Neurological Research A Journal of Progress in Neurosurgery, Neurology and Neurosciences
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura & Keiji Sano To cite this article: Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura & Keiji Sano (1992) Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia, Neurological Research, 14:4, 321-324, DOI: 10.1080/01616412.1992.11740077 To link to this article: http://dx.doi.org/10.1080/01616412.1992.11740077
Published online: 20 Jul 2016.
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Date: 16 April 2017, At: 13:33
Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura and Keiji Sano Department of Neurosurgery, Teikyo University School of Medicine, Tokyo 173, Japan
We studied the activities of xanthine dehydrogenase and xanthine oxidase in rat forebrain after complete ischaemia. Complete ischaemia was induced by decapitation after transcardiac infusion with saline. The activities of xanthine dehydrogenase and xanthine oxidase immediately after ischaemia were 93.3 ± 38.7 and 18.8 ± 7.7 Ji.U / mg protein, respectively, and at 24 h after ischaemia were 183.5 ± 75.1 and 60.8 ± 15.2 Ji.U / mg protein, respectively. The ratios of xanthine dehydrogenase / xanthin oxidase immediately and 24 h after ischaemia were 5.04 ± 1.03 and 3.04 ± 0.99, respectively. These data indicate that xanthine dehydrogenase and xanthine oxidase activities were maintained even 24 h after complete ischaemia. Conversion of xanthine dehydrogenase to xanthine oxidase proceeds slowly during complete ischaemia. [Neurol Res
1992; 14: 321-324 J
Keywords: Hypoxanthine; ischaemia; oxypurine; rat brain; uric acid; xanthine oxidase INTRODUCTION Since little xanthine oxidase (XO) activity in mammalian brain was detected in earlier reports 1, the major end product of A TP degradation in the brain has been believed to be hypoxanthine2 • Our recent experimental study, however, has indicated the presence of uric acid (UA) in the rat brain subjected to focal ischaemia or complete ischaemia' - 6 • Oxygen-derived free radicals such as superoxide anion ( 0 2 - ) , hydrogen peroxide (HP 2 l and hydroxyl radicals ( · OH) may play an important role in cerebral ischaemia and trauma7 - 10 • However, the source of these free radicals remains unknown. One major source of free radicals is known to be the XO system, which generates superoxide anion radicaP 1 . In the brain, infusion of XO, hypoxanthine and ADP-Fe2 + results in lipid peroxidation, oedema and swelling12 • Of particular significance, the enzyme, XO, has been demonstrated in brain parenchyma13 and in vascular endothelial cells14 . Recently, XO activity was recognized in rat caudate by Mueller et al. 15 and in brain capillaries and in the cerebral cortex including the capillary network by Betz16• Our recent works have demonstrated that the oxypurines (hypoxanthine, xanthine and UA) in the ischaemic hemisphere appear in a chronological order which corresponds to the order in the metabolic map, and that the UA increase is inhibited by allopurinol'- 6 • In this paper, we describe the activities of xanthine dehydrogenase (XD) and XO in rat forebrain immediately and 24 h after complete ischaemia. MATERIALS AND METHODS Adult male Sprague Dawley (50 ) rats (300-350 g) that had been permitted free access to food and water until Correspondence to: Or H. Kanemitsu, Department of Neurosurgery, Teikyo University School of Medicine, 2-11 -1, Kaga, ltabashi-ku, Tokyo 173, Japan. Accepted for publication January 1992.
