(Received for publication, May 8, 1995; and in revised form, June 8, 1995)
From the
While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation or that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alterations in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurement of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia.
Oxygen free radical generation has been proposed to be an important mechanism of cellular injury in ischemic and reperfused tissues(1, 2) . Studies in a variety of tissues, including heart, lung, kidney, and brain, have demonstrated that intravascular administration of antioxidant enzymes or free radical-scavenging drugs can prevent reperfusion damage and improve postischemic function(3, 4) . These studies have provided indirect evidence of free radical generation in postischemic tissues. Recently, free radical generation has been measured in ischemic tissues with electron paramagnetic resonance (EPR) spectroscopy. Both direct and spin trapping EPR techniques have demonstrated that there is a burst of oxygen free radical generation in the heart after postischemic reperfusion(5, 6, 7, 8) . It was further demonstrated that the administration of copper-zinc superoxide dismutase, solely upon reperfusion, can prevent contractile dysfunction and quench free radical generation(6, 9) .
Although
several mechanisms have been demonstrated to be involved in the
generation of oxygen free radicals, xanthine oxidase has been proposed
to be a central mechanism in a variety of postischemic cells and
tissues(10, 11, 12) . Although there has been
controversy regarding whether this mechanism occurs in human cells and
tissues, recent studies have shown that the enzyme is present in human
endothelial cells and is responsible for free radical generation in
reoxygenated human endothelial cells(13) . In reperfused
tissues, xanthine oxidase in the presence of its substrates
hypoxanthine or xanthine reduces molecular oxygen to
O and H
O
, which
can further react to form the more reactive
OH(14, 15) . The OH and O
radicals produced by the enzyme can then in turn react with
cellular proteins and membranes inducing cellular injury. In
particular, it was hypothesized that in ischemic tissues xanthine
dehydrogenase, which reduces NAD to NADH, is converted via proteolytic
cleavage to xanthine oxidase(10, 16) . This increase
in xanthine oxidase activity has been proposed to play a key role in
triggering postischemic free radical generation. It has been
demonstrated that the enzyme and its substrates are present and give
rise to a burst of free radical generation upon postischemic
reperfusion in the isolated rat heart, as well as in bovine and human
endothelial cells, but considerable controversy still remains regarding
the process by which ischemia and reperfusion triggers xanthine
oxidase-mediated free radical
generation(12, 13, 15) . Despite extensive
study over the last decade, the regulation and control of this
important mechanism of free radical generation and cellular injury is
largely unknown. Questions remain regarding whether it is enzyme or
substrate formation that triggers the burst in free radical generation
which occurs upon postischemic
reperfusion(10, 16, 17, 18, 19, 20, 21, 22) .
The precise effect of alterations in myocardial enzyme and substrate
concentrations in controlling the time course of free radical
generation is also unknown. Therefore, controversy has remained
regarding the role of xanthine oxidase and its radical-generating
substrates in triggering the presence and time course of free radical
generation in the postischemic heart.
In the present study, experiments were performed to evaluate the role of alterations in xanthine oxidase and its substrates hypoxanthine and xanthine during myocardial ischemia and reperfusion in triggering the presence and controlling the time course of free radical generation in the postischemic rat heart. Experiments were performed correlating the time course of free radical generation, with alterations in xanthine oxidase activity and the concentrations of its free radical-generating substrates, during the process of myocardial ischemia and reperfusion. These studies indicate that substrate availability is the primary factor in triggering and controlling xanthine oxidase-mediated free radical generation in the postischemic heart.
EPR spectra were recorded in flat cells at room temperature with a Bruker-IBM ER 300 spectrometer operating at X-band with a TM 110 cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20 milliwatts, microwave frequency of 9.77 GHz, and acquisition of 10 1-min scans. Quantitation of the free radical signals was performed by comparing the double integral of the observed signal to that of a known concentration of the 2,2,6,6-tetramethylpiperidinoxy free radical in aqueous solution as described previously(12, 13) .
A separate series of hearts was studied with collection of effluent perfusate prior to ischemia and after reflow to determine the time course of xanthine oxidase substrate and product release in the coronary effluent. 30-s collections of perfusate were performed at each sampling time, and these perfusate samples were simply passed through 0.45-µm Millipore filters prior to HPLC analysis.
