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(Chest. 2001;120:1599-1608.)
© 2001 American College of Chest Physicians

Antioxidant and Antiprotease Status in Peripheral Blood and BAL Fluid After Cardiopulmonary Bypass*

Oliver M. Frass, MD; Frank Bühling, MD; Michael Täger, MD; Heidrun Frass, MD; Siegfried Ansorge, MD; Christof Huth, MD and Tobias Welte, MD

* From the Department of Cardiac and Thoracic Surgery (Drs. O. Frass, H. Frass, and Huth), Institute of Immunology (Drs. Bühling and Täger), Institute of Experimental Internal Medicine (Dr. Ansorge), and Department of Cardiology, Angiology, and Pulmology (Dr. Welte), Otto-von-Guericke University, Magdeburg, Germany.

Correspondence to: Oliver M. Frass, MD, Departmenrt of Cardiac and Thoracic Surgery, Otto-von-Guericke University, Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany; e-mail: oliver.frass{at}medizin.uni-magdeburg.de


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: Cardiopulmonary bypass (CPB) triggers systemic inflammation. Recent evidence suggests that metabolic and oxygenation management can affect the outcome of patients after cardiac surgery. We investigated the influence of oxidant/antioxidant and protease/antiprotease imbalance during the course of systemic and pulmonary inflammation.

Methods: In a study of 61 patients, we measured the intracellular thiol concentration, the intracellular activity of cathepsins and elastase, and the concentrations of secreted elastase, soluble {alpha}1-proteinase inhibitor ({alpha}1-PI), and secretory leukoprotease inhibitor (SLPI). Peripheral blood and BAL fluid (BALF) were obtained preoperatively and 2 h after CPB.

Results: A post-CPB depletion of thiol was found in blood granulocytes, lymphocytes, and monocytes, as well as BALF lymphocytes and macrophages. The degree of postoperative depletion correlated with PO2 and blood glucose levels during CPB. Concomitant reduction of FEV1 showed positive correlation with thiol depletion of blood monocytes and granulocytes. Elastase and cathepsin activities were increased in blood cells but not in lymphocytes or macrophages from BALF. The concentrations of secreted elastase were significantly increased in blood plasma but not in BALF. Enhanced antiprotease ({alpha}1-PI, SLPI) concentrations were measured in BALF but not in peripheral blood.

Conclusions: The inflammatory response of the intra-alveolar compartment is clearly distinguishable from systemic inflammation. CPB causes a differentiated impairment of the antioxidant defense system as well as a protease/antiprotease imbalance in blood and BALF. Oxygenation under circumstances of CPB and concomitant pulmonary disease, as well as blood glucose metabolism, influence the antioxidative defense. Individual perioperative management of blood glucose and oxygenation could improve cellular defense systems in the peripheral blood and BALF and therefore result in a more favorable patient outcome.

Key Words: antiproteases • BAL • blood glucose • cardiopulmonary bypass • elastase • glutathione • intracellular thiol • oxygenation • systemic inflammation


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cardiopulmonary bypass (CPB) triggers an inflammatory response that can lead to the development of postoperative organ dysfunction. The importance of leukocytes, mainly neutrophil granulocytes, in the development of pulmonary, renal, and cardiovascular injuries has been demonstrated. However, the molecular mechanisms that trigger, maintain, and regulate the cellular responses of these events have not yet been fully elucidated.1

Hyperoxic CPB, widely used during cardiac operations in adult patients, may cause oxygenation injury induced by reactive oxygen intermediates (ROIs).2 Summarizing the experimental evidence for ROI participation in local and systemic inflammation, it becomes apparent that free radicals derived from neutrophils are responsible for endothelial cell injury and the resulting end-organ damage after cardiovascular operations (for review, see Boyle et al3 ).