the day of the experiment were anaesthetized with nembutal. The activities of XD and XO were measured by the modified method of Engerson et al. 17 • Each animal was transcardially perfused with saline and decapitated. The decapitated head was incubated at 37°( for 24 h. Immediately and 24 h after decapitation, the forebrain was removed and homogenized in 6 ml of an ice cold buffered solution containing 0:05 M potassium phosphate (pH 7.80), 0.3 mM EGTA, 10 mM dithiothreitol (DTI) {to prevent reversible XD to XO conversion ), 1 mM phenylmethylsulphonyl fluoride, 0.2M sucrose and 1 mM salicylate with the aid of a Teflon-glass homogenizer rotating at about 1500 rev / min. The homogenate was centrifuged at 100,000 x g at 4°C for 1 h. The supernatant was dialysed overnight against 0.05 M potassium phosphate buffer (pH 7.80) containing 0.3 mM EGT A. The resultant dialysate was utilized for measurement of XD and XO. The activities of XD and XO were assayed by the measurement of UA formation using an HPLC-ED (electrochemical detector) system 3 . The reaction mixture contained 0.3 ml of 0.05 M potassium phosphate buffer (pH 7.80) containing 0.1 mM EGTA and 10 mM DTI and 0.1 ml of 8 mM xanthine warmed to 37°( in a shaking water bath before 0.2 ml of the dialysed supernatant (sample) was added to start the reaction. Four aliquots of each sample were reacted with xanthine under different conditions. Aliquot 1: to assess the combined act ivities of XD and XO, NAD + (final concentration 1 mM) was included in the reaction mixture. Aliquot 2: identical sample runs in the absence of added NAD + were used to assess XO activity. Aliquots 3 and 4: to assess UA, xanthine was not included in the reaction mixture with and without NAD + . The difference between aliquot 1 and aliquot 3 was used to calculate enzyme activity due to XD and XO. The difference between aliquot 2 and aliquot 4 was used to calculate enzyme activity
Neurological Research, 1992, Volume 14, September
321
XD and XO activities in rat brain after ischaemia: H. Oka et al.
due to XO. Moreover, the difference between the XD + XO and XO, if any, was used to calculate enzyme activity due to XD alone. All the mixtures were incubated in a shaking water bath at 37°C for 15 min. The reaction was stopped by adding 0.1 ml of perchloric acid (final concentration 0.25 M) and the reaction mixture was immediately chilled in ice. The ice-cold reaction mixture was centrifuged at 10,000 x gat 4°C for 10 min. The supernatant was filtered through a 0.45 J.Lm membrane (EKICRODISC 13, Gelman Scientific Instruments Japan, Tokyo, Japan ). An aliquot (20 J.LI ) of the filtrate was injected into the HPLC-ED system, which has been described elsewhere3 . One mU of enzyme activity is defined as the amount of enzyme required to convert 1 nmol of xanthine to UA per minute at 37°C, and specific activity as nmoles of xanthine converted to UA per mg protein. Protein was assayed according to the method of Lowri 8 using bovine serum albumin as a standard. All evaluation were statistically made using Student's t test.
RESUUS The relationship between UA formation and reaction time was linear the course of the first 20 minutes of
4 c
c- 3
.. o!
:;o
Ea. -01
~~
±
±
±
2
c(o :::1 ~
incubation, as shown in Figure 1. The enzyme activity completely disappeared by heat treatment at 70°C for 5 min. The protein concentration and enzyme activity in each group are shown in Table 1. The protein concentration 24 h following complete ischaemia (an ischaemic group) was decreased about half compared with that immediately following ischaemia (a normal group). This may be due to proteolysis. The difference of UA formation by XD between the normal group and the ischaemic group was not statistically significant. However, UA formation by XO in the ischaemic group significantly differed from that in the normal group (p < 0.05 ). A difference in total UA formation was not shown in both groups despite the decrease of protein concentration in the ischaemic goup. The specific activities of XD + XO, XD and XO in the normal group (immediately after decapitation ) were 112.1 ± 45.2, 93.3 ± 38.7 and 18.8 ± 7.7 J.LU / mg protein, respectively. Those of XD + XO and XD 24 h after decapitation were 144.3 ± 80.3 and 183.5 ± 75.1 (p < 0.01 ), respectively, and about twice as high as the corresponding activities in the normal group. The specific activity of 15.2 (p < 0.01) and about three times XO was 60.8 high as compared with the corresponding datum in the normal group. Figure 2 shows the percentages of XD and XO activities, and the ratios of XD / XO. As seen in Figure 2, the percentage of XD and the ratio of XD / XO in the normal group were 83.7 ± 2.6% and 5.30 ± 1.10, respectively. The percentage of XD and the ratio of 7.4% and XD / XO in the ischaemic group were 73.9 0.98, respectively. The activity of XO was 16.3% 3.10 of the XD + XO activity in the normal group. That of
1 Normal
5
10
15
20
25 XD
5.04=1.03
83.7:~:2.6
Reaction time {min)
Figure 1: Time course of uric acid formation by xanthine dehydrogenase (XD ) and xanthine oxidase (XO). All the reaction mixtures were incubated in a shaking water bath at 37°( for 5, 10, 15, 20 or 25 min. The reaction was stopped by adding 0.1 ml of perchloric acid (final concentration 0.25 M) and the reaction mixture was immediately chilled in ice. See the reaction mixture and the UA measurement in Materials and Methods. The regression lines were obtained by least square method (n = 3). 0 h: Immediately after decapitation (a normal group), 24 h; 24 h after decapitation · (an ischaemic group)
Table 1:
24 hr after decapitation
xo
3.04=0.99***
73. 9::7,4**
50
1oo
XD/XO
Figure 2: Changes in percentage of enzyme activity and rat io of xanthine dehydrogenase (XD) to xanthine oxidase (XO ). Normal: immediately after decapitation. Each value 24 h after decapitation was compared with that of the normal group by using two-tailed unpaired Student's t test. ** p < 0.01, *** p < 0.001
The protein concentration and the results of enzyme assay at 37°( for 15 min incubation Immediately after decapitation Normal group (n = 10)
Enzyme activity (J.!U/mg protein)
XD
xo XD +XO
Protein (mg/ g tissue) Uric acid formation (nmol/g tissue)
XD
xo XD + XO
Brain wet weight (g)
93.3 18.8 112.1
± 38.7 ± 7.7
± 45.2
19.55
±
2.54
27.21 5.54 32.75
±
9.69 2.15 11.53
±
± 1.48 ±
0.08
24 h after decapitation Ischemia group (n = 10) 183.5 60.8 244.3
± 75.1 **
± 15.2***
± 80.3** 8.77 ± 1.55*** 23.31 ± 8.80 8.04 ± 2.57* 31.35 1.55
± 10.11 ± 0.05*
XD: xanthine dehydrogenase. XO: xanthine oxidase. Each value 24 h after decapitation was compared with that of the normal group by using two-tailed unpaired Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001.
322
Neurological Research, 1992, Volume 14, September
XD and XO activities in rat brain after ischaemia: H. Oka et al.
XO 24 h after complete ischaemia was 26.1% of the XD + XO activity and showed a significant increase (p < 0.01 ) compared with the correspondi ng datum in the normal group. The ratio of XD /XO 24 h after complete ischaemia (the ischaemic group) was 3.04 0.99 and decreased significantly (p < 0.001 ) compared with that of immediately after decapitation (the normal group ) (5.04 1.03). Decreased activity of XD (p < 0.01 ), increased activity of XO (p < 0.01 ) and a decreased ratio of XD / XO (p < 0.001) were recognized between the normal group and the ischaemic group.
±
±
DISCUSSION It has been believed that XO was not present in mammalian brain tissue 1 and the major end product of purine metabolism in the brain was hypoxanthin e2 • We reported that the UA accumulatio n in rat brain was increased after focal ischaemia, and that the UA increase is due not to dysfunction of the brain blood barrier but to the promotion of purine metabolism in brain tissue3 . Hypoxanthin e, xanthine and UA observed by HPLC after ischaemia appeared in metabolic order6 • Owing to allopurinol pretreatmen t, the hypoxanthin e level increased and the xanthine and UA levels decreased compared with those levels in ischaemic animals 6 • From these results, they concluded that the metaboli c steps from hypoxanthin e to xanthine and then to UA were carried out enzymatical ly. We chose complete ischaemia after decapitation as the simplest experimental system, and measured XO activity in order to confirm the XD and XO activities in the brain. We recognized that: (1) UA production is proportional to reaction time (Figure 1 ); (2) no conversion from xanthine to UA occur when only