Increased Serum Catalase Activity in Septic Patients with the Adult Respiratory Distress Syndrome 1- 3
JONATHAN A. LEFF,4 POLLY E. PARSONS,s CAROLINE E. DAY, ERNEST E. MOORE, FREDRICK A. MOORE, MARTHA A. OPPEGARD, and JOHN E. REPINE
Introduction
Alterations in oxidant-antioxidant balance may be an important cause and/or response in patients with the adult respiratory distresssyndrome (ARDS) (1). Accelerated oxidant generation is suggested becauseARDS patients haveincreased hydrogenperoxide (H 20 2) concentrations in their breath (2, 3); increased concentrations of blood xanthine oxidase (XO), an endothelial cell-based enzyme that makes superoxide anion (0 2,) and H 20 2 (4); and blood neutrophils that make more oxygen metabolites in vitro (5). Indeed, it is likelythat excessH 202 production and related oxygen metabolite derivatives, such as hypochlorous acid (HOCl) or hydroxyl radical (·OH), account for the increased levels of oxidatively inactivated antiproteases in ARDS patients (6). Moreover, H 20 2 and/or its products can synergistically increase the toxicity of neutrophil elastase, cause acute edematous injury in isolated perfused lungs, and induce leak of protein through cultured lung endothelial cell monolayers (7-10). Despite recognition of the potential for increased generation of H 20 2 in patients with ARDS, relevant H 202-combatting antioxidant enzyme activities have not been evaluated, even though they might be an important part of the pathogenesis and could provide markers of prognosis or response to therapy (11). Moreover, exogenously administered catalase and/or enhanced glutathione redox reactions reduce H 202 concentrations and decrease endothelial cell and lung injury in various ARDS models (12, 13). Furthermore, endogenous increases in H 20 rinactivating enzymes can decrease oxidant damage (14-16). In the present investigation, we assessed H 202 scavenging, catalase, and glutathione peroxidase (GPX) activity of serum from septic patients with or without ARDS and control subjects. We found increased degrees of catalase activity in serum from septic patients and then investigated the
SUMMARY Excessive hydrogen peroxide (H,O,) generation appears to contribute to the development of the adult respiratory distress syndrome (ARDS), but H,O,-combattlng antioxidant defenses have not been evaluated. We found that serum from septic patients with ARDS scavenged more (p < 0.05) H,O, in rltro (82.7 ± 3.8%) than did serum from septic patients without ARDS (56.9 ± 3.1%) or control subjects (20.2 ± 2.4%). Serum from septic patients with ARDSalso had more (p < 0.05) catalase activity (54.9 :!: 10.9 U/ml) than did serum from septic patients without ARDS (28.6 ± 3.4 U/ml) or control subjects (7.3 :!: 0.8 U/ml). In contrast, serum from septic patients with or without ARDSand control subjects had the same glutathione peroxidase (GPX)activity. Serum H,O, scavenging activity correlated with serum catalase (r = o.n) but not GPX (r = 0.33) activity and was Inhibitable (> 90%) by sodium azide, a catalase Inhibitor. Increases In serum catalase activity did not appear to be derived from erythrocytes (RBC) because septic patients with or without ARDS and control sUbjects had similar RBC hemolysis In response to osmotic stress In rltro and serum haptoglobin concentrations. Serum from septic patients with ARDS also protected endothelial cells against H,O,-medlated damage (34.5 ± 2.2% 51Cr release) better (p < 0.05) than serum from septic patients without ARDS (47.3 ± 7.4%) or control sUbjects (82.1 ± 10.2%), but killing of bacteria by neutrophlls in r/tro was the same In serum from patients and control sUbJects. Our findings Indicate that patients with sepsis and/or ARDS have Increased serum catalase activity, which may alter H,O,-dependent processes. AM REV RESPIR DIS 1992; 148:885-888
source and possible significance of this alteration. Methods Subject Population After written consent was obtained from the patient or a family member, the patient was subjected to a protocol that had been previously approved by our institutional human subjects review committee. All patients who were identified within 8 h of the initial diagnosis of sepsis were eligible for enrollment. Patients with sepsis had a serious bacterial infection plus rectal or core temperature > 390 C and/or peripheral leukocyte counts of > 12,000 cells/mm" or > 200/0 immature neutrophils, as well as at Jeast one of the foJlowing:a positive blood cuJtureof a commonly accepted pathogen; a strongly suspected or proven source of systemic infection; gross pus in a closed space; unexplained systemic arterial hypotension (systolic blood pressure < 80 mm Hg); systemic vascular resistance < 800 dyne/s/cm'; unexplained metabolic acidosis (17). Patients with ARDS met the following criteria: (1) acute respiratory failure requiring mechanical ventilation, (2) bilateral pulmonary infiltrates, (3) pulmonary capillary wedge pressure < 18 mm Hg, (4) static pulmonary compliance < 50 mllcm H 2 0 , and (5) arterial to alveolar partial pressure of oxy-
gen ratio of < 0.25 (17). Blood samples were obtained at enrollment (zero h) and at 6, 12, 24, and 48 h thereafter. Serum samples were obtained through indwelling arterial or venous catheters and/or by direct venipuncture and stored at -70 0 C. Septic patients with or without ARDS had similar numbers of central venous, arterial, and peripheral blood withdrawals.
(Received in original form January 30, 1992 and in revised form May 15, 1992) I From the Webb-Waring Lung Institute, and the Departments of Medicine and Surgery and Denver General Hospital at the University of Colorado Health Sciences Center, Denver, Colorado. , Supported in part by Grants No. HL 40784 and HL 45582 from the National Institutes of Health, the Colorado Heart Association, the Council for Tobacco Research Inc., Ronald McDonald Children's Charities, Johnson & Johnson, Inc., the Williams Family Foundation, Swan, Hill, Donner, Kleberg, Sachs, and the American Express Foundation. 3 Correspondence and requests for reprints should be addressed to Jonathan A. Leff, M.D., Webb-Waring Institute, 4200 East Ninth Avenue, Box C322, Denver, CO 80262. • Recipient of a Clinician-Scientist Award from the American Heart Association. S Recipient of Clinical Investigator Award No. HL-0I849 from the National Institutes of Health.
