Circulating xanthine oxidase: potential of ischemic injury
mediator
YOSHIFUMI YOKOYAMA, JOSEPH S. BECKMAN, TANYA K. BECKMAN, JOAN K. WHEAT, THOMAS G. CASH, BRUCE A. FREEMAN, AND DALE
A. PARKS
Departments of Anesthesiology, Physiology, and Biophysics and Biochemistry, University of Alabama at Birmingham, Birmingham, Alabama 35233 YOKOYAMA, YOSHIFUMI, JOSEPH S. BECKMAN, TANYA K. BECKMAN, JOAN K. WHEAT, THOMAS G. CASH, BRUCE A. FREEMAN, AND DALE A. PARKS. Circulating xanthine oxidase:
mediator of ischemic injury. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G564-G570,1990.-Reactive oxygen metabolites generated from the enzyme xanthine oxidase (X0) play an important role in the pathogenesis of ischemiainduced tissue injury. The observation that intracellular proteins such as aspartate transaminase (AST) and alcohol dehydrogenase (ADH) are released from the ischemic liver during reperfusion led us to postulate that X0 could be released into the systemic circulation. Livers from fasted rats were extirpated, perfused with oxygenated Krebs-Henseleit buffer, and subjected to 2 h ischemia followed by 2 h reperfusion. Reperfusion increased AST in the perfusate from 1 k 1 to 830 -I- 280 U/l, whereas ADH increased from 0.3 t 0.1 to 95 t 26 U/l. Concomitantly, xanthine dehydrogenase (XDH) + X0 activity in the perfusate increased from 0 to 4.1 k 1.0 mu/ml. A 64% decrease in endogenous tissue XDH + X0 activity paralleled release of XDH + X0. The XDH + X0 activity predicted to appear in the circulation after hepatic ischemia was sufficient, when supplied with substrate, to produce severe vascular endothelial injury in vitro, even in the presence of serum or whole blood. These results suggest that massive quantities of XDH and X0 are released into the circulation after hepatic ischemia and that the resulting reactive oxygen metabolites could produce widespread tissue injury.
potential
ischemia; liver; free radicals; xanthine dehydrogenase; endothelium
REACTIVE OXYGEN metabolites play an important role in the pathogenesis of the structural and functional alterations associated with reperfusion of a wide variety of ischemic tissues. Xanthine oxidase (X0) has been postulated to be a primary source of these cytotoxic oxygen radicals. Evidence supporting involvement of X0 is based primarily on the observation that allopurinol, an inhibitor of xanthine dehydrogenase (XDH) and oxidase, is as effective as oxygen radical scavengers in attenuating the tissue injury associated with ischemia-reperfusion. In accordance with the X0 hypothesis, endogenous XDH is converted to oxygen radical-producing X0 during the ischemic period (12, 22). Concomitantly, cellular ATP is catabolized to hypoxanthine, which accumulates intracellularly. On reperfusion (reoxygenation), X0 can react with hypoxanthine and molecular oxygen to produce the highly reactive oxygen metabolites superoxide, hydrogen G564
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peroxide, and hydroxyl radical. There remains considerable controversy as to the role that X0 and allopurinol play in ischemia-induced tissue injury. In contrast with the X0 hypothesis, conversion of XDH to X0 does not seem to be requisite for tissue injury, since hepatocellular injury, as evidenced by release of cytoplasmic enzymes, is observed before significant conversion of endogenous XDH to X0 (7, 17). It is conceivable that measurements of the rate and extent of XDH-to-X0 conversion in these studies were influenced by the significant XDH + X0 activity of normal rat blood. In addition, the specificity of allopurinol as an inhibitor of X0 has been questioned. Allopurinol has been postulated to directly scavenge free radicals (5, 18, 24) and minimize tissue injury in porcine myocardium where X0 activity is undetectable (25). These data would suggest that allopurinol may exert its effect through a mechanism other than through inhibition of X0. It is well documented that ischemic liver releases cellular enzymes upon reperfusion (1). Since the liver is extremely rich in XDH + X0 activity (6, 29), it is conceivable that XDH and X0 are released into the circulation concomitant with release of other hepatocellular enzymes. Circulating X0 could have tremendous potential for oxygen radical production during postischemic reperfusion and could 1) result in direct injury to the vascular endothelium, 2) activate oxidant-producing inflammatory cells and extend oxidant-induced injury to tissues distal to the site of origin, and 3) produce reactive oxygen metabolites in a relatively antioxidant-poor compartment, the plasma, and result in extension of tissue injury. It was the purpose of the present study to determine whether ischemic liver was capable of releasing XDH or X0 into the circulation, which could serve as a potential source of circulating oxygen radical production resulting in tissue injury at sites distal to its origin. MATERIAL
AND
METHODS
Biochemical Analyses Bovine milk X0 was obtained from Calbiochem (La Jolla, CA). Stock solutions were made daily by centrifuging 25 ,~l of X0 suspended in 2 M (NH&SO4 for 2 min in a microcentrifuge (5,000 g). The supernatant was discarded and the sedimented crystals resuspended in the appropriate buffer. All enzymes and reagents were purchased from Sigma Chemical (St. Louis, MO). Aspar-
0 1990 the American
Physiological
Society
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XANTHINE
tate aminotransferase (AST) was determined using standard assays (13). Alcohol dehydrogenase (ADH) activity was measured using the procedure of Kato et al. (14). Protein was quantified according to Bradford (3) using bovine serum albumin as a standard. The activity of X0 and XDH were assayed in tissue homogenate and perfusate by monitoring formation of urate at 295 nm as previously described (22). Units of enzyme activity were defined as 1 pmol of urate formed/min at 25°C and pH 7.8. Both XDH + X0 and X0 reactions were completely inhibited by 60 FM allopurinol. The extent of conversion of XDH to X0 (%X0) was calculated from the activity of X0 divided by total XDH + X0 activity. Surgical Procedures Male Sprague-Dawley rats (n = 54) weighing 320-380 g were fasted 24 h before surgery but were allowed water ad libitum. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip), a tracheotomy was performed, and the animals were artificially ventilated with room air. The liver was extirpated as previously described (1) and placed in an environmental chamber, which minimized evaporative water loss and maintained liver temperature. Isolated perfused liver studies. The livers were perfused through the portal vein at a constant pressure of 15 cmH20 with a modified, hemoglobin-free Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 27 NaHC03, 2.5 CaCIZ, 1.2 MgSO,, 1.2 KH2P04, 0.05 EDTA, and 11 glucose, plus 300 U heparin). The perfusate was maintained at constant temperature (37°C) and pH (7.4) and equilibrated with 95% OZ-5% CO2 to achieve an influent PO, of 300-500 mmHg. XDH-to-X0 conversion studies. Livers were placed in an environmental chamber immediately after extirpation or after 5 min perfusion through the portal vein with Euro-Collins solution (in mM: 99.3 KCl, 15.1 KH2P04, 9 K2HP04, 10 NaHC03, and 194 glucose, pH 7.4) to remove cellular elements. Vascular endothelial toxicity studies. Bovine aortic endothelial cells were isolated from freshly obtained vessels by gently scraping the lumen of the vessel with a scalpel. Cells were cultured in medium 199, 5% fetal calf serum (Hyclone Labs., Logan, UT), and 5% Ryan’s growth supplement (Dr. Una Ryan, Univ. of Miami, FL) and were maintained at 37°C in 95% air-5% CO,. Endothelial cell purity, assessed by cell uptake of DiL-AC-low density lipoprotein (Biomedical Technologies, Stoughton, MA), was >99%. Cells were passaged by scraping with a rubber policeman and subcultured in a 1 to 3 ratio. Cellular responses to X0 were studied using low population doubling level cultures (2). Experimental
Protocols
Isolated perfused liver studies. For this series of experiments, animals (n = 30) were randomly divided into four groups before hepatectomy. Each liver was perfused at 20 ml/min in a single-pass arrangement for 30 min to rinse the organ free of blood and to permit equilibration. This initial 30-min perfusion served as the control period