xanthine or coenzyme was in contact with air for a short time; (3) no metabolism from xanthine to UA occurs if the sample was preheated at 70°C for 5 min and was reacted with xanthine; (4) UA production is completely inhibited by allopurinol addition. From these results, it is clear that XD and XO exist in SD rat normal brain tissue and are metabolized from hypoxanthin e to UA. The activities of XD and XO, mo reover, still remain 24 h after decapitation ischaemia (Table, Figure 2). The proportion of XO significantly increases 24 h following ischaemia compared with that of XO immediately following ischaemia (Figure 2 ). However, the proportion of XD remains comparative ly high and it is, therefore, thought that the metabolic conversion of hypoxanthin e to UA is, mainly, carried out in vivo by XD. XO activity 24 h after ischaemia is about three times as high as that immediately after ischaemia (Table 1 ). It is said that XD + XO is produced in the form of XD in all tissues and that XO is present in proportion to 10% of XD + XO in normal tissue 19 • Recently, the metaboli c steps f rom hypoxanthin e to UA has been conjectured as a source of free radicals such as superoxide anio n, hyd rogen peroxide and hy droxyl radical; these oxygeo radi cals have been proposed to amplify tissue damage after ischaemia, but free radicals are not produced by XD 20 • It is known that the conversion of XD to XO proceeds in vitro by proteolysis of trypsin or incubation at 37°C. McCord20 proposed that this conversion is carried out in vivo by calcium-acti vated proteases in ischaemic tissue. The proportion of XD /XO is 5.04 1.03 immE;:diately after brain ischaemia (Figure 2), as was shown in
±
ischaemic liver by Suleiman et al. 21 • The increase of XO activity 24 h after ischaemia w as recognized compared with that immediately after ischaemia (Table 1, Figure 2). It is thought that the conversion of XD to XO proceeds during complete ischaemia, and that an increase of XO activity reinforces ischaemic damage in cerebral tissue. Engerson et al.17 reported that the conversion rate of XD to XO is different in organs such as liver, kidney, heart and lung. XO activity increased significantly 24 h after decapitation ischaemia but the proportion of XO was 26.1 ± 7.4% of XD + XO activity (Figure 2 ). It is concluded that the conversion of XD to XO in brain is much slower than other organs.
ACKNOWLEDGEMENTS The authors thank Professo r Brant D. Watson, University of Miami School of Medicine, for helpful discussion and critical reading of the manuscript, and M s N. Tomukai for her skilful assistance in performing the experiments.
REFERENCES 2
3
4
5
6
7 8
9
10
11 12
13
14
AI-Khalidi UAS, Chaglassian TH. The species distribution of xanthine oxidase. Biochem 1 1965; 97: 316-320 Siesjo BK. In: Siesjo BK, ed. Brain Energy Metabolism. New York & London: John Wiley, 1978; pp. 103- 125, 174- 181, 458-470, and 505-515 Kanemitsu H, Tamura A, Sano K, Iwamoto T, Yoshiura M, lriyama K. Changes of uric acid level in rat brain after focal ischemia. 1 Neurochem 1986; 46: 851-853 Kanemitsu H, Tamura A, Ki rino T, Karasawa S, Sano K, Iwamoto T, Yoshiura M, lriyama K. Xanthine and uric acid levels in rat brain following focal ischemia. 1Neurochem 1988;
51: 1882-1885 Kanemitsu H, Tamura A, Kirno T, Sano K, Iwamoto T, Yoshiura M, lriyama K. Allopurinol inhibits uric acid accumulation in the rat brain following focal cerebral ischemia. Brain Res 1989; 499: 367- 370 Nihei H, Kanemitsu H, Tamura A, Oka H, Sano K. Cerebral uric acid, xanthine, and hypoxanthine after ischemia: the effect of allopurinol. Neurosurgery 1989; 25: 613- 617 Flamm ES, Demopoulos HB, Seligman ML, Poser RG, Ransohoff j. Free radicals in cerebral ischemia. Stroke 1978; 9: 445-447 Demopoulos HB, Flamm ES, Seligman M, M itamura JA, Ransohoff j. Membrane perturbations in central nervous system injury: theoretical basis for free radicals damage and a review of the experimental data. In: Popp Aj, Bourke RS, Nelson LR, Kimelberg HK, eds. Neural Trauma. New York: Raven Press, 1979; pp. 63-78 Demopoulos HB, Flamm ES, Seligman M, Pietronigro DD. Oxygen free radicals in central nervous system ischemia and trauma. In: Autor AP, ed. Pathology of Oxygen\ New York: Academic Press, 1982; pp. 127-155 Yoshida 5, Abe K, Busto R, Watson BD, Kogure K, Ginsberg MD. Influence of transient ischemia on lipid-soluble antioxidants, free fatty acids and energy metabolites in rat brain. Brain Res 1982; 245: 307- 316 McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. 