Free thiol moieties are ubiquitous in eucaryotic cells. They play a pivotal role as reducing agents in protection against tissue damage by direct scavenging of ROIs as well as via the attenuation of various cellular defense mechanisms. The reduced form of glutathione represents the most abundant intracellular thiol. It accounts for about 90% of the nonprotein thiols within the cell.4 Thiols, mainly glutathione, have been implicated in many cellular functions, including detoxification and antioxidant processes, regulation of gene expression, protein synthesis, cell-cycle regulation, and apoptosis. Glutathione scavenges free radicals and also detoxifies xenobiotics via the glutathione-S-transferase pathway. It reduces lipid and hydrogen peroxides via glutathione peroxidase.5 6 Deficiencies of glutathione have been documented in various pathologic conditions, including virus infections, poisoning, acute and chronic inflammations, pulmonary diseases such as idiopathic fibrosis and ARDS, reperfusion syndromes, and acute myocardial infarction.7 8 9 10 11 12

A number of studies13 14 15 have described increased liberation of proteolytic enzymes during extracorporeal circulation (ECC), which was associated with membrane injury and increased capillary permeability.16 Elastase, a serine protease that is secreted from neutrophils (polymorphonuclear leukocyte [PMN]-elastase), seems to be the major protease concerning their activity and quantity17 ; cathepsin G shows functional similarities to PMN-elastase.18 Therefore, increased intracellular protease activities, as well as elevated concentrations in plasma or BAL fluid (BALF), could indicate a cellular activation and may have significant influence on postoperative tissue destruction.

Pulmonary dysfunction is the most common clinical manifestation of bypass-induced damage. The efficiency of gas exchange was shown to be impaired,19 and an increase in pulmonary endothelial permeability was demonstrated in animal models16 as well as in patients undergoing CPB.20 The epithelial surface in the normal lung is protected against PMN-elastase by antiproteases such as {alpha}1-proteinase inhibitor ({alpha}1-PI) and secretory leukoprotease inhibitor (SLPI).

The present study focused on changes within the cellular antioxidant and antiprotease defense systems initiated by systemic inflammation that resulted from CPB in cardiac surgery. We demonstrate for the first time (to our knowledge) that the intracellular thiol status of blood leukocytes and BAL cells is depending on perioperative oxygenation and blood glucose metabolism. It becomes evident that CPB with ischemia and reperfusion, which usually results in ROI formation, leads to an impaired antioxidant and antiprotease defense system.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Sixty-one patients undergoing cardiac operations with CPB participated in the study. Written informed consent was obtained from all patients entered into the study, which was approved by the local ethical committee. All patients received preoperative pulmonary function tests (vital capacity [VC], FEV1, expressed as percentage of VC). Exclusion criteria were current nicotine abuse, treatment with immune modulatory drugs (corticosteroids, vaccination, blood products) during the 3 months prior to the study, and current symptoms of an inflammatory process (clinical or abnormal parameters for C-reactive protein [CRP], leukocyte blood count, or body temperature). Emergency operations were also excluded.

Operative Procedures
Operative procedures were as follows: coronary artery bypass grafting (53 patients), aortic valve replacement (6 patients), mitral valve replacement (1 patient), and atrial septum defect closure (1 patient).

Perioperative Management
Anesthesia induction and maintenance were achieved with sufentanil, 50 to 100 µg; propofol, 100 to 400 mg/h; and pancuronium bromide, 8 to 12 mg. For perioperative antibiosis, a single injection of 2 g of cefotiam was given. Dexamethasone, 40 mg, was also administered.21 Blood glucose levels were routinely monitored every 30 min. In diabetic patients, continuous insulin administration was performed IV at a dosage of 4 to 40 IU/h to maintain blood glucose values in the range of 6 to 10 mmol/L. In nondiabetic patients, insulin administration was initiated only when blood glucose values were > 10 mmol/L. All patients received an arterial line, a central venous catheter, and a pulmonary artery catheter.