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for each liver. Group 1 nonischemic control (n = 10) livers were processed immediately after the 30-min equilibration period. Group 2 (n = 10) livers were perfused for 240 min with oxygenated Krebs-Henseleit buffer (nonischemic, perfused). Group 3 (n = 10) livers were subjected to 2 h global normothermic ischemia followed by 2 h reperfusion under conditions identical to the initial 30-min (preischemic) period. Hepatic inflow (portal vein) and outflow (inferior vena cava) perfusion pressures were monitored via pressure transducers (Statham P23 ID) interposed within the circuit and continuously recorded with a physiological recorder (Grass Instrument, Quincy, MA). Aliquots (20 ml) of the perfusate were collected during the control period, immediately after the occlusive period, and every 30 min of the 2-h reperfusion period for appearance of XDH, X0, and hepatocellular enzymes. Blood gases were periodically monitored with a blood gas analyzer (Instrumentation Labs., ILl301). Tissue XDH, X0, and %X0 were determined at the end of the reperfusion period. XDH-to-X0 conuersion studies. In this series of experiments, rats (n = 24) were divided into four groups before hepatectomy. The livers from groups 1 and 2 were maintained at either 37°C or 4°C. Livers of groups 3 and 4 were perfused with either 37°C or 4°C Euro-Collins solution and then maintained at 37°C and 4°C respectively. Biopsies were obtained for XDH and X0 determination after 0, 1, 3,4, 6, and 8 h preservation in EuroCollins solution. XDH and X0 activity were determined in hepatic biopsies. Hepatic tissue biopsies (-1 g) were quickly excised and immersed in 10 ml of ice-cold homogenizing buffer (in mM: 50 K+-phosphate buffer, 10 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and 0.1 EDTA, pH 7.4) supplemented with 0.25 M sucrose. The tissue was homogenized using a motorized Potter-Elvehjem homogenizer and Teflon pestle and then centrifuged at 40,000 g at 4°C for 30 min. The supernatant was decanted and centrifuged for an additional 15 min. Perfusate samples were collected from the inferior caval vein. A portion of the sample was immediately added to the homogenizing buffer and centrifuged at 900 g for 15 min, and the supernatant was processed for XDH and X0 activity. Enzyme determinations were made in the remaining portion of the perfusate. Size exclusion chromatography on prepacked Sephadex G-25 columns (22) of the rat liver homogenate had no effect on total activity, %X0, or inhibition by allopurinol (data not shown). Endothelial toxicity studies. Cells were subcultured to confluency on 12-well tissue plates (Costar, Cambridge, MA) having 2.2 cm2 cell surface area (d-l.5 x 10' cells). Cells were prelabeled for 3 h with 4 PM 8- [“Cl adenine (55.1 mCi/mmol) having no cold carrier in complete medium and washed with Hanks’ balanced salt solution (HBSS). Cells exposed to 500 PM xanthine t 5.0 mu/ml X0 were incubated in HBSS plus varying concentrations of fetal calf serum. In a separate series of experiments, the effect of heparinized whole blood on X0-mediated cytotoxicity was evaluated in cultured endothelial cells. Control-labeled cells were lysed with 0.5% Triton X-100 in distilled water and sonicated for determination of total cellular uptake of 8- [ 14C]adenine.
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XANTHINE
After 4 h incubation under different conditions, aliquots of media were removed (n = 12) and analyzed for release of 8- [“Cl adenine (plus metabolites) by liquid scintillation counting. In studies involving whole blood, cellular 8- [14C]adenine release was assessed after oxidation of hemoglobin with 30% HZOz. Statistics. The effect of ischemia on the release of hepatocellular enzymes, and XDH and X0 activity, was tested by analysis of variance with individual differences between groups analyzed by Scheffe’s post hoc test. The effect of X0 on endothelial release of intracellular adenine metabolites was tested for significance by analysis of variance with individual differences analyzed by Scheffe’s post hoc test. RESULTS
The isolated perfused rat liver preparation was stable for >4 h with no significant increases in AST, ADH, and XDH and X0 during this period (Figs. l-4). During the equilibration period, no significant differences existed between experimental groups. Two hours of hepatic ischemia (group 3) resulted in a significant increase (P c 0.05) in the appearance of all measured cellular enzymes in the perfusate. The activity of AST [glutamic-oxaloacetic transaminase (GOT)] increased from 2 t 2 to 830 t 280 U/l after reperfusion (Fig. 1). The ADH activity 1000
OXIDASE
*
5.0
T
r .'A 'E>0 ag3 F
4.0
-
3.0
-
+ 5. 3
2.0
-
1.0 0.0 Control
1 mm reperfuston
30 mln reperfuslon
60 min reperfusion
90 mm reperfusion
FIG. 3. Release of xanthine dehydrogenase plus xanthine oxidase (XDH + X0) activity from livers after 4 h perfusion and 2 h ischemiareperfusion. * P < 0.05 vs. respective control (4 h perfusion).
3.0 2.5
1 t
Control
1 min reperfusion
30 min reperfusion
60 mln reperfusion
90 mm reperfusion
FIG. 4. Xanthine oxidase (X0) activity is dramatically the perfusate of livers after ischemia-reperfusion. "P < tive control (4 h perfusion).
L6
120 mm reperfuslon
200 i I)
DA
0
. . . . . . ..m
.“.....I).....,...
*- - - - -A- - - - -A- - - - -A- ----A
i I
I I
control
I
I I
1 min reperfusion
I 1
30 min reperfusion
I I
I I
90 min reperfusion
60 min reperfusion
120 min reperfusion
FIG. 1. Effect of ischemia-reperfusion on appearance of aspartate transaminase (AST) activity in the perfusate of livers after 4 h perfusion with oxygenated buffer (A) and 2 h ischemia-reperfusion (H).
100,
I.......
i
25 9 *-
l
I
control FIG.
perfusate reperfusion
I 1
1 min reperfusion
-
-
-
l -A-
. .‘. _
I
30 min reperfusion
-
.‘..)...... - -A-
, I
60 min reperfusion
-
---
. .g-
I
90 min reperfusion
~
~
L
-#
I I
120 min reperfusion
2. Appearance of alcohol dehydrogenase (ADH) activity in the after perfusion with oxygenated buffer (A) or 2 h ischemia(M).
120 mm reperfusion
increased
in
0.05 vs. respec-
increased from 0.2 t 0.1 to 95 t 26 U/l (Fig. 2). Release of hepatocellular enzyme decreased sharply with continued reperfusion and became statistically indistinguishable from the control group after 30 min reperfusion. Total XDH + X0 activity (Fig. 3) in the perfusate was significantly increased from essentially 0 to 4.1 & 1.0 mu/ml during the first minute of reperfusion of the ischemic livers. X0 activity (Fig. 4) was also elevated dramatically in the perfusate during reperfusion of ischemic livers, increasing from undetectable to 2.0 t 0.6 mu/ml. In a constant flow preparation, if the enzyme activity and volume of collection is known, it is possible to estimate the approximate increase in XDH + X0 activity that would appear in the circulation in vivo. It is predicted that the circulating activity of XDH + X0 would increase 2.7 mu/ml above endogenous levels and X0 activity would increase ~1.3 mu/ml. The potential cytotoxicity of circulating X0 was estimated by measuring the release of previously incorporated [‘“Cl adenine from cultured vascular endothelial cells treated with xanthine (500 PM) and X0 (5.0 mU/ ml) (Fig. 5). To determine the total amount of incorporation of [14C]adenine, cells (n = 12) were sonicated in the presence of detergent and the release of intracellular radiolabel measured (18.5 t 0.5 counts/min X 10m4/2.2 cm2 cells). The radiolabeled [14C]adenine plus metabolites are primarily acid soluble under our preincubation
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CIRCULATING
XANTHINE
60
0
X
x0
x+x0
100%
serum
---X+XO+serumd
FIG. 5. Cytotoxicity of exogenously administered xanthine (X, 500 PM), xanthine oxidase (X0, 5 mu/ml), and xanthine plus xanthine oxidase (X + X0) was estimated by release of previously incorporated [14C]adenine. Addition of O-100% serum to the endothelial cells did not attenuate the X-X0 induced cytotoxicity. *P < 0.05 vs. X. "fP
0.05) reduced by the exposure of cultured cells to XOderived reactive oxygen species in the presence of 90% serum (76% of total) or whole blood (67% of total). To determine if the release of XDH + X0 associated with ischemia-reperfusion was significant enough to influence total XDH + X0 activitv of the liver. tissue
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homogenates were assayed for XDH, X0, and %X0. Xanthine oxidase activity constit.uted 17 t 1% of total XDH + X0 activity in liver homogenates immediately after the equilibration period (group 1). Perfusion for 4 h with oxygenated buffer (group 2) significantly increased %X0 to 26 t 1. Reperfusion of ischemic livers (group 3) significantly increased X0 to 26 t 1%. Total XDH + X0 activity was 163 t 11 mU/g wet wt in control (nonperfused) livers and was not significantly decreased (Fig. 6) by either 4 h perfusion with oxygenated KrebsHenseleit buffer (142 t 10 mU/g wet wt). Two-hour ischemia-reperfusion resulted in a 64% reduction (P < 0.05) in total XDH + X0 activity (55 t 6 mU/g wet wt) compared with control livers. The release of massive amounts of XDH + X0 activity (110 mU/g wet wt) from livers subjected to ischemia-reperfusion suggests that sufficient quantities of oxidant-generating enzyme could potentially appear in the circulation to damage a variety of tissues. Although it is possible that the reduction in XDH + X0 activity could’ be partially explained by oxidant-mediated enzyme autoinactivation, the recovery of 82 mU XDH + X0 in the perfusate during just the first minute of reperfusion would suggest that a large amount of the enzyme lost from the liver appears in the circulation. Dehydrogenase- to-Oxidase Conversion Under normal conditions, the liver contains 163 t 11 mU XDH + X0/g tissue with 15-20% of the total activity existing as X0 (Fig. 7). Subjecting the liver to normothermic (37°C) ischemia for O-8 h results in up to 64% of the total activity existing as X0. There was no significant increase in %X0 in liver subjected to up to 8 h ischemia under hypothermic (4°C) conditions. It is
180 160
XDH -I-
140 E
X
XDH
60 t40 0
20 F
iii control
4h perfusion
2h ischemia 2h reDerfusion
.
