Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oxidant generation in anoxia-reoxygenation and ischemia-reperfusion was compared in isolated rat lungs. Anoxia-reoxygenation was produced by N2 ventilation followed by O2 ventilation. After anoxia, lung ATP content was decreased by 59%. Oxygenated ischemia was produced by discontinuing perfusion while ventilation with O2 was maintained. With anoxia-reoxygenation, oxidant generation, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 3.6-fold, lung thiobarbituric acid reactive substances (TBARS) increased 342%, conjugated dienes increased 285%, and protein carbonyl content increased 46%. Pretreatment of lungs with 100 µM allopurinol inhibited the reoxygenation-mediated increase in lung fluorescence by 75% and TBARS by 69%. Oxygenated ischemia resulted in an approximately eightfold increase in lung H2DCF oxidation and a fourfold increase in TBARS, but allopurinol had no effect. On the other hand, 100 µM diphenyliodonium (DPI) inhibited the ischemia-mediated increase in lung fluorescence by 69% and lung TBARS by 70%, but it had no effect on the increase with anoxia-reoxygenation. Therefore, both ischemia-reperfusion and anoxia-reoxygenation result in oxidant generation by the lung, but a comparison of results with a xanthine oxidase inhibitor (allopurinol) and a flavoprotein inhibitor (DPI) indicate that the pathways for oxidant generation are distinctly different.
free radicals; lipid peroxidation; nicotinamide adenine dinucleotide phosphate oxidase; oxidative stress; xanthine oxidase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE BIOCHEMICAL MECHANISMS for cell damage during ischemia and postischemic reperfusion have been described for heart, brain, intestine, kidney, and other organs (13, 26). In these organs, ischemia is invariably accompanied by tissue anoxia, whereas reperfusion reintroduces O2. Therefore, ischemia-reperfusion in essence is equated with anoxia-reoxygenation. A significant biochemical change in tissues during the ischemic phase is the sharp decrease in levels of ATP and phosphocreatine and the corresponding increase in the ATP degradation products hypoxanthine and xanthine. The latter are substrates for the enzyme xanthine oxidase that can generate superoxide when O2 reenters the tissue with reperfusion (26).
It is important to recognize that ischemia-reperfusion in lungs is physiologically different from that in organs with systemic circulation (18). Lung ischemia, for example that seen with pulmonary artery occlusion, does not necessarily result in tissue anoxia because lung ventilation can continue. Supporting evidence is the observation that the ATP content of lung tissue does not change significantly during ischemia in continuously ventilated isolated lungs (18). Therefore, ischemia-reperfusion in lungs does not equate to anoxia-reoxygenation. However, as with anoxia-reoxygenation in systemic organs, lung ischemia-reperfusion does result in oxidant generation and oxidative damage to lung tissue (2-4, 7, 16, 18). Oxidant generation actually occurs during the ischemic phase, provided that tissue oxygenation is maintained through ventilation.
A basic question is whether anoxia with reoxygenation, as distinct from ischemia-reperfusion, results in lung oxidant generation and, if so, whether the mechanisms may differ. Oxidant generation with reoxygenation has been demonstrated in atelectatic lungs after reexpansion (22), although additional factors associated with lung collapse could have contributed to the observed oxidant injury. In the present study, we utilized the isolated rat lung ventilated with gases of varying O2 content to separate anoxia-reoxygenation effects from possible changes due to ischemia or the mechanical effect of atelectasis. We used the isolated lung to evaluate mechanisms and biochemical pathways rather than to simulate pathophysiological conditions. We found generation of oxidants with anoxia-reoxygenation similar to that observed with lung ischemia with or without reperfusion. However, based on response to inhibitors, we found that the pathways for oxidant generation differ in the two models.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
Allopurinol, aprotinin, ATP, butylated hydroxytoluene,
2,4-dinitrophenylhydrazine (DNPH), glucose-6-phosphate dehydrogenase, hexokinase, leupeptin, -NADP, pepstatin, phenylmethylsulfonyl fluoride (PMSF), and thiobarbituric acid (TBA) were purchased from
Sigma Chemical (St. Louis, MO).