Reversed-phase HPLC was performed using a procedure similar to that of Hull-Ryde et al.(24) using a Waters µBondapak C18 column and a Waters the HPLC system (Waters Associates, Milford, MA) with a model 484 UV detector, two model 510 reciprocating pumps, and Maxima software, as described previously(13) .
Figure 1: EPR spectra prior to ischemia (control) and over the time course of reflow measured from the coronary effluent of a heart infused with 40 mM DMPO prior to and after 30 min of global ischemia. Spectra were recorded at room temperature with a microwave frequency of 9.77 GHz using 20 milliwatts of microwave power and a 0.5-G modulation amplitude. While no significant signal was seen prior to ischemia, prominent signals were observed after reflow consisting of a combination of the 1:2:2:1 quartet of DMPO-OH and the 1:1:1:1:1:1 sextet of DMPO-R.
Figure 2: Graph of the time course of total free radical generation in the pre- and postischemic rat heart. Data were obtained from the intensity of the EPR spectra observed from the coronary effluent of hearts perfused with 40 mM DMPO as described in the legend to Fig.1. Data are shown with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the complete time course of free radical generation after the 30-min period of ischemia.
Both control and postischemic heart tissue contained significant xanthine oxidase and xanthine dehydrogenase activity. As shown in the representative tracings (Fig.3), prominent xanthine oxidase activity was present and corresponded to about 7% of the total xanthine oxidase plus xanthine dehydrogenase activity. After 30-min ischemia and 10-min reperfusion the activity of xanthine oxidase was increased by about 30%. A series of hearts were studied to characterize the time course of alterations in xanthine oxidase and xanthine dehydrogenase activity during ischemia and reflow (Fig.4). In preischemic hearts, the activity of xanthine oxidase and xanthine dehydrogenase were 8.4 ± 1.2 and 130 ± 28 milliunits/g of protein, respectively. After 30 min of ischemia, there was no significant change in total xanthine oxidase and xanthine dehydrogenase activity. However, the activity of xanthine oxidase markedly increased to 16.8 ± 1.2 (p < 0.05, versus preischemia) after 1-min reperfusion. Over the subsequent 45-min period of reperfusion the activity of xanthine oxidase remained 30-50% higher than that observed in preischemic control hearts. While some decrease in the activity was seen between 2 and 10 min of reflow, overall the activity of xanthine dehydrogenase was not significantly altered during the time course of reflow. In plots of the ratio of xanthine oxidase activity to xanthine oxidase plus xanthine dehydrogenase activity it was observed that the percent xanthine oxidase increased from base-line values of 7% prior to ischemia to peak values of 11% after reflow. Since only a small increase was seen, this indicates that at most only a small fraction of the xanthine dehydrogenase may have been converted to xanthine oxidase. While the increases in xanthine oxidase activity observed during reperfusion could contribute to the increase in free radical generation which occurs upon reperfusion, these changes are relatively modest and cannot explain the absence of radical generation prior to ischemia or the time course which occurs.
Figure 3:
Kinetics of urate production in the
spectrophotometric assay for xanthine oxidase (XO) and
xanthine dehydrogenase (XD) in representative rat hearts prior
to ischemia (dashed line) or after 30 min of ischemia followed
by 10 min of reflow (solid line). Absorbance at 295 nm is
graphed verses the reaction time. Tissue was homogenized in the
presence of PMSF + DTT, centrifuged at 105,000 g,
and eluted through a Sephadex column. The reaction mixture contained
200 µl of eluent, 50 mM phosphate buffer, and 60
µM xanthine in a final volume of 1 ml for the
determination of xanthine oxidase activity at 37 °C. Measurements
of xanthine dehydrogenase activity were performed in the presence of
0.6 mM NAD
.
Figure 4: Graphs showing the time course of alterations in xanthine oxidase (XO), xanthine dehydrogenase (XD), and the ratio of XO to (XO+XD) in the rat hearts prior to ischemia and after 30-min ischemia with different durations of reperfusion. Data are shown with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the complete time course throughout the 45-min period of reflow.
Figure 5:
Representative chromatograms of
reversed-phase HPLC nucleotide separation. A 10-µm µbondapak
C18 column (3.9 150 mm) was used with a 20-µl injection.