The standardized oxygenation regimen began with preoxygenation with a fraction of inspired oxygen (FIO2) of 1.0 before intubation. The intubated patient then received ventilation with an FIO2 of 0.6. After pericardiotomy until the initiation of ECC, an FIO2 of 1.0 was administered; during ECC, the lungs collapsed completely and the oxygen flow of the CPB was adjusted to a PO2 of 100 to 150 mm Hg. Before discontinuation of ECC, ventilation was started with an FIO2 of 1.0 after complete inflation of both lungs. In the follow-up, the FIO2 was adjusted to come up to a PO2 of 100 to 150 mm Hg.

The closed extracorporeal circuit CPB included a roller pump, a membrane oxygenator (Quadrox; Jostra Medizintechnik; Hirrlingen, Germany), and a 40-µm arterial line filter (Dideco; Puchheim, Germany). The venous drain was routinely established by a two-stage cannula. Therefore, a total bypass was not obtained (except for the one atrial septal defect closure where both venae cava had to be drained separately). Priming was performed with crystalloid solutions, ie, Ringer’s solution, sodium bicarbonate, mannitol, sodium-heparin, and, if necessary, aprotinin. Aprotinin (4 million kallikrein inhibitor units) was administered to 49 patients who had received acetylsalicylic acid during the 10 days prior to the operation and to the patients undergoing valve replacement. Isovolemic hemodilution was applied to achieve a hematocrit of 18 to 24% during CPB. Flow rates of 1.7 to 2.4 L/m2/min were used with a mild-to-moderate hypothermia of 28°C to 30°C; perfusion was nonpulsatile. Bretschneider’s cardioplegia (Custodiol; Köhler Chemie; Alsbach, Germany) was infused in antegrade manner through the aortic root or, in patients with aortic valve insufficiency, directly into both coronary ostia. Operative data are given in Table 1 .


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  		Table 1.. Operative Data (n = 61)*

 
Blood Samples and BAL
Blood samples were collected from the indwelling central venous catheter after induction of anesthesia and 2 h after completion of CPB. At both times, 9 mL of blood was obtained (Vacutainer tubes; Becton-Dickinson; Heidelberg, Germany) containing 0.5 mL of 0.105 mol/L sodium citrate for humoral parameter analysis or 54 µL of 15% ethylenediamintetra-acetic acid for cellular parameter analysis. The sample stabilized with sodium citrate was centrifuged immediately for 10 min at 700g, and the supernatant was centrifuged again for 10 min at 900g. The resulting plasma was stored at - 80°C until analysis.

BAL was performed at both time points through the endotracheal tube, and fluid was acquired preoperatively from the lingula segment and postoperatively from the right middle lobe. One hundred twenty milliliters of 0.9% sodium chloride solution was instilled for each BAL, 60 to 70% of which was recovered.

Proteolytic Enzymes
Concentrations of elastase-inhibitor complex, {alpha}1-PI, and SLPI were determined by commercially available enzyme-immunoassays (Merck; Darmstadt, Germany; R&D Systems; Bad Nauheim, Germany; and IBL; Hamburg, Germany, respectively). Intracellular elastase activity in viable cells was measured by flow cytometry using the substrate Z-(Ala-Ala)2-R110,22 and total cellular cathepsin activity (cathepsins B, L, H, and S) was evaluated using Z-(Phe-Arg)2-R110. The mean fluorescence intensity (MFI) indicated the relative enzymatic activity concerning the specific substrate within the cells of the gated population (lymphocytes, granulocytes, monocytes, and macrophages).