2',7'-Dichlorodihydrofluorescein (H2DCF) diacetate was obtained
from Molecular Probes (Eugene, OR). Diphenyliodonium chloride (DPI) was
obtained from ICN Biochemicals (Cleveland, OH). Protein assay reagent
concentrate and
-globulin were from Bio-Rad Laboratories (Richmond,
CA). All other chemicals used were of analytic grade.
Lung perfusion. The isolated perfused rat lung model used for this study has been described previously (17). Briefly, lungs were isolated from Sprague-Dawley male rats (Charles River Breeding Laboratories, Kingston, NY) weighing 180-200 g and anesthetized with 30 mg/kg intraperitoneal pentobarbital sodium while ventilation was maintained at 60 cycles/min at 2 ml tidal volume and 2 cmH2O end-expiratory pressure. The pulmonary circulation was cleared of blood by gravity flow of perfusate with 25 cmH2O pressure through a cannula inserted in the main pulmonary artery. The perfusate medium, Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose and 3% (wt/vol) fatty acid-free bovine serum albumin, was preincubated with the same gas mixture subsequently used for lung ventilation. The cleared lungs freed of cardiac and other nonpulmonary tissues were suspended in a water-jacketed perfusion chamber maintained at 37°C. Perfusion was maintained using a peristaltic pump at a constant flow rate of 10 ml/min with a recirculating volume of 40 ml. Global ischemia was produced by discontinuing perfusion for 1 h while ventilation was continued. In some experiments, ischemic lungs were reperfused for 1 h after the 1 h of ischemia. To produce anoxia, lungs were ventilated with either 95% N2-5% CO2 or 95% CO-5% CO2 while perfusion was maintained; for all other experimental conditions, lungs were ventilated with 95% O2-5% CO2 (all gases were supplied by BOC Group, Bellmawr, NJ). Perfusate PO2, measured at 30 and 60 min of perfusion, was ~650 mmHg for the oxygenated (control) perfusion and <15 mmHg for N2 ventilation (anoxia). In some experiments, allopurinol or DPI was added to the perfusate 30 min before ischemia or anoxia. At the end of the perfusion experiment, the lungs were rapidly frozen by clamping with aluminum tongs precooled in liquid N2.
Measurement of oxidant generation.
Generation of oxidants in lung tissue was monitored with
H2DCF diacetate fluorescence as
previously described (3). The nonfluorescent molecule is converted by
intracellular esterases to H2DCF,
which serves as a substrate for intracellular oxidants to generate
highly fluorescent dichlorofluorescein (DCF). Briefly, a 5 mM solution
of H2DCF diacetate was prepared in
absolute ethanol, stored under N2
at 20°C in darkness, and added to the lung perfusate to make
a final concentration of 5 µM. Lungs were perfused with the
fluorophore for 30 min before initiation of anoxia or ischemia. The
frozen lungs were homogenized, and fluorescence of the homogenate was
determined using a spectrofluorometer (model MPF-2A; Hitachi Perkin-Elmer) with 488 nm excitation and 530 nm emission.
Determination of lipid peroxidation.
For assay of thiobarbituric acid reactive substances (TBARS), frozen
lung tissue was homogenized under
N2 in 10 volumes of ice-cold 0.9%
sodium chloride containing 0.2% butylated hydroxytoluene. An aliquot
of the homogenate was extracted with trichloroacetic acid (TCA) and was
reacted with TBA at 95°C for 15 min. TBARS were calculated by using
an extinction coefficient of 1.56 × 105
M1 · cm
1 (11). To
validate the TBARS assay, high-performance liquid chromatography
(HPLC), using a LKB 2150 pump and LKB 2140 spectral detector system
(LKB-Produkter, Bromma, Sweden), was performed in two
experiments to separate TBA-malondialdehyde adducts from chromogens
absorbing at 532 nm that may arise from the reaction of TBA with other
components of the lung homogenate (12). The TBA reaction mixture (250 µl) was injected onto a C-18 column (Pharmacia Biotech, Piscataway,
NJ) and was eluted with 65% 50 mM
KH2PO4-KOH
(pH 7.0)-35% methanol at a flow rate of 1 ml/min. The two assay
methods gave essentially the same results for these two experiments for
both control and ischemic reperfused rat lungs. Under control
conditions, TBARS were 39.7 ± 2.8 pmol/mg protein (mean ± range) after TCA extraction and 42.4 ± 4.1 pmol/mg protein with
HPLC. For ischemia-reperfusion, TBARS were 144 ± 7.6 pmol/mg protein after TCA extraction and 147 ± 3.4 pmol/mg protein with HPLC. For assay of conjugated dienes, a portion of the lung homogenate was extracted (9) and assayed as previously described (16). Briefly,
the organic fraction was evaporated under
N2, and the lipid residue was
dissolved in chloroform-methanol (1:6, vol/vol) for measurement
of absorbance at 233 nm. Concentration of conjugated dienes is
expressed as milli-optical density units per milligram protein.