The peaks are labeled as follows: A, uric acid; B,
ATP; C, ADP; D, hypoxanthine; E, xanthine; F, AMP. The top inset shows a chromatogram of a
mixture of standards containing a 100 µM concentration of
each compound (a). The chromatograms from extracts of
preischemic, 30-minute ischemic and 5-minute reflow hearts are shown in insets b, c, and d, respectively. In the
preischemic heart, no hypoxanthine or xanthine peaks are seen. After
30-min ischemia, the ATP peak is markedly decreased and hypoxanthine
and xanthine peaks appear. No significant hypoxanthine and xanthine
peaks were observed after 5 min of
reperfusion.
Figure 6: Time course of the alterations in ATP, hypoxanthine, and xanthine concentrations in isolated rat hearts during ischemia and reperfusion. Data are shown from preischemic hearts, hearts subjected to 30-min global ischemia, and hearts subjected to different durations of reflow. The data are graphed with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the entire time course throughout the 45-min period of reflow. Each point corresponds to the mean ± S.E. values obtained from three hearts.
Two possible pathways may be involved in the rapid decline of hypoxanthine and xanthine in the myocardium during the early period of reperfusion. One possibility is that hypoxanthine and xanthine were converted by xanthine oxidase to form uric acid with accompanying oxygen free radical generation. Another possibility is that reflow may wash out these substrates from the heart. To determine the release of xanthine oxidase substrates and products, coronary effluent was collected in 30-s intervals prior to ischemia and after postischemic reperfusion. 33 samples were studied from a series of 3 hearts, and HPLC measurements were performed on each sample. Representative chromatograms of the effluent from a heart prior to and after reflow are shown in Fig.7. Prior to ischemia no hypoxanthine and xanthine peaks were seen, and no significant uric acid peak was detected in coronary effluent. After reflow, however, large hypoxanthine and xanthine peaks were observed along with a large peak from the xanthine oxidase product uric acid (Fig.7b). Maximum substrate and product peaks were seen over the first 30 s of reflow and then peak height declined over the next 5 min until only trace peaks were seen. The amount of xanthine, hypoxanthine, and uric acid in each 30 s effluent sample was quantitated. In the effluent from the first 30 s of reflow, average values of 1.1 ± 0.3 and 3.1 ± 0.8 nmol were observed for hypoxanthine and xanthine, respectively, with a value of 2.5 ± 0.5 nmol for uric acid. The observed amounts of both the substrates hypoxanthine and xanthine as well as the product uric acid rapidly decreased in the following 2 min of reflow, until after 5-min reperfusion, no hypoxanthine, xanthine, or uric acid was measured in coronary effluent (Fig.8). The time course observed for xanthine oxidase substrate and product formation in the heart tissue and coronary effluent closely correlated with that observed in the EPR spin trapping measurements of free radical generation, suggesting that the presence and time course of free radical generation was triggered and controlled primarily by the availability of enzyme substrates.
Figure 7: Representative chromatograms obtained from samples of coronary effluent prior to or after 30 min of ischemia. Peaks are labeled as in Fig.5. The chromatograms shown are from samples obtained prior to ischemia (a) or after reperfusion for 30 s (b), 60 s (c), 120 s (d), or 5 min (e). Large xanthine oxidase substrate peaks of hypoxanthine (D) and xanthine (E) are observed along with a large product peak of uric acid (A) during the first 5 min of reperfusion.
Figure 8: Graph of the amount of xanthine, hypoxanthine, and uric acid measured in the coronary effluent from preischemic and reperfused hearts by HPLC. Data are shown with a break in the reperfusion time axis between 150 and 300 s, after which the scale is condensed in order to depict the complete time course before ischemia and throughout the 45-min period of reflow. Each point corresponds to the mean ± S.E. value obtained from measurements of three hearts.
Oxidative reperfusion injury is thought to be a central mechanism of the cellular damage affecting all organs and tissues after ischemia; however, the mechanisms which trigger and modulate this damage are still only partially characterized. Xanthine oxidase has been proposed to be a major primary source of free radical generation. This hypothesis was initially supported by many studies which demonstrated that administration of the xanthine oxidase inhibitors allopurinol or oxypurinol reduced infarct size and improved functional recovery(17, 25) ; however, subsequently these results have been questioned by other laboratories who have questioned whether the enzyme xanthine oxidase or its substrates are present and whether it can cause reperfusion injury (26, 27) . More recently, further direct evidence for the presence of xanthine oxidase-mediated free radical generation in postischemic cells and tissues has been provided by EPR studies(12, 13, 15) .