FIG. 6. Effect of ischemia-reperrusion on enaogenous xantmne aehydrogenase + xanthine oxidase (XDH + X0) activity in control (nonischemic) liver, livers perfused for 4 h with oxygenated KrebsHenseleit buffer, and livers after 2 h ischemia and 2 h reperfusion. *P < 0.05 vs. control. tP < 0.05 vs. 4 h nerfusion.
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XANTHINE
80 70
1
60
1 0 -0
I 0
I 12
1I
I, I1 , (, 3 4 5 6 DURATION OF ISCHEMIA (hours)
, 7
, 8
9
FIG. 7. Changes in the relative proportion of xanthine oxidase to total xanthine dehydrogenase + oxidase activity (%X0) after O-8 h ischemia in normothermic perfused (0) or nonperfused liver (A) and hypothermic perfused (0) or nonperfused (A) liver.
assumed that the increase in X0 activity was due to XDH-to-X0 conversion, since total XDH + X0 activity was unchanged during the course of the 8-h period of ischemia in both normothermic and hypothermic groups. The presence of cellular elements did not seem to influence total XDH + X0 activity or the rate of XDH-toX0 conversion, with no significant differences observed between perfused and nonperfused livers. DISCUSSION
The results of the present study indicate that large quantities of X0 are released into the circulation from the ischemic liver upon reperfusion. We postulate that circulating X0 could react with endogenous plasma purine substrates and molecular oxygen to produce intravascular reactive oxygen species as this oxidant-generating system traverses the circulation. Our data would suggest that the XDH + X0 and X0 activity would increase in systemic circulation by 2.7 and 1.3 mu/ml, respectively, over endogenous levels. Since studies with XDH + X0 using blood from normal rats indicate that XDH is rapidly and reversibly converted to X0 after 4 min hypoxia (unpublished observation) and that XDH + X0 in the plasma appears to have a relatively long circulating half-life (26), that predicted elevation of XDH and/or X0 in the plasma would very likely damage the vascular endothelium. This contention is supported by the in vitro studies of vascular endothelium in which exogenously administered xanthine and X0, at levels predicted to exist in the plasma after hepatic ischemia, produced significant cytotoxicity. This cytotoxicity was not significantly reduced (P > 0.05) by the addition of serum or whole blood compared with cells maintained in HBSS. Interestingly, there is no apparent widespread tissue injury associated with the XDH + X0 activity (2-4 mU/ ml) that ordinarily exists in rat blood. Normally there is only l-3 PM xanthine plus hypoxanthine in the circulation (15), far less than the 11 ,uM Michaelis-Menten constant (&-J of X0 and XDH (22). Thus limited substrate concentrations may help to minimize tissue injury from endogenous levels of circulating X0. It is also
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intriguing that elevation of plasma purines up to 5 mM does not induce detectable tissue injury in rats (15). However, xanthine and hypoxanthine concentrations in excess of 50 PM strongly inhibit X0 activity (9). From these kinetic characteristics of X0, it can be predicted that there is a window of plasma purine concentrations that would support potentially excessive production of toxic oxygen species. Under normal conditions, XDH + X0 may be maintained predominantly in the relatively innocuous XDH form with any remaining X0-derived oxidants being adequately detoxified by extracellular antioxidants. Ischemia-reperfusion would release additional XDH or X0 and would approximately double endogenous circulating XDH + X0 activity. Changes in the extracellular environment that occur during the ischemic period could then allow reversible or irreversible conversion of XDH to X0. Antioxidants normally found in the extracellular fluid are also expected to become depleted during a prolonged period of ischemia. Thus the circulating X0 could generate sufficient oxidant to produce extensive endothelial and epithelial injury to a variety of tissues in vivo and potentially be a mediator of the multisystem failure associated with systemic shock states. Oxygen radicals can arise from a number of sources (8); however, XDH + X0 has been frequently postulated to be a primary intracellular generator of toxic oxygen radicals in ischemia-reperfusion injury. The involvement of the enzyme is largely based on the observation that allopurinol and pterin aldehyde, inhibitors of XDH + X0, are as effective as oxygen radical scavengers in attenuating the tissue injury associated with ischemia (11,19,20). A requisite of the hypothesis implicating X0 as a source of these reactive oxygen species is that normally occurring XDH is converted to oxygen radicalproducing X0 during the period of ischemia. Roy and McCord (27) report that 60 min of ischemia is required for 50% conversion of XDH to X0 in a 25°C liver. Stirpe and DellaCorte (29) suggest a much more rapid rate of conversion occurs at 37°C with only 10 min required for nearly complete XDH-to-X0 conversion in liver homogenates. More recently, Engerson et al. (7) reported that 50% irreversible XDH-to-X0 conversion occurred only after 3.6 h at 37OC, 20 h at 25°C and 6 days at 4OC. The results of the present study are consistent with the results of Engerson et al. (7) in that 50% conversion requires 3-4 h at 37°C with no significant conversion being observed in 4°C preserved liver for at least 8 h. McKelvey et al. (17) suggest that the rate of conversion of XDH to X0 may be more rapid in vivo than when the liver is isolated. Rat blood contains significant XDH + X0 activity under basal conditions (-2-4 mu/ml) that conceivably could alter the rate of XDH-to-X0 conversion. However, our studies suggest that the presence or absence of blood does not influence total hepatic XDH + X0 activity or the rate of XDH-to-X0 conversion. Our results are inconsistent with the assumption that endogenous XDH-to-X0 conversion occurs during the 9O- to 12O-min ischemic period used for most studies of ischemia in the isolated perfused liver. Another discordant aspect of the hypothesis invoking
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XANTHINE
a key role for X0 in ischemic tissue injury is the observation of allopurinol-mediated protection against ischemia-induced injury in tissues in which there is no detectable X0 activity. For example, allopurinol attenuates ischemic injury in porcine myocardium in which X0 activity cannot be determined using sensitive assay systems (31). Recent evidence from several studies indicate that allopurinol and its major metabolites may act as antioxidants in vitro, and it is suggested that allopurinol may be beneficial in many models of ischemia-reperfusion because of the enhancement of extracellular antioxidant properties. Das et al. (5) proposed that allopurinol salvages ischemic myocardium by scavenging neutrophilderived oxidants such as hypochlorous acid (HOCl) but not superoxide or hydroxyl radical. Moorhouse et al. (18) demonstrated that allopurinol and oxypurinol scavenge hydroxyl radical and HOCl; however, the minimal allopurinol concentration required for scavenging was 500 PM. Peterson et al. (24) provided evidence that >l,OOO PM allopurinol acts as an electron transfer agent and proposed that tissue injury is attenuated through facilitation of electron transfer during reperfusion. Plasma from animals administered allopurinol by a commonly used dosing regimen results in a lo-20 PM plasma allopurinol-oxypurinol concentration (30). Although this amount of allopurinol-oxypurinol is sufficient to inhibit the catalytic activity of X0 in the plasma, it would not be expected to enhance the antioxidant properties of extracellular fluid (18, 24, 30). The demonstration that ischemic liver, and perhaps small intestine, release XDH and X0 into the circulation upon reperfusion sheds new light on two major concerns regarding the concept that X0-mediated oxygen radical production can be injurious to ischemic tissues: 1) XDHto-X0 conversion is not requisite for formation of oxygen radicals and 2) the putative nonspecific extracellular antioxidant effects of allopurinol. There is supportive evidence in the literature for release of XDH + X0 into the circulation with hepatic dysfunction. Normal human blood contains very low or undetectable levels of XDH + X0 (21). However, XDH + X0 was elevated up to 50 times normal in the serum of patients with infectious hepatitis (10, 16, 26). Slightly elevated values were found in some cases of extrahepatic obstructive jaundice (10, 16) and in some cases of chronic renal failure (10, 16), presumably because of secondary effects on the liver. Serum XDH + X0 was reported to be a sensitive and specific indicator of acute liver damage. Although appearance of XDH + X0 was promoted as simply a passive indicator of viability of the liver and therefore useful for differential diagnosis, the released enzyme is catalytically active and may actually be responsible for amplifying ischemic injury to the liver and other tissues. These observations are consistent with reports that allopurinol is beneficial in various forms of systemic shock (4, 28). We suggest that X0 released into the circulation from ischemic liver can react with purine substrates that accumulate in the plasma during ischemia to produce oxygen radicals. The cytotoxic radicals could result in extensive endothelial and epithelial damage as this locus of
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cytotoxic reactive oxygen species moves through the circulation. It is possible that this hypothesis may implicate oxygen radicals in the pathoetiology of the widespread tissue injury referred to as multisystem failure. The authors thank Kitty Young for her secretarial expertise and Melinda Johansson for her excellent artwork and illustrations. This research was supported by National Institutes of Health Grants DK-38681, NS-24275, and NS-24338 and grant AHAfrom the Alabama Affiliate of the American Heart Association. Address for reprint requests: D. A. Parks, Dept. of Anesthesiology, 619 S. 19th St., Univ. of Alabama at Birmingham, Birmingham, AL 35233. Received
3 January
1989; accepted
in final
form
3 November
1989.