Determination of protein oxidation.
Protein oxidation was determined by reaction with DNPH as described
previously (7). Briefly, frozen lungs were homogenized under
N2 in 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 7.4) containing (in mM) 137 NaCl, 4.6 KCl, 1.1 KH2PO4,
and 0.6 MgSO4; protease inhibitors
(0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 µg/ml aprotinin,
and 40 µg/ml PMSF); and 1.1 mM EDTA. The homogenate was centrifuged
at 100,000 g for 5 min. The soluble
protein in the supernatant was incubated with 10 mM DNPH. The DNPH
derivative of the oxidized protein was separated from the unbound DNPH
by Sephadex G-25 column chromatography and was measured
spectrophotometrically. Protein carbonyl content was calculated by
using an extinction coefficient of 21 mM1 · cm
1 at 360 nm.
Other biochemical assays.
Lung ATP content was measured as previously described (8). Briefly, a
portion of frozen lung tissue was extracted with cold ethanolic
perchloric acid and was assayed enzymatically using reactions coupled
to hexokinase and glucose-6-phosphate dehydrogenase. Xanthine oxidase
activity was measured in lung homogenates (30). Frozen lungs were
homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, and 1 mM PMSF. The homogenate was
centrifuged at 25,000 g at
4°C for 30 min. The resultant supernatants were chromatographed to
remove endogenous substrates on Sephadex G-25 columns (Pharmacia
Biotech) equilibrated with extraction buffer. The column eluate was
incubated at 25°C for 5 min with 100 µM xanthine as the
substrate. Uric acid formation was measured spectroscopically at 295 nm
using the maximum linear rate and a millimolar extinction coefficient
of 12.2. One unit of xanthine oxidase activity represents the
conversion of 1 µmol of xanthine to uric acid/min. The protein
content of the lung homogenate was measured by the Coomassie blue
method using bovine -globulin as the standard (10).
Statistical analysis. Data are summarized as means ± SE. Data were analyzed statistically by one-way analysis of variance for multiple comparisons followed by Bonferroni's test using SigmaStat software (Jandel Scientific, San Rafael, CA). The level of statistical significance was taken as P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATP content. Lung ATP content was significantly decreased during anoxia. At the end of 1 and 2 h of N2 ventilation, the mean level of ATP had decreased by 59 and 70%, respectively (P < 0.05, n = 4; Table 1). After 1 h of reoxygenation following 1 h of anoxia, ATP content was restored by 31% but had not returned to the control level. The change in ATP content when lungs were ventilated for 1 h with 95% CO was similar to that for ventilation with N2 (Table 1). CO and N2 were compared because the former inhibits cytochrome oxidase in addition to replacing O2 in the ventilating gas and precludes an effect of undetected gas leaks (8). The similar ATP change with the two different gases showed that N2 ventilation indeed resulted in tissue anoxia.
|
Oxidant generation with anoxia-reoxygenation. Anoxia-reoxygenation of rat lungs resulted in increased generation of oxidants as evidenced by increased fluorescence of DCF, the oxidized product of H2DCF (Fig. 1). Fluorescence intensity of DCF in homogenate of lungs subjected to 1 h of N2 ventilation followed by 1 h of O2 ventilation increased ~360% compared with control (continuously oxygenated lungs; P < 0.05, n = 4; Fig. 1). Anoxia-reoxygenated lungs perfused in the absence of fluorophore showed no change in fluorescence (data not shown).