Intracellular Thiol Concentration
The intracellular thiol concentration was determined using 5-chloromethylfluoresceindiacetate (CMFDA) in flow cytometry (FACSCalibur; Becton-Dickinson) according to Hedley and Chow,23 Chikahisa et al,24 and Coates and Tripp.25 This method determines the total cellular thiol content on a single cell level. After entering the cell and following fluorescence-inducing hydrolysis of the acetates by cellular esterases, CMFDA does not react exclusively with glutathione but also with other free sulfhydryl groups. Briefly, 100 µL of anticoagulated (ethylenediamintetra-acetic) whole blood samples or BAL preparations was stained with equal volumes CMFDA (Molecular Probes; Eugene, OR) in phosphate-buffered saline solution at a final concentration of 12.5 µM for 15 min at room temperature. The remaining erythrocytes were lysed, and the cells were washed twice and fixed in 1% paraformaldehyde. The staining protocol was optimized with respect to the reproducibility of the measurements. It was shown that glutathione is not taken up spontaneously from the extracellular compartment due to its high polarity, but is rather synthesized intracellularly. Therefore, the very short incubation time and the fixation immediately after the lysis of the erythrocytes did not influence the thiol content. Data analysis was performed on readings from 10,000 cells per sample. The levels of intracellular thiols were indicated by MFI of CMFDA-stained probes vs negative controls.

Statistics
All results are expressed as mean ± SD. The significance of the differences between two groups was tested by Wilcoxon signed-rank test. Correlations between two parameters within groups were tested by Spearman rank-order correlation. A two-tailed p value < 0.05 was considered statistically significant, and a p value < 0.001 was highly significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Demographic Characteristics
The preoperative demographic and anamnestic data of patients were expressed as mean ± SD: age, 62.48 ± 8.99 years; body mass index, 27.11 ± 2.74 kg/m2. The female/male ratio of patients was 19/42. Twenty-five patients had diabetes mellitus or impaired glucose tolerance, 39 patients had hypertension, and 38 patients had at least one myocardial infarction. Twenty-six patients were former smokers who stopped smoking at least 28 days before surgery.

Cell Counts
Differential blood counts before initiation of ECC and 2 h after removal of the heart-lung machine showed significant increases in leukocytes (5.1 ± 1.4 to 14.4 ± 5.2 x 109/L, p < 0.001), a decreasing proportion of monocytes (8.3 ± 3.3% to 4.4 ± 3.4%, p < 0.001) and lymphocytes (33.4 ± 10.1% to 9.5 ± 6.2%, p < 0.001), and an increased proportion of granulocytes (57.4 ± 13.0% to 85.6 ± 7.9%, p < 0.001). There were no significant differences in the differential cell count of BALF macrophages (74.9 ± 18.2% to 78.7 ± 13.9%), lymphocytes (12.5 ± 12.0% to 11.2 ±11.1%), and granulocytes (9.1 ± 12.4% to 8.3 ± 10.1%) before and after CPB.

Blood Glucose
In nondiabetic patients, perioperative blood glucose values changed from 5.03 ± 0.42 to 12.06 ± 2.32 mmol/L (p < 0.001), while diabetics or patients with impaired glucose tolerance had higher preoperative and lower postoperative values (6.9 ± 1.75 mmol/L and 11.3 ± 2.38 mmol/L, respectively) when compared to nondiabetic patients. The resulting relative alterations in blood glucose concentrations of nondiabetic patients (+ 141.6 ± 49.8%) and diabetic patients (+ 72.0 ± 47.2%) were highly significant (p < 0.001).

Intracellular Thiol Concentration
The intracellular thiol concentration of analyzed blood cells showed a highly significant decrease (p < 0.001) in granulocytes (448 ± 126 to 397 ± 119 MFI), monocytes (500 ± 168 to 402 ± 152 MFI), and lymphocytes (393 ± 153 to 302 ± 139 MFI). The thiols in lymphocytes and macrophages from BALF decreased from 1,368 ± 1,249 to 512 ± 764 MFI (p < 0.001) and from 5,112 ± 2,636 to 3,321 ± 2,639 MFI (p = 0.003), respectively (Fig 1 ).



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  		Figure 1.. The intracellular thiol content of cells (left, A) in peripheral blood and (right, B) in BALF recovered preoperatively (light columns) and postoperatively (dark columns), respectively. Values with asterisks are different from preoperative values (*p < 0.001, **p = 0.003).