REFERENCES 1. ADKINSON, D., M. E. HOLLWARTH, J. N. BENOIT, D. A. PARKS, J. M. MCCORD, AND D. N. GRANGER. Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol. Stand. 126, Suppl. 548: 101-107, 1986. ’ 2. BISHOP, C. T., Z. MIRZA, J. D. CRAPO, AND B. A. FREEMAN. Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions. In Vitro Cell. Deu. Biol. 21: 229-235, 1985. 3. BRADFORD, M. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72: 240-254, 1970. 4. CROWELL, J. W., C. E. JONES, AND E. E. SMITH. Effect of allopurinol on hemorrhagic shock. Am. J. Physiol. 216: 744-748, 1969. 5. DAS, D. K., R. M. ENGELMAN, R. CLEMENT, H. OTANI, M. R. PRASAD, AND P. S. RAO. Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage. Biochem. Biophys. Res. Commun. 148: 314-319,1987. 6. DELLACORTE, E., AND F. GOZETTI. Properties of the xanthine oxidase from human liver. Biochim. Biophys. Acta 191: 164-166, 1969. 7. ENGERSON, T. D., T. G. MCKELVEY, D. B. RHYNE, E. B. BOGGIO, S. J. SNYDER, AND H. P. JONES. Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. J. CZin. Invest. 79: 15641570, 1987. 8. FREEMAN, B. A., AND J. D. CRAPO. Biology of disease: free radicals and tissue injury. Lab. Inuest. 47: 412-426, 1982. 9. FRIDOVICH, I. Xanthine oxidase. In: Handbook of Methods for Oxygen RadicaZ Research, edited by R. A. Greenwald. Boca Raton, FL: CRC, 1985, p. 51-54. 10. GILER, S. H., 0. SPERLING, S. BROSK, I. URCA, AND A. DEVRIES. Serum xanthine oxidase in jaundice. Clin. Chim. Acta 63: 37-40, 1975. 11. GRANGER, D. N., J. M. MCCORD, AND D. A. PARKS. Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 90: 80-84, 1986. 12. GRANGER, D. N., G. RUTILLI, AND J. M. MCCORD. Superoxide radicals in feline intestinal ischemia. Gastroenterology 81: 22-29, 1981. 13. HENRY, R. J., N. CHIAMORI, 0. J. GOLUB, AND S. BERKMANN. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am. J. Clin. Pathol. 34: 381-398, 1960. 14. KATO, S., I. ISHII, S. KANO, S. HAGIHARA, T. TODOROKI, S. NAGATA, H. TAKAHASHI, Y. SGIGETA, AND M. TSUCHIYA. Alcohol dehydrogenase: a new sensitive marker of hepatic centrilobular damage. Alcohol 2: 35-38, 1985. 15. SAUGSTAD, 0. D., M. HALLMAN, J. L. ABRAHAM, B. EPSTEIN, C. COCHRANE, AND L. GLUCK. Hypoxanthine and oxygen induced lung injury: a possible basic mechanism of tissue damage? Pediatr. Res. 18: 501-504, 1984. 16. MCHALE, A., H. GRIMES, AND M. P. COUGHLAN. Human serum xanthine oxidase: fluorometric assay applicable to the investigation of liver disorders. Int. J. Biochem. 10: 317-319, 1979. 17. MCKELVEY, T. G., M. E. HOLLWARTH, D. N. GRANGER, T. D.
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CIRCULATING
XANTHINE
ENGERSON, U. LANDLER, AND H. P. JONES. Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G753-G760, 1988. 18. MOORHOUSE, P. C., M. GROOTVELD, B. HALLIWELL, J. G. QUINLAN, AND J. M. C. GUTTERIDGE. Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett. 213: 23-28, 1987. 19. PARKS, D. A., G. B. BULKLEY, D. N. GRANGER, S. R. HAMILTON, AND J. M. MCCORD. Ischemic injury to the cat small intestine: role of superoxide radicals. Gastroenterology 82: 9-15, 1982. 20. PARKS, D. A., AND D. N. GRANGER. Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G285-G289, 1983. 21. PARKS, D. A., AND D. N. GRANGER. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol. Stand. 126, Suppl.
548:87-100,1986. 22.
23. 24.
D. A., T. K. WILLIAMS, AND J. S. BECKMAN. Conversion of xanthine dehydrogenase to oxidase in ischemic rat intestine: a re-evaluation. Am. J. Physiol. 254 (Gastrointest Liver Physiol. 17): G768-G774,1988. PERRY, M. A., S. WADHWA, D. A. PARKS, W. PICKERD, AND D. N. GRANGER. Role of oxygen radicals in ischemia-induced lesion in the cat stomach. Gastroenterology 90: 362-368, 1986. PETERSON, D. A., B. KELLY, AND J. M. GERRARD. Allopurinol can act as an electron transfer agent. Is this relevant during reperfusion injury? Biochem. Biophys. Res. Commun. 137: 76-79, 1986. PARKS,
25.
OXIDASE PODZUMEIT, T., W. BRAUN, A. MULLER, AND W. SCHAPER. Arrhythmias and infarction in the ischemic pig heart are not mediated by xanthine oxidase-derived free radicals. Basic Res. Cardiol. 82:
493-505,1987. 26.
RAMBOER,
30.
ZIMMERMAN, GRANGER.
C., F. PIESSENS, AND J. DEGROOTE. Serum xanthine oxidase and liver disease. Digestion 7: 183-195, 1972. 27. ROY, R.S., AND J. M. MCCORD. Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxygen Radicals and Their Scavenger Systems. Cellular and Medical Aspects, edited by R. A. Greenwald and G. Cohen. New York: Elsevier, 1983, vol. 2, p. 145-153. 28. SAEZ, J. C., B. GUNTHER, P. H. WARD, AND E. VIVALDI. Effect of allopurinol on survival rates of mice and rats submitted to different forms of shock. IRCS Med. Sci. 11: 292-293, 1983. 29. STIRPE, F., AND E. DELLA CORTE. The regulation of rat liver xanthine oxidase. Conversion in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type 0). J. Biol. Chem. 244:
3855-3863,1969. B. J., D. A. PARKS, M. B. GRISHAM, AND D. N. Allopurinol does not enhance the antioxidant properties of extracellular fluid. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): HZOZ-H206,1988. 31. EDDY, L. J., J. R. STEWART, H. P. JONES, T.. D. ENGERSON, J. M. MCCORD, AND J. M. DOWNEY. Free radical-producing enzyme, xanthine oxidase, is undetectable in human hearts. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H709-H711, 1987.