985
SERUM CATALASE ACTIVITY IN ARDS AND SEPSIS
986 TABLE 1 CLINICAL CHARACTERISTICS OF SEPTIC PATIENTS WITH OR WITHOUT ARDS AT STUDY ENTRY·
Parameter Age, yr Gender, mit Hematocrit, % Hemoglobin, gldl Blood leukocyte count, x 1,000 Absolute blood neutrophil, count, x 1,000 Serum SGOT, Ulml Serum bilirubin, mgldl Serum albumin, g1dl Serum uric acid, mgldl APACHE II score Mortality, 0/0
Septic Patients Without ARDS 60 ± 3 (20) 10110 35 ± 3 (13) 12 ± 1 (11) 17±3(11) ± 3 (10) ± 67 (7) ± 1.0 (6) ± 0.2 (16) 5.7 ± 0.8 (12) 15±2{18) 6/20 (30%)
16 191 2.6 2.8
Septic Patients With ARDS 51 ± 4 (9) 7/2 35 ± 3 (5) 12 ± 1 (5) 14 ± 4 (5) ± 5 (4) ± 31 (4) ± 0.1 (4) ± 0.3 (6) ± 0.5 (5) ± 2 (6) 4/9 (44%)
13 52 0.6 2.6 4.5 13
Definition of abbreviation: SGOT = serum glutamic-oxaloacetic transaminase. • Values are the mean ::t: SE. The number of determinations is given in parentheses.
Patients were divided into two groups: those who had sepsis without ARDS (serum withdrawn at 6, 12, 24, and 48 h after the initial diagnosis of sepsis in patients who did not develop ARDS) and those who had sepsis with ARDS (serum withdrawn from septic patients at all time points after the diagnosis of ARDS). Serum samples were assayed individually at each time point, and then all time points were averaged for each patient, yielding one value for each patient. Patients were prospectively and sequentially followed until death or discharge. Patients with sarcoidosis, idiopathic pulmonary fibrosis, a,-antitrypsin deficiency, and cystic fibrosis were also studied using serum kindly provided by Dr. Ronald G. Crystal (National Institutes of Health, Bethesda, MD). Control subjects were healthy individuals who were drug free at the time of study. All assays were performed without knowledge of the diagnoses of the patients.
Source of Reagents Hanks' balanced salt solution (HBSS) was purchased from GIBCO (Grand Island, NY). H,O, was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
Assay of Serum H202 Scavenging Activity In Vitro Samples (10 ul.) of patient or control serum were mixed with H,O, (0.1 ml of 10 mM) in a total reaction mixture of 1.0 ml of HBSS at 37° C for 30 min. In certain experiments, sodium azide (0.1 ml of 10 mM) was added before addition of H,O,. Subsequently, 0.1 ml of 50070 trichloroacetic acid was added to stop H,O, consumption. After samples were centrifuged for 5 min, each supernatant (0.2 ml) was assayed in triplicate for residual H,O, (12) and compared with a serum-free control sample.
Measurements Serum catalase activity was measured po-
larographically as the rate of production of oxygen from H,O, (18).Briefly, serum (10ul.) was added to H,O, (10 mM) in 400 ul, of HBSS in an airtight chamber equipped with a magnetic stirrer and maintained with a circulating water bath at 25° C. The rate of oxygen production was measured with an electrode monitor (Yellow Springs Instrument Co., Yellow Springs, OH) coupled to a chart recorder (Beckman Instruments Inc., Fullerton, CAl. Purified catalase (Worthington Biochemical Corp., Freehold, NJ), of which the specific activity was determined spectrophotometrically, was used to calibrate the polarographic signal. One unit of catalase activity was defined as the amount of catalase that consumed 1 umol H,O,/min at 25° C, pH 7.0. Serum GPX activity was measured as the rate of oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate at 340 nm in glutathione reductase, glutathione, and t-butyl hydro peroxide (19). Albumin concentrations were measured in serum samples spectrophotometrically (20). Serum haptoglobin levels were determined by nephelometry (21) and uric acid by highperformance liquid chromatography (22). Packed erythrocytes (RBC) (20 ul.) were separated from freshly heparinized blood, washed twice with normal saline, and added to 2.0 ml of saline of increasing tonicity (zero to 0.9%). After gentle mixing, incubation at 25° C for 30 min, and centrifugation, supernatant absorbance was measured at 414 nm. The RB fragility index was defined as the tonicity of saline causing 50% hemolysis. s'Cr (0.1 IlCi/well) was added to confluent bovine pulmonary artery endothelial cells (EC) (18) monolayers in 96 well plates. After 16 h, media was removed. Each well was then gently washed three times with HBSS to remove extracellular S'Cr. Subsequently, H,O, in HBSS (10mM final concentration), serum (30 ilL) and/or a sufficient amount of HBSS were added to each well for a final volume of 200 ul.; Plates were incubated for 4 h at
37° C in 5% CO,. Supernatants were removed and EC monolayers were washed with HBSS (200 ul.) to remove extracellular SICr.An additional wash (200 ul.) was performed and discarded. Supernatants and washes were counted in a liquid scintillation counter (Beckman, Irvine, CAl. The EC monolayer was incubated with 2% TritonX-loo(2oo ul, in water) at 37° C for 15 min, washed with HBSS (200 ul.), and counted. Injury was defined as s'Cr counts released into the media as a percentage of total t'Cr counts for a given well. Neutrophil bactericidal activity against Staphylococcus aureus (502a) was assessed using patient or control serum and neutrophils isolated from healthy donors (23). Serum (575 ul.) was combined with neutrophils (400 ilL) and bacteria (25 ilL) in a 1:1 bacteria to neutrophil ratio in a 1.0-ml incubation volume. Bactericidal activity was expressed as the percentage of S. aureus killed after incubation for 60 min (23).
Statistical Analyses Patient groups were compared using an analysis of variance with a Student-NewmanKeuls test of multiple comparisons. Significance was accepted at a p value of less than 0.05. Results
A total of 29 septic patients were studied prospectively; nine eventually developed ARDS. Clinical parameters of the patients are shown in table 1 (24).
Nature and Mechanisms Responsible for Increased Serum /£02 Scavenging Ability in Septic Patients with ARDS Effect ofserum on /£02 concentrations in vitro. Addition of serum from septic patients with or without ARDS decreased H 2 0 2 concentrations (scavenged) more than serum from control subjects in vitro (p < 0.05) (figure 1).Furthermore, serum from septic patients with ARDS scavenged H 2 0 2 better than did serum from septic patients without ARDS in vitro (p < 0.05) (figure 1). The addition of sodium azide inhibited H 20 2 scavenging by serum from septic patients with ARDS (900/0), septic patients without ARDS (91%), and healthy controls (85%). Scavenging of H 2 0 , was the same (p > 0.05) using azide-treated serum from septic patients with or without ARDS and control subjects. Serum catalase and glutathione peroxidase activity. Serum from septic patients with or without ARDS had more (p < 0.05) catalase activity than serum from control subjects (7.3 ± 0.8 Vlml; figure 2A). Serum from patients with sepsis with ARDS had increased catalase activity compared with septic patients with-
987
LEFF, PARSONS, DAY, MOORE, MOORE, OPPEGARD, AND REPINE
100
100
A Sepsis with .........