|
Tissue oxidation with anoxia-reoxygenation. Lipid peroxidation was stimulated by anoxia-reoxygenation in isolated perfused rat lungs. TBARS at the end of the reoxygenation period were increased 240% (P < 0.05, n = 4) above the value for control lungs that were continuously perfused for 2 h (Fig. 2). No change in TBARS was observed after ventilation with N2 for 2 h (Fig. 2). Conjugated dienes with anoxia-reoxygenation essentially paralleled the observations for TBARS and increased by 156% above control (P < 0.05, n = 4; Table 2).
|
|
Oxidant generation and tissue oxidation with ischemia-reperfusion. Compatible with our previous results (3, 4, 18), ischemia resulted in increased oxidant generation and lipid peroxidation as detected by lung DCF fluorescence and TBARS. DCF fluorescence (Fig. 1) and TBARS (Fig. 2) of lung tissue after 1 h of oxygenated ischemia were significantly increased by 8.1- and 3.9-fold, respectively, compared with control. DCF fluorescence and TBARS after reperfusion (1 h after 1 h of ischemia) were essentially unchanged from the end-ischemic values (Figs. 1 and 2).
Effect of allopurinol and DPI. Addition of a xanthine oxidase inhibitor, 100 µM allopurinol, to the perfusate before anoxia-reoxygenation inhibited the subsequent increase in lung DCF fluorescence by 75% and the increase in TBARS by 69% (Fig. 3). By contrast, allopurinol added to the perfusate before ischemia had no effect on the subsequent increase in lung fluorescence or TBARS (Fig. 3). DCF fluorescence at the end of 1 h of reperfusion after 1 h of ischemia also was unchanged by pretreatment with allopurinol (data not shown). Xanthine oxidase activity of lung homogenate after ischemia (1.36 ± 0.14 mU/mg protein; n = 4) and after anoxia-reoxygenation (1.26 ± 0.06 mU/mg protein; n = 4) were not significantly different and essentially were not different from control (1.10 and 1.14 mU/mg protein; n = 2). Xanthine oxidase activity of lung homogenate was inhibited completely by the addition of 50 µM allopurinol. Activity in lungs was not evaluated after perfusion with allopurinol because this low-molecular-weight inhibitor is removed by the column chromatography.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous data from this laboratory have demonstrated that isolated lungs produce oxidants during ischemia, provided that tissue oxygenation is maintained through continued ventilation. Oxidant generation and tissue oxidative injury during ischemia were indicated in isolated lungs by oxidation of fluorophores, by depletion of tissue glutathione, and by tissue lipid and protein oxidation (3, 4, 6, 7, 16, 18). In the oxygenated ischemic model, oxidative injury depended on ventilation gas PO2 and was prevented by ventilation with N2 (3, 4, 7, 18). The present results confirm the oxidation of H2DCF and lipid during oxygenated ischemia.
The present study shows that anoxia-reoxygenation also leads to oxidant generation in isolated rat lungs. As expected, oxidant generation was not detected with anoxia alone but required the reintroduction of O2. DCF fluorescence increased 3.6-fold in lungs subjected to anoxia-reoxygenation, and oxidation of lipid (TBARS and conjugated dienes) and protein also indicated increased oxidant generation. Oxidation of H2DCF can occur via H2O2 (plus intracellular peroxidases), lipid hydroperoxides, peroxynitrite, · OH, and perhaps other oxidants so that this fluorophore is not a specific indicator but can be considered a general index for generation of reactive O2 species (ROS; see Refs. 3 and 14). Compared with oxygenated ischemia, the changes in lung fluorescence and protein oxidation observed with anoxia-reoxygenation were somewhat less, although lipid peroxidation was similar. The increase in TBARS may reflect, in part, flux through cyclooxygenase/lipoxygenase pathways in addition to nonspecific lipid peroxidation (7). Therefore, both ischemia and anoxia-reoxygenation result in oxidant generation and tissue oxidant injury. Our previous results with combined ischemia and anoxia-reoxygenation are compatible with an additive effect on tissue TBARS (16).