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mediator
YOSHIFUMI YOKOYAMA, JOSEPH S. BECKMAN, TANYA K. BECKMAN, JOAN K. WHEAT, THOMAS G. CASH, BRUCE A. FREEMAN, AND DALE
A. PARKS
Departments of Anesthesiology, Physiology, and Biophysics and Biochemistry, University of Alabama at Birmingham, Birmingham, Alabama 35233 YOKOYAMA, YOSHIFUMI, JOSEPH S. BECKMAN, TANYA K. BECKMAN, JOAN K. WHEAT, THOMAS G. CASH, BRUCE A. FREEMAN, AND DALE A. PARKS. Circulating xanthine oxidase:
mediator of ischemic injury. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G564-G570,1990.-Reactive oxygen metabolites generated from the enzyme xanthine oxidase (X0) play an important role in the pathogenesis of ischemiainduced tissue injury. The observation that intracellular proteins such as aspartate transaminase (AST) and alcohol dehydrogenase (ADH) are released from the ischemic liver during reperfusion led us to postulate that X0 could be released into the systemic circulation. Livers from fasted rats were extirpated, perfused with oxygenated Krebs-Henseleit buffer, and subjected to 2 h ischemia followed by 2 h reperfusion. Reperfusion increased AST in the perfusate from 1 k 1 to 830 -I- 280 U/l, whereas ADH increased from 0.3 t 0.1 to 95 t 26 U/l. Concomitantly, xanthine dehydrogenase (XDH) + X0 activity in the perfusate increased from 0 to 4.1 k 1.0 mu/ml. A 64% decrease in endogenous tissue XDH + X0 activity paralleled release of XDH + X0. The XDH + X0 activity predicted to appear in the circulation after hepatic ischemia was sufficient, when supplied with substrate, to produce severe vascular endothelial injury in vitro, even in the presence of serum or whole blood. These results suggest that massive quantities of XDH and X0 are released into the circulation after hepatic ischemia and that the resulting reactive oxygen metabolites could produce widespread tissue injury.
potential
ischemia; liver; free radicals; xanthine dehydrogenase; endothelium
REACTIVE OXYGEN metabolites play an important role in the pathogenesis of the structural and functional alterations associated with reperfusion of a wide variety of ischemic tissues. Xanthine oxidase (X0) has been postulated to be a primary source of these cytotoxic oxygen radicals. Evidence supporting involvement of X0 is based primarily on the observation that allopurinol, an inhibitor of xanthine dehydrogenase (XDH) and oxidase, is as effective as oxygen radical scavengers in attenuating the tissue injury associated with ischemia-reperfusion. In accordance with the X0 hypothesis, endogenous XDH is converted to oxygen radical-producing X0 during the ischemic period (12, 22). Concomitantly, cellular ATP is catabolized to hypoxanthine, which accumulates intracellularly. On reperfusion (reoxygenation), X0 can react with hypoxanthine and molecular oxygen to produce the highly reactive oxygen metabolites superoxide, hydrogen G564
0193-1857/90
$1.50
Copyright
peroxide, and hydroxyl radical. There remains considerable controversy as to the role that X0 and allopurinol play in ischemia-induced tissue injury. In contrast with the X0 hypothesis, conversion of XDH to X0 does not seem to be requisite for tissue injury, since hepatocellular injury, as evidenced by release of cytoplasmic enzymes, is observed before significant conversion of endogenous XDH to X0 (7, 17). It is conceivable that measurements of the rate and extent of XDH-to-X0 conversion in these studies were influenced by the significant XDH + X0 activity of normal rat blood. In addition, the specificity of allopurinol as an inhibitor of X0 has been questioned. Allopurinol has been postulated to directly scavenge free radicals (5, 18, 24) and minimize tissue injury in porcine myocardium where X0 activity is undetectable (25). These data would suggest that allopurinol may exert its effect through a mechanism other than through inhibition of X0. It is well documented that ischemic liver releases cellular enzymes upon reperfusion (1). Since the liver is extremely rich in XDH + X0 activity (6, 29), it is conceivable that XDH and X0 are released into the circulation concomitant with release of other hepatocellular enzymes. Circulating X0 could have tremendous potential for oxygen radical production during postischemic reperfusion and could 1) result in direct injury to the vascular endothelium, 2) activate oxidant-producing inflammatory cells and extend oxidant-induced injury to tissues distal to the site of origin, and 3) produce reactive oxygen metabolites in a relatively antioxidant-poor compartment, the plasma, and result in extension of tissue injury. It was the purpose of the present study to determine whether ischemic liver was capable of releasing XDH or X0 into the circulation, which could serve as a potential source of circulating oxygen radical production resulting in tissue injury at sites distal to its origin. MATERIAL
AND
METHODS
Biochemical Analyses Bovine milk X0 was obtained from Calbiochem (La Jolla, CA). Stock solutions were made daily by centrifuging 25 ,~l of X0 suspended in 2 M (NH&SO4 for 2 min in a microcentrifuge (5,000 g). The supernatant was discarded and the sedimented crystals resuspended in the appropriate buffer. All enzymes and reagents were purchased from Sigma Chemical (St. Louis, MO). Aspar-
0 1990 the American
Physiological
Society
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CIRCULATING
XANTHINE
tate aminotransferase (AST) was determined using standard assays (13). Alcohol dehydrogenase (ADH) activity was measured using the procedure of Kato et al. (14). Protein was quantified according to Bradford (3) using bovine serum albumin as a standard. The activity of X0 and XDH were assayed in tissue homogenate and perfusate by monitoring formation of urate at 295 nm as previously described (22). Units of enzyme activity were defined as 1 pmol of urate formed/min at 25°C and pH 7.8. Both XDH + X0 and X0 reactions were completely inhibited by 60 FM allopurinol. The extent of conversion of XDH to X0 (%X0) was calculated from the activity of X0 divided by total XDH + X0 activity. Surgical Procedures Male Sprague-Dawley rats (n = 54) weighing 320-380 g were fasted 24 h before surgery but were allowed water ad libitum. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip), a tracheotomy was performed, and the animals were artificially ventilated with room air. The liver was extirpated as previously described (1) and placed in an environmental chamber, which minimized evaporative water loss and maintained liver temperature. Isolated perfused liver studies. The livers were perfused through the portal vein at a constant pressure of 15 cmH20 with a modified, hemoglobin-free Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 27 NaHC03, 2.5 CaCIZ, 1.2 MgSO,, 1.2 KH2P04, 0.05 EDTA, and 11 glucose, plus 300 U heparin). The perfusate was maintained at constant temperature (37°C) and pH (7.4) and equilibrated with 95% OZ-5% CO2 to achieve an influent PO, of 300-500 mmHg. XDH-to-X0 conversion studies. Livers were placed in an environmental chamber immediately after extirpation or after 5 min perfusion through the portal vein with Euro-Collins solution (in mM: 99.3 KCl, 15.1 KH2P04, 9 K2HP04, 10 NaHC03, and 194 glucose, pH 7.4) to remove cellular elements. Vascular endothelial toxicity studies. Bovine aortic endothelial cells were isolated from freshly obtained vessels by gently scraping the lumen of the vessel with a scalpel. Cells were cultured in medium 199, 5% fetal calf serum (Hyclone Labs., Logan, UT), and 5% Ryan’s growth supplement (Dr. Una Ryan, Univ. of Miami, FL) and were maintained at 37°C in 95% air-5% CO,. Endothelial cell purity, assessed by cell uptake of DiL-AC-low density lipoprotein (Biomedical Technologies, Stoughton, MA), was >99%. Cells were passaged by scraping with a rubber policeman and subcultured in a 1 to 3 ratio. Cellular responses to X0 were studied using low population doubling level cultures (2). Experimental
Protocols
Isolated perfused liver studies. For this series of experiments, animals (n = 30) were randomly divided into four groups before hepatectomy. Each liver was perfused at 20 ml/min in a single-pass arrangement for 30 min to rinse the organ free of blood and to permit equilibration. This initial 30-min perfusion served as the control period
OXIDASE
G565