Fig. 1. Serum from septic patients with ARDS (n = 9) decreased H,O, concentrations more (p < 0.05) invitro in 30 min than serum from septic patients without ARDS (n = 20). Serum from healthy control subjects (n = 7) scavenged less H,O, than serum from septic patients with or without ARDS (p < 0.05), Values are mean ± SEM,
Serum H 202 Scavenging Activity
Serum Catalase Activity (U!ml)
50
A
25
B
0.75
0,50
Sepsis With ARDS
Sepsis Without ARDS
• •• .... • • B
Serum Glutathione Peroxidase Activity (Vlml)
Sepsis With ARDS
Fig. 2. (A) Serum catalase activity was increased (p < 0.05) in septic patients with ARDS (n = 9) compared with septic patients without ARDS (n = 20). Serum from healthy control subjects (n = 15) had lower serum catalase actiVity (p < 0.05) compared with serum from septic patients with or without ARDS. (B) Serum glutathione peroxidase activity was similar (p > 0.05) in septic patients with (n = 9) or without (n = 20) ARDS and healthy control subjects (n = 15). Each value is the mean ± SEM.
• •
.6
• • •
• • • •• • • •
0.50
..
•
•
••
25
50
75
100
Serum H202 Scavenging Activity (%
scavenged)
Fig. 3. (A) Serum catalase activity correlated with serum H,O, scavenging activity both in septic patients without ARDS (r' = 0.60, n = 21; P < 0.05) and in septic patients with ARDS (r' = 0.47, n = 9; P < 0.05). (B) Serum GPX activity did not correlate with serum H2 0 2 scavenging activity in septic patients without ARDS (r2 = 0.12, n = 21) or septic patients with ARDS (r 2 = 0.14, n = 9).
S. aureus (502a) by neutrophils was similar in serum from septic patients with or without ARDS and control subjects (p > 0.05) (figure 7). Discussion
Wefound that serum from septic patients with ARDS had more catalase activity, scavenged more H 2 0 2 in vitro, and protected EC against H 2 0 2-mediated damage better than did serum from septic patients without ARDS and control subjects. In contrast, serum from septic patients with ARDS had the same GPX activity and uric acid, bilirubin, and albumin concentrations (25, 26), and the same effect on neutrophil bactericidal activity in vitro as serum from septic patients without ARDS. Catalase appeared to be responsible for the increased scavenging of H 2 0 2 by se-
75
50
Activity
(U!ml)
0.75
a
100
Serum Catalase
•••• • • • •
o+--~--~-~-~
Potential Consequences of Increased Serum Catalase Activity in Septic Patients with ARDS Cultured EC treated with H 2 0 2 released more (p < 0.05) 51Cr (79.3 ± 2.4%) than HBSS treated EC (22.4 ± 1.8%, figure 6). The addition of serum from control subjects did not decrease 51er release from EC treated with H 20 2 (p > 0.05). However, the addition of serum from septic patients with or without ARDS decreased 51Cr release from H 2 0 2-treated EC more than serum from control subjects (p < 0.05) (figure 6). Moreover, the addition of serum from septic patients with ARDS decreased 51Cr release from H 2 0 2-treated EC more than serum from septic patients without ARDS (p < 0.05) (figure 6). Effect ofserum on bactericidal activity ofneutrophils in vitro. The killing of
Fig. 4. Serum catalase activity was increased (p < 0.05)in septic patients with or without ARDS compared with healthy controls and patients with idiopathic pul· monary fibrosis (IPF), a,-antitrypsin defi· ciency (A1AT), sarcoidosis (Sarcoid), and cystic fibrosis (CF). Each value represents the mean ± SEM of 5 to 20 patients and 5 control subjects.
•••
0.25
Evaluation of RBC Hemolysis as a Possible Source of Increased Serum Catalase Activity in Septic Patients with ARDS Septic patients with or without ARDS and control subjects had the same serum haptoglobin concentrations (p > 0.05) (figure SA). Likewise, the fragility index (concentration of sodium chloride causing 50070 lysis) of RBC from septic patients with (0.39%) or without (0.39%) ARDS and control subjects (0.44%) was similar (figure 5B).
••
• • •
1.00
0.25
Control Subjects
ARDS,
a
•
•
Sepsis without 40 20
Control Subjects
•
•• •
60
1.25
a
Serum Glutathione Peroxidase Activity (U!ml)
Serum Catalase Activity (U/ml)
25
50
1,00
ARDS
75
(% scavenged)
out ARDS or control subjects (p < 0.05) (figure 2A). In contrast, serum from septic patients with or without ARDS had the same GPX activity as control subjects (p > 0.05) (figure 2B). In septic patients with or without ARDS, serum catalase activity correlated with serum H 2 0 2 scavenging activity (r = 0.77, P < 0.05) (figure 3A) but not GPX activity (r = 0.33, p < 0.05) (figure 3B). Serum from septic patients with or without ARDS also had increased catalase activity (p < 0.05) compared with serum from patients with idiopathic pulmonary fibrosis, (11antitrypsin deficiency, sarcoidosis, or cystic fibrosis, which had the same catalase activity as serum from control subjects (figure 4).
75
80
25
988
SERUM CATALASE ACTIVITY IN ARDS AND SEPSIS
A 250
Serum
200
Haptoglobin 150
(mg/dl)
100 50 0 0.5
B
0.4
RBC 0.3
Fragility Index
0.2 0.1 0
Control Subjects
Sepsis Without ARDS
Sepsis With ARDS
Fig. 5. (A) Serum haptoglobin concentrations were similar (p > 0.05) in septic patients with (n = 9) or without (n = 20) ARDS and healthy control subjects (n = 8). (B) The RBC fragility index (susceptibility to hypotonic lysis) was similar (p > 0.05) in septic patients with (n = 5) or without (n = 2) ARDS and healthy control subjects (n = 7). Each value is the mean ± SEM.
rum from septic patients with or without ARDS. First, H 202 scavengingby serum from septic patients with or without ARDS and control subjects was similarly abolished by treatment with azide, an inhibitor of catalase, but not GPX or other potential H 202 scavengers. Second, serum H 202 scavenging ability correlated with serum catalase, but not GPX, activity in septic patients with or without ARDS. It is also known that the H 20 2 scavenging activity of normal serum is heat and trichloroacetic acid labile, inhibitable by azide, and contained in molecular weight fractions of approximately 240,000 D, findings that are consistent with catalase which also has a molecular weight of 240,000 D (18).
