tissue.

• Toxicological Research
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• v.11 no.2
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• pp.279-288
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• 1995

To apply the serum xanthine oxidase (XO) determining for the index of the toluene intoxication, the serum XO activity was compared with the other parameters, the activities of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), 5'-nucleotidase(5'-NT), alkaline phosphatase(ALP), guanase(GDA) and $\gamma$-gIutamyl transpeptidase(T-GTP). Concomitantly, the cause of increased level of serum XO was clarified in the present experimental conditions. Although the other serum enzyme activities, ALT, AST, 5'-NT, ALP, GDA and $\gamma$-GTP were respectively not found to be different between control group and toluene-treated group, the serum XO activity in toluene-treated group showed the higher levels than that in the control group. These suggested that the determination of serum XO activity could be used for monitoring the intoxication of toluene. On the other hand, the activities of XO both in the serum and liver were higher in toluene-treated or benzaldehyde-treated rats than those in each control group. In the pooled liver XO from each group, toluene-treated or benzaldehyde-treated group showed the higher $V_{max}$ value than the control group, whereas no changes were observed in liver XO activities between the control liver specimen and that preincubated with bertzaldehyde in vitro. The present results indicate that the increased level of XO in toluene-treated rats is due to the result of enzyme protein induction in liver cell by the benzaldehyde metabolized from toluene. All the more, the benzaldehyde may be acted as a substrate for XO, since the benzaldehyde induced the increased activity of both liver and serum XO, and no changes were found in purine catabolite, uric acid in serum or urine and liver purine catabolizing enzymes, adenosine deaminase, GDA, uricuse except XO in toluene-treated rats.

• Biomedical Science Letters
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• v.12 no.4
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• pp.439-442
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• 2006

To evaluate the postmortem changes in activities of oxygen free radical metabolizing enzymes, the rats were sacrificed with cervical dislocation and were kept in an incubator at $25^{\circ}C$, 70% of humidity for 12 hours. The activities of aniline hydroxylase, catalase, glutathione-S-transferase and superoxlde dismutase were decreased with the time. On the other hand, the activity and type conversion ratio (type D ${\to}$type O) of hepatic xanthine oxidase (XO) were gradually increased. From these changes of XO, the estimation of death time (mathematical equation) could be determined with the least square method. To clarify the cause of increasing XO activity, enzyme kinetics were examined. The Km values of XO were decreased with the time. In conclusion, the determination of liver XO activity might be used for the estimation of death time in the early postmortem period.

• The Journal of Korean Medicine
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• v.21 no.1
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• pp.29-39
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• 2000

In order to elucidate the toxic mechanism of oxygen radicals in cultured mouse spinal sensory neurons, cytotoxic effect of oxygen radicals was evaluated by M1T assay and NR assay. In addition, protective effect of Sopunghwalhyultang(SPHHT) water extract on oxidant-induced neurotoxicity was investigated on these cultures. Spinal sensory neurons derived from mice were cultured in mediums containing various concentrations of Xanthine Oxidase / Hypoxanthine(XO/HX). Cell viability was measured by MTT assay and NR assay. XO/HX-mediated oxygen radicals remarkably decreased cell viability of cultured spinal sensory neurons in a dose-and time-dependent manner. And also, SPHHT blocked XO/HX-induced neurotoxicity in these cultures. These results suggest that oxygen radicals are toxic and SPHHT are effective in blocking against the oxidant-induced neurotoxicity in cultures of spinal sensory neurons of mice.

• Biomedical Science Letters
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• v.1 no.1
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• pp.37-43
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• 1995

To evaluate an effect of xanthine oxidase(XO) reaction system on the carbon tetrachloride($CCl_4$) metabolism, $CCl_4$ was given twice at 0.1ml/100g body wt. at intervals of 18 hour to the rats and those pretreated with allopurinol (50mg/kg. body wt.). The influence of XO on the metabolism of $CCl_4$ was focused on the degree of liver damage and the activities of a $CCl_4$ metabolizing marker enzyme, glucose-6-phosphatase. The increasing rate of liver weight per body weight and the levels of serum alanine aminotransferase to the control group were more decreased in allopurinol-pretreated rats than in those treated with $CCl_4$ alone. The liver XO activities were more increased in $CCl_4$-treated rats than the control group and the $CCl_4$-treated rats pretreated with allopurinol showed a decreased activities of XO compared to the $CCl_4$-treated rats. The type conversion (type D --> type O) rate was more decreased tendency in allopurinol pretreated rats than those treated $CCl_4$ alone. In dialyzed liver enzyme preparations, all of the xanthine oxidase activities: $CCl_4$-treated, allopurinol and $CCl_4$-treated rats pretreated with allopurinol showed the more increased Vmax value than the control group, but similar Km value. Moreover, $CCl_4$-treated rats pretreated with allopurinol showed the more increased Vmax value than the group treated with $CCl_4$ alone. In conclusion, it can not be negate the possibility of metabolism of $CCl_4$ by the xanthine oxidase enzyme system.

• Biomedical Science Letters
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• v.8 no.3
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• pp.155-159
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• 2002

To evaluate the physiological significance of xanthine oxidase (XO) in rat skin, the activity of XO (type O) in skin was compared with that of small intestine or liver. Concomitantly, XO activities in some partial portions (scalp, leg, dorsal and ventral part) of skin were determined and then compared with each partial portion. XO activity of skin was lower than that of small intestine and rather higher than that of liver. Furthermore, the activity of XO in skin, after clipping of hairs and then in 5 days, was more increased than that of rat which was clipped before having been sacrificed. As for activities of free radical scavenging system (GPx, GST, SOD), skin is lower than liver and small intestine. Although it is hewn that the oxygen free radical generated by XO system lead to injurious effect on the cell, the XO activity of ventral part which is to be less exposed to xenobiotics and biological agents was the lowest among those of ventral, dorsal, leg and scalp parts in skin. In conclusion, it may be hypothesized that XO system in skin act on defence mechanism.