for each liver. Group 1 nonischemic control (n = 10) livers were processed immediately after the 30-min equilibration period. Group 2 (n = 10) livers were perfused for 240 min with oxygenated Krebs-Henseleit buffer (nonischemic, perfused). Group 3 (n = 10) livers were subjected to 2 h global normothermic ischemia followed by 2 h reperfusion under conditions identical to the initial 30-min (preischemic) period. Hepatic inflow (portal vein) and outflow (inferior vena cava) perfusion pressures were monitored via pressure transducers (Statham P23 ID) interposed within the circuit and continuously recorded with a physiological recorder (Grass Instrument, Quincy, MA). Aliquots (20 ml) of the perfusate were collected during the control period, immediately after the occlusive period, and every 30 min of the 2-h reperfusion period for appearance of XDH, X0, and hepatocellular enzymes. Blood gases were periodically monitored with a blood gas analyzer (Instrumentation Labs., ILl301). Tissue XDH, X0, and %X0 were determined at the end of the reperfusion period. XDH-to-X0 conuersion studies. In this series of experiments, rats (n = 24) were divided into four groups before hepatectomy. The livers from groups 1 and 2 were maintained at either 37°C or 4°C. Livers of groups 3 and 4 were perfused with either 37°C or 4°C Euro-Collins solution and then maintained at 37°C and 4°C respectively. Biopsies were obtained for XDH and X0 determination after 0, 1, 3,4, 6, and 8 h preservation in EuroCollins solution. XDH and X0 activity were determined in hepatic biopsies. Hepatic tissue biopsies (-1 g) were quickly excised and immersed in 10 ml of ice-cold homogenizing buffer (in mM: 50 K+-phosphate buffer, 10 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and 0.1 EDTA, pH 7.4) supplemented with 0.25 M sucrose. The tissue was homogenized using a motorized Potter-Elvehjem homogenizer and Teflon pestle and then centrifuged at 40,000 g at 4°C for 30 min. The supernatant was decanted and centrifuged for an additional 15 min. Perfusate samples were collected from the inferior caval vein. A portion of the sample was immediately added to the homogenizing buffer and centrifuged at 900 g for 15 min, and the supernatant was processed for XDH and X0 activity. Enzyme determinations were made in the remaining portion of the perfusate. Size exclusion chromatography on prepacked Sephadex G-25 columns (22) of the rat liver homogenate had no effect on total activity, %X0, or inhibition by allopurinol (data not shown). Endothelial toxicity studies. Cells were subcultured to confluency on 12-well tissue plates (Costar, Cambridge, MA) having 2.2 cm2 cell surface area (d-l.5 x 10' cells). Cells were prelabeled for 3 h with 4 PM 8- [“Cl adenine (55.1 mCi/mmol) having no cold carrier in complete medium and washed with Hanks’ balanced salt solution (HBSS). Cells exposed to 500 PM xanthine t 5.0 mu/ml X0 were incubated in HBSS plus varying concentrations of fetal calf serum. In a separate series of experiments, the effect of heparinized whole blood on X0-mediated cytotoxicity was evaluated in cultured endothelial cells. Control-labeled cells were lysed with 0.5% Triton X-100 in distilled water and sonicated for determination of total cellular uptake of 8- [ 14C]adenine.
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G566
CIRCULATING
XANTHINE
After 4 h incubation under different conditions, aliquots of media were removed (n = 12) and analyzed for release of 8- [“Cl adenine (plus metabolites) by liquid scintillation counting. In studies involving whole blood, cellular 8- [14C]adenine release was assessed after oxidation of hemoglobin with 30% HZOz. Statistics. The effect of ischemia on the release of hepatocellular enzymes, and XDH and X0 activity, was tested by analysis of variance with individual differences between groups analyzed by Scheffe’s post hoc test. The effect of X0 on endothelial release of intracellular adenine metabolites was tested for significance by analysis of variance with individual differences analyzed by Scheffe’s post hoc test. RESULTS
The isolated perfused rat liver preparation was stable for >4 h with no significant increases in AST, ADH, and XDH and X0 during this period (Figs. l-4). During the equilibration period, no significant differences existed between experimental groups. Two hours of hepatic ischemia (group 3) resulted in a significant increase (P c 0.05) in the appearance of all measured cellular enzymes in the perfusate. The activity of AST [glutamic-oxaloacetic transaminase (GOT)] increased from 2 t 2 to 830 t 280 U/l after reperfusion (Fig. 1). The ADH activity 1000
OXIDASE
*
5.0
T
r .'A 'E>0 ag3 F
4.0
-
3.0
-
+ 5. 3
2.0
-
1.0 0.0 Control
1 mm reperfuston
30 mln reperfuslon
60 min reperfusion
90 mm reperfusion
FIG. 3. Release of xanthine dehydrogenase plus xanthine oxidase (XDH + X0) activity from livers after 4 h perfusion and 2 h ischemiareperfusion. * P < 0.05 vs. respective control (4 h perfusion).
3.0 2.5
1 t
Control
1 min reperfusion
30 min reperfusion
60 mln reperfusion
90 mm reperfusion
FIG. 4. Xanthine oxidase (X0) activity is dramatically the perfusate of livers after ischemia-reperfusion. "P < tive control (4 h perfusion).
L6
120 mm reperfuslon
200 i I)
DA
0
. . . . . . ..m
.“.....I).....,...
*- - - - -A- - - - -A- - - - -A- ----A
i I
I I
control
I
I I
1 min reperfusion
I 1
30 min reperfusion
I I
I I
90 min reperfusion
60 min reperfusion
120 min reperfusion
FIG. 1. Effect of ischemia-reperfusion on appearance of aspartate transaminase (AST) activity in the perfusate of livers after 4 h perfusion with oxygenated buffer (A) and 2 h ischemia-reperfusion (H).
100,
I.......
i
25 9 *-
l
I
control FIG.
perfusate reperfusion
I 1
1 min reperfusion
-
-
-
l -A-
. .‘. _
I
30 min reperfusion
-
.‘..)...... - -A-
, I
60 min reperfusion
-
---
. .g-
I
90 min reperfusion
~
~
L
-#
I I
120 min reperfusion
2. Appearance of alcohol dehydrogenase (ADH) activity in the after perfusion with oxygenated buffer (A) or 2 h ischemia(M).
120 mm reperfusion
increased
in
0.05 vs. respec-
increased from 0.2 t 0.1 to 95 t 26 U/l (Fig. 2). Release of hepatocellular enzyme decreased sharply with continued reperfusion and became statistically indistinguishable from the control group after 30 min reperfusion. Total XDH + X0 activity (Fig. 3) in the perfusate was significantly increased from essentially 0 to 4.1 & 1.0 mu/ml during the first minute of reperfusion of the ischemic livers. X0 activity (Fig. 4) was also elevated dramatically in the perfusate during reperfusion of ischemic livers, increasing from undetectable to 2.0 t 0.6 mu/ml. In a constant flow preparation, if the enzyme activity and volume of collection is known, it is possible to estimate the approximate increase in XDH + X0 activity that would appear in the circulation in vivo. It is predicted that the circulating activity of XDH + X0 would increase 2.7 mu/ml above endogenous levels and X0 activity would increase ~1.3 mu/ml. The potential cytotoxicity of circulating X0 was estimated by measuring the release of previously incorporated [‘“Cl adenine from cultured vascular endothelial cells treated with xanthine (500 PM) and X0 (5.0 mU/ ml) (Fig. 5). To determine the total amount of incorporation of [14C]adenine, cells (n = 12) were sonicated in the presence of detergent and the release of intracellular radiolabel measured (18.5 t 0.5 counts/min X 10m4/2.2 cm2 cells). The radiolabeled [14C]adenine plus metabolites are primarily acid soluble under our preincubation
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CIRCULATING
XANTHINE
60
0
X
x0
x+x0
100%
serum
---X+XO+serumd
FIG. 5. Cytotoxicity of exogenously administered xanthine (X, 500 PM), xanthine oxidase (X0, 5 mu/ml), and xanthine plus xanthine oxidase (X + X0) was estimated by release of previously incorporated [14C]adenine. Addition of O-100% serum to the endothelial cells did not attenuate the X-X0 induced cytotoxicity. *P < 0.05 vs. X. "fP
0.05) reduced by the exposure of cultured cells to XOderived reactive oxygen species in the presence of 90% serum (76% of total) or whole blood (67% of total). To determine if the release of XDH + X0 associated with ischemia-reperfusion was significant enough to influence total XDH + X0 activitv of the liver. tissue
G567
OXIDASE
homogenates were assayed for XDH, X0, and %X0. Xanthine oxidase activity constit.uted 17 t 1% of total XDH + X0 activity in liver homogenates immediately after the equilibration period (group 1). Perfusion for 4 h with oxygenated buffer (group 2) significantly increased %X0 to 26 t 1. Reperfusion of ischemic livers (group 3) significantly increased X0 to 26 t 1%. Total XDH + X0 activity was 163 t 11 mU/g wet wt in control (nonperfused) livers and was not significantly decreased (Fig. 6) by either 4 h perfusion with oxygenated KrebsHenseleit buffer (142 t 10 mU/g wet wt). Two-hour ischemia-reperfusion resulted in a 64% reduction (P < 0.05) in total XDH + X0 activity (55 t 6 mU/g wet wt) compared with control livers. The release of massive amounts of XDH + X0 activity (110 mU/g wet wt) from livers subjected to ischemia-reperfusion suggests that sufficient quantities of oxidant-generating enzyme could potentially appear in the circulation to damage a variety of tissues. Although it is possible that the reduction in XDH + X0 activity could’ be partially explained by oxidant-mediated enzyme autoinactivation, the recovery of 82 mU XDH + X0 in the perfusate during just the first minute of reperfusion would suggest that a large amount of the enzyme lost from the liver appears in the circulation. Dehydrogenase- to-Oxidase Conversion Under normal conditions, the liver contains 163 t 11 mU XDH + X0/g tissue with 15-20% of the total activity existing as X0 (Fig. 7). Subjecting the liver to normothermic (37°C) ischemia for O-8 h results in up to 64% of the total activity existing as X0. There was no significant increase in %X0 in liver subjected to up to 8 h ischemia under hypothermic (4°C) conditions. It is
180 160
XDH -I-
140 E
X
XDH
60 t40 0
20 F
iii control
4h perfusion
2h ischemia 2h reDerfusion
.