In the present study, the expected decrease in lung ATP content with anoxia was observed. Degradation of ATP can provide the substrate to xanthine oxidase for generation of oxidants. Evidence that the generation of oxidants with anoxia-reoxygenation occurs, at least in part, through the xanthine oxidase pathway was obtained by addition of allopurinol to the perfusate before anoxia. This agent markedly attenuated the increase in DCF fluorescence and TBARS with reoxygenation. At the concentration used in this study, allopurinol is a xanthine oxidase inhibitor that has been studied widely for protection against oxidant-mediated ischemia-reperfusion injury in various organs and tissues such as heart (19), kidney (31), liver (24), skeletal muscle (5), and intestine (32). Allopurinol also has been studied in several models of lung ischemia (anoxia)-reperfusion (reoxygenation). Kennedy et al. (25) and Adkins and Taylor (1) observed in isolated rabbit lungs that ischemia (2-3 h) in the absence of ventilation followed by reperfusion (with reventilation) resulted in an oxidant-mediated microvascular injury that was significantly attenuated by 100 µM allopurinol. These published reports indicate that xanthine oxidase is involved in lung damage associated with a nonventilated lung model of ischemia-reperfusion. The present findings suggest that it is the tissue anoxia component with nonventilated ischemia that is responsible for increased flux through the xanthine oxidase pathway with reoxygenation and the protective effect of allopurinol. A recent immunohistochemical study has demonstrated a widespread distribution of xanthine oxidase in bronchial and alveolar tissues of the rat lung (27). Therefore, various cellular sites may be involved in the allopurinol-sensitive generation of oxidants with anoxia-reoxygenation, although the precise cells involved remain to be determined.
Several findings indicate that the pathways for oxidant generation in anoxia-reoxygenation and oxygenated ischemia differ. First, the effect of ischemia is not due to ATP depletion because previous data from this laboratory have shown that lung tissue ATP content is not significantly changed with ischemia when ventilation is maintained (2, 18). Second, in contrast to anoxia-reoxygenation, oxidant generation during oxygenated ischemia was not inhibited by allopurinol. Finally, addition of DPI to the perfusate before oxygenated ischemia significantly attenuated the increase in DCF fluorescence and TBARS but had no significant effect on oxidant generation with anoxia-reoxygenation.
DPI is a probe that binds and inhibits flavoproteins and has been
widely used for assessing flavin-linked NADPH oxidase activity in both
phagocytic and nonphagocytic cells (15, 20, 23, 28). The observed
inhibitory effect of DPI on oxidant production in the isolated lung
suggests a role for NADPH oxidase in oxidant generation by rat lungs
subjected to oxygenated ischemia. The presence of this enzyme
complex in endothelium has been suggested, and the presence of several
key components has been demonstrated in human umbilical vein
endothelial cells (23). Al-Mehdi et al. (2-4) previously have
demonstrated that pulmonary capillary endothelium is a major site for
ischemia-mediated oxidant generation. Therefore, we postulate that
endothelial generation of ROS occurs through the NADPH oxidase pathway.
Alternatively, DPI could limit H2DCF and tissue oxidation, at
least in part, through inhibition of NO synthase (29), thereby
inhibiting the formation of
ONOO. The latter has been
shown to contribute to oxidative stress in lung ischemia-reperfusion
(21). Other flavin-linked oxidases also represent potential
DPI-sensitive pathways for generation of oxidants with lung ischemia
and cannot as yet be excluded.
Although endothelium appears to be the predominant cellular source of oxidants with lung ischemia, other cells types, such as the macrophage that is known to have NADPH oxidase activity (20), may contribute to the lung oxidant burden. As discussed previously (18), polymorphonuclear leukocytes (PMN) are not present in significant numbers and therefore do not appear to play a significant role in oxidant generation in this isolated perfused lung preparation. However, PMN could contribute to O2-derived radical production via the NADPH oxidase or other pathways in vivo or in isolated blood-perfused lungs.