100
Endothelial
100
A
75
8
15
Cell Injury (51Cr release) 50
50
25
25
Addition: Serum Added:
o .l-..I_...L---l_...LHBSS None
"2°1
Control Subjects
100 The source of the increased serum catalase in septic patients with and with75 out ARDS is unclear. Increases in serum Bactericidal Activity catalase could reflect release of catalase 50 from damaged cells. We found no evi- (% S. aureus killed) 25 dence to suggest leakage of catalase from RBC. RBC from septic patients with or without ARDS resisted osmotic stress in Control Sepsis Sepsis Subjects With Without vitro as well as RBC from control subARDS ARDS jects. Moreover, septic patients with or without ARDS had the same haptoglobin Fig. 7. Neutrophils from healthy control subjects killed S. aureus (502a) comparably (p > 0.05) in serum from concentrations (which decrease during healthy control SUbjects(n = 4) and septic patients with hemolysis) as control subjects. While ar- (n = 4) or without (n = 4) ARDS. Values are the mean guing against hemolysis as a source of ± SEM. increased serum catalase, the normal RBC fragility and serum haptoglobin concentrations do not completely exclude increased in patients with the acquired this possibility inasmuch as haptoglobin immunodeficiency syndrome (AIDS), levels may be artifactually elevated as an and it is interesting that glutathione conacute-phase reactant and small amounts centrations are depressed in both ARDS of hemolysisthat are not detectable using and AIDS patients (28, 29). The latter these techniques may have occurred. is consistent with the possibility that an RBC hemolysis may, in some models, increased oxidative stress decreases glumediate protection. In one system, an an- tathione concentrations and, as a result, tecedent oxidant stress, like that which increases serum catalase activity. The consequences of increased catamay be occurring in ARDS patients, increases plasma catalase activity and is as- lase activity in serum from septic patients sociated with protection against a sub- with ARDS are most likely multiple and sequent oxidative insult via a hemolytic diverse and obviously cannot all be studmechanism (14). A second possibility is ied. Serum from septic patients with that an increased oxidant stress occurred ARDS had more catalase activity and in septic patients in whom ARDS devel- protected EC against H 202-mediated inoped and initiated processes that in- jury better than serum with lower catacreased catalase synthesis and release. lase activity from septic patients withThird, clearance of catalase from the out ARDS or control subjects. Although bloodstream by the liver could have been it seems paradoxical that patients in altered in septic patients with or without whom ARDS developed had higher seARDS (27). Fourth, serum from septic rum catalase activity and protected EC patients with or without ARDS also had against H 202 better than serum with lowincreased catalase activity compared with er catalase activity from septic patients serum from patients with other lung dis- who did not develop ARDS, there are a eases, such as idiopathic pulmonary number of possible explanations. First, fibrosis, ai-antitrypsin deficiency, sar- regardlessof the antioxidant status of any coidosis, or cysticfibrosis, indicating that cell, it most likely can be overwhelmed the response is not merely a nonspecific by excess generation of oxidants. For reflection of lung injury. Recently, we example, death is delayed but not prehave found that serum catalase is also vented by increased antioxidant enzyme activities in lungs of rats exposed to hyperoxia (30, 31). Second, if injury is occurring due toH 202 that is generated in inaccessible locations, such as within Fig. 6. (A) The addition of 10 mM H.O. cells or at imperrneant neutrophil-endoto EC increased (p < 0.05) release of thelial cell interfaces, serum catalase may preincorporated 5'Cr as an index of cell not effectively penetrate and protect. We injury over baseline levels in EC treated with HBSS. (B) The addition of serum also examined the effect of serum from from septic patients with (n = 7) or withseptic patients with and without ARDS out (n = 5) ARDS decreased (p < 0.05) on the bactericidal action of neutrophils 5'Cr release from EC compared with the in vitro. Because killing of bacteria by addition of serum from healthy control neutrophils is dependent on H 202 and subjects (n = 5). The addition of serum from septic patients with ARDS deinhibited by large amounts of exogenous H,O, creased (p < 0.05) injury more than secatalase (32) in vitro, it is possible that Sepsis rum from septic patients without ARDS. With elevationsin serum catalase could depress Values are the mean ± SEM normalARDS the bactericidal activity of neutrophils ized to the degree of H.O.-induced injury; data were evaluated in triplicate. and increase susceptibility to infection.
LEFF, PARSONS, DAY, MOORE, MOORE, OPPEGARD, AND REPINE
However, we found that incubation of normal neutrophils in serum from septic patients with or without ARDS had no effect on the bactericidal activity of neutrophils compared with incubation in control serum. Although we evaluated only two examples that might be relevant in septic patients with ARDS, increased serum catalase may also affect a wide variety of other essential H 202-dependent functions in both beneficial and deleterious ways with respect to the development of ARDS. These may include alterations in vasoreactivity, hemodynamics, immune function, and/or repair processes (9, 32-35). Acknowledgment The writers thank Jacqueline Smith for graphics, the patient care staff at Denver General Hospital, Michael Owens and May Gillespie for their expert technical assistance, and Lance S. Terada, M.D., for helpful discussions.