 
Correlation of Intracellular Thiol Concentration and Pulmonary Parameters
Among the preoperative pulmonary parameters, FEV1, but not VC, was negatively correlated to the relative alterations of intracellular thiol concentration in blood monocytes (r = 0.2; p = 0.041) and granulocytes (r = 0.2; p = 0.024). Monocytes and granulocytes of patients with a pathologically reduced FEV1 (< 70% of VC) lose nearly three times and five times more thiols postoperatively, respectively, than cells of patients with normal preoperative spirometric parameters (Fig 2 ).



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  		Figure 2.. The intracellular thiol content in monocytes (hatched columns) and granulocytes (dotted columns) from peripheral blood is dependent on preoperative FEV1 (%VC). Preoperative data are shown above. As shown below, perioperative depletion of intracellular glutathione is more marked in patients with bronchial obstruction. *p = 0.024, **p = 0.041 compared with the group without bronchial obstruction.

 
Importantly, we found a negative correlation in intraoperative patient oxygenation, ie PO2, to changes in mean intracellular thiol concentration of macrophages (r = 0.41; p = 0.012) in BALF. Classifying patients into two groups according to the mean PO2 (196 mm Hg), the influence of intraoperative oxygenation becomes more apparent. Mean thiol concentration of macrophages in patients with PO2 < 196 mm Hg was almost unchanged postoperatively vs a postoperative decrease to 66.5% in patients with PO2 > 196 mm Hg (p < 0.01; Fig 3 ).



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  		Figure 3.. The changes of intracellular thiol levels in BAL macrophages and approximate PO2 values during CPB. Patients were classified into two groups (PO2 approximately < 196 mm Hg and PO2 > 196 mm Hg, which is the median of all values).*p = 0.012, ** p = 0.018 compared with the group with PO2 < 196 mm Hg. GSH = glutathione.

 
Correlation of Thiol and Blood Glucose Concentrations
A negative correlation of postoperative blood glucose concentration to changes in the intracellular thiol concentration of blood lymphocytes (r = 0.29; p = 0.025) and monocytes (r = 0.29; p = 0.018) as well as BALF lymphocytes (r = 0.31; p = 0.031) and macrophages (r = 0.34; p = 0.005) was found. In patients with blood glucose concentrations below the median, the thiol depletion was found to be less than in patients with blood glucose levels above the median (Table 2 ).


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  		Table 2.. Changes in Intracellular Thiol Concentration Related to the Intraoperative Blood Glucose Concentration

 
Intracellular Activity of Elastase and Cathepsins
Perioperatively, significant increases (p < 0.001) were observed in intracellular elastase and cathepsin activities in blood granulocytes. Enzymatic activities of granulocytes obtained from BALF were unchanged (Fig 4 ).



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  		Figure 4.. The intracellular activities of elastase and cathepsins in peripheral blood and BALF-derived granulocytes preoperatively (light columns) and postoperatively (dark columns) are depicted. *p < 0.001 vs preoperative values.

 
Concentration of Neutrophil Elastase in Blood Plasma and BALF
The concentration of neutrophil elastase increased by approximately ninefold in peripheral blood (p < 0.001) and 1.5-fold in BALF (p = 0.041; Fig 5 ). The increase in soluble PMN-elastase in blood 2 h after CPB correlated with CRP levels on the fifth postoperative day (r = 0.4; p < 0.001; Fig 6 ).



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  		Figure 5.. The levels of secreted elastase in peripheral blood and BALF preoperatively (light columns) and postoperatively (dark columns) are demonstrated. *p < 0.001 and **p = 0.041 vs preoperative values.

 


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  		Figure 6.. The correlation of perioperative changes in soluble elastase levels with CRP on the fifth postoperative day (left, A), and the correlation of perioperative changes in SLPI levels with CRP on the fifth postoperative day (right, B) are shown. The regression lines for peripheral blood elastase and CRP as well as BALF SLPI and CRP are bold and solid. The nonsignificant correlations of BALF elastase and peripheral blood SLPI with CRP are shown as light signs and the regression lines are plain and solid.