FIG. 6. Effect of ischemia-reperrusion on enaogenous xantmne aehydrogenase + xanthine oxidase (XDH + X0) activity in control (nonischemic) liver, livers perfused for 4 h with oxygenated KrebsHenseleit buffer, and livers after 2 h ischemia and 2 h reperfusion. *P < 0.05 vs. control. tP < 0.05 vs. 4 h nerfusion.
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CIRCULATING
XANTHINE
80 70
1
60
1 0 -0
I 0
I 12
1I
I, I1 , (, 3 4 5 6 DURATION OF ISCHEMIA (hours)
, 7
, 8
9
FIG. 7. Changes in the relative proportion of xanthine oxidase to total xanthine dehydrogenase + oxidase activity (%X0) after O-8 h ischemia in normothermic perfused (0) or nonperfused liver (A) and hypothermic perfused (0) or nonperfused (A) liver.
assumed that the increase in X0 activity was due to XDH-to-X0 conversion, since total XDH + X0 activity was unchanged during the course of the 8-h period of ischemia in both normothermic and hypothermic groups. The presence of cellular elements did not seem to influence total XDH + X0 activity or the rate of XDH-toX0 conversion, with no significant differences observed between perfused and nonperfused livers. DISCUSSION
The results of the present study indicate that large quantities of X0 are released into the circulation from the ischemic liver upon reperfusion. We postulate that circulating X0 could react with endogenous plasma purine substrates and molecular oxygen to produce intravascular reactive oxygen species as this oxidant-generating system traverses the circulation. Our data would suggest that the XDH + X0 and X0 activity would increase in systemic circulation by 2.7 and 1.3 mu/ml, respectively, over endogenous levels. Since studies with XDH + X0 using blood from normal rats indicate that XDH is rapidly and reversibly converted to X0 after 4 min hypoxia (unpublished observation) and that XDH + X0 in the plasma appears to have a relatively long circulating half-life (26), that predicted elevation of XDH and/or X0 in the plasma would very likely damage the vascular endothelium. This contention is supported by the in vitro studies of vascular endothelium in which exogenously administered xanthine and X0, at levels predicted to exist in the plasma after hepatic ischemia, produced significant cytotoxicity. This cytotoxicity was not significantly reduced (P > 0.05) by the addition of serum or whole blood compared with cells maintained in HBSS. Interestingly, there is no apparent widespread tissue injury associated with the XDH + X0 activity (2-4 mU/ ml) that ordinarily exists in rat blood. Normally there is only l-3 PM xanthine plus hypoxanthine in the circulation (15), far less than the 11 ,uM Michaelis-Menten constant (&-J of X0 and XDH (22). Thus limited substrate concentrations may help to minimize tissue injury from endogenous levels of circulating X0. It is also
OXIDASE
intriguing that elevation of plasma purines up to 5 mM does not induce detectable tissue injury in rats (15). However, xanthine and hypoxanthine concentrations in excess of 50 PM strongly inhibit X0 activity (9). From these kinetic characteristics of X0, it can be predicted that there is a window of plasma purine concentrations that would support potentially excessive production of toxic oxygen species. Under normal conditions, XDH + X0 may be maintained predominantly in the relatively innocuous XDH form with any remaining X0-derived oxidants being adequately detoxified by extracellular antioxidants. Ischemia-reperfusion would release additional XDH or X0 and would approximately double endogenous circulating XDH + X0 activity. Changes in the extracellular environment that occur during the ischemic period could then allow reversible or irreversible conversion of XDH to X0. Antioxidants normally found in the extracellular fluid are also expected to become depleted during a prolonged period of ischemia. Thus the circulating X0 could generate sufficient oxidant to produce extensive endothelial and epithelial injury to a variety of tissues in vivo and potentially be a mediator of the multisystem failure associated with systemic shock states. Oxygen radicals can arise from a number of sources (8); however, XDH + X0 has been frequently postulated to be a primary intracellular generator of toxic oxygen radicals in ischemia-reperfusion injury. The involvement of the enzyme is largely based on the observation that allopurinol and pterin aldehyde, inhibitors of XDH + X0, are as effective as oxygen radical scavengers in attenuating the tissue injury associated with ischemia (11,19,20). A requisite of the hypothesis implicating X0 as a source of these reactive oxygen species is that normally occurring XDH is converted to oxygen radicalproducing X0 during the period of ischemia. Roy and McCord (27) report that 60 min of ischemia is required for 50% conversion of XDH to X0 in a 25°C liver. Stirpe and DellaCorte (29) suggest a much more rapid rate of conversion occurs at 37°C with only 10 min required for nearly complete XDH-to-X0 conversion in liver homogenates. More recently, Engerson et al. (7) reported that 50% irreversible XDH-to-X0 conversion occurred only after 3.6 h at 37OC, 20 h at 25°C and 6 days at 4OC. The results of the present study are consistent with the results of Engerson et al. (7) in that 50% conversion requires 3-4 h at 37°C with no significant conversion being observed in 4°C preserved liver for at least 8 h. McKelvey et al. (17) suggest that the rate of conversion of XDH to X0 may be more rapid in vivo than when the liver is isolated. Rat blood contains significant XDH + X0 activity under basal conditions (-2-4 mu/ml) that conceivably could alter the rate of XDH-to-X0 conversion. However, our studies suggest that the presence or absence of blood does not influence total hepatic XDH + X0 activity or the rate of XDH-to-X0 conversion. Our results are inconsistent with the assumption that endogenous XDH-to-X0 conversion occurs during the 9O- to 12O-min ischemic period used for most studies of ischemia in the isolated perfused liver. Another discordant aspect of the hypothesis invoking
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a key role for X0 in ischemic tissue injury is the observation of allopurinol-mediated protection against ischemia-induced injury in tissues in which there is no detectable X0 activity. For example, allopurinol attenuates ischemic injury in porcine myocardium in which X0 activity cannot be determined using sensitive assay systems (31). Recent evidence from several studies indicate that allopurinol and its major metabolites may act as antioxidants in vitro, and it is suggested that allopurinol may be beneficial in many models of ischemia-reperfusion because of the enhancement of extracellular antioxidant properties. Das et al. (5) proposed that allopurinol salvages ischemic myocardium by scavenging neutrophilderived oxidants such as hypochlorous acid (HOCl) but not superoxide or hydroxyl radical. Moorhouse et al. (18) demonstrated that allopurinol and oxypurinol scavenge hydroxyl radical and HOCl; however, the minimal allopurinol concentration required for scavenging was 500 PM. Peterson et al. (24) provided evidence that >l,OOO PM allopurinol acts as an electron transfer agent and proposed that tissue injury is attenuated through facilitation of electron transfer during reperfusion. Plasma from animals administered allopurinol by a commonly used dosing regimen results in a lo-20 PM plasma allopurinol-oxypurinol concentration (30). Although this amount of allopurinol-oxypurinol is sufficient to inhibit the catalytic activity of X0 in the plasma, it would not be expected to enhance the antioxidant properties of extracellular fluid (18, 24, 30). The demonstration that ischemic liver, and perhaps small intestine, release XDH and X0 into the circulation upon reperfusion sheds new light on two major concerns regarding the concept that X0-mediated oxygen radical production can be injurious to ischemic tissues: 1) XDHto-X0 conversion is not requisite for formation of oxygen radicals and 2) the putative nonspecific extracellular antioxidant effects of allopurinol. There is supportive evidence in the literature for release of XDH + X0 into the circulation with hepatic dysfunction. Normal human blood contains very low or undetectable levels of XDH + X0 (21). However, XDH + X0 was elevated up to 50 times normal in the serum of patients with infectious hepatitis (10, 16, 26). Slightly elevated values were found in some cases of extrahepatic obstructive jaundice (10, 16) and in some cases of chronic renal failure (10, 16), presumably because of secondary effects on the liver. Serum XDH + X0 was reported to be a sensitive and specific indicator of acute liver damage. Although appearance of XDH + X0 was promoted as simply a passive indicator of viability of the liver and therefore useful for differential diagnosis, the released enzyme is catalytically active and may actually be responsible for amplifying ischemic injury to the liver and other tissues. These observations are consistent with reports that allopurinol is beneficial in various forms of systemic shock (4, 28). We suggest that X0 released into the circulation from ischemic liver can react with purine substrates that accumulate in the plasma during ischemia to produce oxygen radicals. The cytotoxic radicals could result in extensive endothelial and epithelial damage as this locus of
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cytotoxic reactive oxygen species moves through the circulation. It is possible that this hypothesis may implicate oxygen radicals in the pathoetiology of the widespread tissue injury referred to as multisystem failure. The authors thank Kitty Young for her secretarial expertise and Melinda Johansson for her excellent artwork and illustrations. This research was supported by National Institutes of Health Grants DK-38681, NS-24275, and NS-24338 and grant AHAfrom the Alabama Affiliate of the American Heart Association. Address for reprint requests: D. A. Parks, Dept. of Anesthesiology, 619 S. 19th St., Univ. of Alabama at Birmingham, Birmingham, AL 35233. Received
3 January
1989; accepted
in final
form
3 November
1989.