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Shasby SS, McMurtry IF, Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygenradicals. Am Rev Respir Dis 1982; 125:443-7. 9. Tate RM, Morris HG, Schroeder WR, Repine JE. Oxygen metabolites stimulate thromboxane production and vasoconstriction in isolated salineperfused rabbit lungs. 1 Clin Invest 1984;74:608-13. 10. Shasby DM, Shasby SS, Peach MI. Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygenradicals and granulocyteadherence. Am Rev Respir Dis 1983; 127:72-6. 11. Rinaldo JE, Rogers RM. Adult respiratory distress syndrome: changing concepts of lung injury and repair. N Engl J Med 1982; 306:900-9. 12. Weiss SJ, Young 1, LoBuglio AF, Slivka A, Nimeh NF. Role of hydrogenperoxide in neutrophilmediated destruction of cultured endothelial cells. J Clin Invest 1981; 68:714-21. 13. Till GO, Johnson KJ, Kunkel R, Ward PA. Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites. J Clin Invest 1982;69:1126-35. 14. Leff JA, Kennedy DA, Terada LS, et al. Reperfusion of ischemic skeletal muscle causes erythrocyte hemolysis and decreases subsequent oxidant mediated lung injury. J Lab Clin Med 1991; 118: 352-8. 15. Yano S, Tierney DF. Butyrate increases catalase activity and protects rat pulmonary artery smooth muscle cells against hyperoxia. Biochem Biophys Res Commun 1989; 164:1143-8. 16. Brown JM, Grosso MA, Terada LS, et al. Endotoxin pretreatment increases endogenous myocardial catalase activity and decreases ischemiareperfusion injury of isolated rat hearts. Proc Natl Acad Sci USA 1989; 86:2516-20. 17. Parsons PE, Worthen GS, Moore EE, TateRM, Henson PM. The association of circulating endotoxin with the development of the adult respiratory distress syndrome. Am Rev Respir Dis 1989; 140:294-301. 18. Leff JA, Oppegard MA, Terada LS, McCarty EC, Repine lE. Human serum catalase decreases endothelial cell injury from hydrogen peroxide. J Appl Physiol 1991; 71:1903-6. 19. Beutler E. Red cell metabolism: a manual of biochemical methods. 3rd ed. Orlando, FL: Grune & Stratton, 1984; 74-6. 20. Corcoran R, Duran S. Albumin determination by a modified bromcresol green method. Clin Chern 1977; 23:765-6. 21. Ritzmann SE, Daniels JC. Serum protein abnormalities-diagnostic and clinical aspects. Bos-
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JONATHAN A. LEFF,4 POLLY E. PARSONS,s CAROLINE E. DAY, ERNEST E. MOORE, FREDRICK A. MOORE, MARTHA A. OPPEGARD, and JOHN E. REPINE
Introduction
Alterations in oxidant-antioxidant balance may be an important cause and/or response in patients with the adult respiratory distresssyndrome (ARDS) (1). Accelerated oxidant generation is suggested becauseARDS patients haveincreased hydrogenperoxide (H 20 2) concentrations in their breath (2, 3); increased concentrations of blood xanthine oxidase (XO), an endothelial cell-based enzyme that makes superoxide anion (0 2,) and H 20 2 (4); and blood neutrophils that make more oxygen metabolites in vitro (5). Indeed, it is likelythat excessH 202 production and related oxygen metabolite derivatives, such as hypochlorous acid (HOCl) or hydroxyl radical (·OH), account for the increased levels of oxidatively inactivated antiproteases in ARDS patients (6). Moreover, H 20 2 and/or its products can synergistically increase the toxicity of neutrophil elastase, cause acute edematous injury in isolated perfused lungs, and induce leak of protein through cultured lung endothelial cell monolayers (7-10). Despite recognition of the potential for increased generation of H 20 2 in patients with ARDS, relevant H 202-combatting antioxidant enzyme activities have not been evaluated, even though they might be an important part of the pathogenesis and could provide markers of prognosis or response to therapy (11). Moreover, exogenously administered catalase and/or enhanced glutathione redox reactions reduce H 202 concentrations and decrease endothelial cell and lung injury in various ARDS models (12, 13). Furthermore, endogenous increases in H 20 rinactivating enzymes can decrease oxidant damage (14-16). In the present investigation, we assessed H 202 scavenging, catalase, and glutathione peroxidase (GPX) activity of serum from septic patients with or without ARDS and control subjects. We found increased degrees of catalase activity in serum from septic patients and then investigated the
SUMMARY Excessive hydrogen peroxide (H,O,) generation appears to contribute to the development of the adult respiratory distress syndrome (ARDS), but H,O,-combattlng antioxidant defenses have not been evaluated. We found that serum from septic patients with ARDS scavenged more (p < 0.05) H,O, in rltro (82.7 ± 3.8%) than did serum from septic patients without ARDS (56.9 ± 3.1%) or control subjects (20.2 ± 2.4%). Serum from septic patients with ARDSalso had more (p < 0.05) catalase activity (54.9 :!: 10.9 U/ml) than did serum from septic patients without ARDS (28.6 ± 3.4 U/ml) or control subjects (7.3 :!: 0.8 U/ml). In contrast, serum from septic patients with or without ARDSand control subjects had the same glutathione peroxidase (GPX)activity. Serum H,O, scavenging activity correlated with serum catalase (r = o.n) but not GPX (r = 0.33) activity and was Inhibitable (> 90%) by sodium azide, a catalase Inhibitor. Increases In serum catalase activity did not appear to be derived from erythrocytes (RBC) because septic patients with or without ARDS and control sUbjects had similar RBC hemolysis In response to osmotic stress In rltro and serum haptoglobin concentrations. Serum from septic patients with ARDS also protected endothelial cells against H,O,-medlated damage (34.5 ± 2.2% 51Cr release) better (p < 0.05) than serum from septic patients without ARDS (47.3 ± 7.4%) or control sUbjects (82.1 ± 10.2%), but killing of bacteria by neutrophlls in r/tro was the same In serum from patients and control sUbJects. Our findings Indicate that patients with sepsis and/or ARDS have Increased serum catalase activity, which may alter H,O,-dependent processes. AM REV RESPIR DIS 1992; 148:885-888
source and possible significance of this alteration. Methods Subject Population After written consent was obtained from the patient or a family member, the patient was subjected to a protocol that had been previously approved by our institutional human subjects review committee. All patients who were identified within 8 h of the initial diagnosis of sepsis were eligible for enrollment. Patients with sepsis had a serious bacterial infection plus rectal or core temperature > 390 C and/or peripheral leukocyte counts of > 12,000 cells/mm" or > 200/0 immature neutrophils, as well as at Jeast one of the foJlowing:a positive blood cuJtureof a commonly accepted pathogen; a strongly suspected or proven source of systemic infection; gross pus in a closed space; unexplained systemic arterial hypotension (systolic blood pressure < 80 mm Hg); systemic vascular resistance < 800 dyne/s/cm'; unexplained metabolic acidosis (17). Patients with ARDS met the following criteria: (1) acute respiratory failure requiring mechanical ventilation, (2) bilateral pulmonary infiltrates, (3) pulmonary capillary wedge pressure < 18 mm Hg, (4) static pulmonary compliance < 50 mllcm H 2 0 , and (5) arterial to alveolar partial pressure of oxy-
gen ratio of < 0.25 (17). Blood samples were obtained at enrollment (zero h) and at 6, 12, 24, and 48 h thereafter. Serum samples were obtained through indwelling arterial or venous catheters and/or by direct venipuncture and stored at -70 0 C. Septic patients with or without ARDS had similar numbers of central venous, arterial, and peripheral blood withdrawals.