In summary, our studies demonstrate that lung ATP content decreases during anoxia but not during oxygenated ischemia. In isolated blood-free lungs, both ischemia with or without reperfusion and reoxygenation after anoxia lead to oxidant generation and tissue oxidation. Inhibition of xanthine oxidase with allopurinol attenuates this effect in reoxygenated lungs but not in ischemic lungs. On the other hand, DPI, an inhibitor of flavoproteins such as NADPH oxidase, attenuates oxidant generation with ischemia but has no significant effect with anoxia-reoxygenation. We conclude that the generation of oxidants occurs through distinctly different pathways with ischemia and with reoxygenation after anoxia.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Chandra Dodia and Dr. Raymond Foust III for assistance with the high-performance liquid chromatography assay for thiobarbituric acid reactive substances, Dr. S. Lahiri for assistance with perfusate gas measurements, and E. Primerano for typing the manuscript.
![]() |
FOOTNOTES |
---|
This research was supported by National Heart, Lung, and Blood Institute Grant HL-41939 and was presented in part at the Experimental Biology '97 meeting in New Orleans, Louisiana, April 6-10, 1997.
Address for reprint requests: A. B. Fisher, Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068.
Received 5 May 1997; accepted in final form 22 August 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adkins, W. K.,
and
A. E. Taylor.
Role of xanthine oxidase and neutrophils in ischemia-reperfusion injury in rabbit lung.
J. Appl. Physiol.
69:
2012-2018,
1990
2.
Al-Mehdi, A.,
H. Shuman,
and
A. B. Fisher.
Endothelial depolarization as an initiator of lung ischemic injury (Abstract).
Am. J. Respir. Crit. Care Med.
149:
A552,
1994.
3.
Al-Mehdi, A.,
H. Shuman,
and
A. B. Fisher.
Fluorescence microtopography of oxidative stress in lung ischemia-reperfusion.
Lab. Invest.
70:
579-587,
1994[Medline].
4.
Al-Mehdi, A. B.,
H. Shuman,
and
A. B. Fisher.
Intracellular generation of reactive oxygen species during nonhypoxic lung ischemia.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L294-L300,
1997
5.
Asami, A.,
M. Orii,
N. Shirasugi,
M. Yamazaki,
Y. Akiyama,
and
M. Kitajima.
The effect of allopurinol on interstitial purine metabolism and tissue damage in skeletal muscle I-R injury.
J. Card. Surg.
37:
209-216,
1996.
6.
Ayene, I. S.,
A. Al-Mehdi,
and
A. B. Fisher.
Inhibition of lung tissue oxidation during ischemia/reperfusion by 2-mercaptopropionylglycine.
Arch. Biochem. Biophys.
303:
307-312,
1993[Medline].
7.
Ayene, I. S.,
C. Dodia,
and
A. B. Fisher.
Role of oxygen in oxidation of lipid and protein during ischemia/reperfusion in isolated perfused rat lung.
Arch. Biochem. Biophys.
296:
183-189,
1992[Medline].
8.
Bassett, D. J. P.,
and
A. B. Fisher.
Metabolic response to carbon monoxide by isolated lungs.
Am. J. Physiol.
230:
658-663,
1976[Medline].
9.
Bligh, E. B.,
and
W. J. Dyer.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37:
911-917,
1959.
10.
Bradford, M. M.
A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
11.
Buege, J. A.,
and
S. D. Aust.
Microsomal lipid peroxidation.
Methods Enzymol.
52:
302-310,
1978[Medline].
12.
Chirico, S.
High-performance liquid chromatography-based thiobarbituric acid test.
Methods Enzymol.
233:
314-318,
1994[Medline].
13.
Cross, C. E.,
B. Halliwell,
E. T. Borish,
W. A. Pryor,
B. N. Ames,
R. L. Saul,
J. M. McCord,
and
D. Harman.
Oxygen radicals and human disease.
Ann. Intern. Med.
107:
526-545,
1987[Medline].
14.
Crow, J. P.
Dichlorodihydrofluorscein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species.
Nitric Oxide: Biol. Chem.
1:
145-157,
1997.[Medline]
15.
Doussiere, J.,
and
P. V. Vignais.
Inhibition of generating oxidase of neutrophils by iodonium biphenyl in a cell free system: effect of the redox state of the oxidase complex.
Biochem. Biophys. Res. Commun.