It has been demonstrated that a burst of xanthine oxidase-mediated radical generation occurs upon reperfusion and is the major source of free radicals in the isolated rat heart. This burst of radical generation can be blocked by the xanthine oxidase inhibitor oxypurinol (15) . In the present study we observe that the burst of radical generation peaks during the first minute of reperfusion with little, if any, radical generation continuing after 5 min. A number of studies have shown that endothelial cells from animal species as well as humans contain xanthine oxidase and similarly give rise to the formation of oxygen free radicals at the time of reoxygenation(12, 13, 15) . In human aortic endothelial cells we have demonstrated that free radical generation occurs due to xanthine oxidase substrate formation in the presence of the enzyme. However, in these isolated cellular preparations the observed free radical generation persists for more than 30 min after reoxygenation. Therefore, while xanthine oxidase-mediated free radical generation has been observed both in isolated endothelial cells and whole tissues such as the heart, clear differences in the time course of radical generation are present that could not be explained previously. Although there is considerable evidence that xanthine oxidase-mediated free radical generation occurs in reperfused tissues, the precise nature of the enzyme and substrate control of the magnitude and time course of this process had not been elucidated.
Both the
formation and modulation of tissue concentrations of xanthine oxidase
and its requisite substrates are factors that would be expected to
limit and regulate the generation of free radicals in the pathogenesis
of reperfusion injury. McCord (10, 11) proposed that
in ischemic tissues xanthine dehydrogenase undergoes proteolytic
conversion to the oxidase that uses O as its electron
acceptor with the formation of superoxide and hydrogen peroxide. This
protease-mediated increase in xanthine oxidase activity was
hypothesized to play a major role in triggering the generation of
oxygen free radicals. Subsequently, it was proposed that this
protease-dependent xanthine oxidase conversion was the major event
triggering xanthine oxidase-mediated reperfusion injury (16, 17, 18) . However, subsequent reports (28, 29) have shown little if any conversion of
dehydrogenase to oxidase in perfused rat hearts following periods of
global ischemia. Similarly, in the present study we did observe that
xanthine oxidase is present in the preischemic rat heart, with 7% of
the dehydrogenase plus oxidase pool present as oxidase. While a
50-100% increase in xanthine oxidase activity was observed upon
reperfusion, still only a small proportion of the xanthine
dehydrogenase appeared to be converted to xanthine oxidase. Our results
clearly demonstrate that although xanthine oxidase activity is somewhat
increased in the postischemic heart, these alterations of xanthine
oxidase activity alone do not explain the time course of free radical
generation that is observed in the normal and postischemic heart. Prior
to ischemia, xanthine oxidase is present, but no free radical
generation is observed. Although the enzyme activity is increased after
ischemia, it is largely unchanged throughout the time course of reflow;
however, free radical generation is only observed during the first 5
min of reperfusion. These data indicate that although the presence of
xanthine oxidase is an important requisite factor in the process of
free radical generation, alterations in xanthine oxidase activity are
not sufficient to account for the observed time course of radical
generation and are not the major limiting factor in the process of free
radical generation during postischemic reperfusion.
It was also well known that during ischemia ATP is degraded to ADP, AMP, and adenosine, which could be further metabolized to inosine, hypoxanthine, and xanthine(21, 30) . McCord (10) also proposed that this process of increased substrate formation could be an important factor in triggering xanthine oxidase-mediated free radical generation. In general, however, the presence and importance of this substrate control has been less emphasized in the literature. Abd-Elfattah et al.(21, 22, 31) have noted with in vivo surgical canine models of global ischemia and reflow that xanthine oxidase substrate formation is an important factor in the process of postischemic injury and have shown that pharmacological interventions aimed at decreasing substrate concentrations can decrease the severity of this injury.
In the present study, we have observed that the
alterations in myocardial xanthine oxidase substrate concentrations can
explain the time course of radical generation which is observed. In
preischemic hearts, no hypoxanthine and xanthine can be detected.