1 Bioi Chem 1968; 243: 5753- 5760 Chan PH, Schmidley JW, Fishman RA, Longar SM. Brain injury, edema, and vascular permeability changes induced by oxygen-derived free radicals. Neurology 1984; 34: 315-320 Villela GG. Xanthine oxidase activity in the brain. Experientia 1968; 24: 1101-1102 jarasch ED, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW. Localization of xanthine oxidase in mammarygland epithelium and capillary endothelium. Cell 1981; 25: 67-82
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15
16
17
18
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Mueller K, Palmour R, Andrews CD, Knott Pj. In vivo voltammetric evidence of production of uric acid by rat caudate. Brain Res 1985; 335: 231-235 8etz AL. identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries. 1 Neurochem 1985; 44: 574-579 Engerson TD, MacKelvey TG, Rhyne DB, Boggio EB, Sayder SJ, Jones HP. Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. j Clin Invest 1987; 79: 1564-1570 Lowry OH, Rosebrough NJ, Farr AL, Randall Rj. Protein measurement with the folin phenol reagent. J Bioi Chem 1951;
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20 21
193: 265-277 Roy RS, McCord JM. Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In: Greenwald R, Cohen G, eds. Amsterdam: Elsevier. Oxygen Radicals and Their Scavenger Systems, Vol. II: Cellular and Medical Aspects 1983; pp. 145-153 McCord JM. Oxygen-derived free radicals in postischemic tissue injury. New Eng! 1 Med 1985; 312: 159-163 Suleiman SA, Stevens JB. Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields. Arch Biochem Biophys 1987; 258: 219-225
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura & Keiji Sano To cite this article: Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura & Keiji Sano (1992) Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia, Neurological Research, 14:4, 321-324, DOI: 10.1080/01616412.1992.11740077 To link to this article: http://dx.doi.org/10.1080/01616412.1992.11740077
Published online: 20 Jul 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yner20 Download by: [University of Toronto Libraries]
Date: 16 April 2017, At: 13:33
Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia Hidemune Oka, Hideaki Kanemitsu, Hiroshi Nihei, Hitoshi Nakayama, Akira Tamura and Keiji Sano Department of Neurosurgery, Teikyo University School of Medicine, Tokyo 173, Japan
We studied the activities of xanthine dehydrogenase and xanthine oxidase in rat forebrain after complete ischaemia. Complete ischaemia was induced by decapitation after transcardiac infusion with saline. The activities of xanthine dehydrogenase and xanthine oxidase immediately after ischaemia were 93.3 ± 38.7 and 18.8 ± 7.7 Ji.U / mg protein, respectively, and at 24 h after ischaemia were 183.5 ± 75.1 and 60.8 ± 15.2 Ji.U / mg protein, respectively. The ratios of xanthine dehydrogenase / xanthin oxidase immediately and 24 h after ischaemia were 5.04 ± 1.03 and 3.04 ± 0.99, respectively. These data indicate that xanthine dehydrogenase and xanthine oxidase activities were maintained even 24 h after complete ischaemia. Conversion of xanthine dehydrogenase to xanthine oxidase proceeds slowly during complete ischaemia. [Neurol Res
1992; 14: 321-324 J
Keywords: Hypoxanthine; ischaemia; oxypurine; rat brain; uric acid; xanthine oxidase INTRODUCTION Since little xanthine oxidase (XO) activity in mammalian brain was detected in earlier reports 1, the major end product of A TP degradation in the brain has been believed to be hypoxanthine2 • Our recent experimental study, however, has indicated the presence of uric acid (UA) in the rat brain subjected to focal ischaemia or complete ischaemia' - 6 • Oxygen-derived free radicals such as superoxide anion ( 0 2 - ) , hydrogen peroxide (HP 2 l and hydroxyl radicals ( · OH) may play an important role in cerebral ischaemia and trauma7 - 10 • However, the source of these free radicals remains unknown. One major source of free radicals is known to