(Received in original form January 30, 1992 and in revised form May 15, 1992) I From the Webb-Waring Lung Institute, and the Departments of Medicine and Surgery and Denver General Hospital at the University of Colorado Health Sciences Center, Denver, Colorado. , Supported in part by Grants No. HL 40784 and HL 45582 from the National Institutes of Health, the Colorado Heart Association, the Council for Tobacco Research Inc., Ronald McDonald Children's Charities, Johnson & Johnson, Inc., the Williams Family Foundation, Swan, Hill, Donner, Kleberg, Sachs, and the American Express Foundation. 3 Correspondence and requests for reprints should be addressed to Jonathan A. Leff, M.D., Webb-Waring Institute, 4200 East Ninth Avenue, Box C322, Denver, CO 80262. • Recipient of a Clinician-Scientist Award from the American Heart Association. S Recipient of Clinical Investigator Award No. HL-0I849 from the National Institutes of Health.
985
SERUM CATALASE ACTIVITY IN ARDS AND SEPSIS
986 TABLE 1 CLINICAL CHARACTERISTICS OF SEPTIC PATIENTS WITH OR WITHOUT ARDS AT STUDY ENTRY·
Parameter Age, yr Gender, mit Hematocrit, % Hemoglobin, gldl Blood leukocyte count, x 1,000 Absolute blood neutrophil, count, x 1,000 Serum SGOT, Ulml Serum bilirubin, mgldl Serum albumin, g1dl Serum uric acid, mgldl APACHE II score Mortality, 0/0
Septic Patients Without ARDS 60 ± 3 (20) 10110 35 ± 3 (13) 12 ± 1 (11) 17±3(11) ± 3 (10) ± 67 (7) ± 1.0 (6) ± 0.2 (16) 5.7 ± 0.8 (12) 15±2{18) 6/20 (30%)
16 191 2.6 2.8
Septic Patients With ARDS 51 ± 4 (9) 7/2 35 ± 3 (5) 12 ± 1 (5) 14 ± 4 (5) ± 5 (4) ± 31 (4) ± 0.1 (4) ± 0.3 (6) ± 0.5 (5) ± 2 (6) 4/9 (44%)
13 52 0.6 2.6 4.5 13
Definition of abbreviation: SGOT = serum glutamic-oxaloacetic transaminase. • Values are the mean ::t: SE. The number of determinations is given in parentheses.
Patients were divided into two groups: those who had sepsis without ARDS (serum withdrawn at 6, 12, 24, and 48 h after the initial diagnosis of sepsis in patients who did not develop ARDS) and those who had sepsis with ARDS (serum withdrawn from septic patients at all time points after the diagnosis of ARDS). Serum samples were assayed individually at each time point, and then all time points were averaged for each patient, yielding one value for each patient. Patients were prospectively and sequentially followed until death or discharge. Patients with sarcoidosis, idiopathic pulmonary fibrosis, a,-antitrypsin deficiency, and cystic fibrosis were also studied using serum kindly provided by Dr. Ronald G. Crystal (National Institutes of Health, Bethesda, MD). Control subjects were healthy individuals who were drug free at the time of study. All assays were performed without knowledge of the diagnoses of the patients.
Source of Reagents Hanks' balanced salt solution (HBSS) was purchased from GIBCO (Grand Island, NY). H,O, was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
Assay of Serum H202 Scavenging Activity In Vitro Samples (10 ul.) of patient or control serum were mixed with H,O, (0.1 ml of 10 mM) in a total reaction mixture of 1.0 ml of HBSS at 37° C for 30 min. In certain experiments, sodium azide (0.1 ml of 10 mM) was added before addition of H,O,. Subsequently, 0.1 ml of 50070 trichloroacetic acid was added to stop H,O, consumption. After samples were centrifuged for 5 min, each supernatant (0.2 ml) was assayed in triplicate for residual H,O, (12) and compared with a serum-free control sample.
Measurements Serum catalase activity was measured po-
larographically as the rate of production of oxygen from H,O, (18).Briefly, serum (10ul.) was added to H,O, (10 mM) in 400 ul, of HBSS in an airtight chamber equipped with a magnetic stirrer and maintained with a circulating water bath at 25° C. The rate of oxygen production was measured with an electrode monitor (Yellow Springs Instrument Co., Yellow Springs, OH) coupled to a chart recorder (Beckman Instruments Inc., Fullerton, CAl. Purified catalase (Worthington Biochemical Corp., Freehold, NJ), of which the specific activity was determined spectrophotometrically, was used to calibrate the polarographic signal. One unit of catalase activity was defined as the amount of catalase that consumed 1 umol H,O,/min at 25° C, pH 7.0. Serum GPX activity was measured as the rate of oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate at 340 nm in glutathione reductase, glutathione, and t-butyl hydro peroxide (19). Albumin concentrations were measured in serum samples spectrophotometrically (20). Serum haptoglobin levels were determined by nephelometry (21) and uric acid by highperformance liquid chromatography (22). Packed erythrocytes (RBC) (20 ul.) were separated from freshly heparinized blood, washed twice with normal saline, and added to 2.0 ml of saline of increasing tonicity (zero to 0.9%). After gentle mixing, incubation at 25° C for 30 min, and centrifugation, supernatant absorbance was measured at 414 nm. The RB fragility index was defined as the tonicity of saline causing 50% hemolysis. s'Cr (0.1 IlCi/well) was added to confluent bovine pulmonary artery endothelial cells (EC) (18) monolayers in 96 well plates. After 16 h, media was removed. Each well was then gently washed three times with HBSS to remove extracellular S'Cr. Subsequently, H,O, in HBSS (10mM final concentration), serum (30 ilL) and/or a sufficient amount of HBSS were added to each well for a final volume of 200 ul.; Plates were incubated for 4 h at
37° C in 5% CO,. Supernatants were removed and EC monolayers were washed with HBSS (200 ul.) to remove extracellular SICr.An additional wash (200 ul.) was performed and discarded. Supernatants and washes were counted in a liquid scintillation counter (Beckman, Irvine, CAl. The EC monolayer was incubated with 2% TritonX-loo(2oo ul, in water) at 37° C for 15 min, washed with HBSS (200 ul.), and counted. Injury was defined as s'Cr counts released into the media as a percentage of total t'Cr counts for a given well. Neutrophil bactericidal activity against Staphylococcus aureus (502a) was assessed using patient or control serum and neutrophils isolated from healthy donors (23). Serum (575 ul.) was combined with neutrophils (400 ilL) and bacteria (25 ilL) in a 1:1 bacteria to neutrophil ratio in a 1.0-ml incubation volume. Bactericidal activity was expressed as the percentage of S. aureus killed after incubation for 60 min (23).