During ischemia, hypoxanthine and xanthine accumulated due to the
breakdown of ATP to AMP, adenosine, inosine, and finally hypoxanthine.
At the end of ischemia, radical-generating substrate concentrations
reach a maximal level in the myocardium. Upon reflow, O is
reintroduced, then xanthine oxidase metabolizes xanthine and
hypoxanthine with the formation of uric acid and the reduction of
O
to superoxide and hydrogen peroxide. Myocardial substrate
levels then rapidly decrease after the start of reflow, due to both
substrate metabolism and washout, with the presence of the product uric
acid as well as the substrates hypoxanthine and xanthine in the
coronary effluent. Therefore, substrate limitation results in the
gradual decline of free radical generation that is observed, despite
the presence of xanthine oxidase. These results demonstrate that the
burst of xanthine oxidase-mediated free radical generation that occurs
in the postischemic rat heart is largely triggered and controlled by
substrate availability.
From the data obtained in this study it is
possible to consider the roles of in vivo substrate control in
the process of postischemic free radical generation. Metabolite
concentrations within the heart can be estimated from the measured
number of micromoles per gram of cell water, to increase by from near
0, less than 0.5 µM, prior to ischemia, to values of 576
or 628 µM for hypoxanthine and xanthine, respectively,
after 30-min ischemia. This more than 1000-fold increase would be
expected to have a marked effect on radical generation from the enzyme.
The K of xanthine oxidase for xanthine
has been reported to be in the range of 11-36
µM(33, 34, 35) , therefore at
least a 44-144-fold increase in radical generation would be
expected. EPR spin trapping studies demonstrated that there was a
marked increase in radical generation in the reperfused heart with more
than a 40-fold increase seen. In studies of plasma concentrations of
xanthine oxidase substrates in normal human plasma, similar low
concentrations of hypoxanthine or xanthine have been reported with
values in the range of 0.3-0.9
µM(36, 37) . Thus, in in vivo myocardial ischemia as occurs in human patients with coronary
artery disease, xanthine oxidase substrate concentrations would be
expected to have a similarly important role in modulating the process
of radical generation from the enzyme.
In the present experiments, myocardial flow was observed to be an important factor that effects the time course of xanthine oxidase-mediated free radical generation in the postischemic heart. Although it has been previously recognized that reflow is a critical event, since it results in reoxygenation and triggers the onset of oxygen free radical generation after the near anoxic conditions which occur during ischemia, it had not been considered that sustained flow could subsequently limit the duration of radical generation. In the present study, it was observed that myocardial flow is an important factor that effects the time course of substrate clearance and limits the duration of xanthine oxidase mediated free radical generation by decreasing the pool of available substrates. After postischemic reflow, large amounts of xanthine and hypoxanthine were present in the coronary effluent from the heart, and this substrate clearance paralleled the decrease in myocardial substrate concentration that was seen. This important effect of reflow may explain why xanthine oxidase-mediated free radical generation persists for a longer time in suspensions of reoxygenated endothelial cells than in the reperfused heart. In these static endothelial cell preparations, the high substrate concentrations observed (13) would be expected to remain elevated for prolonged periods of time in the absence of flow.
Since substrate availability plays a limiting role in the process of free radical generation within postischemic myocardium, pharmacological approaches that decrease substrate formation may provide an alternative approach to that of radical scavenging compounds or direct xanthine oxidase blockers at preventing free radical generation and subsequent oxidant-mediated myocardial reperfusion injury. These approaches may also exert other beneficial effects, including salvage of high energy phosphates and increasing adenosine concentrations within the heart (32) . Conversely, increased xanthine oxide substrate formation that would occur from increased ATP degradation due to increased severity of supply demand imbalance during myocardial ischemia would be expected to result in increased xanthine oxidase-mediated free radical generation and postischemic injury, further exacerbating the process of myocardial injury which occurs secondary to ATP depletion.
In conclusion, we have characterized the presence and time course of xanthine oxidase-mediated free radical generation in the postischemic heart, as well as the nature of enzyme or substrate control which occurs. Xanthine oxidase activity was present in the preischemic heart and significantly increased during ischemia and reperfusion. However, the observed time course of radical generation was largely triggered and controlled by the formation of the radical-generating substrates hypoxanthine and xanthine that accumulated during ischemia.