be the XO system, which generates superoxide anion radicaP 1 . In the brain, infusion of XO, hypoxanthine and ADP-Fe2 + results in lipid peroxidation, oedema and swelling12 • Of particular significance, the enzyme, XO, has been demonstrated in brain parenchyma13 and in vascular endothelial cells14 . Recently, XO activity was recognized in rat caudate by Mueller et al. 15 and in brain capillaries and in the cerebral cortex including the capillary network by Betz16• Our recent works have demonstrated that the oxypurines (hypoxanthine, xanthine and UA) in the ischaemic hemisphere appear in a chronological order which corresponds to the order in the metabolic map, and that the UA increase is inhibited by allopurinol'- 6 • In this paper, we describe the activities of xanthine dehydrogenase (XD) and XO in rat forebrain immediately and 24 h after complete ischaemia. MATERIALS AND METHODS Adult male Sprague Dawley (50 ) rats (300-350 g) that had been permitted free access to food and water until Correspondence to: Or H. Kanemitsu, Department of Neurosurgery, Teikyo University School of Medicine, 2-11 -1, Kaga, ltabashi-ku, Tokyo 173, Japan. Accepted for publication January 1992.
the day of the experiment were anaesthetized with nembutal. The activities of XD and XO were measured by the modified method of Engerson et al. 17 • Each animal was transcardially perfused with saline and decapitated. The decapitated head was incubated at 37°( for 24 h. Immediately and 24 h after decapitation, the forebrain was removed and homogenized in 6 ml of an ice cold buffered solution containing 0:05 M potassium phosphate (pH 7.80), 0.3 mM EGTA, 10 mM dithiothreitol (DTI) {to prevent reversible XD to XO conversion ), 1 mM phenylmethylsulphonyl fluoride, 0.2M sucrose and 1 mM salicylate with the aid of a Teflon-glass homogenizer rotating at about 1500 rev / min. The homogenate was centrifuged at 100,000 x g at 4°C for 1 h. The supernatant was dialysed overnight against 0.05 M potassium phosphate buffer (pH 7.80) containing 0.3 mM EGT A. The resultant dialysate was utilized for measurement of XD and XO. The activities of XD and XO were assayed by the measurement of UA formation using an HPLC-ED (electrochemical detector) system 3 . The reaction mixture contained 0.3 ml of 0.05 M potassium phosphate buffer (pH 7.80) containing 0.1 mM EGTA and 10 mM DTI and 0.1 ml of 8 mM xanthine warmed to 37°( in a shaking water bath before 0.2 ml of the dialysed supernatant (sample) was added to start the reaction. Four aliquots of each sample were reacted with xanthine under different conditions. Aliquot 1: to assess the combined act ivities of XD and XO, NAD + (final concentration 1 mM) was included in the reaction mixture. Aliquot 2: identical sample runs in the absence of added NAD + were used to assess XO activity. Aliquots 3 and 4: to assess UA, xanthine was not included in the reaction mixture with and without NAD + . The difference between aliquot 1 and aliquot 3 was used to calculate enzyme activity due to XD and XO. The difference between aliquot 2 and aliquot 4 was used to calculate enzyme activity
Neurological Research, 1992, Volume 14, September
321
XD and XO activities in rat brain after ischaemia: H. Oka et al.
due to XO. Moreover, the difference between the XD + XO and XO, if any, was used to calculate enzyme activity due to XD alone. All the mixtures were incubated in a shaking water bath at 37°C for 15 min. The reaction was stopped by adding 0.1 ml of perchloric acid (final concentration 0.25 M) and the reaction mixture was immediately chilled in ice. The ice-cold reaction mixture was centrifuged at 10,000 x gat 4°C for 10 min. The supernatant was filtered through a 0.45 J.Lm membrane (EKICRODISC 13, Gelman Scientific Instruments Japan, Tokyo, Japan ). An aliquot (20 J.LI ) of the filtrate was injected into the HPLC-ED system, which has been described elsewhere3 . One mU of enzyme activity is defined as the amount of enzyme required to convert 1 nmol of xanthine to UA per minute at 37°C, and specific activity as nmoles of xanthine converted to UA per mg protein. Protein was assayed according to the method of Lowri 8 using bovine serum albumin as a standard. All evaluation were statistically made using Student's t test.