 
Concentration of Protease Inhibitors
Interestingly, while the changes of protease levels were more marked in blood, the alterations of {alpha}1-PI and SLPI were significantly increased in BALF only. {alpha}1-PI and SLPI levels increased in the BALF from 5.43 ± 0.8 to 8.9 ± 0.95 µg/mL (p = 0.002) and from 38,968 ± 12,047 to 51,369 ± 2,230 pg/mL (p = 0.043), respectively. SLPI elevation in BALF correlated significantly with CRP levels 5 days after operation (r = 0.47; p = 0.009). The type of operation (coronary artery bypass grafting, valvular disease, and atrial septum defect) had no influence on any of the aforementioned parameters or correlations.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the relationship of antioxidants, proteases, and antiproteases to CPB in cardiac surgery. We found intracellular thiol depletion in peripheral blood and pulmonary cells. Enhanced intracellular elastase and cathepsin activities and elevated elastase concentrations occurred in plasma and BALF. We also found increased concentrations of soluble {alpha}1-PI and SLPI in BALF. The clinical significance of these alterations in the context of the pulmonary situation and oxygen and glucose metabolism will be discussed.

Intracellular Total Thiol Content
Reduced glutathione, the most abundant intracellular nonprotein thiol, plays a critical role as an intracellular redox regulator.10 11 26 Plasma glutathione levels are approximately three orders of magnitude lower than tissue-specific cellular glutathione concentrations, and the half-life of glutathione in human plasma (1.6 min) is very short.27 The predominant role of glutathione in pulmonary homeostasis is emphasized by its high intra-alveolar concentration.28

Changes in thiol concentrations have been reported in several pathophysiologic conditions. Persistent exposure of rat lungs to inspired oxygen concentrations of 80% results in marked increases in tissue and BALF glutathione concentrations.29 This may reflect either lung injury with liberation of intracellular glutathione or an adaptive response. These elevated glutathione levels in epithelial lining fluid are associated with decreased glutathione concentrations in type-II pneumocytes.30 Oxidative stress, such as cigarette smoke, leads to altered glutathione concentrations in epithelial lining fluid. However, several studies31 32 33 34 35 36 37 38 demonstrated either a reduction or an increase of the glutathione concentration. It is thought that changes in the metabolism of the alveolar macrophages caused these effects.31 32 33 34 35 36 37 38 In patients with idiopathic pulmonary fibrosis, soluble glutathione levels are depressed whereas cellular glutathione levels remain normal.11 In contrast, increased soluble and decreased cellular glutathione levels are found in patients with COPD.33

In summary, information about the intracellular thiol concentration and the condition of the redox system within the cells is limited or is subject to controversy. This could be explained by the different analytical methods used. In addition, it could depend on the preparative limitations of pulmonary tissues and fluids. Therefore, we decided to determine the intracellular thiol content at the single cell level in distinct separated cell populations.

To measure the total cellular thiol content, we used a flow cytometric approach. Although it plays a central role, glutathione represents only part of the cellular thiol status. Therefore, a sensitive determination of cellular thiol content can reflect the cellular defense capacities better than the isolated measurement of glutathione or glutathione disulfide. Furthermore, with the flow cytometry technique, we were able to determine the cellular thiol content in the cells independently of their size. Cellular thiol levels of lymphocytes, neutrophils, and monocytes from peripheral blood were significantly decreased during CPB. To our knowledge, we were able to demonstrate for the first time an identical pattern of cellular thiol alterations in intra-alveolar lymphocytes as well as macrophages after CPB.