175:
143-151,
1991[Medline].
16.
Eckenhoff, R. C.,
C. Dodia,
Z. Tan,
and
A. B. Fisher.
Oxygen-dependent reperfusion injury in the isolated rat lung.
J. Appl. Physiol.
72:
1454-1460,
1992
17.
Fisher, A. B.
The isolated perfused lung.
Handb. Exp. Pharmacol.
75:
149-179,
1985.
18.
Fisher, A. B.,
C. Dodia,
Z. Tan,
I. Ayene,
and
R. G. Eckenhoff.
Oxygen-dependent lipid peroxidation during lung ischemia.
J. Clin. Invest.
88:
674-679,
1991[Medline].
19.
Gimpel, J. A.,
J. R. Lahpor,
A. J. van der Molen,
J. Damen,
and
J. F. Hitchcock.
Reduction of reperfusion injury of human myocardium by allopurinol: a clinical study.
Free Radic. Biol. Med.
19:
251-255,
1995[Medline].
20.
Hancock, J. T.,
and
O. T. Jones.
The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.
Biochem. J.
242:
103-107,
1987[Medline].
21.
Ischiropoulos, H.,
A. Al-Mehdi,
and
A. B. Fisher.
Reactive species in ischemic rat lung injury: contribution of peroxynitrite.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L158-L164,
1995
22.
Jackson, R. M.,
W. J. Russell,
and
C. F. Veal.
Endogenous and exogenous catalase in reoxygenation lung injury.
J. Appl. Physiol.
72:
858-864,
1992
23.
Jones, S. A.,
V. B. O'Donnell,
J. D. Wood,
J. P. Broughton,
E. J. Hughes,
and
O. T. G. Jones.
Expression of phagocyte NADPH oxidase components in human endothelial cells.
Am. J. Physiol.
271 (Heart Circ. Physiol. 15):
H1626-H1634,
1996
24.
Karwinski, W.,
R. Garcia,
and
W. S. Helton.
Allopurinol dose is important for attenuation of liver dysfunction after normothermic ischemia: correlation between bile flow and liver enzymes in circulation.
Res. Exp. Med. (Berl.)
194:
321-327,
1994[Medline].
25.
Kennedy, T. P.,
N. V. Rao,
C. Hopkins,
L. Pennington,
E. Tolley,
and
J. R. Hoidal.
Role of reactive oxygen species in reperfusion injury of the rabbit lung.
J. Clin. Invest.
83:
1326-1335,
1989[Medline].
26.
McCord, J. M.
Oxygen-derived free radicals in postischemic tissue injury.
N. Engl. J. Med.
312:
159-163,
1985[Abstract].
27.
Moriwaki, Y.,
T. Yamamoto,
K. Yamaguchi,
S. Takahashi,
and
K. Higashino.
Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies.
Histochem. Cell Biol.
105:
71-79,
1996[Medline].
28.
O'Donnell, B. V.,
D. G. Tew,
O. T. Jones,
and
P. J. England.
Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.
Biochem. J.
290:
41-49,
1993[Medline].
29.
Stuehr, D. J.,
O. A. Fasehun,
N. S. Kwon,
S. S. Gross,
J. A. Gonzalez,
R. Levi,
and
C. F. Nathan.
Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs.
FASEB J.
5:
98-103,
1991
30.
Terada, L. S.,
J. A. Leff,
and
J. E. Repine.
Measurement of xanthine oxidase in biological tissues.
Methods Enzymol.
186:
651-656,
1990[Medline].
31.
Vaughan, D. L.,
Y. A. Wickramasinghe,
G. I. Russell,
M. S. Thorniley,
R. F. Houston,
E. Ruban,
and
P. Rolfe.
Is allopurinol beneficial in the prevention of renal ischemia-reperfusion injury in the rat? Evaluation by near-infrared spectroscopy.
Clin. Sci. (Lond.)
88:
359-364,
1995[Medline].
32.
Vaughan, W. G.,
J. W. Horton,
and
P. B. Walker.
Allopurinol prevents intestinal permeability changes after ischemia-reperfusion injury.
J. Pediatr. Surg.
27:
968-973,
1992[Medline].