RESUUS The relationship between UA formation and reaction time was linear the course of the first 20 minutes of
4 c
c- 3
.. o!
:;o
Ea. -01
~~
±
±
±
2
c(o :::1 ~
incubation, as shown in Figure 1. The enzyme activity completely disappeared by heat treatment at 70°C for 5 min. The protein concentration and enzyme activity in each group are shown in Table 1. The protein concentration 24 h following complete ischaemia (an ischaemic group) was decreased about half compared with that immediately following ischaemia (a normal group). This may be due to proteolysis. The difference of UA formation by XD between the normal group and the ischaemic group was not statistically significant. However, UA formation by XO in the ischaemic group significantly differed from that in the normal group (p < 0.05 ). A difference in total UA formation was not shown in both groups despite the decrease of protein concentration in the ischaemic goup. The specific activities of XD + XO, XD and XO in the normal group (immediately after decapitation ) were 112.1 ± 45.2, 93.3 ± 38.7 and 18.8 ± 7.7 J.LU / mg protein, respectively. Those of XD + XO and XD 24 h after decapitation were 144.3 ± 80.3 and 183.5 ± 75.1 (p < 0.01 ), respectively, and about twice as high as the corresponding activities in the normal group. The specific activity of 15.2 (p < 0.01) and about three times XO was 60.8 high as compared with the corresponding datum in the normal group. Figure 2 shows the percentages of XD and XO activities, and the ratios of XD / XO. As seen in Figure 2, the percentage of XD and the ratio of XD / XO in the normal group were 83.7 ± 2.6% and 5.30 ± 1.10, respectively. The percentage of XD and the ratio of 7.4% and XD / XO in the ischaemic group were 73.9 0.98, respectively. The activity of XO was 16.3% 3.10 of the XD + XO activity in the normal group. That of
1 Normal
5
10
15
20
25 XD
5.04=1.03
83.7:~:2.6
Reaction time {min)
Figure 1: Time course of uric acid formation by xanthine dehydrogenase (XD ) and xanthine oxidase (XO). All the reaction mixtures were incubated in a shaking water bath at 37°( for 5, 10, 15, 20 or 25 min. The reaction was stopped by adding 0.1 ml of perchloric acid (final concentration 0.25 M) and the reaction mixture was immediately chilled in ice. See the reaction mixture and the UA measurement in Materials and Methods. The regression lines were obtained by least square method (n = 3). 0 h: Immediately after decapitation (a normal group), 24 h; 24 h after decapitation · (an ischaemic group)
Table 1:
24 hr after decapitation
xo
3.04=0.99***
73. 9::7,4**
50
1oo
XD/XO
Figure 2: Changes in percentage of enzyme activity and rat io of xanthine dehydrogenase (XD) to xanthine oxidase (XO ). Normal: immediately after decapitation. Each value 24 h after decapitation was compared with that of the normal group by using two-tailed unpaired Student's t test. ** p < 0.01, *** p < 0.001
The protein concentration and the results of enzyme assay at 37°( for 15 min incubation Immediately after decapitation Normal group (n = 10)
Enzyme activity (J.!U/mg protein)
XD
xo XD +XO
Protein (mg/ g tissue) Uric acid formation (nmol/g tissue)
XD
xo XD + XO
Brain wet weight (g)
93.3 18.8 112.1
± 38.7 ± 7.7
± 45.2
19.55
±
2.54
27.21 5.54 32.75
±
9.69 2.15 11.53
±
± 1.48 ±
0.08
24 h after decapitation Ischemia group (n = 10) 183.5 60.8 244.3
± 75.1 **
± 15.2***
± 80.3** 8.77 ± 1.55*** 23.31 ± 8.80 8.04 ± 2.57* 31.35 1.55
± 10.11 ± 0.05*
XD: xanthine dehydrogenase. XO: xanthine oxidase. Each value 24 h after decapitation was compared with that of the normal group by using two-tailed unpaired Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001.