REFERENCES 1. ADKINSON, D., M. E. HOLLWARTH, J. N. BENOIT, D. A. PARKS, J. M. MCCORD, AND D. N. GRANGER. Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol. Stand. 126, Suppl. 548: 101-107, 1986. ’ 2. BISHOP, C. T., Z. MIRZA, J. D. CRAPO, AND B. A. FREEMAN. Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions. In Vitro Cell. Deu. Biol. 21: 229-235, 1985. 3. BRADFORD, M. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72: 240-254, 1970. 4. CROWELL, J. W., C. E. JONES, AND E. E. SMITH. Effect of allopurinol on hemorrhagic shock. Am. J. Physiol. 216: 744-748, 1969. 5. DAS, D. K., R. M. ENGELMAN, R. CLEMENT, H. OTANI, M. R. PRASAD, AND P. S. RAO. Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage. Biochem. Biophys. Res. Commun. 148: 314-319,1987. 6. DELLACORTE, E., AND F. GOZETTI. Properties of the xanthine oxidase from human liver. Biochim. Biophys. Acta 191: 164-166, 1969. 7. ENGERSON, T. D., T. G. MCKELVEY, D. B. RHYNE, E. B. BOGGIO, S. J. SNYDER, AND H. P. JONES. Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. J. CZin. Invest. 79: 15641570, 1987. 8. FREEMAN, B. A., AND J. D. CRAPO. Biology of disease: free radicals and tissue injury. Lab. Inuest. 47: 412-426, 1982. 9. FRIDOVICH, I. Xanthine oxidase. In: Handbook of Methods for Oxygen RadicaZ Research, edited by R. A. Greenwald. Boca Raton, FL: CRC, 1985, p. 51-54. 10. GILER, S. H., 0. SPERLING, S. BROSK, I. URCA, AND A. DEVRIES. Serum xanthine oxidase in jaundice. Clin. Chim. Acta 63: 37-40, 1975. 11. GRANGER, D. N., J. M. MCCORD, AND D. A. PARKS. Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 90: 80-84, 1986. 12. GRANGER, D. N., G. RUTILLI, AND J. M. MCCORD. Superoxide radicals in feline intestinal ischemia. Gastroenterology 81: 22-29, 1981. 13. HENRY, R. J., N. CHIAMORI, 0. J. GOLUB, AND S. BERKMANN. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am. J. Clin. Pathol. 34: 381-398, 1960. 14. KATO, S., I. ISHII, S. KANO, S. HAGIHARA, T. TODOROKI, S. NAGATA, H. TAKAHASHI, Y. SGIGETA, AND M. TSUCHIYA. Alcohol dehydrogenase: a new sensitive marker of hepatic centrilobular damage. Alcohol 2: 35-38, 1985. 15. SAUGSTAD, 0. D., M. HALLMAN, J. L. ABRAHAM, B. EPSTEIN, C. COCHRANE, AND L. GLUCK. Hypoxanthine and oxygen induced lung injury: a possible basic mechanism of tissue damage? Pediatr. Res. 18: 501-504, 1984. 16. MCHALE, A., H. GRIMES, AND M. P. COUGHLAN. Human serum xanthine oxidase: fluorometric assay applicable to the investigation of liver disorders. Int. J. Biochem. 10: 317-319, 1979. 17. MCKELVEY, T. G., M. E. HOLLWARTH, D. N. GRANGER, T. D.
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ENGERSON, U. LANDLER, AND H. P. JONES. Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G753-G760, 1988. 18. MOORHOUSE, P. C., M. GROOTVELD, B. HALLIWELL, J. G. QUINLAN, AND J. M. C. GUTTERIDGE. Allopurinol and oxypurinol are hydroxyl radical scavengers. FEBS Lett. 213: 23-28, 1987. 19. PARKS, D. A., G. B. BULKLEY, D. N. GRANGER, S. R. HAMILTON, AND J. M. MCCORD. Ischemic injury to the cat small intestine: role of superoxide radicals. Gastroenterology 82: 9-15, 1982. 20. PARKS, D. A., AND D. N. GRANGER. Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G285-G289, 1983. 21. PARKS, D. A., AND D. N. GRANGER. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol. Stand. 126, Suppl.
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D. A., T. K. WILLIAMS, AND J. S. BECKMAN. Conversion of xanthine dehydrogenase to oxidase in ischemic rat intestine: a re-evaluation. Am. J. Physiol. 254 (Gastrointest Liver Physiol. 17): G768-G774,1988. PERRY, M. A., S. WADHWA, D. A. PARKS, W. PICKERD, AND D. N. GRANGER. Role of oxygen radicals in ischemia-induced lesion in the cat stomach. Gastroenterology 90: 362-368, 1986. PETERSON, D. A., B. KELLY, AND J. M. GERRARD. Allopurinol can act as an electron transfer agent. Is this relevant during reperfusion injury? Biochem. Biophys. Res. Commun. 137: 76-79, 1986. PARKS,
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OXIDASE PODZUMEIT, T., W. BRAUN, A. MULLER, AND W. SCHAPER. Arrhythmias and infarction in the ischemic pig heart are not mediated by xanthine oxidase-derived free radicals. Basic Res. Cardiol. 82:
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C., F. PIESSENS, AND J. DEGROOTE. Serum xanthine oxidase and liver disease. Digestion 7: 183-195, 1972. 27. ROY, R.S., AND J. M. MCCORD. Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxygen Radicals and Their Scavenger Systems. Cellular and Medical Aspects, edited by R. A. Greenwald and G. Cohen. New York: Elsevier, 1983, vol. 2, p. 145-153. 28. SAEZ, J. C., B. GUNTHER, P. H. WARD, AND E. VIVALDI. Effect of allopurinol on survival rates of mice and rats submitted to different forms of shock. IRCS Med. Sci. 11: 292-293, 1983. 29. STIRPE, F., AND E. DELLA CORTE. The regulation of rat liver xanthine oxidase. Conversion in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type 0). J. Biol. Chem. 244:
3855-3863,1969. B. J., D. A. PARKS, M. B. GRISHAM, AND D. N. Allopurinol does not enhance the antioxidant properties of extracellular fluid. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): HZOZ-H206,1988. 31. EDDY, L. J., J. R. STEWART, H. P. JONES, T.. D. ENGERSON, J. M. MCCORD, AND J. M. DOWNEY. Free radical-producing enzyme, xanthine oxidase, is undetectable in human hearts. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H709-H711, 1987.
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