Statistical Analyses Patient groups were compared using an analysis of variance with a Student-NewmanKeuls test of multiple comparisons. Significance was accepted at a p value of less than 0.05. Results
A total of 29 septic patients were studied prospectively; nine eventually developed ARDS. Clinical parameters of the patients are shown in table 1 (24).
Nature and Mechanisms Responsible for Increased Serum /£02 Scavenging Ability in Septic Patients with ARDS Effect ofserum on /£02 concentrations in vitro. Addition of serum from septic patients with or without ARDS decreased H 2 0 2 concentrations (scavenged) more than serum from control subjects in vitro (p < 0.05) (figure 1).Furthermore, serum from septic patients with ARDS scavenged H 2 0 2 better than did serum from septic patients without ARDS in vitro (p < 0.05) (figure 1). The addition of sodium azide inhibited H 20 2 scavenging by serum from septic patients with ARDS (900/0), septic patients without ARDS (91%), and healthy controls (85%). Scavenging of H 2 0 , was the same (p > 0.05) using azide-treated serum from septic patients with or without ARDS and control subjects. Serum catalase and glutathione peroxidase activity. Serum from septic patients with or without ARDS had more (p < 0.05) catalase activity than serum from control subjects (7.3 ± 0.8 Vlml; figure 2A). Serum from patients with sepsis with ARDS had increased catalase activity compared with septic patients with-
987
LEFF, PARSONS, DAY, MOORE, MOORE, OPPEGARD, AND REPINE
100
100
A Sepsis with .........
Fig. 1. Serum from septic patients with ARDS (n = 9) decreased H,O, concentrations more (p < 0.05) invitro in 30 min than serum from septic patients without ARDS (n = 20). Serum from healthy control subjects (n = 7) scavenged less H,O, than serum from septic patients with or without ARDS (p < 0.05), Values are mean ± SEM,
Serum H 202 Scavenging Activity
Serum Catalase Activity (U!ml)
50
A
25
B
0.75
0,50
Sepsis With ARDS
Sepsis Without ARDS
• •• .... • • B
Serum Glutathione Peroxidase Activity (Vlml)
Sepsis With ARDS
Fig. 2. (A) Serum catalase activity was increased (p < 0.05) in septic patients with ARDS (n = 9) compared with septic patients without ARDS (n = 20). Serum from healthy control subjects (n = 15) had lower serum catalase actiVity (p < 0.05) compared with serum from septic patients with or without ARDS. (B) Serum glutathione peroxidase activity was similar (p > 0.05) in septic patients with (n = 9) or without (n = 20) ARDS and healthy control subjects (n = 15). Each value is the mean ± SEM.
• •
.6
• • •
• • • •• • • •
0.50
..
•
•
••
25
50
75
100
Serum H202 Scavenging Activity (%
scavenged)
Fig. 3. (A) Serum catalase activity correlated with serum H,O, scavenging activity both in septic patients without ARDS (r' = 0.60, n = 21; P < 0.05) and in septic patients with ARDS (r' = 0.47, n = 9; P < 0.05). (B) Serum GPX activity did not correlate with serum H2 0 2 scavenging activity in septic patients without ARDS (r2 = 0.12, n = 21) or septic patients with ARDS (r 2 = 0.14, n = 9).
S. aureus (502a) by neutrophils was similar in serum from septic patients with or without ARDS and control subjects (p > 0.05) (figure 7). Discussion
Wefound that serum from septic patients with ARDS had more catalase activity, scavenged more H 2 0 2 in vitro, and protected EC against H 2 0 2-mediated damage better than did serum from septic patients without ARDS and control subjects. In contrast, serum from septic patients with ARDS had the same GPX activity and uric acid, bilirubin, and albumin concentrations (25, 26), and the same effect on neutrophil bactericidal activity in vitro as serum from septic patients without ARDS. Catalase appeared to be responsible for the increased scavenging of H 2 0 2 by se-
75
50
Activity
(U!ml)
0.75
a
100
Serum Catalase
•••• • • • •
o+--~--~-~-~
Potential Consequences of Increased Serum Catalase Activity in Septic Patients with ARDS Cultured EC treated with H 2 0 2 released more (p < 0.05) 51Cr (79.3 ± 2.4%) than HBSS treated EC (22.4 ± 1.8%, figure 6). The addition of serum from control subjects did not decrease 51er release from EC treated with H 20 2 (p > 0.05). However, the addition of serum from septic patients with or without ARDS decreased 51Cr release from H 2 0 2-treated EC more than serum from control subjects (p < 0.05) (figure 6). Moreover, the addition of serum from septic patients with ARDS decreased 51Cr release from H 2 0 2-treated EC more than serum from septic patients without ARDS (p < 0.05) (figure 6). Effect ofserum on bactericidal activity ofneutrophils in vitro. The killing of
Fig. 4. Serum catalase activity was increased (p < 0.05)in septic patients with or without ARDS compared with healthy controls and patients with idiopathic pul· monary fibrosis (IPF), a,-antitrypsin defi· ciency (A1AT), sarcoidosis (Sarcoid), and cystic fibrosis (CF). Each value represents the mean ± SEM of 5 to 20 patients and 5 control subjects.
•••
0.25
Evaluation of RBC Hemolysis as a Possible Source of Increased Serum Catalase Activity in Septic Patients with ARDS Septic patients with or without ARDS and control subjects had the same serum haptoglobin concentrations (p > 0.05) (figure SA). Likewise, the fragility index (concentration of sodium chloride causing 50070 lysis) of RBC from septic patients with (0.39%) or without (0.39%) ARDS and control subjects (0.44%) was similar (figure 5B).
••
• • •
1.00
0.25
Control Subjects
ARDS,
a
•
•
Sepsis without 40 20
Control Subjects
•
•• •
60
1.25
a
Serum Glutathione Peroxidase Activity (U!ml)
Serum Catalase Activity (U/ml)
25
50
1,00
ARDS
75
(% scavenged)
out ARDS or control subjects (p < 0.05) (figure 2A). In contrast, serum from septic patients with or without ARDS had the same GPX activity as control subjects (p > 0.05) (figure 2B). In septic patients with or without ARDS, serum catalase activity correlated with serum H 2 0 2 scavenging activity (r = 0.77, P < 0.05) (figure 3A) but not GPX activity (r = 0.33, p < 0.05) (figure 3B). Serum from septic patients with or without ARDS also had increased catalase activity (p < 0.05) compared with serum from patients with idiopathic pulmonary fibrosis, (11antitrypsin deficiency, sarcoidosis, or cystic fibrosis, which had the same catalase activity as serum from control subjects (figure 4).