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XD and XO activities in rat brain after ischaemia: H. Oka et al.
XO 24 h after complete ischaemia was 26.1% of the XD + XO activity and showed a significant increase (p < 0.01 ) compared with the correspondi ng datum in the normal group. The ratio of XD /XO 24 h after complete ischaemia (the ischaemic group) was 3.04 0.99 and decreased significantly (p < 0.001 ) compared with that of immediately after decapitation (the normal group ) (5.04 1.03). Decreased activity of XD (p < 0.01 ), increased activity of XO (p < 0.01 ) and a decreased ratio of XD / XO (p < 0.001) were recognized between the normal group and the ischaemic group.
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DISCUSSION It has been believed that XO was not present in mammalian brain tissue 1 and the major end product of purine metabolism in the brain was hypoxanthin e2 • We reported that the UA accumulatio n in rat brain was increased after focal ischaemia, and that the UA increase is due not to dysfunction of the brain blood barrier but to the promotion of purine metabolism in brain tissue3 . Hypoxanthin e, xanthine and UA observed by HPLC after ischaemia appeared in metabolic order6 • Owing to allopurinol pretreatmen t, the hypoxanthin e level increased and the xanthine and UA levels decreased compared with those levels in ischaemic animals 6 • From these results, they concluded that the metaboli c steps from hypoxanthin e to xanthine and then to UA were carried out enzymatical ly. We chose complete ischaemia after decapitation as the simplest experimental system, and measured XO activity in order to confirm the XD and XO activities in the brain. We recognized that: (1) UA production is proportional to reaction time (Figure 1 ); (2) no conversion from xanthine to UA occur when only xanthine or coenzyme was in contact with air for a short time; (3) no metabolism from xanthine to UA occurs if the sample was preheated at 70°C for 5 min and was reacted with xanthine; (4) UA production is completely inhibited by allopurinol addition. From these results, it is clear that XD and XO exist in SD rat normal brain tissue and are metabolized from hypoxanthin e to UA. The activities of XD and XO, mo reover, still remain 24 h after decapitation ischaemia (Table, Figure 2). The proportion of XO significantly increases 24 h following ischaemia compared with that of XO immediately following ischaemia (Figure 2 ). However, the proportion of XD remains comparative ly high and it is, therefore, thought that the metabolic conversion of hypoxanthin e to UA is, mainly, carried out in vivo by XD. XO activity 24 h after ischaemia is about three times as high as that immediately after ischaemia (Table 1 ). It is said that XD + XO is produced in the form of XD in all tissues and that XO is present in proportion to 10% of XD + XO in normal tissue 19 • Recently, the metaboli c steps f rom hypoxanthin e to UA has been conjectured as a source of free radicals such as superoxide anio n, hyd rogen peroxide and hy droxyl radical; these oxygeo radi cals have been proposed to amplify tissue damage after ischaemia, but free radicals are not produced by XD 20 • It is known that the conversion of XD to XO proceeds in vitro by proteolysis of trypsin or incubation at 37°C. McCord20 proposed that this conversion is carried out in vivo by calcium-acti vated proteases in ischaemic tissue. The proportion of XD /XO is 5.04 1.03 immE;:diately after brain ischaemia (Figure 2), as was shown in
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ischaemic liver by Suleiman et al. 21 • The increase of XO activity 24 h after ischaemia w as recognized compared with that immediately after ischaemia (Table 1, Figure 2). It is thought that the conversion of XD to XO proceeds during complete ischaemia, and that an increase of XO activity reinforces ischaemic damage in cerebral tissue. Engerson et al.17 reported that the conversion rate of XD to XO is different in organs such as liver, kidney, heart and lung. XO activity increased significantly 24 h after decapitation ischaemia but the proportion of XO was 26.1 ± 7.4% of XD + XO activity (Figure 2 ). It is concluded that the conversion of XD to XO in brain is much slower than other organs.
ACKNOWLEDGEMENTS The authors thank Professo r Brant D. Watson, University of Miami School of Medicine, for helpful discussion and critical reading of the manuscript, and M s N. Tomukai for her skilful assistance in performing the experiments.
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