In contrast to other signs of systemic inflammation, eg, differential cell counts, interleukin-6 concentration and neutrophil elastase levels, changes in thiol levels were identical in BAL cells as well as in blood leukocytes. This suggests that oxidative stress plays a major pathophysiologic role in the lung during ECC. The immunologic function of all cell populations in the alveolar compartment is essentially determined by a sufficient antioxidative potential. For this reason, the observed shortage of thiols could interfere with defense against various toxins, especially infections. In accordance with our results, episodes of pneumonia after operations with CPB have been found to be more severe and carry higher mortality rates than after operations on any other organ.3

Oxygenation and Total Thiol Content
The oxygenation state of the patients correlated to the perioperative cellular thiol depletion in both peripheral blood and BAL cells. This correlation suggests that thiol depletion is caused by systemic oxygen radical stress. A significant negative correlation of PO2 to thiol depletion could be demonstrated even in lymphocytes and macrophages from the lung, which is collapsed during ECC. Obviously, in addition to the inspired oxygen, the flooding of the bronchial arteries with an excess of oxygen suppresses the antioxidative capacity.

Enhanced thiol consumption under conditions of oxidative stress during anesthesia and surgery could be partly responsible for the cellular glutathione depletion described. In addition, Adams et al39 and Sies and Akerboom40 observed glutathione disulfide export from cells in response to oxidative stress; therefore, it is no longer available for intracellular reduction to glutathione. Our results give rise to consequences for artificial ventilation as well as the management of CPB: long-lasting excess oxygenation impairs the antioxidative potential of intra-alveolar cells. Nevertheless, a comfortable oxygen supply is common in clinical practice because the oxygen levels that would avoid tissue hypoxia and acidosis have not yet been defined.

In patients with bronchial obstruction, measured by an FEV1 of < 70% of the expected volume, the correlation of thiol depletion with the intraoperative PO2 was even stronger. The intracellular thiol depletion correlated with the extent of airway obstruction. Long-term cigarette smoke inhalation leads to considerable intracellular glutathione depletion due to the production of ROIs.32 In susceptible people, nicotine abuse correlated with the development of a COPD and a reduction of the FEV1,41 which did not recover after they stopped smoking. In our study, 32 patients were cigarette smokers. We suggest that a compensated depletion of thiol sources that is present in patients with obstructive disease and/or former smokers before surgery is made worse by the ROIs. These patients seem to be unable to restore their intracellular glutathione levels under the influence of further toxins. Patients undergoing coronary artery bypass grafting for ischemic heart disease were shown42 to have a higher mortality rate not only with reduced left ventricular function, but also with an impaired preoperative FEV1. The worsening in the immunologic function of phagocytosing cells, as shown in our study, could explain the postoperative problems of patients with obstructive lung disease.

Blood Glucose and Total Thiol Content
Malmberg et al43 showed an improvement in the long-term prognosis of diabetic patients with acute myocardial infarction when treated with an intensified insulin-glucose management. Earlier, Clark et al44 suggested that improved metabolic control should reduce the complication rate and mortality in diabetic patients with acute myocardial infarction.

Due to transient cardiac ischemia in cardioplegic arrest and reperfusion, the pathophysiology of ECC resembles, to some extent, the circumstances of a myocardial infarction. In our study, perioperative hyperglycemia correlates with intracellular thiol depletion of blood and BALF lymphocytes as well as monocytes and macrophages. The clinical relevance of increased thiol depletion with higher blood glucose is becoming more apparent, as suggested by the study of Golden et al.45 They demonstrated an increased predisposition to postoperative infections after cardiac operations in patients with high blood glucose values. The impairment of the immunologic function of phagocytosing cells could explain these particular problems in hyperglycemic patients.

In a recent study by Matata and Galinanes,46 oxidative stress in terms of lipid and protein oxidation was found to be increased during CPB in patients with diabetes. These findings seem to be the logical consequence of our results, since an impaired antioxidant defense is expected to lead to more structural damage.

Concerning possible interference of oxygenation parameters and blood glucose values, we could not find any correlation of blood glucose values intraoperatively or postoperatively with either preoperative FEV1 or intraoperative PO2 values. For this reason, we postulate two processes that are not directly dependent.