75
80
25
988
SERUM CATALASE ACTIVITY IN ARDS AND SEPSIS
A 250
Serum
200
Haptoglobin 150
(mg/dl)
100 50 0 0.5
B
0.4
RBC 0.3
Fragility Index
0.2 0.1 0
Control Subjects
Sepsis Without ARDS
Sepsis With ARDS
Fig. 5. (A) Serum haptoglobin concentrations were similar (p > 0.05) in septic patients with (n = 9) or without (n = 20) ARDS and healthy control subjects (n = 8). (B) The RBC fragility index (susceptibility to hypotonic lysis) was similar (p > 0.05) in septic patients with (n = 5) or without (n = 2) ARDS and healthy control subjects (n = 7). Each value is the mean ± SEM.
rum from septic patients with or without ARDS. First, H 202 scavengingby serum from septic patients with or without ARDS and control subjects was similarly abolished by treatment with azide, an inhibitor of catalase, but not GPX or other potential H 202 scavengers. Second, serum H 202 scavenging ability correlated with serum catalase, but not GPX, activity in septic patients with or without ARDS. It is also known that the H 20 2 scavenging activity of normal serum is heat and trichloroacetic acid labile, inhibitable by azide, and contained in molecular weight fractions of approximately 240,000 D, findings that are consistent with catalase which also has a molecular weight of 240,000 D (18).
100
Endothelial
100
A
75
8
15
Cell Injury (51Cr release) 50
50
25
25
Addition: Serum Added:
o .l-..I_...L---l_...LHBSS None
"2°1
Control Subjects
100 The source of the increased serum catalase in septic patients with and with75 out ARDS is unclear. Increases in serum Bactericidal Activity catalase could reflect release of catalase 50 from damaged cells. We found no evi- (% S. aureus killed) 25 dence to suggest leakage of catalase from RBC. RBC from septic patients with or without ARDS resisted osmotic stress in Control Sepsis Sepsis Subjects With Without vitro as well as RBC from control subARDS ARDS jects. Moreover, septic patients with or without ARDS had the same haptoglobin Fig. 7. Neutrophils from healthy control subjects killed S. aureus (502a) comparably (p > 0.05) in serum from concentrations (which decrease during healthy control SUbjects(n = 4) and septic patients with hemolysis) as control subjects. While ar- (n = 4) or without (n = 4) ARDS. Values are the mean guing against hemolysis as a source of ± SEM. increased serum catalase, the normal RBC fragility and serum haptoglobin concentrations do not completely exclude increased in patients with the acquired this possibility inasmuch as haptoglobin immunodeficiency syndrome (AIDS), levels may be artifactually elevated as an and it is interesting that glutathione conacute-phase reactant and small amounts centrations are depressed in both ARDS of hemolysisthat are not detectable using and AIDS patients (28, 29). The latter these techniques may have occurred. is consistent with the possibility that an RBC hemolysis may, in some models, increased oxidative stress decreases glumediate protection. In one system, an an- tathione concentrations and, as a result, tecedent oxidant stress, like that which increases serum catalase activity. The consequences of increased catamay be occurring in ARDS patients, increases plasma catalase activity and is as- lase activity in serum from septic patients sociated with protection against a sub- with ARDS are most likely multiple and sequent oxidative insult via a hemolytic diverse and obviously cannot all be studmechanism (14). A second possibility is ied. Serum from septic patients with that an increased oxidant stress occurred ARDS had more catalase activity and in septic patients in whom ARDS devel- protected EC against H 202-mediated inoped and initiated processes that in- jury better than serum with lower catacreased catalase synthesis and release. lase activity from septic patients withThird, clearance of catalase from the out ARDS or control subjects. Although bloodstream by the liver could have been it seems paradoxical that patients in altered in septic patients with or without whom ARDS developed had higher seARDS (27). Fourth, serum from septic rum catalase activity and protected EC patients with or without ARDS also had against H 202 better than serum with lowincreased catalase activity compared with er catalase activity from septic patients serum from patients with other lung dis- who did not develop ARDS, there are a eases, such as idiopathic pulmonary number of possible explanations. First, fibrosis, ai-antitrypsin deficiency, sar- regardlessof the antioxidant status of any coidosis, or cysticfibrosis, indicating that cell, it most likely can be overwhelmed the response is not merely a nonspecific by excess generation of oxidants. For reflection of lung injury. Recently, we example, death is delayed but not prehave found that serum catalase is also vented by increased antioxidant enzyme activities in lungs of rats exposed to hyperoxia (30, 31). Second, if injury is occurring due toH 202 that is generated in inaccessible locations, such as within Fig. 6. (A) The addition of 10 mM H.O. cells or at imperrneant neutrophil-endoto EC increased (p < 0.05) release of thelial cell interfaces, serum catalase may preincorporated 5'Cr as an index of cell not effectively penetrate and protect. We injury over baseline levels in EC treated with HBSS. (B) The addition of serum also examined the effect of serum from from septic patients with (n = 7) or withseptic patients with and without ARDS out (n = 5) ARDS decreased (p < 0.05) on the bactericidal action of neutrophils 5'Cr release from EC compared with the in vitro. Because killing of bacteria by addition of serum from healthy control neutrophils is dependent on H 202 and subjects (n = 5). The addition of serum from septic patients with ARDS deinhibited by large amounts of exogenous H,O, creased (p < 0.05) injury more than secatalase (32) in vitro, it is possible that Sepsis rum from septic patients without ARDS. With elevationsin serum catalase could depress Values are the mean ± SEM normalARDS the bactericidal activity of neutrophils ized to the degree of H.O.-induced injury; data were evaluated in triplicate. and increase susceptibility to infection.
LEFF, PARSONS, DAY, MOORE, MOORE, OPPEGARD, AND REPINE
However, we found that incubation of normal neutrophils in serum from septic patients with or without ARDS had no effect on the bactericidal activity of neutrophils compared with incubation in control serum. Although we evaluated only two examples that might be relevant in septic patients with ARDS, increased serum catalase may also affect a wide variety of other essential H 202-dependent functions in both beneficial and deleterious ways with respect to the development of ARDS. These may include alterations in vasoreactivity, hemodynamics, immune function, and/or repair processes (9, 32-35). Acknowledgment The writers thank Jacqueline Smith for graphics, the patient care staff at Denver General Hospital, Michael Owens and May Gillespie for their expert technical assistance, and Lance S. Terada, M.D., for helpful discussions.
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