Proteases and Antiproteases
Systemic markers of inflammatory response, such as the impairment of the protease-antiprotease balance, may not adequately reflect organ-related, eg, intrapulmonary, events after CPB. A discrepancy between systemic data (no significant changes in serum antiprotease levels, but highly significant eightfold increases in free PMN-elastase levels) and intra-alveolar data (significant but moderate increases in both free PMN-elastase and antiprotease amounts in BALF) supports our hypothesis of the existence of distinct compartments with distinct patterns of inflammatory responses. These patterns of expression were also reflected by the flow cytometric data concerning the intracellular PMN-elastase and cathepsin activities. While intracellular PMN-elastase and cathepsin activities were increased in granulocytes from peripheral blood, no changes could be found in cells from BALF.

Fosse et al47 could not detect intra-alveolar elastase 4 h after the conclusion of CPB. It was thought that the obtaining of the lavage sample was probably not well timed. In our study, we demonstrated that PMN-elastase was only slightly increased in BALF 2 h after CPB. These increases were much smaller than the perioperative changes found in blood plasma. Previous studies by Weiland et al48 and Rocker et al,49 who postulated a close relationship between intrapulmonary tissue injury and intra-alveolar PMN-elastase release despite peripheral neutrophil activation, were not in accordance with our data. Alternatively, our data support the primary role of PMN-elastase in pulmonary injury on the capillary endothelial level rather than on the intra-alveolar epithelial side, where PMN-elastase is buffered by an adequate antiprotease response. The endothelial damage promotes the invasion of inflammatory cells followed by liberation of cytokines.

Aprotinin is an antifibrinolytic and protease inhibitor that inhibits serine proteases via formation of reversible enzyme-inhibitor complexes. High-dose aprotinin was administered to 49 patients in our study who had received acetylsalicylic acid during the 10 days prior to the operation. Interestingly, we found that aprotinin had no influence on the parameters of protease/antiprotease balance. Hill et al50 found that low-dose aprotinin and methylprednisolone administration had similar anti-inflammatory effects in moderating specific parameters of systemic inflammation. However, it could also be the case that the application of 40 mg of dexamethasone to all patients in our study reduced the antiproteatic effects of aprotinin.

Because all of the patients in our study had subclinical organ dysfunction, our data may also have restricted significance concerning exaggerating proinflammatory developments. But even in clinically healthy patients, the extent of blood and intra-alveolar protease and antiprotease alterations correlated with inflammatory parameters (eg, CRP level on the fifth postoperative day) that have not yet been reported. The usefulness of our postoperative parameters for infection and necessity for antibiotic therapy after CPB has to be questioned.

Under the influence of systemic inflammation due to the artificial membrane contact, a considerable alteration in intrapulmonary antioxidative processes can develop. The magnitude of this alteration is determined by preceding conditions of the lung, oxygenation during CPB, and metabolic parameters (in particular, the blood glucose level). Even though it cannot be proven, our data clearly suggest a causal connection between postoperative complications after cardiac surgery and our immunologic findings. COPD and an insufficiently treated hyperglycemia are risk factors in the development of a ventilation-associated pneumonia. This makes it probable that the inflammatory mechanisms associated with CPB are comparable to those associated with ventilation-associated pneumonia. Therefore, the "model CPB" appears to be suitable for studying protection against and therapy for inflammation.


   Acknowledgements
 
We thank Annelore Ittenson, Bärbel Rösler, Marianne Blichmann, and Anke Nehring for technical assistance.


   Footnotes
 
Abbreviations: {alpha}1-PI = {alpha}1-proteinase inhibitor; BALF = BAL fluid; CMFDA = 5-chloromethylfluoresceindiacetate; CPB = cardiopulmonary bypass; CRP = C-reactive protein; ECC = extracorporeal circulation; FIO2 = fraction of inspired oxygen; MFI = mean fluorescence intensity; PMN = polymorphonuclear leukocyte; ROI = reactive oxygen intermediate; SLPI = secretory leukoprotease inhibitor; VC = vital capacity

Received for publication June 2, 2000. Accepted for publication May 2, 2001.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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